carbocyanines and 3-3--dioctadecylindocarbocyanine

The endoplasmic reticulum (ER) of eggs is a major internal store of calcium ions that must be properly mobilized at fertilization for development to proceed. In most species, the ER develops distinct clusters in the cortical ooplasm as the oocyte matures into a fertilizable egg. Following fertilization, the structure of the ER rapidly reorganizes in eggs that produce a single fertilization-induced calcium wave, whereas ER clusters persist for relatively long periods in eggs that generate multiple calcium oscillations. This review considers such pre- and post-fertilization reorganizations of the ER and what effects these changes might have on calcium signaling patterns.

Topics: Androstadienes; Animals; Calcium Signaling; Carbocyanines; Cell Cycle; Endoplasmic Reticulum; Enzyme Inhibitors; Fertilization; Fluorescent Dyes; Maturation-Promoting Factor; Ovum; Phosphoinositide-3 Kinase Inhibitors; Wortmannin

Since the first morphological description of the gap junctions use electron microscopy, a considerable number of techniques has been introduced to evaluate gap junction channel functionality, many of which use dye transfer techniques, such as dye injection and fluorescent dye transfer, analyzed by flow cytometry.. To analyze dye transfer, generally one population of cells is incubated with calcein-AM (0.5 microM) for 30 min at 37 degrees C, and the other population was incubated with the lipophilic dye DiIC(18) (3) (10 microM) for 1 h at 37 degrees C; after incubation, these cells were washed five times with PBS and cocultured for different times, and then the dye transfer was analyzed by flow cytometry.. In this short overview, we focus on some advantages and disadvantages of flow cytometry as a technique to investigate gap junction-mediated intercellular communication (GJIC). In addition, we point out some technical pitfalls that we have encountered when applying this technique to study gap junctions in immune system cells.. Analysis of fluorescent dye transfer by flow cytometry is a useful tool to investigate GJIC. However, some points must be taken into consideration before using this methodology, which are discussed herein.

Topics: Animals; Carbocyanines; Cell Communication; Cells, Cultured; Flow Cytometry; Fluoresceins; Fluorescent Dyes; Gap Junctions; Humans; Intercellular Junctions; Models, Biological

Topics: Animals; Birds; Carbocyanines; Coloring Agents; Dextrans; Embryo, Nonmammalian; Fluorescent Dyes; Iontophoresis; Microinjections

Topics: Animals; Carbocyanines; Chick Embryo; Fluorescent Dyes; Gene Expression; In Situ Hybridization; Proteins; RNA

Neural crest cells are remarkable in their extensive and stereotypic patterns of migration. The pathways of neural crest migration have been documented by cell marking techniques, including interspecific neural tube grafts, immunocytochemistry and DiI-labelling. In the trunk, neural crest cells migrate dorsally under the skin or ventrally through the somites, where they move in a segmental fashion through the rostral half of each sclerotome. The segmental migration of neural crest cells appears to be prescribed by the somites, perhaps by an inhibitory cue from the caudal half. Within the rostral sclerotome, neural crest cells fill the available space except for a region around the notochord, suggesting the notochord may inhibit neural crest cells in its vicinity. In the cranial region, antibody perturbation experiments suggest that multiple cell-matrix interactions are required for proper in vivo migration of neural crest cells. Neural crest cells utilize integrin receptors to bind to a number of extracellular matrix molecules. Substrate selective inhibition of neural crest cell attachment in vitro by integrin antibodies and antisense oligonucleotides has demonstrated that they possess at least three integrins, one being an alpha 1 beta 1 integrin which functions in the absence of divalent cations. Thus, neural crest cells utilize complex sets of interactions which may differ at different axial levels.

Topics: Animals; Carbocyanines; Cell Movement; Chick Embryo; Chimera; Embryonic and Fetal Development; Extracellular Matrix Proteins; Head; Integrins; Morphogenesis; Neural Crest; Notochord; Quail; Substrate Specificity

The fluorescent carbocyanine dyes dil and diO have an extensive history of use in cell biology, but their use as neuronal tracers is relatively recent. We found in 1985 that these molecules were excellent retrograde and anterograde tracers in the developing nervous system. We went on to show that these dyes were retained in neurons placed in culture, that they initially labelled the processes as well as the cell bodies of cultured neurons, and that they were seemingly non-toxic. We suggested that the major mechanism of translocation for these molecules was lateral diffusion in the membrane, rather than fast axonal transport. This suggestion was recently confirmed in a striking manner by Godement et al., when they showed that these dyes can be used to label axonal projections in fixed tissues. Labelling with carbocyanine dyes has already allowed several exciting advances in developmental neurobiology. In this article we review the properties of carbocyanine dyes and point out some of their uses and advantages.

Topics: Animals; Axonal Transport; Axons; Carbocyanines; Cell Membrane; Fluorescent Dyes; Microscopy, Fluorescence; Neurons; Quinolines

To test the effects of fluvastatin on low-density lipoprotein (LDL) receptor activity in patients with heterozygous familial hypercholesterolemia, the authors measured LDL receptor activity in stimulated T-lymphocytes prepared from 34 patients before and after treatment with 40 mg fluvastatin daily for 12 weeks. Maximally induced pretreatment LDL receptor activities did not correlate with pretreatment plasma cholesterol levels or with changes in plasma cholesterol levels during treatment, and there were no significant changes in LDL receptor activity during treatment. Barring methodological problems, two explanations are possible. Insofar that LDL receptor activity in lymphocytes reflects LDL receptor activity in the liver, the results suggest that the primary response to treatment with fluvastatin in heterozygous familial hypercholesterolemia (FH) patients is not enhanced LDL receptor activity. Alternatively, fluvastatin increases LDL receptor activity in hepatocytes but has little effect on receptor-dependent lipoprotein catabolism in extrahepatic tissues in vivo.

Topics: Amino Acid Substitution; Antibodies, Monoclonal; Anticholesteremic Agents; Carbocyanines; Cholesterol; Cholesterol, HDL; Cholesterol, LDL; Cross-Over Studies; Data Interpretation, Statistical; Fatty Acids, Monounsaturated; Flow Cytometry; Fluorescein-5-isothiocyanate; Fluvastatin; Heterozygote; Humans; Hyperlipoproteinemia Type II; Indoles; Lipoproteins, LDL; Mutation; Protein Binding; Receptors, LDL; T-Lymphocytes; Triglycerides

After establishing the general possibility of real-time impedance monitoring during stereotactic procedures in five postmortem human brains, the authors proceeded to analyze the value of impedance measurements in intact cerebral structures.. The authors performed impedance measurements at 24 selected points in the brain of a human cadaver and labeled the points by using 1,1'-dilinoleyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI). The texture of the nerve fibers was visualized using confocal laser scanning microscopy, which allowed classification of distinct anatomical regions in the white and gray matter according to their fiber structure (density, direction, structure, and diameter). These regions displayed characteristic differences in impedances according to their fiber structure at frequencies between 8000 and 10,000 Hz. Differences in tissue impedance are not removed, although they are shifted to higher values by fixation with formalin. The reference frequency used in clinical environments (50,000 Hz) is not useful for distinct localization purposes. Finally, the orientation of the electrode in relation to the tissue is a crucial factor.. It should be possible to draw conclusions about the correct position of the stereotactic needle on the basis of the proposed location, the texture of the fibers (orientation and density), and the orientation of the electrode.

Topics: Carbocyanines; Electric Impedance; Fluorescent Dyes; Humans; Microscopy, Confocal; Monitoring, Intraoperative; Nerve Fibers; Stereotaxic Techniques; Telencephalon

Specific interactions between viruses and host cells provide essential insights into material science-based strategies to combat emerging viral diseases. pH-triggered viral fusion is ubiquitous to multiple viral families and is important for understanding the viral infection cycle. Inspired by this process, virus detection has been achieved using nanomaterials with host-mimetic membranes, enabling interactions with amphiphilic hemagglutinin fusion peptides of viruses. Most research has been on designing functional nanoparticles with fusogenic capability for virus detection, and there has been little exploitation of the kinetic stability to alter the ability of nanoparticles to interact with viral membranes and improve their sensing performance. In this study, a homogeneous fluorescent assay using self-assembled polymeric nanoparticles (PNPs) with tunable responsiveness to external stimuli is developed for rapid and straightforward detection of an activated influenza A virus. Dissociation of PNPs induced by virus insertion can be readily controlled by varying the fraction of hydrophilic segments in copolymers constituting PNPs, giving rise to fluorescence signals within 30 min and detection of various influenza viruses, including H9N2, CA04(H1N1), H4N6, and H6N8. Therefore, the designs demonstrated in this study propose underlying approaches for utilizing engineered PNPs through modulation of their kinetic stability for direct and sensitive identification of infectious viruses.

Topics: Animals; Carbocyanines; Chickens; Eggs; Fluorescence Resonance Energy Transfer; Fluorescent Dyes; Influenza A virus; Limit of Detection; Membrane Fusion; Membranes, Artificial; Nanoparticles; Peptides; Polyethylene Glycols; Viral Fusion Proteins

Assessment of the coronary microcirculation remains challenging.. we explored the feasibility of evaluating the coronary microvasculature in rats with myocardial infarction (MI) using a three-dimensional visualization technique.. Animals were divided into the sham operation group (S), MI 45 min group (M45), and MI 180 min group (M180). Opened microvessels were labelled with the fluorescent dye DiI (1, 1'-dioctadecyl-3, 3, 3'3'-tetramethylindo carbocyanine perchlorate) using a heart perfusion method. The microvascular distribution and opening status were observed under laser scanning confocal microscopy, which was adjusted to facilitate evaluation of subjects around 6 to 20 μm.. Microvascular vessels (6-20 μm) were successfully labelled by DiI. Intact and clear three-dimensional microvascular structures were observed in myocardium of sham rats and remote non-infarct myocardial tissue of MI rats, while there was almost no microvascular structure in the infarct area of the M45 group, and only a small amount of microvascular visualization was visualized in the infarct area of the M180 group. The microvascular area and microvascular density in M45 group and M180 group in the infarct border zone were significantly lower than corresponding area in S group.. Three-dimensional visualization of opened coronary microvascular vessels is feasible in DiI-labelled myocardium in this rat MI model. This novel technique might be useful for defining the underlying mechanisms of coronary microvascular diseases and observe the efficacy of various therapy strategies on coronary microvessels.

Topics: Animals; Carbocyanines; Coronary Vessels; Disease Models, Animal; Feasibility Studies; Fluorescent Dyes; Imaging, Three-Dimensional; Microscopy, Confocal; Microscopy, Fluorescence; Microvessels; Myocardial Infarction; Predictive Value of Tests; Rats, Sprague-Dawley

PLGA (poly(lactic-co-glycolic acid))-based nanoparticles (NPs) are promising drug carrier systems because of their excellent biocompatibility and ability for sustained drug release. However, it is not well understood how the kinetics of such drug delivery system perform in the retinal blood circulation as imaged in vivo and in real time. To answer this question, PLGA NPs were loaded either with lipophilic carbocyanine perchlorate (DiI) or hydrophilic Rhodamine 123 (Rho123) and coated with poloxamer 188 (P188): PLGA-DiI/P188 and PLGA-Rho123/P188. All particles had narrow size distributions around 130 nm, spherical shape and negative potential. Subsequently, we performed in vivo real-time imaging of retinal blood vessels, combined with ex vivo microscopy to monitor the kinetics and to detect location of those two fluorescent markers. We found that DiI signals were long lasting, detectable >90 min in blood vessels after intravenous injection as visible by homogeneous labelling of the vessel wall as well as by spots in the lumen of blood vessels. In contrast, Rho123 signals mostly disappeared after 15 min post intravenous injection in such compartment. To explore how PLGA NP-loaded cargoes are released in the retina in vivo, we thereafter monitored the Cyanine5.5 amine (Cy5.5) covalently linked PLGA polymer (Cy5.5-PLGA) in parallel to DiI and Rho123. The Cy5.5 signal from PLGA polymer was detectable in the retina vessels >90 min for both, the Cy5.5-PLGA-DiI/P188 and Cy5.5-PLGA-Rho123/P188 groups. Microscopy of the ex vivo retina tissue revealed partial level of colocalization of PLGA with DiI but no colocalization between PLGA and Rho123 at 2 h post injection. This indicates that at least a fraction of the lipophilic DiI was preserved within NPs, whereas no hydrophilic Rho123 was associated with NPs at that time point. In conclusion, the properties of PLGA carrier-cargo system in the blood circulation of the retina might be strongly influenced by the combination of factors, including the individual properties of loaded compounds and blood milieu. Thus, it is unlikely that a single nanoparticle formulation will be identified that is universally effective for the delivery of different compounds.

Topics: Animals; Carbocyanines; Drug Carriers; Drug Compounding; Fluorescent Dyes; Kinetics; Male; Microscopy, Confocal; Microscopy, Fluorescence; Nanoparticles; Poloxamer; Polylactic Acid-Polyglycolic Acid Copolymer; Rats; Regional Blood Flow; Retinal Vessels; Rhodamine 123

Fluorescent organic nanoparticles (FONs) are emerging as an attractive alternative to the well-established fluorescent inorganic nanoparticles or small organic dyes. Their proper design allows one to obtain biocompatible probes with superior brightness and high photostability, although usually affected by low colloidal stability. Herein, we present a type of FONs with outstanding photophysical and physicochemical properties in-line with the stringent requirements for biomedical applications. These FONs are based on quatsome (QS) nanovesicles containing a pair of fluorescent carbocyanine molecules that give rise to Förster resonance energy transfer (FRET). Structural homogeneity, high brightness, photostability, and high FRET efficiency make these FONs a promising class of optical bioprobes. Loaded QSs have been used for in vitro bioimaging, demonstrating the nanovesicle membrane integrity after cell internalization, and the possibility to monitor the intracellular vesicle fate. Taken together, the proposed QSs loaded with a FRET pair constitute a promising platform for bioimaging and theranostics.

Topics: Animals; Carbocyanines; CHO Cells; Cholesterol; Cricetulus; Fluorescence Resonance Energy Transfer; Fluorescent Dyes; HeLa Cells; Humans; Light; Nanoparticles; Quaternary Ammonium Compounds

Midbrain dopaminergic (DA) axons make long longitudinal projections towards the striatum. Despite the importance of DA striatal innervation, processes involved in establishment of DA axonal connectivity remain largely unknown. Here we demonstrate a striatal-specific requirement of transcriptional regulator Nolz1 in establishing DA circuitry formation. DA projections are misguided and fail to innervate the striatum in both constitutive and striatal-specific Nolz1 mutant embryos. The lack of striatal Nolz1 expression results in nigral to pallidal lineage conversion of striatal projection neuron subtypes. This lineage switch alters the composition of secreted factors influencing DA axonal tract formation and renders the striatum non-permissive for dopaminergic and other forebrain tracts. Furthermore, transcriptomic analysis of wild-type and Nolz1

Topics: Animals; Axon Guidance; Axons; Carbocyanines; Corpus Striatum; Dopaminergic Neurons; Embryo, Mammalian; Female; Fluorescent Dyes; Intracellular Signaling Peptides and Proteins; Intravital Microscopy; Mice, Knockout; Microfluidic Analytical Techniques; Microinjections; Microscopy, Confocal; Nerve Net; Nerve Tissue Proteins; Tissue Culture Techniques

Here, we describe a new immersion-based clearing method suitable for optical clearing of thick adult human brain samples while preserving its lipids and lipophilic labels such as 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI). This clearing procedure is simple, easy to implement, and allowed for clearing of 5 mm thick human brain tissue samples within 12 days. Furthermore, we show for the first time the advantageous effect of the Periodate-Lysine-Paraformaldehyde (PLP) fixation as compared to the more commonly used 4% paraformaldehyde (PFA) on clearing performance.

Topics: Affinity Labels; Animals; Brain; Carbocyanines; Fluorescent Dyes; Formaldehyde; Humans; Lipids; Lysine; Mice; Periodic Acid; Swine; Tissue Fixation

Visualization of membrane domains like lipid rafts in natural or artificial membranes is a crucial task for cell biology. For this purpose, fluorescence microscopy is often used. Since fluorescing probes in lipid membranes partition specifically in e.g. local liquid disordered or liquid ordered environments, the consequent changes in their orientation and location are both theoretically and experimentally of interest. Here we focused on a liquid disordered membrane phase and performed molecular dynamics (MD) simulations of the indocarbocyanine DiD probes by varying the length of the attached alkyl tails and also the length of the cyanine backbone. From the probed compounds in a DOPC lipid bilayer at ambient temperature, a varying orientation of the transition dipole moment was observed, which is crucial for fluorescence microscopy and which, through photoselection, was found to be surprisingly more effective for asymmetric probes than for the symmetric ones. Furthermore, we observed that the orientation of the probes was dependent on the tail length; with the methyls or propyls attached, DiD oriented with its tails facing the water, contrary to the ones with longer tails. With advanced hybrid QM/MM calculations we show that the different local environment for differently oriented probes affected the one-photon absorption spectra, that was blue-shifted for the short-tailed DiD with respect to the DiDs with longer tails. We show here that the presented probes can be successfully used for fluorescence microscopy and we believe that the described properties bring further insight for the experimental use of these probes.

Topics: Carbocyanines; Fluorescent Dyes; Lipid Bilayers; Molecular Dynamics Simulation; Spectrometry, Fluorescence

Synchronized bio-distribution of combination therapies has several merits such as synergistic effects and reduced side-effects. Co-delivery of a protein and small molecule drug using a single nanocarrier is challenging because they possess totally different characteristics. Herein, we report the development of sophisticated nanoparticles composed of lipids, calcium carbonate and RGD peptide ligands for the co-delivery of a protein and small molecule drug combination via a simple preparation method. A 'one-step' ethanol injection method was employed to prepare the highly organized nanoparticles. The nanoparticles exhibited a spherical shape with ca. 130 nm diameter, and clearly had an integrated lipid layer covering the periphery. As a ligand, an RGD-modified lipid was post-inserted into the nanoparticles, which was important to overcome the 'PEG dilemma'. The pH-sensitivity of the targeted nanoparticles contributed to the efficient intracellular co-delivery of a protein and drug combination in Colon26 tumor cells, and noticeably improved their accumulation in the tumor region of xenograft mice. Synchronized bio-distribution of the protein and drug was achieved, which was the foundation for the synergistic effects of the combination. The targeting capability of the nanoparticles along with their pH-sensitive drug release and the synchronized bio-distribution of their cargos led to the significant antitumor activity of the SOD and paclitaxel combination in mice. This study provides novel information for the design and preparation of functionalized nanoparticles for the delivery of a protein/drug combination in vivo.

Topics: Animals; Antineoplastic Agents; Calcium Carbonate; Carbocyanines; Cell Line, Tumor; Cell Membrane Permeability; Drug Liberation; Drug Therapy, Combination; Fluorescent Dyes; Humans; Hydrogen-Ion Concentration; Lipids; Male; Mice; Molecular Targeted Therapy; Nanocapsules; Neoplasms, Experimental; Oligopeptides; Optical Imaging; Paclitaxel; Phosphatidylethanolamines; Serum Albumin, Bovine; Superoxide Dismutase; Surface Properties

Zebrafish thrombocytes are similar to mammalian platelets. Mammals have young platelets (also called reticulated platelets) and mature platelets. Likewise, zebrafish have 2 populations of thrombocytes; one is DiI-C

Topics: Animals; Animals, Genetically Modified; Blood Platelets; Carbocyanines; Flow Cytometry; Green Fluorescent Proteins; Lipids; Luminescent Proteins; Microscopy, Fluorescence; Phosphatidylcholines; Phosphatidylethanolamines; Red Fluorescent Protein; Single-Cell Analysis; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Zebrafish

We compared the lateral structure of giant unilamellar vesicles (GUVs) composed of three pseudo binary mixtures of different glycosphingolipid (GSL), i.e. sulfatide, asialo-GM1 or GM1, with POPC. These sphingolipids possess similar hydrophobic residues but differ in the size and charge of their polar head group. Fluorescence microscopy experiments using LAURDAN and DiIC

Topics: 2-Naphthylamine; Carbocyanines; Fluorescent Dyes; G(M1) Ganglioside; Laurates; Lipid Bilayers; Microscopy, Fluorescence; Molecular Structure; Phosphatidylcholines; Sulfoglycosphingolipids; Unilamellar Liposomes

A novel and versatile design of DNA-lipid conjugates is presented. The assembly of the DNA headgroups into G-quadruplex structures is essential for the formation of micelles and their stability. By hybridization with a complementary oligonucleotide the micelles were destabilized, resulting in cargo release. In combination with a hairpin DNA aptamer as complementary strand, the release is obtained selectively by the presence of ATP.

Topics: Adenosine Triphosphate; Animals; Aptamers, Nucleotide; Carbocyanines; Cattle; DNA; Drug Carriers; Drug Liberation; Fluorescent Dyes; G-Quadruplexes; Lipids; Micelles; Nucleic Acid Hybridization; Oxazines; Serum Albumin, Bovine

Visceral pain is initiated by activation of primary afferent neurons among which the capsaicin-sensitive (TRPV1-positive) neurons play an important role. The stomach is a common source of visceral pain. Similar to other organs, the stomach receives dual spinal and vagal afferent innervation. Developmentally, spinal dorsal root ganglia (DRG) and vagal jugular neurons originate from embryonic neural crest and vagal nodose neurons originate from placodes. In thoracic organs the neural crest- and placodes-derived TRPV1-positive neurons have distinct phenotypes differing in activation profile, neurotrophic regulation and reflex responses. It is unknown to whether such distinction exists in the stomach.. We hypothesized that gastric neural crest- and placodes-derived TRPV1-positive neurons express phenotypic markers indicative of placodes and neural crest phenotypes.. Gastric DRG and vagal neurons were retrogradely traced by DiI injected into the rat stomach wall. Single-cell RT-PCR was performed on traced gastric neurons.. Retrograde tracing demonstrated that vagal gastric neurons locate exclusively into the nodose portion of the rat jugular/petrosal/nodose complex. Gastric DRG TRPV1-positive neurons preferentially expressed markers PPT-A, TrkA and GFRα. There are phenotypic distinctions between the neural crest-derived DRG and placodes-derived vagal nodose TRPV1-positive neurons innervating the rat stomach that are similar to those described in thoracic organs.

Topics: Animals; Biomarkers; Carbocyanines; Gene Expression Regulation; Male; Neural Crest; Neurons, Afferent; Rats; Rats, Sprague-Dawley; Stomach; TRPV Cation Channels; Vagus Nerve

Kisspeptin (KP) synthesizing neurons of the hypothalamic infundibular region are critically involved in the central regulation of fertility; these cells regulate pulsatile gonadotropin-releasing hormone (GnRH) secretion and mediate sex steroid feedback signals to GnRH neurons. Fine structural analysis of the human KP system is complicated by the use of post mortem tissues. To gain better insight into the neuroanatomy of the somato-dendritic cellular compartment, we introduced the diolistic labeling of immunohistochemically identified KP neurons using a gene gun loaded with the lipophilic dye, DiI. Confocal microscopic studies of primary dendrites in 100-µm-thick tissue sections established that 79.3% of KP cells were bipolar, 14.1% were tripolar, and 6.6% were unipolar. Primary dendrites branched sparsely, contained numerous appendages (9.1 ± 1.1 spines/100 µm dendrite), and received rich innervation from GABAergic, glutamatergic, and KP-containing terminals. KP neuron synaptology was analyzed with immunoelectron microscopy on perfusion-fixed specimens. KP axons established frequent contacts and classical synapses on unlabeled, and on KP-immunoreactive somata, dendrites, and spines. Synapses were asymmetric and the presynaptic structures contained round and regular synaptic vesicles, in addition to dense-core granules. Although immunofluorescent studies failed to detect vesicular glutamate transporter isoforms in KP axons, ultrastructural characteristics of synaptic terminals suggested use of glutamatergic, in addition to peptidergic, neurotransmission. In summary, immunofluorescent and DiI labeling of KP neurons in thick hypothalamic sections and immunoelectron microscopic studies of KP-immunoreactive neurons in brains perfusion-fixed shortly post mortem allowed us to identify previously unexplored fine structural features of KP neurons in the mediobasal hypothalamus of humans.

Topics: Aged; Aged, 80 and over; Autopsy; Axons; Carbocyanines; Cell Body; Dendrites; gamma-Aminobutyric Acid; Glutamic Acid; Humans; Hypothalamus; Imaging, Three-Dimensional; Kisspeptins; Lysine; Male; Microscopy, Confocal; Microscopy, Immunoelectron; Middle Aged; Nerve Net; Neurons; Synapses; Vesicular Glutamate Transport Protein 2; Vesicular Inhibitory Amino Acid Transport Proteins

The present in-vivo study investigated the behavior and performance of differently charged poly(lactic‑co‑glycolic) acid microparticles (PLGA MP) as vaccination platform. For this purpose, particles loaded with ovalbumin (OVA) as model antigen were subcutaneously (s.c.) injected in SKH1 mice. The utilized SKH1 hairless mice exhibit a fully operative immune system and allow parallel imaging investigations due to the lack of hair. Usage of this species enabled the combination of two investigations within a single study protocol, namely noninvasive in-vivo imaging and immune responses directed towards the antigen. All treatments were well tolerated, no safety drop-outs occurred. The fate of the model antigen OVA as well as the PLGA particles was monitored using a dual dye approach (CF660C & DiR) by multispectral fluorescence imaging (msFI). A depot effect for the OVA antigen adsorbed to the MP surface could be observed for the positively charged MPs. The immune response against OVA was then analyzed. OVA alone did not induce an immune response, whereas the positively charged as well as the neutral MP induced a strong and consistent humoral immune response with a clear favor of IgG1 over IgG2a subclass antibodies. In contrast, negatively charged MP were not able to induce measurable antibody responses. Cellular immune response was weak and inconsistent for all treated groups, which verifies previous in-vitro results conducted with the herein described microparticulate antigen platform. In conclusion, the characterization of the in-vivo performance yielded valuable information about antigen and carrier fate after application. The presented adjuvant platform is capable of inducing strong T

Topics: Adjuvants, Immunologic; Animals; Antigens; Carbocyanines; Cytokines; Drug Carriers; Drug Evaluation, Preclinical; Fluorescent Dyes; Immunoglobulin G; Lactic Acid; Male; Mice, Hairless; Ovalbumin; Polyglycolic Acid; Polylactic Acid-Polyglycolic Acid Copolymer

The percutaneous passage of poorly skin absorbed molecules can be improved using nanocarriers, particularly biodegradable polymeric nanospheres (NSs) or nanocapsules (NCs). However, penetration of the encapsulated molecules may be affected by other factors than the nanocarrier properties. To gain insight information on the skin absorption of two fluorescent cargos, DiIC

Topics: Administration, Cutaneous; Animals; Carbocyanines; Coumarins; Drug Carriers; Fluorescent Dyes; Humans; Nanocapsules; Nanospheres; Skin; Skin Absorption; Swine

Topical administration is the preferred route of drug delivery for ophthalmic ailments. However, poor permeation through ocular surface and significant systemic absorption, makes the topical drug delivery challenging. Furthermore, distribution of topically delivered drugs varies with their physicochemical properties and the type of formulation used. Hence, this study was done to understand the pattern of ocular drug distribution of topically applied hydrophilic and lipophilic substances in two different formulations.. 5-Carboxyfluorescein and 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate were used as representative candidates for hydrophilic and lipophilic substances, respectively. They were formulated in solution and liposomes. Single drop of either formulation containing hydrophilic or lipophilic substance was instilled topically, unilaterally to rat eyes. Subsequently, rats were sacrificed at 10, 30 and 120 min post-instillation. Eyes were cryosectioned and examined under confocal microscope to determine the fluorescence intensity in ocular tissues.. Corneal permeation of hydrophilic and lipophilic substances in both formulations peaked at 30 min post-instillation. Liposomal-lipophilic dye and non-liposomal-hydrophilic dye showed better corneal distribution. Fluorescence was absent in contralateral eyes of non-liposomal-hydrophilic dye-treated animals but was present in contralateral eyes of liposomal-hydrophilic dye-treated animals. Fluorescence in contralateral eyes of liposomal-lipophilic dye-treated animals was significantly higher compared to non-liposomal-lipophilic dye-treated animals.. Topically applied liposomal formulation of lipophilic substance provides higher corneal concentration of drug with lesser systemic absorption compared to its solution. For hydrophilic substance, topical use of solution provides greater corneal concentration compared to liposomes which is more likely to be absorbed systemically.

Topics: Administration, Topical; Animals; Carbocyanines; Chemistry, Pharmaceutical; Cornea; Drug Carriers; Drug Delivery Systems; Fluoresceins; Hydrophobic and Hydrophilic Interactions; Liposomes; Ophthalmic Solutions; Permeability; Rats; Rats, Sprague-Dawley

We synthesized multifunctional activatible microbubbles (MAMs) for ultrasound mediated delivery of oxygen and drugs with both ultrasound and fluorescence imaging guidance. Oxygen enriched perfluorocarbon (PFC) compound was encapsulated in liposome microbubbles (MBs) by a modified emulsification process. DiI dye was loaded as a model drug. The ultrasound targeted microbubble destruction (UTMD) process was guided by both ultrasonography and fluorescence imaging modalities. The process was validated in both a dialysis membrane tube model and a porcine carotid artery model. Our experiment results show that the UTMD process effectively facilitates the controlled delivery of oxygen and drug at the disease site and that the MAM agent enables ultrasound and fluorescence imaging guidance of the UTMD process. The proposed MAM agent can be potentially used for UTMD-mediated combination therapy in hypoxic ovarian cancer.

Topics: Animals; Carbocyanines; Carotid Arteries; Drug Delivery Systems; Microbubbles; Optical Imaging; Oxygen; Phantoms, Imaging; Swine; Ultrasonography

Peripheral nerve injury leads to a regenerative state. However, the reinnervation process is highly non-selective. Growing axons are often misrouted and establish aberrant synapsis to abductor or adductor muscles. Determining the complex properties of abductor and adductor motoneurons in a neuron culture, may lay the groundwork for future studies on axon guidance, leading to a clinical treatment for a selective reinnervation.. In the present study we develop a neuron culture protocol to isolate recurrent laryngeal nerve abductor and adductor motoneurons in order to study their unique properties. Comparison with existing methods the best period to perform the present protocol for postnatal rat cranial motoneurons isolation was determined. In addition, the method allows identification of specific motoneurons from other primary motoneurons and interneurons within brainstem.. The present protocol will allow investigators to perform targeted and novel studies of the mechanisms of peripheral nerve regeneration.

Topics: Age Factors; Animals; Animals, Newborn; Carbocyanines; Cell Culture Techniques; Cells, Cultured; Efferent Pathways; Facial Nerve; Fluorescence; Medulla Oblongata; Motor Neurons; Muscles; Rats; Rats, Sprague-Dawley; Time Factors

Phase-sensitive in situ forming implants (ISFI) are a promising platform for the controlled release of therapeutic agents. The simple manufacturing, ease of placement, and diverse payload capacity make these implants an appealing delivery system for a wide range of applications. Tailoring the release profile is paramount for effective treatment of disease. In this study, three innovative formulation modifications were used to control drug release. Specifically, water, 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI), and bovine serum albumin (BSA) were incorporated into an ISFI solution containing the small molecular weight mock drug, sodium fluorescein. The effects of these additives on drug release, swelling, phase inversion, erosion, and implant microstructure were evaluated. Diagnostic ultrasound was used to monitor changes in swelling and phase inversion over time noninvasively. Water, DiI, and the combination of BSA/DiI functioned to reduce burst release 47.6%, 76.6%, and 59.0%, respectively. Incorporation of water into the casting solution also enhanced the release of drug during the diffusion period of release by 165.2% relative to the excipient free control. Incorporation of BSA into the polymer solution did not significantly alter the burst release (p < 0.05); however, the onset of degradation facilitated release was delayed relative to the excipient-free control by 5 days. This study demonstrates that the use of excipients provides a facile method to tailor the release profile and degradation rate of implants without changing the polymer or solvent used in the implant formulation, providing fine control of drug dissolution during distinct phases of release.

Topics: Carbocyanines; Chemistry, Pharmaceutical; Drug Implants; Excipients; Fluorescein; Molecular Weight; Serum Albumin, Bovine; Solubility; Ultrasonics

Major pelvic ganglia (MPG) are relay centers for autonomic reflexes such as micturition and penile erection. MPG innervate the urogenital system, including bladder. γ-Aminobutyric acid (GABA) is the major inhibitory neurotransmitter in the mammalian central nervous system, and may also play an important role in some peripheral autonomic ganglia, including MPG. However, the electrophysiological properties and function of GABAA receptor in MPG neurons innervating bladder remain unknown. This study examined the electrophysiological properties and functional roles of GABAA receptors in bladder-innervating neurons identified by retrograde Dil tracing. Neurons innervating bladder showed previously established parasympathetic properties, including small membrane capacitance, lack of T-type Ca(2+) channel expression, and tyrosine-hydroxylase immunoreactivity. GABAA receptors were functionally expressed in bladder innervating neurons, but GABAC receptors were not. GABA elicited strong depolarization followed by increase of intracellular Ca(2+) in neurons innervating bladder, supporting the hypothesis GABA may play an important role in bladder function. These results provide useful information about the autonomic function of bladder in physiological and pathological conditions.

Topics: Animals; Calcium; Carbocyanines; gamma-Aminobutyric Acid; Ganglia, Sympathetic; Male; Neurons; Patch-Clamp Techniques; Pelvis; Rats; Receptors, GABA-A; Urinary Bladder

The ability of materials to define the architecture and microenvironment experienced by cells provides new opportunities to direct the fate of human pluripotent stem cells (HPSCs) [Robinton DA, Daley GQ (2012) Nature 481(7381):295-305]. However, the conditions required for self-renewal vs. differentiation of HPSCs are different, and a single system that efficiently achieves both outcomes is not available [Giobbe GG, et al. (2012) Biotechnol Bioeng 109(12):3119-3132]. We have addressed this dual need by developing a hydrogel-based material that uses ionic de-cross-linking to remove a self-renewal permissive hydrogel (alginate) and switch to a differentiation-permissive microenvironment (collagen). Adjusting the timing of this switch can preferentially steer the HPSC differentiation to mimic lineage commitment during gastrulation to ectoderm (early switch) or mesoderm/endoderm (late switch). As an exemplar differentiated cell type, we showed that directing early lineage specification using this single system can promote cardiogenesis with increased gene expression in high-density cell populations. This work will facilitate regenerative medicine by allowing in situ HPSC expansion to be coupled with early lineage specification within defined tissue geometries.

Topics: Alginates; Carbocyanines; Cell Differentiation; Collagen; Glucuronic Acid; Hexuronic Acids; Humans; Hydrogels; Pluripotent Stem Cells; Regenerative Medicine; Spectrum Analysis; Stem Cell Niche

Mesenchymal stem cells (MSCs) have generated a great deal of promise as a potential source of cells for cell-based therapies. Various labeling techniques have been developed to trace MSC survival, migration, and behavior in vitro or in vivo. In the present study, we labeled MSCs derived from rat bone marrow (rMSCs) with florescent membrane dyes PKH67 and DiI, and with nuclear labeling using 5 μM BrdU and 10 μM BrdU. The cells were then cultured for 6 d or passaged (1-3 passages). The viability of rMSCs, efficacy of fluorescent expression, and transfer of the dyes were assessed. Intense fluorescence in rMSCs was found immediately after membrane labeling (99.3 ± 1.6% PKH67+ and 98.4 ± 1.7% DiI+) or after 2 d when tracing of nuclei was applied (91.2 ± 4.6% 10 μM BrdU+ and 77.6 ± 4.6% 5 μM BrdU+), which remained high for 6 d. Viability of labeled cells was 91 ± 3.8% PKH67+, 90 ± 1.5% DiI+, 91 ± 0.8% 5 μM BrdU+, and 76.9 ± 0.9% 10 μM BrdU+. The number of labeled rMSCs gradually decreased during the passages, with almost no BrdU+ nuclei left at final passage 3. Direct cocultures of labeled rMSCs (PKH67+ or DiI+) with unlabeled rMSCs revealed almost no dye transfer from donor to unlabeled recipient cells. Our results confirm that labeling of rMSCs with PKH67 or DiI represents a non-toxic, highly stable, and efficient method suitable for steady tracing of cells, while BrdU tracing is more appropriate for temporary labeling due to decreasing signal over time.

Topics: Animals; Bromodeoxyuridine; Carbocyanines; Flow Cytometry; Immunohistochemistry; Mesenchymal Stem Cells; Microscopy, Fluorescence; Organic Chemicals; Rats; Staining and Labeling

Multispectral optoacoustic tomography (MSOT) offers the potential to image in high-resolution cells tagged with optical labels. In contrast to single wavelength imaging, multispectral excitation and spectral unmixing can differentiate labeled moieties over tissue absorption in the absence of background measurements. This feature can enable longitudinal cellular biology studies well beyond the depths reached by optical microscopy. However, the relation between spectrally resolved fluorescently labeled cells and optoacoustic detection has not been systematically investigated. Herein, we measured titrations of fluorescently labeled cells and establish the optoacoustic signal generated by these cells as a function of cell number and across different cell types. We then assess the MSOT sensitivity to resolve cells implanted in animals.

Topics: Animals; Carbocyanines; Cell Line; Fluorescent Dyes; Humans; Immune System; Jurkat Cells; Macrophages; Mice; Optical Phenomena; Phantoms, Imaging; Photoacoustic Techniques; T-Lymphocytes; Tomography, Optical

Circulating angiogenic cells (CACs) are a heterogeneous cell population of bone marrow (BM) origin. These cells are most commonly derived from the peripheral blood, bone marrow, and cord blood, and are one of the leading candidates for promoting vascularization in tissue engineering therapies. CACs can be isolated by culturing peripheral blood mononuclear cells (PBMCs) on fibronectin or by flow cytometry to obtain more specific subpopulations. Here we will describe how to generate a population of CACs, and how to characterize the cells and confirm their phenotype. Also, we will provide select methods that can be used to assess the angiogenic and endothelial cell-like properties of the CACs.

Topics: Carbocyanines; Cell Separation; Fibronectins; Flow Cytometry; Human Umbilical Vein Endothelial Cells; Humans; Lectins; Leukocytes, Mononuclear; Neovascularization, Physiologic; Phenotype; Tissue Engineering

This study was carried out to investigate the expression of large-conductance Ca(2+)-activated potassium (BK) channels and to explore the possible modulation of BK channel activities by calcium-sensing receptors (CaSR) in rat bronchopulmonary sensory neurons. The expression of BK channels was demonstrated by immunohistochemistry and RT-PCR. Results from whole-cell patch-clamp recordings demonstrated that activation of CaSR with its agonist spermine or NPS R-568 showed a dual regulating effect on BK channel activities: it potentiated BK currents in cells exhibiting low baseline BK activity while slightly inhibited BK currents in cells with high baseline BK activity. Blocking CaSR with its antagonist NPS 2143 significantly inhibited BK currents. Our results further showed that the modulation of BK currents by CaSR activation or blockade was completely abolished when the intracellular Ca(2+) was chelated by BAPTA-AM. In summary, our data suggest that CaSR plays an integrative role in bronchopulmonary afferent signaling, at least partially through the regulation of BK channel activities.

Topics: Action Potentials; Animals; Biophysical Phenomena; Calcium; Carbocyanines; Chelating Agents; Egtazic Acid; Electric Stimulation; Ganglia, Sensory; Glomus Jugulare; Large-Conductance Calcium-Activated Potassium Channels; Naphthalenes; Patch-Clamp Techniques; Potassium Channel Blockers; Rats; Rats, Sprague-Dawley; Receptors, Calcium-Sensing; Sensory Receptor Cells; TRPV Cation Channels

The overall objective of this study is to non-invasively image and assess tumor targeting and retention of directly labeled T-lymphocytes following their adoptive transfer in mice. T-lymphocytes obtained from draining lymph nodes of 4T1 (murine breast cancer cell) sensitized BALB/C mice were activated in-vitro with Bryostatin/Ionomycin for 18 hours, and were grown in the presence of Interleukin-2 for 6 days. T-lymphocytes were then directly labeled with 1,1-dioctadecyltetramethyl indotricarbocyanine Iodide (DiR), a lipophilic near infrared fluorescent dye that labels the cell membrane. Assays for viability, proliferation, and function of labeled T-lymphocytes showed that they were unaffected by DiR labeling. The DiR labeled cells were injected via tail vein in mice bearing 4T1 tumors in the flank. In some cases labeled 4T1 specific T-lymphocytes were injected a week before 4T1 tumor cell implantation. Multi-spectral in vivo fluorescence imaging was done to subtract the autofluorescence and isolate the near infrared signal carried by the T-lymphocytes. In recipient mice with established 4T1 tumors, labeled 4T1 specific T-lymphocytes showed marked tumor retention, which peaked 6 days post infusion and persisted at the tumor site for up to 3 weeks. When 4T1 tumor cells were implanted 1-week post-infusion of labeled T-lymphocytes, T-lymphocytes responded to the immunologic challenge and accumulated at the site of 4T1 cell implantation within two hours and the signal persisted for 2 more weeks. Tumor accumulation of labeled 4T1 specific T-lymphocytes was absent in mice bearing Meth A sarcoma tumors. When lysate of 4T1 specific labeled T-lymphocytes was injected into 4T1 tumor bearing mice the near infrared signal was not detected at the tumor site. In conclusion, our validated results confirm that the near infrared signal detected at the tumor site represents the DiR labeled 4T1 specific viable T-lymphocytes and their response to immunologic challenge can be imaged in vivo.

Topics: Animals; Breast Neoplasms; Carbocyanines; Cell Line, Tumor; Cell Membrane; Cell Survival; Cell Tracking; Disease Models, Animal; Female; Immunohistochemistry; Immunotherapy, Adoptive; Interferon-gamma; Interleukin-2; Ionomycin; Lymphocyte Activation; Mice; Mice, Inbred BALB C; Spectroscopy, Near-Infrared; T-Lymphocytes; Transplantation, Homologous

Sensitization of esophageal afferents plays an important role in esophageal nociception, but the mechanism is less clear. Our previous studies demonstrated that mast cell (MC) activation releases the preformed mediators histamine and tryptase, which play important roles in sensitization of esophageal vagal nociceptive C fibers. PGD2 is a lipid mediator released by activated MCs. Whether PGD2 plays a role in this sensitization process has yet to be determined. Expression of the PGD2 DP1 and DP2 receptors in nodose ganglion neurons was determined by immunofluorescence staining, Western blotting, and RT-PCR. Extracellular recordings were performed in ex vivo esophageal-vagal preparations. Action potentials evoked by esophageal distension were compared before and after perfusion of PGD2, DP1 and DP2 receptor agonists, and MC activation, with or without pretreatment with antagonists. The effect of PGD2 on 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI)-labeled esophageal nodose neurons was determined by patch-clamp recording. Our results demonstrate that DP1 and DP2 receptor mRNA and protein were expressed mainly in small- and medium-diameter neurons in nodose ganglia. PGD2 significantly increased esophageal distension-evoked action potential discharges in esophageal nodose C fibers. The DP1 receptor agonist BW 245C mimicked this effect. PGD2 directly sensitized DiI-labeled esophageal nodose neurons by decreasing the action potential threshold. Pretreatment with the DP1 receptor antagonist BW A868C significantly inhibited PGD2 perfusion- or MC activation-induced increases in esophageal distension-evoked action potential discharges in esophageal nodose C fibers. In conclusion, PGD2 plays an important role in MC activation-induced sensitization of esophageal nodose C fibers. This adds a novel mechanism of visceral afferent sensitization.

Topics: Action Potentials; Animals; Blotting, Western; Carbocyanines; Data Interpretation, Statistical; Esophagus; Fluorescent Antibody Technique; Guinea Pigs; Male; Mast Cells; Nerve Fibers; Nerve Fibers, Unmyelinated; Neurons, Afferent; Nodose Ganglion; Patch-Clamp Techniques; Physical Stimulation; Prostaglandin D2; Real-Time Polymerase Chain Reaction; Receptors, Immunologic; Receptors, Prostaglandin; Vagus Nerve

Adipose tissue-derived stem cells (ASCs) have become the primary focus of tissue engineering research. To understand their functions and behavior in in vitro and in vivo models, it is mandatory to track the implanted cells and distinguish them from the resident or host cells. A common labeling method is the use of fluorescent dyes, e.g. the lipophilic carbocyanine dye, DiI. This study aimed to analyze potential DNA damage, toxicity and impairment of the functional properties of human ASCs after labeling with DiI.. Cytotoxicity was measured using the MTT assay and DNA damage was determined by means of the comet assay. Potential apoptotic effects were determined using the annexin V-propidium iodide test. Differentiation potential was evaluated by trilineage differentiation procedures in labeled and unlabeled ASCs. Proliferation as well as migration capability was analyzed, and the duration and stability of DiI labeling in ASCs during in vitro expansion was observed over a period of 35 days.. DiI labeling did not cause genotoxic effects 15, or 30 min or 24 h after the labeling procedure, and there were no cytotoxic effects until 72 h afterwards. No impairment of proliferation or migration capability or differentiation potential could be determined. However, after 35 days, only 37% of labeled cells could be detected using the fluorescence microscope, which indicates a decrease in staining stability during in vitro expansion.. DiI is a convenient method for ASCs labeling which causes no toxic effects and does not impair the proliferation, migration or differentiation potential of ASCs after the labeling procedure.

Topics: Adipose Tissue; Annexin A5; Carbocyanines; Cell Death; Cell Differentiation; Cell Lineage; Cell Movement; Cell Proliferation; Cell Survival; Comet Assay; DNA Damage; Humans; Phenotype; Propidium; Staining and Labeling; Stem Cells; Trypan Blue

Recent advances within materials science and its interdisciplinary applications in biomedicine have emphasized the potential of using a single multifunctional composite material for concurrent drug delivery and biomedical imaging. Here we present a novel composite material consisting of a photoluminescent nanodiamond (ND) core with a porous silica (SiO2) shell. This novel multifunctional probe serves as an alternative nanomaterial to address the existing problems with delivery and subsequent tracing of the particles. Whereas the unique optical properties of ND allows for long-term live cell imaging and tracking of cellular processes, mesoporous silica nanoparticles (MSNs) have proven to be efficient drug carriers. The advantages of both ND and MSNs were hereby integrated in the new composite material, ND@MSN. The optical properties provided by the ND core rendered the nanocomposite suitable for microscopy imaging in fluorescence and reflectance mode, as well as super-resolution microscopy as a STED label; whereas the porous silica coating provided efficient intracellular delivery capacity, especially in surface-functionalized form. This study serves as a demonstration how this novel nanomaterial can be exploited for both bioimaging and drug delivery for future theranostic applications.

Topics: Carbocyanines; Cell Survival; Drug Carriers; Fluorescent Dyes; HeLa Cells; Humans; Microscopy, Confocal; Nanodiamonds; Nanoparticles; Organophosphates; Oxazoles; Particle Size; Polyethylene Glycols; Polyethyleneimine; Porosity; Silicon Dioxide

Visceral pain is common symptom involved in many gastrointestinal disorders such as inflammatory bowel disease. The underlying molecular mechanisms remain elusive. We investigated the molecular mechanisms and the role for voltage gated calcium channel (VGCC) in the pathogenesis in a rat model of 2,4,6-trinitrobenzenesulfonic acid (TNBS) induced visceral inflammatory hypersensitivity.. Using Agilent cDNA arrays, we found 172 genes changed significantly in dorsal root ganglia (DRG) of TNBS treated rats. Among these changed genes, Cav1.2 and Cav2.3 were significantly up-regulated. Then the RT-PCR and Western blot further confirmed the up-regulation of Cav1.2 and Cav2.3. The whole cell patch clamp recording of acutely dissociated colonic specific DRG neurons showed that the peak IBa density was significantly increased in colonic neurons of TNBS treated rats compared with control rats (-127.82 ± 20.82 pA/pF Vs -91.67 ± 19.02 pA/pF, n = 9, *P < 0.05). To distinguish the different type of calcium currents with the corresponding selective channel blockers, we found that L-type (-38.56 ± 3.97 pA/pF Vs -25.75 ± 3.35 pA/pF, n = 9, * P < 0.05) and R-type (-13.31 ± 1.36 pA/pF Vs -8.60 ± 1.25 pA/pF, n = 9, * P < 0.05) calcium current density were significantly increased in colonic DRG neurons of TNBS treated rats compared with control rats. In addition, pharmacological blockade with L-type antagonist (nimodipine) and R-type antagonist (SNX-482) with intrathecal injection attenuates visceral pain in TNBS induced inflammatory visceral hypersensitivity.. Cav1.2 and Cav2.3 in colonic primary sensory neurons play an important role in visceral inflammatory hyperalgesia, which maybe the potential therapeutic targets.

Topics: Animals; Blotting, Western; Calcium Channels, L-Type; Calcium Channels, R-Type; Carbocyanines; Cation Transport Proteins; Colon; Ganglia, Spinal; Gene Expression Profiling; Hypersensitivity; Injections, Spinal; Male; Nimodipine; Protein Subunits; Rats; Rats, Sprague-Dawley; Real-Time Polymerase Chain Reaction; Spider Venoms; Trinitrobenzenesulfonic Acid; Viscera

HIV-associated neurocognitive disorders (HAND) is characterized by development of cognitive, behavioral and motor abnormalities, and occur in approximately 50% of HIV infected individuals. Our current understanding of HAND emanates mainly from HIV-1 subtype B (clade B), which is prevalent in USA and Western countries. However very little information is available on neuropathogenesis of HIV-1 subtype C (clade C) that exists in Sub-Saharan Africa and Asia. Therefore, studies to identify specific neuropathogenic mechanisms associated with HAND are worth pursuing to dissect the mechanisms underlying this modulation and to prevent HAND particularly in clade B infection. In this study, we have investigated 84 key human synaptic plasticity genes differential expression profile in clade B and clade C infected primary human astrocytes by using RT(2) Profile PCR Array human Synaptic Plasticity kit. Among these, 31 and 21 synaptic genes were significantly (≥3 fold) down-regulated and 5 genes were significantly (≥3 fold) up-regulated in clade B and clade C infected cells, respectively compared to the uninfected control astrocytes. In flow-cytometry analysis, down-regulation of postsynaptic density and dendrite spine morphology regulatory proteins (ARC, NMDAR1 and GRM1) was confirmed in both clade B and C infected primary human astrocytes and SK-N-MC neuroblastoma cells. Further, spine density and dendrite morphology changes by confocal microscopic analysis indicates significantly decreased spine density, loss of spines and decreased dendrite diameter, total dendrite and spine area in clade B infected SK-N-MC neuroblastoma cells compared to uninfected and clade C infected cells. We have also observed that, in clade B infected astrocytes, induction of apoptosis was significantly higher than in the clade C infected astrocytes. In conclusion, this study suggests that down-regulation of synaptic plasticity genes, decreased dendritic spine density and induction of apoptosis in astrocytes may contribute to the severe neuropathogenesis in clade B infection.

Topics: AIDS Dementia Complex; Apoptosis; Astrocytes; Carbocyanines; Cells, Cultured; Central Nervous System; Dendritic Spines; Down-Regulation; Flow Cytometry; HIV-1; Humans; Membrane Proteins; Microscopy, Confocal; Neuronal Plasticity; Synapses; Transcriptome

Methods to monitor transplanted stem cells in vivo are of great importance for potential therapeutic applications. Of particular interest are methods allowing noninvasive detection of stem cells throughout the body. Magnetic resonance imaging (MRI) is a tool that would allow detection of cells in nearly any tissue in the body and is already commonly used in the clinic. MRI tracking of stem cells is therefore feasible and likely to be easily adapted to patients receiving donor cells. Patients with Duchenne muscular dystrophy are good candidates for stem cell therapy, given the naturally regenerative nature of skeletal muscle, which repairs damage by employing endogenous stem cells from the muscle interstitium to regenerate muscle fibers throughout adulthood. We describe methods for labeling stem cells with superparamagnetic iron oxide nanoparticles (SPIO) to enhance MRI contrast, injecting them locally into skeletal and cardiac muscle, or systemically in mouse models for Duchenne muscular dystrophy, and tracking them in muscle tissue of live mice following injection. We focus on the use of whole body MRI to detect stem cells, as this is necessary for conditions such as muscular dystrophy, in which affected tissues are present throughout the body and systemic delivery of stem cells may be necessary. Emphasis is placed on the development of an MRI coil that is field of view (FOV) adjustable and can be used for both whole body imaging to determine stem cell localization as well as subsequent focusing on smaller, local regions where stem cells are present to obtain high-resolution images. We discuss the coil design and its significance for stem cell tracking. We also describe methods for labeling stem cells with a fluorescent dye and for tracking them in postmortem tissue specimens with fluorescent microscopy to correlate, compare, and contrast with results of whole body MRI in preclinical studies.

Topics: Animals; Carbocyanines; Cell Tracking; Cell- and Tissue-Based Therapy; Dextrans; Ferric Compounds; Injections, Intramuscular; Magnetic Resonance Imaging; Magnetite Nanoparticles; Metal Nanoparticles; Mice; Microscopy, Fluorescence; Muscle, Skeletal; Muscular Dystrophy, Duchenne; Stem Cell Transplantation; Stem Cells; Whole Body Imaging

To compare tissue distribution of dye-drug surrogates after intravitreal (IVT) and suprachoroidal (SCS) delivery to determine the influence of drug lipophilicity and choroidal circulation.. Thirty-two pig eyes were collected immediately after euthanasia. Sixteen eyes were perfused for 30 min through one long posterior ciliary artery with nondye containing nutrient media. An IVT or SCS injection was performed with either a 100 μL balanced salt solution (BSS, n=8), 1% sodium fluorescein (NaF, n=12) or 0.12% lipophilic carbocyanine dye (DiI, n=12). Globes were maintained at 37°C for 15 min, and then snap-frozen and dissected. Aqueous extraction and measurement of NaF or DiI concentration was performed using spectrophotometry and spectrofluorometry, respectively.. After SCS delivery of NaF scleral, iris-ciliary body, choroidal and vitreous dye levels were higher in nonperfused eyes compared to perfused eyes. After DiI SCS or IVT delivery, no significant differences were found in dye tissue concentrations in perfused eyes compared to nonperfused eyes. Following perfusion, a better and even drug distribution was found in the retinal pigmented epithelium (RPE)-choroid following IVT and SCS delivery of the hydrophilic drug and after IVT injection of the lipophilic drug compared to nonperfused eyes.. Choroidal circulation reduces the tissue drug concentration of the hydrophilic drug suggesting an early clearance mechanism after SCS delivery. SCS injections of lipid and hydrophilic drugs allowed direct drug delivery to the retina and RPE-choroid with limited exposition to the anterior segment.

Topics: Animals; Carbocyanines; Choroid; Ciliary Arteries; Drug Delivery Systems; Female; Fluorescein; In Vitro Techniques; Intravitreal Injections; Male; Metabolic Clearance Rate; Microcirculation; Microscopy, Fluorescence; Perfusion; Regional Blood Flow; Swine; Tissue Distribution; Vitreous Body

Influenza virus attaches to sialic acid residues on the surface of host cells via the hemagglutinin (HA), a glycoprotein expressed on the viral envelope, and enters into the cytoplasm by receptor-mediated endocytosis. The viral genome is released and transported in to the nucleus, where transcription and replication take place. However, cellular factors affecting the influenza virus infection such as the cell cycle remain uncharacterized.. To resolve the influence of cell cycle on influenza virus infection, we performed a single-virus infection analysis using optical tweezers. Using this newly developed single-virus infection system, the fluorescence-labeled influenza virus was trapped on a microchip using a laser (1064 nm) at 0.6 W, transported, and released onto individual H292 human lung epithelial cells. Interestingly, the influenza virus attached selectively to cells in the G1-phase. To clarify the molecular differences between cells in G1- and S/G2/M-phase, we performed several physical and chemical assays. Results indicated that: 1) the membranes of cells in G1-phase contained greater amounts of sialic acids (glycoproteins) than the membranes of cells in S/G2/M-phase; 2) the membrane stiffness of cells in S/G2/M-phase is more rigid than those in G1-phase by measurement using optical tweezers; and 3) S/G2/M-phase cells contained higher content of Gb3, Gb4 and GlcCer than G1-phase cells by an assay for lipid composition.. A novel single-virus infection system was developed to characterize the difference in influenza virus susceptibility between G1- and S/G2/M-phase cells. Differences in virus binding specificity were associated with alterations in the lipid composition, sialic acid content, and membrane stiffness. This single-virus infection system will be useful for studying the infection mechanisms of other viruses.

Topics: Carbocyanines; Cell Line, Tumor; Chromatography, Thin Layer; DNA Primers; Fluorescence; G1 Phase; Humans; Influenza A virus; Influenza, Human; Microchip Analytical Procedures; N-Acetylneuraminic Acid; Optical Tweezers; Reverse Transcriptase Polymerase Chain Reaction; Virus Internalization

Egg size is a correlate of larval evolution in marine embryos. Comparing species with different egg sizes that develop via similar larvae reveals the flexibility and the constraints underlying larval forms. Clypeaster rosaceus is an echinoid that develops via a facultatively planktotrophic pluteus larva. Unlike most echinoids that develop via plutei, C. rosaceus (1) has a larger egg, with a correspondingly smaller ratio of surface area to volume, and (2) forms a large left coelom early in development. Given these characteristics, we predicted underlying changes in the allocation of embryonic tissues to germ layers. With a low surface-to-volume ratio, the C. rosaceus pluteus likely requires relatively less ectoderm than a typical pluteus, whereas the early formation of a large left coelom likely requires relatively more mesoderm than a typical pluteus. We tested this hypothesis by examining the cell lineage of C. rosaceus. We found that the boundary between ectoderm and endoderm in C. rosaceus has shifted relative to echinoids with more typical planktotrophic plutei and extends to or above the third cleavage plane at the equator of the embryo. This indicates a smaller proportional allocation to ectoderm and a larger proportional allocation to endomesoderm compared to echinoids with smaller egg sizes. On the basis of this observation, we develop a new model for the transition from obligate planktotrophy to lecithotrophy. We argue that species with larger eggs may allocate proportionally more tissue to structures selected for accelerated development. In the case of C. rosaceus, the larval cell lineage apportions more cells to endomesoderm and less to ectoderm due to the smaller surface-to-volume ratio of its larger eggs and the early formation of a large left coelom.

Topics: Animals; Biological Evolution; Carbocyanines; Cell Lineage; Embryo, Nonmammalian; Feeding Behavior; Female; Fluorescent Dyes; Germ Layers; Larva; Microscopy, Confocal; Ovum; Sea Urchins; Xanthenes

In order to clarify the peripheral mechanisms of ectopic persistent pain in a tooth pulp following pulpal inflammation of an adjacent tooth, masseter muscle activity, phosphorylated extracellular signal-regulated protein kinase (pERK) and TRPV1 immunohistochemistries and satellite cell activation using glial fibrillary acidic protein (GFAP) immunohistochemistry in the trigeminal ganglion (TG) were studied in the rats with molar tooth-pulp inflammation. And, Fluorogold (FG) and DiI were also used in a neuronal tracing study to analyze if some TG neurons innervate more than one tooth pulp. Complete Freund's adjuvant (CFA) or saline was applied into the upper first molar tooth pulp (M1) in pentobarbital-anesthetized rats, and capsaicin was applied into the upper second molar tooth pulp (M2) on day 3 after the CFA or saline application. Mean EMG activity elicited in the masseter muscle by capsaicin application to M2 was significantly larger in M1 CFA-applied rats compared with M1 vehicle-applied rats. The mean number of pERK-immunoreactive (IR) TG cells was significantly larger in M1 CFA-applied rats compared with M1 vehicle-applied rats. Application of the satellite cell inhibitor fluorocitrate (FC) into TG caused a significant depression of capsaicin-induced masseter muscle activity and a significant reduction of satellite cell activation. The number of TRPV1-IR TG cells innervating M2 was significantly larger in M1 CFA-applied rats compared with M1 vehicle-applied rats, and that was decreased following FC injection into TG. Furthermore, 6% of TG neurons innervating M1 and/or M2 innervated both M1 and M2. These findings suggest that satellite cell activation following tooth pulp inflammation and innervation of multiple tooth pulps by single TG neurons may be involved in the enhancement of the activity of TG neurons innervating adjacent non-inflamed teeth that also show enhancement of TRPV1 expression in TG neurons, resulting in the ectopic persistent tooth-pulp pain following pulpal inflammation of adjacent teeth.

Topics: Animals; Capsaicin; Carbocyanines; Citrates; Dental Pulp; Extracellular Signal-Regulated MAP Kinases; Freund's Adjuvant; Glial Fibrillary Acidic Protein; Inflammation; Male; Masseter Muscle; Models, Biological; Molar; Neurons; Phosphorylation; Rats; Rats, Sprague-Dawley; Satellite Cells, Skeletal Muscle; Stilbamidines; Toothache; Trigeminal Ganglion; TRPV Cation Channels

Targeted nano-particulate systems hold extraordinary potential for delivery of therapeutics across blood brain barrier (BBB). In this work, we investigated the potential of novel bi-ligand (transferrin-poly-l-arginine) liposomal vector for delivery of desired gene to brain, in vivo. The in vivo evaluation of the delivery vectors is essential for clinical translation. We followed an innovative approach of combining transferrin receptor targeting with enhanced cell penetration to design liposomal vectors for improving the transport of molecules into brain. The biodistribution profile of 1, 1'-dioctadecyl-3,3,3',3'-tetramethyl-indocarbocyanine iodide(DiR)-labeled liposomes was evaluated in adult rats after single intravenous injection at dose of 15.2μmoles of phospholipids/kg body weight. We demonstrated that bi-ligand liposomes accumulated in rat brain at significantly (p<0.05) higher concentrations as compared to the single-ligand (transferrin) or plain liposomes. In addition, the bi-ligand liposomes resulted in increased expression of β-galactosidase(β-gal) plasmid in rat brain tissue in comparison to the single-ligand liposomes. Histological examination of the transfected tissues did not show any signs of tissue necrosis or inflammation. Hemolysis assay further authenticated the biocompatibility of bi-ligand liposomes in blood up to 600 nmoles of phospholipids/1.4×10(7) erythrocytes. The findings of this study provide important and detailed information regarding the distribution of bi-ligand liposomes in vivo and accentuate their ability to demonstrate improved brain penetration and transfection potential over single-ligand liposomes.

Topics: Animals; beta-Galactosidase; Brain; Carbocyanines; Cell-Penetrating Peptides; Erythrocytes; Fluorescent Dyes; Hemolysis; Ligands; Liposomes; Peptides; Rats; Rats, Sprague-Dawley; Tissue Distribution; Transfection; Transferrin

The microvasculature is the network of blood vessels involved in delivering nutrients and gases necessary for tissue survival. Study of the microvasculature often involves immunohistological methods. While useful for visualizing microvasculature at the µm scale in specific regions of interest, immunohistology is not well suited to visualize the global microvascular architecture in an organ. Hence, use of immunohistology precludes visualization of the entire microvasculature of an organ, and thus impedes study of global changes in the microvasculature that occur in concert with changes in tissue due to various disease states. Therefore, there is a critical need for a simple, relatively rapid technique that will facilitate visualization of the microvascular network of an entire tissue.. The systemic vasculature of a mouse is stained with the fluorescent lipophilic dye DiI using a method called "vessel painting". The brain, or other organ of interest, is harvested and fixed in 4% paraformaldehyde. The organ is then sliced into 1 mm sections and optically cleared, or made transparent, using FocusClear, a proprietary optical clearing agent. After optical clearing, the DiI-labeled tissue microvasculature is imaged using confocal fluorescence microscopy and adjacent image stacks tiled together to produce a depth-encoded map of the microvasculature in the tissue slice. We demonstrated that the use of optical clearing enhances both the tissue imaging depth and the estimate of the vascular density. Using our "optical histology" technique, we visualized microvasculature in the mouse brain to a depth of 850 µm.. Presented here are maps of the microvasculature in 1 mm thick slices of mouse brain. Using combined optical clearing and optical imaging techniques, we devised a methodology to enhance the visualization of the microvasculature in thick tissues. We believe this technique could potentially be used to generate a three-dimensional map of the microvasculature in an entire organ.

Topics: Animals; Brain; Carbocyanines; Fixatives; Fluorescent Dyes; Formaldehyde; Imaging, Three-Dimensional; Male; Mice; Microscopy, Confocal; Microscopy, Fluorescence; Microtomy; Microvessels; Polymers; Tissue Fixation

The availability of transgenic disease backgrounds and the accessibility of molecular research reagents have contributed to make the mouse ischemic hindlimb the model of choice for many studies of angiogenesis, and to investigate new treatments for peripheral artery disease. A limitation of these models involves our inability to easily visualize the regenerated vascular architecture. Approaches such as micro-computed tomography and micro-angiography are expensive, technically demanding and not available to many laboratories. Here we describe a rapid and inexpensive adaptation of a vascular staining procedure for precise imaging of the mouse hindlimb vasculature. We introduced two technical modifications and an analytical extension to the original method including (i) pre-skinning of the muscle prior to fixation that preserves tissue integrity, (ii) mild pressure-desiccation subsequent to fixing that enhances resolution and image penetration, and (iii) reconstruction of confocal data into 3D images. The procedure provides resolution that is equivalent or superior to other approaches at a fraction of the cost, time and technology required.

Topics: Animals; Carbocyanines; Hindlimb; Imaging, Three-Dimensional; Ischemia; Mice; Mice, Inbred BALB C; Microscopy, Confocal

Design of an efficient site-specific drug delivery system based on degradable functional polymers still remains challenging. In this work, we synthesized and characterized three degradable functional polyesters belonging to the poly(malic acid) family: the poly(benzyl malate) (PMLABe), the poly(ethylene glycol)-b-poly(benzyl malate) (PEG(42)-b-PMLABe), the biotin-poly(ethylene glycol)-b-poly(benzyl malate) (Biot-PEG(62)-PMLABe). Starting from these building blocks, we were able to prepare the corresponding well-defined degradable functional nanoparticles whose toxicity was evaluated in vitro on normal and cancer cell lines. Results have evidenced that the prepared nanoparticles did not show any significant cytotoxicity even at high concentrations. A model anti-cancer drug (doxorubicin, Dox) or a fluorescent probe (1,1'-dioctadecyl-3,3,3',3'-tetramethylindodicarbocyanine, DiD oil) has been encapsulated into PMLABe, PEG(42)-PMLABe or Biot-PEG(62)-PMLABe based nanoparticles in order to evaluate, respectively, the in vitro cytotoxicity of Dox-loaded nanoparticles on normal and cancer cell lines and the ligand (biotin) effect on cellular uptake in vitro using mmt 060562 cell line. Dox-loaded PMLABe, PEG(42)-PMLABe or Biot-PEG(62)-PMLABe nanoparticles showed an in vitro cytotoxicity similar to that of free Dox. Moreover, the DiD oil loaded Biot-PEG(62)-PMLABe based nanoparticles showed a better in vitro cellular uptake than ligand-free DiD oil loaded nanoparticles. Both results evidence the great potential of such degradable functional poly(malic acid) derivatives for the design of highly efficient site-specific anti-cancer nanovectors.

Topics: Animals; Antineoplastic Agents; Biotin; Carbocyanines; Cell Line, Transformed; Cell Line, Tumor; Cell Survival; Doxorubicin; Drug Delivery Systems; Fibroblasts; Fluorescent Dyes; Humans; Malates; Mice; Molecular Structure; Nanoparticles; Particle Size; Polyethylene Glycols; Polymers; Static Electricity; Surface Properties

Noninvasive near-infrared (NIR) fluorescence imaging is a promising technique for the intraoperative assessment of solid tumor removal. We incorporated a lipophilic NIR probe, 1,1'-dioctadecyltetramethyl indotricarbocyanine iodide (DiR), in poly(ethylene glycol)-b-poly(ɛ-caprolactone) (PEG-b-PCL) micelles, resulting in DiR solubilization in water, occupying nanoscopic PEG-b-PCL micelles. DiR in a self-quenched or nonquenched state showed different kinetics of release from PEG-b-PCL micelles in vitro; however, both obtained high tumor delineation (tumor-to-muscle ratio of 30-43 from collected organs). These results suggest that PEG-b-PCL micelles with DiR are a promising nanosized imaging agent that will provide a basis for enhanced surgical guidance via NIR visualization of tumors.. In this paper, noninvasive near-infrared fluorescence imaging coupled with specific lipophilic probes is discussed as a promising technique for intraoperative assessment of solid tumor removal, leading to optimized outcomes for in toto removal of tumors.

Topics: Carbocyanines; Diagnostic Imaging; Fluorescent Dyes; Humans; Micelles; Neoplasms; Particle Size; Polyesters; Polyethylene Glycols; Solubility; Water

The biodistribution of intravenously injected DNA lipid nanocapsules (DNA LNCs), encapsulating pHSV-tk, was analysed by in vivo imaging on an orthotopic melanoma mouse model and by a subsequent treatment with ganciclovir (GCV), using the gene-directed enzyme prodrug therapy (GDEPT) approach. Luminescent melanoma cells, implanted subcutaneously in the right flank of the mice, allowed us to follow tumour growth and tumour localisation with in vivo bioluminescence imaging (BLI). In parallel, DNA LNCs or PEG DNA LNCs (DNA LNCs recovered with PEG(2000)) encapsulating a fluorescent probe, DiD, allowed us to follow their biodistribution with in vivo biofluorescence imaging (BFI). The BF-images confirmed a prolonged circulation-time for PEG DNA LNCs as was previously observed on an ectotopic model of glioma; comparison with BL-images evidenced the colocalisation of PEG DNA LNCs and melanoma cells. After these promising results, treatment with PEG DNA LNCs and GCV on a few animals was performed and the treatment efficacy measured by BLI. The first results showed tumour growth reduction tendency and, once optimised, this therapy strategy could become a new option for melanoma treatment.

Topics: Animals; Benzothiazoles; Carbocyanines; DNA; Electrophoresis, Agar Gel; Fatty Acids, Monounsaturated; Female; Fluorescent Dyes; Ganciclovir; Gene Transfer Techniques; Genes, Transgenic, Suicide; Glycerol; Herpes Simplex; Humans; Lipids; Luciferases; Melanoma-Specific Antigens; Melanoma, Experimental; Mice; Mice, Nude; Molecular Imaging; Nanocapsules; Octoxynol; Oleic Acids; Particle Size; Phosphatidylethanolamines; Plasmids; Polyethylene Glycols; Quaternary Ammonium Compounds; Static Electricity; Stearic Acids; Surface Properties; Thymidine Kinase; Tissue Distribution; Treatment Outcome; Triglycerides; Xenograft Model Antitumor Assays

Mesenchymal stem cells (MSCs) have been shown to improve cardiac electrophysiology when administered in the setting of acute myocardial infarction. However, the electrophysiological phenotype of MSCs in situ is not clear. We hypothesize that MSCs delivered intramyocardially to cryoinjured myocardium can engraft, but will not actively generate, action potentials. Cryoinjury-induced scar was created on the left ventricular epicardial surface of adult rat hearts. Within 30 min, hearts were injected with saline (sham, n = 11) or bone marrow-derived MSCs (2 × 10(6)) labeled with 1,1'-dioctadecyl-3,3,3,3'-tetramethylindocarbocyanine percholate (DiI; n = 16). At 3 wk, optical mapping and cell isolation were used to measure optical action potentials and calcium transients, respectively. Histological analysis confirmed subepicardial scar thickness and the presence of DiI-positive cells that express connexin-43. Optical action potential amplitude within the scar at MSC-positive sites (53.8 ± 14.3%) was larger compared with sites devoid of MSCs (35.3 ± 14.2%, P < 0.05) and sites within the scar of shams (33.5 ± 6.9%, P < 0.05). Evidence of simultaneous action potential upstroke, the loss of action potential activity following ablation of adjacent viable myocardium, and no rapid calcium transient response in isolated DiI+ cells suggest that the electrophysiological influence of engrafted MSCs is electrotonic. MSCs can engraft when directly injected into a cryoinjury and are associated with evidence of action potential activity. However, our results suggest that this activity is not due to generation of action potentials, but rather passive influence coupled from neighboring viable myocardium.

Topics: Action Potentials; Animals; Calcium Signaling; Carbocyanines; Cell Communication; Connexin 43; Cryosurgery; Disease Models, Animal; Fluorescent Dyes; Heart Conduction System; Heart Ventricles; Male; Mesenchymal Stem Cell Transplantation; Myocardial Infarction; Myocardium; Rats; Rats, Inbred Lew; Regeneration; Time Factors; Voltage-Sensitive Dye Imaging

Mutations have been identified in a non-canonical Wnt signalling cascade (the planar cell polarity pathway) in several mouse genetic models of severe neural tube defects. In each of these models, neurulation fails to be initiated at the 3-4 somite stage, leading to an almost entirely open neural tube (termed craniorachischisis). Studies in whole embryo culture have identified a defect in the morphogenetic process of convergent extension during gastrulation, preceding the onset of neural tube closure. The principal defect is a failure of midline extension, both in the neural plate and axial mesoderm. This leads to an abnormally wide neural plate in which the elevating neural folds are too far apart to achieve closure. In this chapter, we provide details of several experimental methods that can be used to evaluate convergent extension in cultured mouse embryos. We describe analytical methods that can reveal the abnormalities that characterise neurulation-stage embryos with defective planar cell polarity signalling, in particular the loop-tail (Lp; Vangl2) mutant.

Topics: Animals; Carbocyanines; Cell Polarity; Dissection; Electroporation; Embryo Culture Techniques; Embryo, Mammalian; Female; Genetic Vectors; Green Fluorescent Proteins; Mice; Mutation; Nerve Tissue Proteins; Neural Plate; Neural Tube; Neurulation; Paraffin Embedding; Phenotype; Pregnancy

To understand a possible role for transient potential receptor vanilloid 1 (TRPV1) ion channels in sumatriptan relief of pain mediated by trigeminal nociceptors.. TRPV1 channels are expressed in small nociceptive sensory neurons. In dorsal root ganglia, TRPV1-containing nociceptors mediate certain types of inflammatory pain. Neurogenic inflammation of cerebral dura and blood vessels in the trigeminal nociceptive system is thought to be important in migraine pain, but the ion channels important in transducing migraine pain are not known. Sumatriptan is an agent effective in treatment of migraine and cluster headache. We hypothesized that sumatriptan might modulate activity of TRPV1 channels found in the trigeminal nociceptive system.. We used immunohistochemistry to detect the presence of TRPV1 channel protein, whole-cell recording in acutely dissociated trigeminal ganglia (TG) to detect functionality of TRPV1 channels, and whole-cell recording in trigeminal nucleus caudalis (TNC) to detect effects on release of neurotransmitters from trigeminal neurons onto second order sensory neurons. Effects specifically on TG neurons that project to cerebral dura were assessed by labeling dural nociceptors with DiI.. Immunohistochemistry demonstrated that TRPV1 channels are present in cerebral dura, in trigeminal ganglion, and in the TNC. Capsaicin, a TRPV1 agonist, produced depolarization and repetitive action potential firing in current clamp recordings, and large inward currents in voltage clamp recordings from acutely dissociated TG neurons, demonstrating that TRPV1 channels are functional in trigeminal neurons. Capsaicin increased spontaneous excitatory postsynaptic currents in neurons of layer II in TNC slices, showing that these channels have a physiological effect on central synaptic transmission. Sumatriptan (10 µM), a selective antimigraine drug, inhibited TRPV1-mediated inward currents in TG and capsaicin-elicited spontaneous excitatory postsynaptic currents in TNC slices. The same effects of capsaicin and sumatriptan were found in acutely dissociated DiI-labeled TG neurons innervating cerebral dura.. Our results build on previous work indicating that TRPV1 channels in trigeminal nociceptors play a role in craniofacial pain. Our findings that TRPV1 is inhibited by the specific antimigraine drug sumatriptan, and that TRPV1 channels are functional in neurons projecting to cerebral dura suggests a specific role for these channels in migraine or cluster headache.

Topics: Action Potentials; Analysis of Variance; Animals; Animals, Newborn; Capsaicin; Carbocyanines; Dura Mater; Electric Stimulation; Excitatory Postsynaptic Potentials; Female; Gene Expression Regulation; In Vitro Techniques; Male; Neurons; Patch-Clamp Techniques; Rats; Rats, Sprague-Dawley; Serotonin 5-HT1 Receptor Agonists; Sumatriptan; Trigeminal Ganglion; TRPV Cation Channels

The cuticle of C. elegans is a highly resistant structure that surrounds the exterior of the animal(1-4). The cuticle not only protects the animal from the environment, but also determines body shape and plays a role in motility(4-6). Several layers secreted by epidermal cells comprise the cuticle, including an outermost lipid layer(7). Circumferential ridges in the cuticle called annuli pattern the length of the animal and are present during all stages of development(8). Alae are longitudinal ridges that are present during specific stages of development, including L1, dauer, and adult stages(2,9). Mutations in genes that affect cuticular collagen organization can alter cuticular structure and animal body morphology(5,6,10,11). While cuticular imaging using compound microscopy with DIC optics is possible, current methods that highlight cuticular structures include fluorescent transgene expression(12), antibody staining(13), and electron microscopy(1). Labeled wheat germ agglutinin (WGA) has also been used to visualize cuticular glycoproteins, but is limited in resolving finer cuticular structures(14). Staining of cuticular surface using fluorescent dye has been observed, but never characterized in detail(15). We present a method to visualize cuticle in live C. elegans using the red fluorescent lipophilic dye DiI (1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate), which is commonly used in C. elegans to visualize environmentally exposed neurons. This optimized protocol for DiI staining is a simple, robust method for high resolution fluorescent visualization of annuli, alae, vulva, male tail, and hermaphrodite tail spike in C. elegans.

Topics: Animals; Animals, Genetically Modified; Caenorhabditis elegans; Carbocyanines; Female; Fluorescent Dyes; Male; Staining and Labeling

Topics: Carbocyanines; Cell Membrane; Erythrocytes; Fluorescence; Fluorescent Dyes; HeLa Cells; Humans; Lasers

Fluorescence correlation spectroscopy (FCS) measurements are widely used for determination of diffusion coefficients of lipids and proteins in biological membranes. In recent years, several variants of FCS have been introduced. However, a comprehensive comparison of these methods on identical systems has so far been lacking. In addition, there exist no consistent values of already determined diffusion coefficients for well-known or widely used membrane systems. This study aims to contribute to a better comparability of FCS experiments on membranes by determining the absolute diffusion coefficient of the fluorescent lipid analog 1,1'-dioctadecyl-3,3,3',3'-tetramethylindodicarbocyanine (DiD) in giant unilamellar vesicles (GUVs) made of dioleoylphosphatidylcholine (DOPC), which can in future studies be used as a reference value. For this purpose, five FCS variants, employing different calibration methods, were compared. Potential error sources for each particular FCS method and strategies to avoid them are discussed. The obtained absolute diffusion coefficients for DiD in DOPC were in good agreement for all investigated FCS variants. An average diffusion coefficient of D = 10.0 ± 0.4 μm(2) s(-1) at 23.5 ± 1.5 °C was obtained. The independent confirmation with different methods indicates that this value can be safely used for calibration purposes. Moreover, the comparability of the methods also in the case of slow diffusion was verified by measuring diffusion coefficients of DiD in GUVs consisting of DOPC and cholesterol.

Topics: Carbocyanines; Diffusion; Phosphatidylcholines; Spectrometry, Fluorescence

Alterations in dendritic spines have been documented in numerous neurodevelopmental disorders, including Rett Syndrome (RTT). RTT, an X chromosome-linked disorder associated with mutations in MECP2, is the leading cause of intellectual disabilities in women. Neurons in Mecp2-deficient mice show lower dendritic spine density in several brain regions. To better understand the role of MeCP2 on excitatory spine synapses, we analyzed dendritic spines of CA1 pyramidal neurons in the hippocampus of Mecp2(tm1.1Jae) male mutant mice by either confocal microscopy or electron microscopy (EM). At postnatal-day 7 (P7), well before the onset of RTT-like symptoms, CA1 pyramidal neurons from mutant mice showed lower dendritic spine density than those from wildtype littermates. On the other hand, at P15 or later showing characteristic RTT-like symptoms, dendritic spine density did not differ between mutant and wildtype neurons. Consistently, stereological analyses at the EM level revealed similar densities of asymmetric spine synapses in CA1 stratum radiatum of symptomatic mutant and wildtype littermates. These results raise caution regarding the use of dendritic spine density in hippocampal neurons as a phenotypic endpoint for the evaluation of therapeutic interventions in symptomatic Mecp2-deficient mice. However, they underscore the potential role of MeCP2 in the maintenance of excitatory spine synapses.

Topics: Animals; CA1 Region, Hippocampal; Carbocyanines; Cell Count; Coloring Agents; Dendritic Spines; Endpoint Determination; Male; Methyl-CpG-Binding Protein 2; Mice; Mice, Inbred C57BL; Mice, Knockout; Microscopy, Confocal; Microscopy, Electron; Pyramidal Cells; Rett Syndrome; Synapses; X Chromosome

The aim of this research is to study the dynamics and efficiency of liposome accumulation in sensitive and resistant human breast cancer cells.. Methods of fluorescence microscopy, fluorescence microspectroscopy and MTT-test have been used.. The liposome-to-cell interaction and dye cellular uptake in sensitive, cisplatin-resistant and doxorubicin-resistant MCF-7 human breast cancer cells have been analyzed using time changes in both fluorescence resonance energy transfer signal from the donor probe DiO to the acceptor one DiI preloaded in liposomes and cell image brightness.. Obtained results show that resistant cells accumulate dye-loaded liposomes more effectively and reveal more effective dye molecule cellular uptake.

Topics: Antineoplastic Agents; Breast Neoplasms; Carbocyanines; Cisplatin; Doxorubicin; Drug Carriers; Drug Resistance, Neoplasm; Fluorescence Resonance Energy Transfer; Fluorescent Dyes; Humans; Lipid Bilayers; Liposomes; MCF-7 Cells; Microscopy, Fluorescence; Molecular Structure; Phosphatidylcholines; Spectrometry, Fluorescence; Tissue Distribution

Commissural dI1 neurons have been extensively studied to elucidate the mechanisms underlying axon guidance during development(1,2). These neurons are located in the dorsal spinal cord and send their axons along stereotyped trajectories. Commissural axons initially project ventrally towards and then across the floorplate. After crossing the midline, these axons make a sharp rostral turn and project longitudinally towards the brain. Each of these steps is regulated by the coordinated activities of attractive and repulsive guidance cues. The correct interpretation of these cues is crucial to the guidance of axons along their demarcated pathway. Thus, the physiological contribution of a particular molecule to commissural axon guidance is ideally investigated in the context of the living embryo. Accordingly, gene knockdown in vivo must be precisely controlled in order to carefully distinguish axon guidance activities of genes that may play multiple roles during development. Here, we describe a method to knockdown gene expression in the chicken neural tube in a cell type-specific, traceable manner. We use novel plasmid vectors(3) harboring cell type-specific promoters/enhancers that drive the expression of a fluorescent protein marker, followed directly by a miR30-RNAi transcript(4) (located within the 3'-UTR of the cDNA encoding the fluorescent protein) (Figure 1). When electroporated into the developing neural tube, these vectors elicit efficient downregulation of gene expression and express bright fluorescent marker proteins to enable direct tracing of the cells experiencing knockdown(3). Mixing different RNAi vectors prior to electroporation allows the simultaneous knockdown of two or more genes in independent regions of the spinal cord. This permits complex cellular and molecular interactions to be examined during development, in a manner that is fast, simple, precise and inexpensive. In combination with DiI tracing of commissural axon trajectories in open-book preparations(5), this method is a useful tool for in vivo studies of the cellular and molecular mechanisms of commissural axon growth and guidance. In principle, any promoter/enhancer could be used, potentially making the technique more widely applicable for in vivo studies of gene function during development(6). This video first demonstrates how to handle and window eggs, the injection of DNA plasmids into the neural tube and the electroporation procedure. To investigate commissural axon guidance,

Topics: Animals; Carbocyanines; Chick Embryo; DNA; Electroporation; Fluorescent Dyes; Gene Knockdown Techniques; MicroRNAs; Neural Tube; Phenotype; Plasmids

Integumentary sensory organs (ISOs) are densely distributed on the jaws of crocodilians and on body scales of members of the families Crocodilidae and Gavialidae. We examined the distribution, anatomy, innervation and response properties of ISOs on the face and body of crocodilians and documented related behaviors for an alligatorid (Alligator mississippiensis) and a crocodylid (Crocodylus niloticus). Each of the ISOs (roughly 4000 in A. mississippiensis and 9000 in C. niloticus) was innervated by networks of afferents supplying multiple different mechanoreceptors. Electrophysiological recordings from the trigeminal ganglion and peripheral nerves were made to isolate single-unit receptive fields and to test possible osmoreceptive and electroreceptive functions. Multiple small (<0.1 mm(2)) receptive fields, often from a single ISO, were recorded from the premaxilla, the rostral dentary, the gingivae and the distal digits. These responded to a median threshold of 0.08 mN. The less densely innervated caudal margins of the jaws had larger receptive fields (>100 mm(2)) and higher thresholds (13.725 mN). Rapidly adapting, slowly adapting type I and slowly adapting type II responses were identified based on neuronal responses. Several rapidly adapting units responded maximally to vibrations at 20-35 Hz, consistent with reports of the ISOs' role in detecting prey-generated water surface ripples. Despite crocodilians' armored bodies, the ISOs imparted a mechanical sensitivity exceeding that of primate fingertips. We conclude that crocodilian ISOs have diverse functions, including detection of water movements, indicating when to bite based on direct contact of pursued prey, and fine tactile discrimination of items held in the jaws.

Topics: Alligators and Crocodiles; Animals; Azo Compounds; Carbocyanines; Mechanotransduction, Cellular; Microscopy, Confocal; Microscopy, Electron, Scanning; Naphthalenes; Neurons, Afferent; Peripheral Nerves; Predatory Behavior; Sense Organs; Skin; Skin Physiological Phenomena; Trigeminal Ganglion

On the basis of the fact of the overexpression of integrins in malignant tumor cells and neovasculature, and the advantage of polymeric micelles (PM) as the drug carriers, a cyclic RGD peptide (cRGDyK) was anchored on the surface of polyethylene glycol-b-poly(lactic-co-glycolic acid) (PEG-b-PLGA) micelles as a ligand of integrins in order to enhance the intracellular delivery of encapsulated hydrophobic drug into the tumor cells and its neovasculature. Toward this goal, PEG-b-PLGA micelles without or with cRGDyK conjugation loaded with paclitaxel (PTX) or DiI were prepared and characterized. The results revealed that drug-loaded micelles were stable in solution, with small diameters (<80 nm) and a low critical micelle concentration. Spectrophotofluorometry, confocal microscopy, and flow cytometry showed that cRGDyK-conjugated micelles (TPM) facilitated the cell-specific uptake of DiI into the murine melanoma B16-F10 cells and human umbilical vein endothelial cells (HUVEC) via integrin-mediated endocytosis compared with cRGDyK-free micelles (NPM), and the uptake was proportional to the ratio of cRGDyK modification in certain range. Meanwhile, PTX-loaded TPM displayed higher cytotoxicity and antiproliferation activities against both cells than PTX-loaded NPM. These results suggest that cRGDyK-coupled PEG-b-PLGA micelles may be the promising intracellular targeting carriers for efficient delivery of chemotherapeutic agents into tumor cells and neovasculature.

Topics: Animals; Carbocyanines; Cell Line, Tumor; Drug Carriers; Drug Delivery Systems; Endocytosis; Endothelial Cells; Flow Cytometry; Fluorescent Dyes; Humans; Integrins; Melanoma; Mice; Micelles; Microscopy, Confocal; Paclitaxel; Particle Size; Peptides, Cyclic; Polyethylene Glycols; Polyglactin 910

Membrane tension modulates cellular processes by initiating changes in the dynamics of its molecular constituents. To quantify the precise relationship between tension, structural properties of the membrane, and the dynamics of lipids and a lipophilic reporter dye, we performed atomistic molecular dynamics (MD) simulations of DiI-labeled dipalmitoylphosphatidylcholine (DPPC) lipid bilayers under physiological lateral tensions ranging from -2.6 mN m(-1) to 15.9 mN m(-1). Simulations showed that the bilayer thickness decreased linearly with tension consistent with volume-incompressibility, and this thinning was facilitated by a significant increase in acyl chain interdigitation at the bilayer midplane and spreading of the acyl chains. Tension caused a significant drop in the bilayer's peak electrostatic potential, which correlated with the strong reordering of water and lipid dipoles. For the low tension regime, the DPPC lateral diffusion coefficient increased with increasing tension in accordance with free-area theory. For larger tensions, free area theory broke down due to tension-induced changes in molecular shape and friction. Simulated DiI rotational and lateral diffusion coefficients were lower than those of DPPC but increased with tension in a manner similar to DPPC. Direct correlation of membrane order and viscosity near the DiI chromophore, which was just under the DPPC headgroup, indicated that measured DiI fluorescence lifetime, which is reported to decrease with decreasing lipid order, is likely to be a good reporter of tension-induced decreases in lipid headgroup viscosity. Together, these results offer new molecular-level insights into membrane tension-related mechanotransduction and into the utility of DiI in characterizing tension-induced changes in lipid packing.

Topics: 1,2-Dipalmitoylphosphatidylcholine; Biomechanical Phenomena; Carbocyanines; Cell Membrane; Diffusion; Lipid Bilayers; Mechanical Phenomena; Mechanotransduction, Cellular; Molecular Conformation; Molecular Dynamics Simulation; Spectrometry, Fluorescence; Static Electricity; Viscosity

To clarify whether use of angiogenic vessel-homing peptide, Ala-Pro-Arg-Pro-Gly (APRPG)-modified liposomes encapsulating 3-([2,4-dimethylpyrrhol-5-yl] methylidenyl)-indolin-2-one (SU5416), an angiogenesis inhibitor, can inhibit the development of experimental choroidal neovascularization (CNV) in rats.. Liposomes were prepared using the thin-film hydration method. To set up the rat experimental CNV model, intense fundus laser photocoagulation at 6 spots per eye was performed on pigmented rats. After photocoagulation, the rats were divided into 4 groups (6 rats in each group): an APRPG-liposomal SU5416 treatment group and control groups treated with a balanced salt solution, APRPG liposomes, or soluble SU5416. Each rat received a single intravitreal injection immediately after the injury. One week or 2 weeks after laser injury, the extent of CNV was evaluated by perfusion with high-molecular-weight fluorescein isothiocyanate-dextran.. Two weeks after injection, the CNV area was significantly (P < .05) smaller in the APRPG-liposomal SU5416-treated group compared with the CNV area in the balanced salt solution-and APRPG liposome-treated groups.. Liposomes modified by APRPG and encapsulating SU5416 constitute a potential drug formulation for CNV treatment that would require only a single intravitreal injection.. This liposomal delivery may enable the sustained release of small molecules and be a new treatment modality for CNV.

Topics: Angiogenesis Inhibitors; Animals; Carbocyanines; Choroidal Neovascularization; Dextrans; Disease Models, Animal; Fluorescein-5-isothiocyanate; Fluorescent Dyes; Indoles; Intravitreal Injections; Liposomes; Male; Oligopeptides; Protein-Tyrosine Kinases; Pyrroles; Rats; Rats, Inbred BN

Neutrophil recruitment to sites of inflammation involves P-selectin-dependent rolling. qDF is a useful tool to visualize the topography of the neutrophil footprint as it interacts with the substrate. However, elucidating the role of specific proteins in addition to topography requires simultaneous visualization of two fluorochromes.. To validate DqDF, mouse neutrophils were labeled with the membrane dyes DiO and DiI and perfused into microchannels coated with P-selectin-Fc. Footprints of rolling neutrophils were recorded as two separate images, one for each fluorochrome. To assess the localization of the cytoskeletal protein paxillin, we applied DqDF to DiO-stained neutrophils of mice expressing an mCherry-paxillin fusion protein.. The footprint topographies obtained from DiO and DiI in the plasma membrane were identical. The z-coordinates of the microvilli tips obtained with the two fluorochromes in the footprint were also identical. Paxillin was found to be localized to some, but not all ridges in the neutrophil footprint.. Our data suggest that the spectral properties of the fluorochrome do not affect the results. DqDF will be useful for simultaneous visualization of two fluorochromes in the footprint of rolling cells.

Topics: Animals; Carbocyanines; Fluorescent Dyes; Leukocyte Rolling; Mice; Mice, Transgenic; Microscopy, Fluorescence; Neutrophils; Paxillin; Recombinant Fusion Proteins

Atherosclerosis (AS) is a common cardiovascular disease. Transformation of macrophages to form foam cells by internalizing modified low density-lipoprotein (LDL) via scavenger receptor (SR) is a key pathogenic process in the onset of AS. It has been demonstrated that SR-PSOX functions as either a scavenger receptor for uptake of atherogenic lipoproteins and bacteria or a membrane-anchored chemokine for adhesion of macrophages and T-cells to the endothelium. Therefore, SR-PSOX plays an important role in the development of AS. In this study the key basic amino acids in the chemokine domain of SR-PSOX have been identified for its functions.. A cell model to study the functions of SR-PSOX was successfully established. Based on the cell model, a series of mutants of human SR-PSOX were constructed by replacing the single basic amino acid residue in the non-conservative region of the chemokine domain (arginine 62, arginine 78, histidine 80, arginine 82, histidine 85, lysine 105, lysine 119, histidine 123) with alanine (designated as R62A, R78A, H80A, R82A, H85A, K105A, K119A and H123A, respectively). Functional studies showed that the mutants with H80A, H85A, and K105A significantly increased the activities of oxLDL uptake and bacterial phagocytosis compared with the wild-type SR-PSOX. In addition, we have also found that mutagenesis of either of those amino acids strongly reduced the adhesive activity of SR-PSOX by using a highly non-overlapping set of basic amino acid residues.. Our study demonstrates that basic amino acid residues in the non-conservative region of the chemokine domain of SR-PSOX are critical for its functions. Mutation of H80, H85, and K105 is responsible for increasing SR-PSOX binding with oxLDL and bacteria. All the basic amino acids in this region are important in the cells adhesion via SR-PSOX. These findings suggest that mutagenesis of the basic amino acids in the chemokine domain of SR-PSOX may contribute to atherogenesis.

Topics: Amino Acid Substitution; Amino Acids, Basic; Carbocyanines; Cell Adhesion; Cell Line; Cell Membrane; Chemokine CXCL16; Chemokines, CXC; Escherichia coli; Genetic Vectors; Green Fluorescent Proteins; Humans; Lipoproteins, LDL; Mutant Proteins; Phagocytosis; Point Mutation; Receptors, Scavenger; Transfection

Idiopathic pulmonary fibrosis is a devastating disease characterized by a progressive, irreversible, and ultimately lethal form of lung fibrosis. Except for lung transplantation, no effective treatment options currently exist. The bleomycin animal model is one of the best studied models of lung injury and fibrosis. A previous study using mouse tumor models observed that liposome-encapsulated bleomycin exhibited reduced lung toxicity. Therefore, we hypothesized that airway delivery of synthetic phosphatidylcholine-containing liposomes alone would protect mice from bleomycin-induced lung toxicity. C57BL/6 mice were administered uncharged multilamellar liposomes (100 μl) or PBS vehicle on day 0 by airway delivery. Bleomycin (3.33 U/kg) or saline vehicle was then given intratracheally on day 1 followed by four additional separate doses of liposomes on days 4, 8, 12, and 16. Fluorescent images of liposomes labeled with 1,1'-dioctadecyl-3,3,3',3' tetramethylindocarbocyanine perchlorate confirmed effective and widespread delivery of liposomes to the lower respiratory tract as well as uptake primarily by alveolar macrophages and to a lesser extent by type II alveolar epithelial cells. Results at day 22, 3 wk after bleomycin treatment, showed that airway delivery of liposomes before and after intratracheal administration of bleomycin significantly reduced bleomycin-induced lung toxicity as evidenced by less body weight loss, chronic lung inflammation, and fibrosis as well as improved lung compliance compared with controls. These data indicate that airway-delivered synthetic liposomes represent a novel treatment strategy to reduce the lung toxicity associated with bleomycin in a mouse model.

Topics: Administration, Inhalation; Animals; Bleomycin; Carbocyanines; Chronic Disease; Female; Fluorescent Dyes; Intubation, Intratracheal; Liposomes; Lung; Mice; Mice, Inbred C57BL; Pneumonia; Pulmonary Fibrosis; Weight Loss

The cell-impermeant oligomer-(e.g. beta-amyloid-, or tubulin-) specific fluorescent dye, bis-ANS (4,4'-bis-1-anilinonaphtalene-8-sulfonate), was successfully used for labeling mechanically damaged but still viable neuron bodies, neurites and neurite cross sections in acute brain slices. Acute hippocampal brain slices of rats were co-stained with bis-ANS and the cell-impermeant, DNA-specific dye propidium iodide (PI) and were then analyzed using fluorescence and confocal microscopes. Both the neuron bodies and the neurites were found to exhibit increased fluorescence intensities, suggesting that using this method they can be detected more easily. In addition, bis-ANS showed good region - but not cell specific co-localization with the neuron-specific fluorescent dye Dil (1,1'-Dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate). These two dyes label different neuronal structures: Dil binds specifically to intact cell membranes while bis-ANS can enter cells with compromised cell membranes and then stain the microtubules in the cytoplasm. For a quick (10min) staining of acute brain slices with bis-ANS both HEPES and NaHCO(3) were needed in order to achieve high signal intensity. Labeling with bis-ANS fluorescent dye is an easy method for imaging the neuronal structures on the surface of acute brain slices.

Topics: Anilino Naphthalenesulfonates; Animals; Brain; Carbocyanines; Cell Line, Tumor; Cell Membrane; Coloring Agents; Cytoplasm; Fluorescent Dyes; Male; Microscopy, Confocal; Microscopy, Fluorescence; Microtubules; Neurons; Propidium; Rats; Rats, Wistar; Staining and Labeling; Tubulin

A variety of approaches can be applied to investigate the multiple steps and interactions that occur during virus entry into the host cell. Single-virus tracking is a powerful real-time imaging technique that offers the possibility to monitor virus-cell binding, internalization, intracellular trafficking behavior, and the moment of membrane fusion of single virus particles in living cells. Here we describe the development and applications of a single-virus tracking assay based on the use of DiD-labeled dengue virus (DENV) in BS-C-1 cells. In addition - and using the same experimental setup - we present a binding and fusion assay that can be used to obtain a rapid insight into the relative extent of virus binding to the cell surface and membrane fusion. Details of virus labeling and characterization, microscopy setup, protocols, data analysis, and hints for troubleshooting are described throughout the paper.

Topics: Animals; Carbocyanines; Cells, Cultured; Dengue Virus; Endocytosis; Fluorescent Dyes; Microscopy, Fluorescence; Microscopy, Video; Staining and Labeling; Virion; Virus Internalization

Plasma lipoproteins are thought to transport cholesterol, vitamins and carotenoids to the retinal pigment epithelium (RPE) for ultimate use by the photoreceptors. However, to reach the RPE, these lipoprotein particles must cross Bruch's membrane. We examined the reflection coefficient of Bruch's membrane (BrM) to low-density lipoprotein (LDL). Bruch's membrane and choroid were removed from 47 bovine eyes. Specimens were placed in a Ussing chamber and perfused with phosphate-buffered saline (PBS) with (31 specimens) or without (16 specimens) fluorescent low-density lipoproteins (DiI-LDL). The hydraulic conductivity of the tissue was determined for both calf and cow eyes. In the perfusions with DiI-LDL, the fluorescence intensity emitted by DiI-LDL in the efflux was measured and the reflection coefficient of BrM/choroid preparations to DiI-LDL determined. Leakage tests were done to confirm tissue integrity. Several specimens were examined using scanning electron microscopy (SEM) to examine tissue integrity before and after perfusion. Leak testing confirmed that BrM was intact both before and after perfusion. The average hydraulic conductivity of BrM/choroid perfusion of calf eyes with PBS alone was 1.42 ± 0.55 × 10(-9) m/s/Pa (mean ± SD, n = 11). The average hydraulic conductivity of the cow eyes was 4.94 ± 1.48 × 10(-10) m/s/Pa (n = 5), nearly a 3-fold decrease with age. While the flow rate remained constant during the PBS perfusions, it decreased as a function of time during perfusion with DiI-LDLs. Our major finding was of fluorescence in the effluent collected in all perfusions with DiI-LDLs, demonstrating passage of LDL through the tissue. The average reflection coefficient of calf BrM/choroid preparations to DiI-LDL was 0.58 ± 0.25 (n = 23); a similar distribution of reflection coefficients was seen in tissue from cow eyes (0.51 ± 0.33, n = 8). Our data suggested that the DiI-LDL was modestly hindered and/or captured by the tissue. This might explain the progressive decrease of hydraulic conductivity with continued perfusion of DiI-LDL.

Topics: Animals; Biological Transport; Bruch Membrane; Carbocyanines; Cattle; Choroid; Fluorescent Dyes; Kinetics; Lipoproteins, LDL; Microscopy, Electron, Scanning; Models, Biological; Perfusion; Permeability; Spectrometry, Fluorescence

Here we present a technique to label the trajectories of small groups of DRG neurons into the embryonic spinal cord by diffusive staining using the lipophilic tracer 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI). The comparison of axonal pathways of wild-type with those of mouse lines in which genes are mutated allows testing for a functional role of candidate proteins in the control of axonal branching which is an essential mechanism in the wiring of the nervous system. Axonal branching enables an individual neuron to connect with multiple targets, thereby providing the physical basis for the parallel processing of information. Ramifications at intermediate target regions of axonal growth may be distinguished from terminal arborization. Furthermore, different modes of axonal branch formation may be classified depending on whether branching results from the activities of the growth cone (splitting or delayed branching) or from the budding of collaterals from the axon shaft in a process called interstitial branching (Fig. 1). The central projections of neurons from the DRG offer a useful experimental system to study both types of axonal branching: when their afferent axons reach the dorsal root entry zone (DREZ) of the spinal cord between embryonic days 10 to 13 (E10-E13) they display a stereotyped pattern of T- or Y-shaped bifurcation. The two resulting daughter axons then proceed in rostral or caudal directions, respectively, at the dorsolateral margin of the cord and only after a waiting period collaterals sprout from these stem axons to penetrate the gray matter (interstitial branching) and project to relay neurons in specific laminae of the spinal cord where they further arborize (terminal branching). DiI tracings have revealed growth cones at the dorsal root entry zone of the spinal cord that appeared to be in the process of splitting suggesting that bifurcation is caused by splitting of the growth cone itself (Fig. 2), however, other options have been discussed as well. This video demonstrates first how to dissect the spinal cord of E12.5 mice leaving the DRG attached. Following fixation of the specimen tiny amounts of DiI are applied to DRG using glass needles pulled from capillary tubes. After an incubation step, the labeled spinal cord is mounted as an inverted open-book preparation to analyze individual axons using fluorescence microscopy.

Topics: Animals; Axons; Carbocyanines; Fluorescent Dyes; Ganglia, Spinal; Mice; Microscopy, Fluorescence; Sensory Receptor Cells; Spinal Cord; Staining and Labeling

The corneal epithelium is one of the most highly innervated structures in the body, and proper innervation is necessary for corneal maintenance and sensation. However, little is known about how these nerves function and how innervation occurs developmentally. The authors have examined certain aspects of corneal innervation in the developing chicken embryo.. DiI was used to determine the source of the neurons responsible for innervating the cornea. Immunohistochemistry, electron microscopy, and immunoelectron microscopy were used to examine corneal innervation and the relationships that develop between nerves and corneal epithelial cells.. Corneal nerves in the embryonic chicken originate entirely from the ophthalmic lobe of the trigeminal ganglion. Within the cornea the nerves interact with apical corneal epithelial (ACE) cells to form specialized structures that are synapse-like because they contain accumulations of vesicles and have the SV2 synaptic vesicle protein. These ACE cells themselves have unique characteristics, including transient expression of the neuronal isoform of class III beta-tubulin and formation of extensive intercellular channels and clefts that contain these specialized synapse-like structures and nerves; in addition, they are mitotically active. Given that these ACE cells react with a monoclonal antibody against this neuronal isoform of beta-tubulin (the TuJ-1 antibody), we have termed them TuJ-1(+)ACE cells.. During avian corneal development the nerves make close associations with a specialized type of ACE cell. There they form synapse-like structures, suggesting that not all nerves within the CE terminate as free nerve endings.

Topics: Animals; Biomarkers; Carbocyanines; Chick Embryo; Cornea; Epithelium, Corneal; Fluorescent Antibody Technique, Indirect; Fluorescent Dyes; Microscopy, Confocal; Microscopy, Immunoelectron; Nerve Fibers; Nerve Tissue Proteins; Reverse Transcriptase Polymerase Chain Reaction; Synapses; Trigeminal Ganglion; Tubulin

Lipid nanoparticles and liposomal carrier systems are of growing interest for intravenous drug delivery due to their biocompatibility and targetability. It is, however, difficult to investigate their release behavior for lipophilic drugs under physiological conditions. This study describes a novel flow cytometric method studying drug transfer from such carrier systems to particles simulating physiological receptor sites. For this purpose, liquid and solid trimyristin nanoparticles or soybean phospholipid liposomes were loaded with the lipophilic fluorescent substances Nile red, temoporfin, and DiI. The transfer of these model drugs to large emulsion droplets was examined by flow cytometry. Transfer of DiI to differently sized acceptor emulsions was also monitored by separating donor and acceptor particles using ultracentrifugation. Flow cytometry revealed a completion of transfer within a few minutes for Nile red and temoporfin at considerable amounts of transferred dye. In contrast, the highly lipophilic DiI transferred over a period of weeks only for a small percentage of the dye. Ultracentrifugation results confirmed this for DiI and indicated a dependence of transfer characteristics on the acceptor surface area. Nile red transfer into a bulk oil phase as alternative acceptor system was also very slow. Flow cytometry seems to be well suited to study the intrinsic transfer of fluorescent lipophilic substances, as no kinetic hindrances like dialysis bags nor separation steps are required. Additional detailed experiments will, however, be necessary to elucidate the prevalent transfer mechanisms completely.

Topics: Carbocyanines; Drug Carriers; Emulsions; Flow Cytometry; Liposomes; Mesoporphyrins; Nanoparticles; Oxazines; Particle Size

Three-dimensional molecular orientations of single fluorescence molecules in polymeric thin films were measured by focused azimuthally and radially polarized light, in which we found that the fluorescence intensity was dependent on the depth position of the molecule with respect to the film surface. We found that the fluorescence intensity for a molecule which is 80 nm deep in the film excited by radial polarization is appreciably larger when compared with the fluorescence intensity for a molecule which is also excited by radial polarization but which is closer to the polymer/air interface, a feature which leads to different fluorescence intensities, under excitation by radial polarization, for molecules with the same polar orientation but with different depths inside the film. We also found that the variation of fluorescence intensity from a molecule inside an 80 nm film in radial polarization is appreciably larger compared with one in azimuthal polarization. These findings were confirmed by comparing experiments using different thickness films with theoretically calculated electric field distributions.

Topics: Carbocyanines; Fluorescence; Free Radicals; Membranes, Artificial; Optics and Photonics; Surface Properties

Topics: Animals; Blastocyst; Carbocyanines; Cell Culture Techniques; Cell Proliferation; Embryo, Mammalian; Female; Fluorescent Dyes; Gastrula; Rabbits; Staining and Labeling; Uterus; Zona Pellucida

By counteracting the shortening of chromosome telomeres, telomerase reverse transcriptase (hTERT) prevents senescence and age-related cell death. Embryonic cells display a high telomerase activity that declines rapidly with cell differentiation. Conversely, de-differentiated tumor cells tend to re-express telomerase. In view of the controversial data on the reciprocal correlation between cell proliferation and differentiation, we questioned whether telomerase overexpression and the resulting immortalization would affect the functional phenotype of human endothelial cells. Our comparative analysis addressed (1) distinct cell adhesion to different ECM-proteins analyzed on miniaturized multisubstrate arrays (MSA), (2) protein expression of diverse markers, (3) the uptake of DiI-Ac-LDL, (4) the inflammatory response based on upregulation of ICAM-1, (5) tube formation, and (6) the barrier properties of cell monolayers in transfilter cultures. Our results, based on some 40 data sets, demonstrate that immortalization of primary endothelial cells by hTERT maintains the typical endothelial characteristics without any sign of functional de-differentiation.

Topics: Carbocyanines; Cell Adhesion Molecules; Cell Dedifferentiation; Cell Movement; Cell Transformation, Neoplastic; Cells, Cultured; Cloning, Molecular; Endothelial Cells; Humans; Intercellular Adhesion Molecule-1; Interferon-gamma; Lipoproteins, LDL; Permeability; Proteins; Telomerase; Transduction, Genetic; Tumor Necrosis Factor-alpha; Vascular Endothelial Growth Factor A; von Willebrand Factor

Here we evaluate an improved orthotopic rat bladder tumor model, to be used for the evaluation of the therapeutic potential of novel cancer therapeutics. Before instilling AY-27 tumor cells into chemically denudated rat bladders, AY-27 cells were labeled with the fluorescent carbocyanine dye 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine (DiI). We found that the presence of Dil did not alter the in vitro AY-27 cell proliferation and that the Dil label was strongly associated with the cells. We further provide evidence that the use of fluorescently labeled AY-27 tumor cells allows the visualization and hence the validation of the orthotopic tumor inoculation process. Using this technique it was possible to track down the tumor cells after inoculation into the bladder, which makes it straightforward to distinguish tumor cells from remaining or regenerated normal urothelium over a period of 5 d. The results also demonstrated that malignant AY-27 tissue exists as an intact non-muscle invasive bladder tumor only for 1-3 d after cell implantation. Accordingly the AY-27 bladder tumor model was used to evaluate the antitumoral effect of a single intravesical MM-C instillation. All rats instilled with 1 mM MM-C survived the final endpoint of 30 d after intravesical MM-C. Moreover, 10 and 30 d after treatment the urothelium of the MM-C-treated animals was completely restored. Remarkably, after MM-C treatment distinct patchy fluorescent dots were found into the submucosa and the regenerated urothelium, suggesting that dye retention is secondary to the digestion of Dil-loaded AY-27 cells and cellular debris by macrophages and related immune cells.

Topics: Animals; Antibiotics, Antineoplastic; Carbocyanines; Cell Line, Tumor; Disease Models, Animal; Female; Fluorescence; Fluorescent Dyes; Mitomycin; Rats; Rats, Inbred F344; Staining and Labeling; Urinary Bladder Neoplasms; Xenograft Model Antitumor Assays

Several studies have shown functional improvements, neuroprotective, and neuroregenerative effects after mesenchymal stem cells transplantation to parkinsonian animal models. However, questions remain about the safety, feasibility, and long-term efficacy of this approach. In this study, we investigated migration, therapeutic, tumorigenesis, and epileptogenic effects of human umbilical cord mesenchymal stromal cells (HUMSCs) 1 year after transplantation into rotenone-induced hemiparkinsonian rats. Our data indicated that DiI-labeled HUMSCs migrated in the lesioned hemisphere, from corpus striatum (CPu) to substantia nigra. By integrating with host cells and differentiating into NSE, GFAP, Nestin, and tyrosine hydroxylase-positive cells, HUMSCs prevented 48.4% dopamine neurons from degeneration and 56.9% dopamine terminals from loss, both correlating with improvement of apomorphine-induced rotations. The CD50 and CD97 value of pentylenetetrazol and semiquantitative immunohistochemical analysis of proliferating cell nuclear antigen (PCNA), β-catenin, C-myc, and NF-κB expression showed no significant difference between HUMSCs transplanted and untransplanted groups, whereas the expressions of Bcl-2 and P53 in the grafted CPu were upregulated by 281% and 200% compared to ungrafted CPu. The results of this long-term study suggest that HUMSCs transplantation, 1 of the most potential treatments for Parkinson's disease, is an effective and safe approach.

Topics: Adaptor Proteins, Signal Transducing; Animals; Apomorphine; Brain; Carbocyanines; Cell Differentiation; Cell Movement; Corpus Striatum; Dose-Response Relationship, Drug; Epilepsy; Female; Humans; Intermediate Filament Proteins; Locomotion; Mesenchymal Stem Cell Transplantation; Mesenchymal Stem Cells; Neoplasm Proteins; Nerve Tissue Proteins; Nestin; Neurons; Parkinsonian Disorders; Pentylenetetrazole; Phosphopyruvate Hydratase; Rats; Rats, Sprague-Dawley; Rotation; Rotenone; Staining and Labeling; Substantia Nigra; Treatment Outcome; Tyrosine 3-Monooxygenase; Umbilical Cord; Ventral Tegmental Area

The aim of this study was to elucidate which of the following two factors plays a more important role in the localization of atherogenesis: the barrier function of the arterial endothelium modulated by wall shear stress or flow-dependent low-density lipoprotein (LDL) concentration at the blood/wall interface. To determine this, the rabbit aorto-iliac bifurcation was perfused with 3,3'-dioctadecylindocarbocyanine (DiI)-LDL solution under three different flow conditions: (i) forward flow (perfused in the in vivo flow direction); (ii) backward flow (perfused in a reversed flow direction); and (iii) static group (no flow). The results showed that there was a peak in the curve of DiI-LDL uptake distribution along the lateral wall of the bifurcation for all three groups, which was located in the branching areas where the endothelial cells were round and polygonal with no preferred orientation. Nevertheless, the peak of the forward flow group was much sharper than those of the other two groups. The overall DiI-LDL uptake was the highest for the static group. The present experimental study supports the concept that both the barrier function of the endothelium modulated by wall shear stress and the mass transport phenomenon of LDL concentration polarization are involved in the infiltration/accumulation of atherogenic lipids within the arterial wall. Nevertheless, the latter might play a larger role in the localization of atherogenesis.

Topics: Animals; Aorta; Aorta, Abdominal; Atherosclerosis; Biological Transport; Carbocyanines; Cholesterol, LDL; Endothelial Cells; Endothelium, Vascular; Lipoproteins, LDL; Male; Perfusion; Rabbits; Stress, Mechanical

The intracellular vesicle-mediated degradation of extracellular cargo is an essential cellular function. Using two-color single particle tracking fluorescence microscopy, we have probed the intracellular degradation of low-density lipoprotein (LDL) in living cells. To detect degradation, individual LDL particles were heavily labeled with multiple fluorophores resulting in a quenched fluorescent signal. The degradation of the LDL particle then resulted in an increase in fluorescence. Endocytic vesicles were fluorescently labeled with variants of GFP. We imaged the transient colocalization of LDL with endocytic vesicles while simultaneously measuring the intensity of the LDL particle as an indicator of degradation. These studies demonstrate that late endosomes are active sites of degradation for LDL. Measurement of the time from colocalization with lysosome-associated membrane protein 1 (LAMP1) vesicles to degradation suggests that LAMP1-vesicles initiate the degradative event. Observing degradation as it occurs in living cells makes it possible to describe the complete endocytic pathway of LDL from internalization to degradation. More generally, this research provides a model for the intracellular degradation of extracellular cargo and a method for its study in living cells.

Topics: Androstadienes; Animals; Carbocyanines; Cathepsin B; Cell Line; Chlorocebus aethiops; Endocytosis; Endosomes; Epithelial Cells; Humans; Intracellular Space; Kinetics; Lipoproteins, LDL; Luminescent Proteins; Lysosomal-Associated Membrane Protein 1; Lysosomes; Microscopy, Fluorescence; Protein Transport; rab GTP-Binding Proteins; rab5 GTP-Binding Proteins; rab7 GTP-Binding Proteins; Transfection; Trypsin; Wortmannin

Quantitative assessment of microvascular structure is relevant to the investigations of ischemic injury, reparative angiogenesis and tumor revascularization. In light microscopy applications, thick tissue specimens are necessary to characterize microvascular networks; however, thick tissue leads to image distortions due to out-of-focus light. Structured illumination confocal microscopy is an optical sectioning technique that improves contrast and resolution by using a grid pattern to identify the plane-of-focus within the specimen. Because structured illumination can be applied to wide-field (nonscanning) microscopes, the microcirculation can be studied by sequential intravital and confocal microscopy. To assess the application of structured illumination confocal microscopy to microvessel imaging, we studied cell-sized microspheres and fused silica microcapillary tissue phantoms. As expected, structured illumination produced highly accurate images in the lateral (X-Y) plane, but demonstrated a loss of resolution in the Z-Y plane. Because the magnitude of Z-axis distortion was variable in complex tissues, the silica microcapillaries were used as spatial calibration standards. Morphometric parameters, such as shape factor, were used to empirically optimize Z-axis software compression. We conclude that the silica microcapillaries provide a useful tissue phantom for in vitro studies as well as spatial calibration standard for in vivo morphometry of the microcirculation.

Topics: Animals; Calibration; Capillaries; Carbocyanines; Data Interpretation, Statistical; Image Processing, Computer-Assisted; Male; Mice; Mice, Inbred C57BL; Microcirculation; Microscopy, Confocal; Microscopy, Fluorescence; Microspheres; Microvessels; Nonlinear Dynamics; Phantoms, Imaging; Silicon Dioxide; Software

To substantiate our hypothesis that concentration polarization of low-density lipoprotein (LDL) plays an important role in the localization of atherogenesis, we investigated the effects of wall shear stress and water filtration rate (or perfusion pressure) on the luminal surface LDL concentration (c(w)) and the LDL uptake by human vascular endothelial cells and smooth muscle cells co-cultured on a permeable membrane using a parallel-plate flow chamber technique and a flow cytometry method. The results indicated that the uptake of fluorescent labeled LDL (DiI-LDL) by the co-cultured cells was positively correlated with c(w) in a non-linear fashion. When c(w) was low, the uptake increased very sharply with increasing c(w). Then the increase became gradual and the uptake was seemingly leveled out when c(w) reached beyond 160 microg/ml. The present study therefore has provided further experimental evidence that concentration polarization may occur in the arterial system and have a positive correlation with the uptake of LDLs by the arterial wall, which gives support to our hypothesis regarding the localization of atherogenesis.

Topics: Atherosclerosis; Carbocyanines; Coculture Techniques; Endothelial Cells; Flow Cytometry; Fluorescent Dyes; Humans; Lipoproteins, LDL; Models, Cardiovascular; Myocytes, Smooth Muscle; Perfusion; Pressure; Rheology; Stress, Mechanical; Tunica Intima

We investigated the pathways of afferent fibers innervating the lumbar spine. The neurotracer DiI was applied to reference sites at the L5 level in rats. One of 4 surgeries was performed before DiI application: (1) transaction of the dorsal ramus of the L2 spinal nerve, (2) transaction of the ventral ramus of the L2 spinal nerve, (3) transaction of the psoas major muscle at L3-L4, or (4) removal of the paravertebral sympathetic trunks from L3-L5. The number of DiI-labeled neurons in the dorsal root ganglia after surgery was compared with neuron numbers in surgery-naïve rats. The number of DiI-labeled neurons decreased drastically with transection of the L2 ventral ramus or psoas major muscle for the ventral and lateral portions of the disc and vertebral body and after transection of the L2 dorsal ramus for the facet joint and spinous process. Removal of the sympathetic trunks did not reduce the number of DiI-labeled neurons significantly in the extra-spinal canal sites. In contrast, significant reductions occurred after the removal of the paravertebral sympathetic trunks in the intra-spinal canal sites. Extra-spinal canal sites received afferent fibers primarily through somatic routes, but intra-spinal canal sites received afferent fibers via the sympathetic trunks.. Extra-spinal canal sites of the lumbar spine received afferent fibers from muscles originating in the site. Intra-spinal canal sites received a considerable number of afferent fibers via the paravertebral sympathetic trunks. These results may provide new insights for nerve block treatment of low back pain.

Topics: Afferent Pathways; Affinity Labels; Animals; Carbocyanines; Cell Count; Ganglia, Spinal; Lumbar Vertebrae; Male; Neurons, Afferent; Peripheral Nerves; Rats; Rats, Sprague-Dawley; Spinal Nerves

After partial optic nerve (ON) injury, intact retinal ganglion cells (RGCs) undergo secondary death, but the topographic distribution of this death is unknown, and it is unclear which cell death pathways are involved. Although the calcium channel blocker lomerizine reduces RGC death after partial ON injury, it is unknown whether this drug alleviates necrotic or apoptotic death.. The dorsal ON was transected in adult Piebald-Virol-Glaxo (PVG) rats, and the site of secondary RGC death was determined using anterograde and retrograde DiI tracing. RGC death was assessed at 2 and 3 weeks. Retrograde tracing with fluorogold injected into the superior colliculus 3 days before euthanatization was used to identify RGCs undergoing secondary death. Overall cell loss was quantified using betaIII-tubulin immunohistochemistry. Lomerizine (30 mg/kg, oral) or vehicle was given twice daily, and retinal wholemounts were analyzed for necrotic morphology (nucleic acid stain) or anticleaved caspase-3 expression at 2 and 3 weeks.. Ventral retina was identified as the site of secondary RGC death, and central and dorsal retinae were defined as sites of both primary and secondary death. Overall RGC loss occurred by 2 weeks in central and ventral retina (P < 0.05) and by 3 weeks in dorsal retina (P < 0.05). Secondary RGC death was characterized mainly by necrotic morphology, with caspase-3 expression in some RGCs. Lomerizine reduced secondary necrosis at 2 weeks and secondary caspase-3 expression at 3 weeks.. Lomerizine had differential effects on necrotic and apoptotic death with time, but its inability to completely prevent secondary death suggests that full neuroprotection will require combinatorial treatments.

Topics: Animals; Calcium Channel Blockers; Carbocyanines; Caspase 3; Cell Count; Cell Death; Cell Survival; Female; Fluorescent Antibody Technique, Indirect; Fluorescent Dyes; Neuroprotective Agents; Optic Nerve Injuries; Piperazines; Rats; Retinal Diseases; Retinal Ganglion Cells; Tubulin

The uptake of the 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI)-labeled sex-unspecific Nereis lipoprotein was investigated in oocytes of the nereidid polychaetes Nereis virens and Platynereis dumerilii. The fluorescence label was first observed in endocytic vesicles (<1 microm diameter), which later fused to larger vesicles (2-3 microm); these were finally incorporated into existing unlabeled yolk granules (5-6 microm). In Platynereis oocytes, the fusion of endocytic vesicles was delayed in oocytes at their final stage of development compared with those at an early stage of development. Lipoprotein double-labeled with fluorescein isothiocyanate (FITC) and DiI revealed that both the protein and the lipid moiety remained co-localized during incorporation into the yolk granules of the oocyte. No labeling of the cytoplasmic lipid droplets was observed. In N. virens, unlabeled Nereis lipoprotein was effective as a competitive inhibitor of DiI-labeled Nereis lipoprotein. Ligand blot experiments demonstrated the presence of a lipoprotein receptor with an apparent molecular mass of 120 kDa, which is different from that of the known yolk protein receptor. This indicates the presence, in the polychaete oocyte, of two distinct receptors mediating yolk protein and lipoprotein uptake, respectively. Thus, the sex-unspecific lipoprotein contributes to the lipid supply of the growing oocyte in addition to the known uptake of the yolk-protein-associated lipids. The absence of label in the cytoplasmic lipid droplets, even after prolonged incubation with labeled lipoprotein, suggests that these lipids arise either by the breakdown and resynthesis of lipoprotein-derived lipids and/or by de novo synthesis within the oocyte.

Topics: Animals; Annelida; Carbocyanines; Fluorescent Dyes; Lipoproteins; Oocytes; Receptors, Lipoprotein

Intertaxonomic comparisons are important for understanding neurogenesis and evolution of nervous systems, but high-resolution, cellular studies of early central nervous system development and the molecular mechanisms controlling this process in lophotrochozoans are still lacking. We provide a detailed cellular and molecular description of early brain neurogenesis in a lophotrochozoan annelid, Capitella sp. I. We utilized different approaches including DiI lineage tracing, immunohistochemistry, BrdU labeling, and gene expression analyses to characterize neural precursor cells in Capitella sp. I. Brain neurogenesis proceeds by the ingression of single cells from the anterior ectoderm to generate a stratified epithelial layer. Most cell divisions are restricted to apically localized cells with mitotic spindles oriented parallel to the epithelial layer. Prior to and during this process, an achaete-scute complex homolog, CapI-ash1, is expressed in clusters of surface cells in the anterior ectoderm, consistent with a proneural function for CapI-ash1. In contrast, a homolog of the neural differentiation marker elav, CapI-elav1, is restricted to basally localized cells within the forming brain. Unlike insects, Capitella sp. I does not have morphologically obvious enlarged neuroblasts, although Capitella sp. I brain neurogenesis displays several similarities with non-insect arthropod and vertebrate neurogenesis, providing a useful lophotrochozoan model for comparison.

Topics: Animals; Body Patterning; Brain; Carbocyanines; Cell Division; Cell Lineage; Cell Proliferation; Fluorescent Dyes; Intracellular Signaling Peptides and Proteins; Membrane Proteins; Neurogenesis; Neurons; Polychaeta; Receptors, Notch; Spindle Apparatus; Stem Cells

The epicardium contributes to the majority of nonmyocardial cells in the adult heart. Recent studies have reported that the epicardium is derived from Nkx2.5-positive progenitors and can differentiate into cardiomyocytes. Not much is known about the relation between the myocardial and epicardial lineage during development, whereas insights into these embryonic mechanisms could facilitate the design of future regenerative strategies.. Acquiring insight into the signaling pathways involved in the lineage separation leading to the differentiation of myocardial and (pro)epicardial cells at the inflow of the developing heart.. We made 3D reconstructions of Tbx18 gene expression patterns to give insight into the developing epicardium in relation to the developing myocardium. Next, using DiI tracing, we show that the (pro)epicardium separates from the same precursor pool as the inflow myocardium. In vitro, we show that this lineage separation is regulated by a crosstalk between bone morphogenetic protein (BMP) signaling and fibroblast growth factor (FGF) signaling. BMP signaling via Smad drives differentiation toward the myocardial lineage, which is inhibited by FGF signaling via mitogen-activated protein kinase kinase (Mek)1/2. Embryos exposed to recombinant FGF2 in vivo show enhanced epicardium formation, whereas a misbalance between FGF and BMP by Mek1/2 inhibition and BMP stimulation causes a developmental arrest of the epicardium and enhances myocardium formation at the inflow of the heart.. Our data show that FGF signaling via Mek1/2 is dominant over BMP signaling via Smad and is required to separate the epicardial lineage from precardiac mesoderm. Consequently, myocardial differentiation requires BMP signaling via Smad and inhibition of FGF signaling at the level of Mek1/2. These findings are of clinical interest for the development of regeneration-based therapies for heart disease.

Topics: Animals; Apoptosis; Bone Morphogenetic Protein 2; Bone Morphogenetic Proteins; Butadienes; Carbocyanines; Cell Differentiation; Cell Line; Cell Lineage; Cell Proliferation; Chick Embryo; Extracellular Signal-Regulated MAP Kinases; Fibroblast Growth Factor 2; Fibroblast Growth Factors; Fluorescent Dyes; Gene Expression Regulation, Developmental; Heart; Image Processing, Computer-Assisted; Imaging, Three-Dimensional; MAP Kinase Kinase 1; MAP Kinase Kinase 2; Microscopy, Fluorescence; Myocardium; Nitriles; Pericardium; Phenotype; Phosphorylation; Protein Kinase Inhibitors; Rats; Recombinant Proteins; Signal Transduction; Smad Proteins; T-Box Domain Proteins

The aim of this study is to conduct in vivo, noninvasive magnetic resonance imaging of labeled rat bone mesenchymal stem cells (BMSCs) as they home into the site of injured common carotid artery following allograft transplantation. Our study was approved by the Institutional Committee on Animal Research. Purified rat BMSCs were dual labeled with superparamagnetic iron oxide (SPIO) particle and fluorescent DiI dye, and subsequently transplanted into recipient rats injured in the left common carotid arteries. Immediately before and 3 hr, 3, 7 and 12 days after transplantation, the labeled cells were monitored in vivo using a 7T micromagnetic resonance imaging (7T micro-MRI) scanner. The signal-to-noise ratios (SNRs) at the injured sites were corroborated with histological examination using Prussian blue staining and fluorescent imaging. Rat BMSCs were labeled with SPIO and DiI at 100% efficiency. When compared with the baseline level before transplantation, the SNR decreased significantly on Days 3 and 7 after injection in the experimental group (Dunnet t test, P < 0.05), whereas insignificant differences were observed after 3 hr and 12 days (Dunnet t test, P > 0.05). In the control group, no significant differences in SNR were found among different time points (ANOVA, P > 0.05). Histological analyses illustrated that red fluorescence and Prussian blue-positive cells were mainly distributed around the lesion areas of injured common carotid arteries. Rat BMSCs can be efficiently labeled with SPIO and DiI, and the directional homing of labeled cells to the site of injured common carotid arteries after intravascular transplantation could be tracked in vivo with 7T micro-MRI.

Topics: Animals; Carbocyanines; Carotid Artery Injuries; Carotid Artery, Common; Cell Survival; Ferrocyanides; Fluorescent Dyes; Magnetic Resonance Imaging; Male; Mesenchymal Stem Cell Transplantation; Mesenchymal Stem Cells; Metal Nanoparticles; Microscopy, Fluorescence; Rats; Rats, Sprague-Dawley; Transplantation, Homologous

Lampreys are one of the most basal animals in which many of the true vertebrate characteristics (e.g., neural crest, placodes, segmented brain, skull, paired sensory organs, pharyngeal skeleton) are present. Studying the molecular and developmental mechanisms responsible for the formation of these structures in lamprey and higher vertebrates can provide insight into how these vertebrate characteristics evolved. The relative ease of obtaining mature adults and embryos makes this animal an ideal model for investigations into early vertebrate evolution. In addition, studies of features that are unique to lampreys can provide insights into mechanisms of parallel evolution. Lamprey embryos are particularly amenable to injection techniques. Like zebrafish and Xenopus embryos, they have double chorions and are resistant to surface-tension-induced rupture when removed from liquid. They can therefore be injected in a dry dish; this eliminates the need to support the embryo while performing injections and makes the procedure very rapid. Also, a single ovulating female can contain up to 100,000 eggs, so the number of injectable embryos per fertilization is not a limiting factor. This protocol describes how to label lamprey embryo cells by microinjecting the fluorescent dye DiI (1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate) to study cell fate during development.

Topics: Animals; Carbocyanines; Cell Biology; Cell Lineage; Chorion; Developmental Biology; Fluorescent Dyes; Lampreys; Microscopy, Fluorescence; Surface Properties

Novel thread-like structures and corpuscles, designated Bonghan ducts (BHDs) and corpuscles (BHCs), are known to form a system of networked channels. Here, we tested the effectiveness of a fluorescent carbocyanine dye, DiI (1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate), in staining BHDs and BHCs. DiI solution was infused into a BHC on the surface of a rat abdominal organ at a steady rate and the resulting labeling of neighboring BHCs connected via BHDs was examined, as identified by the red fluorescence of DiI. BHDs diameters tapered away from BHCs and formed tree-like branches with fine arborizations embedded in the membranous tissues at their terminal parts. In the proximal parts, DiI fluorescence appeared as continuous lines within BHDs, but a large portion of BHDs remained unstained. In the distal parts of BHDs, discontinuous elongated DiI microparticles were identified along the sinuses within BHDs. The results showed that inner spaces within the BHDs allowed DiI to flow and that BHDs have tree-like branches and terminal arborizations. In conclusion, DiI can be used in visualizing BHDs fine structures.

Topics: Abdominal Cavity; Acupuncture Points; Animals; Carbocyanines; Fluorescent Dyes; Injections; Male; Rats; Rats, Wistar; Staining and Labeling

The uptake of OxLDLs (oxidized low density lipoproteins) by CD36-expressing macrophages in the arterial intima and the subsequent 'foam cell' formation represents a crucial step in the initiation and development of atherosclerotic plaques. The present study has addressed the function of the CD36 N-terminal cytoplasmic domain in the binding and internalization of OxLDL. A selection of CD36 N-terminal cytoplasmic domain mutants were generated and stably expressed in HEK-293 (human embryonic kidney) cells. The capacity of three mutants [CD36_C3/7-A (CD36-C3A/C7A), CD36_D4/R5-A (CD36-D4A/R5A) and CD36_nCPD(-) (CD36 lacking the N-terminal cytoplasmic domain)] to bind and endocytose OxLDL was then studied using immunofluorescence microscopy and quantitative fluorimetry. Each of the CD36 constructs was expressed at differing levels at the cell surface, as measured by flow cytometry and Western blotting. Following incubation with DiI (1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate)-OxLDL, cells bearing the CD36_wt (wild-type CD36), CD36_C3/7-A, CD36_D4/R5-A and CD36_nCPD(-) constructs all internalized DiI-OxLDL into endosomal structures, whereas empty-vector-transfected cells failed to do so, indicating that, unlike the C-terminal cytoplasmic domain, the N-terminal cytoplasmic domain is not essential for the endocytosis of OxLDL. In conclusion, the uptake of OxLDL by CD36 is not reliant on the presence of the CD36 N-terminal cytoplasmic domain. However, the N-terminal cytoplasmic domain may conceivably be implicated in the maturation of CD36.

Topics: Carbocyanines; CD36 Antigens; Cell Line; Endocytosis; Fluorescent Dyes; Fluorometry; Humans; Lipoproteins, LDL; Microscopy, Fluorescence; Protein Binding; Protein Interaction Mapping; Protein Structure, Tertiary; Recombinant Fusion Proteins

Ternary lipid compositions in model membranes segregate into large-scale liquid-ordered (L(o)) and liquid-disordered (L(d)) phases. Here, we show mum-sized lipid domain separation leading to vesicle formation in unperturbed human HaCaT keratinocytes. Budding vesicles in the apical portion of the plasma membrane were predominantly labelled with L(d) markers 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate, 1,1'-dilinoleyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate, 1,1'-didodecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate and weakly stained by L(o) marker fluorescein-labeled cholera toxin B subunit which labels ganglioside GM(1) enriched plasma membrane rafts. Cholesterol depletion with methyl-beta-cyclodextrin enhanced DiI vesiculation, GM(1)/DiI domain separation and was accompanied by a detachment of the subcortical cytoskeleton from the plasma membrane. Based on these observations we describe the energetic requirements for plasma membrane vesiculation. We propose that the decrease in total 'L(o)/L(d)' boundary line tension arising from the coalescence of smaller L(d)-like domains makes it energetically favourable for L(d)-like domains to bend from flat mum-sized surfaces to cap-like budding vesicles. Thus living cells may utilize membrane line tension energies as a control mechanism of exocytic events.

Topics: Adhesiveness; beta-Cyclodextrins; Biophysical Phenomena; Carbocyanines; Cell Line; Cell Survival; Cytoplasmic Vesicles; Cytoskeleton; Exocytosis; Humans; Keratinocytes; Lipids

Recent studies have shown that neuroblasts migrate from the subventricular zone (SVZ) into the injured area after ischemic brain insults. However, it is not well understood which mechanism mediates this ectopic migration and which types of cells migrate into the damaged region from the SVZ. The present study was designed to investigate the characteristics of the migration of nestin-positive neural stem cells toward the region of ischemic injury after focal cortical ischemia. Nestin-eGFP transgenic mice were used to effectively model the migration of SVZ cells. Photothrombotic ischemia was induced by injection of rose bengal (30 mg/kg) and exposure to cold light. Migration of nestin-positive cells was examined using 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI) and bromodeoxyuridine (BrdU) labeling. The number of nestin-positive cells was increased significantly in the peri-infarct area at 5 and 7 days after photothrombosis. A subset of nestin-positive cells was co-labeled with DiI or BrdU. Some of the nestin-positive cells co-expressed doublecortin (DCX) and only a few nestin-positive cells co-labeled with anti-epidermal growth factor receptor (EGFr) antibody. However, no nestin-positive cells were immunoreactive for glial fibrillary acidic protein (GFAP). The inhibition of matrix metalloproteinases (MMPs) using the MMP inhibitor, FN-439, decreased nestin-positive cells in the peri-infarct region at 7 days after photothrombosis. Although MMP-9 was not co-expressed in the nestin-positive cells in the peri-infarct cortex, MMP-9 did co-localize with GFAP-positive astrocytes. These results suggest that nestin-positive neural progenitor cells migrate into the peri-infarct cortex after photothrombotic ischemia and that MMP-9 is involved in the migration.

Topics: Animals; Brain Infarction; Brain Ischemia; Bromodeoxyuridine; Carbocyanines; Cell Movement; Doublecortin Domain Proteins; Doublecortin Protein; ErbB Receptors; Glial Fibrillary Acidic Protein; Green Fluorescent Proteins; Hydroxamic Acids; Immunohistochemistry; Intermediate Filament Proteins; Matrix Metalloproteinase 9; Matrix Metalloproteinase Inhibitors; Mice; Mice, Transgenic; Microinjections; Microtubule-Associated Proteins; Nerve Tissue Proteins; Nestin; Neuroglia; Neurons; Neuropeptides; Oligopeptides; Rose Bengal; Stem Cells

To visualize retinal ganglion cells (RGCs) and their gradual loss in the living mouse.. With the use of B6.Cg-Tg(Thy1-CFP)23Jrs/J mice, which express cyan fluorescent protein (CFP) in RGCs, and a commercially available mydriatic retinal camera attached with a 5 million-pixel digital camera to visualize RGCs in vivo, the authors recorded fundus photographs longitudinally in the ischemia reperfusion model group and the untreated group to evaluate longitudinal changes in the number of RGCs in experimental models. Moreover, RGCs expressing CFP were evaluated histologically by a retrograde-labeling method and retinal whole mount or sections.. The authors devised an in vivo imaging technique using a conventional retinal camera and visualized RGCs at the single-cell level. In the ischemia reperfusion model, a longitudinal reduction in the number of RGCs was demonstrated in each mouse eye. The number of RGCs and the fluorescence intensity of the nerve fiber decreased considerably during the first week. The percentages of RGCs decreased to 34.2% +/- 7.5%, 24.1% +/- 9.1%, 23.0% +/- 9.3%, and 22.2% +/- 8.4% (mean +/- SD, n = 5) of the percentages before injury at 1, 2, 3, and 4 weeks after injury, respectively (P < 0.001). In this transgenic mouse, 97% of CFP-expressing cells were RGCs and 73% of RGCs expressed CFP.. This in vivo technique allows noninvasive, repeated, and longitudinal evaluation of RGCs for investigation of retinal neurodegenerative diseases and new therapeutic modalities for them.

Topics: Animals; Carbocyanines; Cell Count; Disease Models, Animal; Female; Fluorescein Angiography; Fundus Oculi; Gene Expression; Green Fluorescent Proteins; Male; Mice; Mice, Inbred C57BL; Mice, Transgenic; Photography; Reperfusion Injury; Retinal Diseases; Retinal Ganglion Cells

Sensory organs are composed of neurons, which convert environmental stimuli to electrical signals, and glia-like cells, whose functions are not well understood. To decipher glial roles in sensory organs, we ablated the sheath glial cell of the major sensory organ of Caenorhabditis elegans. We found that glia-ablated animals exhibit profound sensory deficits and that glia provide activities that affect neuronal morphology, behavior generation, and neuronal uptake of lipophilic dyes. To understand the molecular bases of these activities, we identified 298 genes whose messenger RNAs are glia-enriched. One gene, fig-1, encodes a labile protein with conserved thrombospondin TSP1 domains. FIG-1 protein functions extracellularly, is essential for neuronal dye uptake, and also affects behavior. Our results suggest that glia are required for multiple aspects of sensory organ function.

Topics: Animals; Caenorhabditis elegans; Caenorhabditis elegans Proteins; Calcium; Carbocyanines; Chemotaxis; Cilia; Fluorescent Dyes; Gene Expression; Genes, Helminth; Neuroglia; Odorants; Oligonucleotide Array Sequence Analysis; Osmolar Concentration; Sense Organs; Sensory Receptor Cells; Signal Transduction; Sodium Chloride; Temperature; Thrombospondins; Transcription, Genetic

The cranial neural crest has been shown to give rise to a diversity of cells and tissues, including cartilage, bone and connective tissue, in a variety of tetrapods and in the zebrafish. It has been claimed, however, that in the Australian lungfish these tissues are not derived from the cranial neural crest, and even that no migrating cranial neural crest cells exist in this species. We have earlier documented that cranial neural crest cells do migrate, although they emerge late, in the Australian lungfish. Here, we have used the lipophilic fluorescent dye, DiI, to label premigratory cranial neural crest cells and follow their fate until stage 43, when several cranial skeletal elements have started to differentiate. The timing and extent of their migration was investigated, and formation of mandibular, hyoid and branchial streams documented. Cranial neural crest was shown to contribute cells to several parts of the head skeleton, including the trabecula cranii and derivatives of the mandibular arch (e.g., Meckel's cartilage, quadrate), the hyoid arch (e.g., the ceratohyal) and the branchial arches (ceratobranchials I-IV), as well as to the connective tissue surrounding the myofibers in cranial muscles. We conclude that cranial neural crest migration and fate in the Australian lungfish follow the stereotyped pattern documented in other vertebrates.

Topics: Animals; Carbocyanines; Cell Differentiation; Cell Lineage; Cell Movement; Embryo, Nonmammalian; Fishes; Head; Immunohistochemistry; Models, Biological; Neural Crest; Species Specificity

The purpose of this study was to investigate the effect of a polymer coating using modified polyvinyl alcohol (PVA-R) on the interaction between liposomes and macrophage cells (J774 cells). The PVA-R-coated liposomes, which were labeled with 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocianin perchlorate (DiI) as a fluorescence reagent, were prepared with the conventional hydration method followed by extrusion and surface modification with PVA-R. The effects of polymer coating on the interaction with J774 cells were evaluated by using flow cytometry and confocal laser scanning microscopy (CLSM). When liposomes with or without PVA-R coating were incubated with J744 cells, the fluorescence emission intensity of DiI from J774 cells was significantly smaller than in the case of non-coated liposomes. This result indicates that decreased interaction of PVA-R-coated liposomes with macrophage cells could be well detected using flow cytometry and CLSM. These in vitro tests explained the differences in blood circulation of polymer-coated liposomes having different lipid formulations in rats.

Topics: Animals; Carbocyanines; Cells, Cultured; Flow Cytometry; Fluorescent Dyes; Hypromellose Derivatives; In Vitro Techniques; Liposomes; Macrophages; Methylcellulose; Mice; Microscopy, Confocal; Polymers; Polyvinyl Alcohol; Surface Properties

Topics: Carbocyanines; Electrolytes; Fluorescence; Fluorescent Dyes; Liposomes; Polyvinyls

It has been shown that airway exposure to eosinophil-derived cationic proteins stimulated vagal pulmonary C fibers and markedly potentiated their responses to lung inflation in anesthetized rats (Lee LY, Gu Q, Gleich GJ, J Appl Physiol 91: 1318-1326, 2001). However, whether the effects resulted from a direct action of these proteins on the sensory nerves was not known. The present study was therefore carried out to determine the effects of these proteins on isolated rat vagal pulmonary sensory neurons. Our results obtained from perforated whole cell patch-clamp recordings showed that pretreatment with eosinophil major basic protein (MBP; 2 microM, 60 s) significantly increased the capsaicin-evoked inward current in these neurons; this effect peaked approximately 10 min after MBP and lasted for >60 min; in current-clamp mode, MBP substantially increased the number of action potentials evoked by both capsaicin and electrical stimulation. Pretreatment with MBP did not significantly alter the input resistance of these sensory neurons. In addition, the sensitizing effect of MBP was completely abolished when its cationic charge was neutralized by mixing with a polyanion, such as low-molecular-weight heparin or poly-L-glutamic or poly-L-aspartic acid, before its delivery to the neurons. Moreover, a similar sensitizing effect was also generated by other eosinophil granule-derived proteins (e.g., eosinophil peroxidase). These results demonstrate a direct, charge-dependent, and long-lasting sensitizing effect of cationic proteins on pulmonary sensory neurons, which may contribute to the airway hyperresponsiveness associated with airway infiltration of eosinophils under pathophysiological conditions.

Topics: Action Potentials; Animals; Capsaicin; Carbocyanines; Drug Synergism; Electric Stimulation; Eosinophil Cationic Protein; Eosinophil Major Basic Protein; Eosinophil Peroxidase; Heparin, Low-Molecular-Weight; Lung; Neurons, Afferent; Patch-Clamp Techniques; Pneumonia; Rats; Rats, Sprague-Dawley; Vagus Nerve

We have examined the innervation of the gut-associated lymphoid system of the sheep ileum, with a view to identifying potential sites for neuroinvasion by pathogens, such as prions (PrP(Sc)). Special attention has been paid to the follicles of Peyer's patches (PPs), which are major sites of PrP(Sc) accumulation during infection. Evidence exists that the enteric nervous system, together with the parasympathetic and sympathetic pathways projecting to the intestine, are important for PrP(Sc) entry into the central nervous system. Thus, PrP(Sc) might move from PPs to the neurons and nerve fibres that innervate them. We investigated, by immunohistochemistry and retrograde tracing (DiI) from the follicles, the distribution and phenotype of enteric neurons innervating the follicles. Antibodies against protein gene product 9.5, tyrosine hydroxylase, dopamine beta hydroxylase, choline acetyltransferase, calbindin (CALB), calcitonin gene-related peptide (CGRP), and nitric oxide synthase were used to characterise the neurons. Immunoreactivity for each of these was observed in fibres around and inside PP follicles. CGRP-immunoreactive fibres were mainly seen at the follicular dome. Retrograde tracing revealed submucosal neurons that contributed to the innervation of PPs, including Dogiel type II neurons and neurons immunoreactive for CALB and CGRP. The major source of the adrenergic fibres are the sympathetic ganglia. Our results thus suggest that enteric and sympathetic neurons are involved during the first stage of neuroinvasion, with neurons connecting to them acting as potential carriers of PrP(Sc) to the central nervous system.

Topics: Animals; Calbindins; Carbocyanines; Cattle; Choline O-Acetyltransferase; Dopamine beta-Hydroxylase; Enteric Nervous System; Fluorescent Dyes; Ileum; Nerve Fibers; Neurons; Nitric Oxide Synthase; Organ Culture Techniques; Peyer's Patches; Prions; S100 Calcium Binding Protein G; Sheep; Tyrosine 3-Monooxygenase; Ubiquitin Thiolesterase

To investigate whether human hepatocytes could proliferate after transplantation to normal immunocompetent rats treated with 2-acetaminofluorene or retrorsine and partial hepatectomy.. L02 hepatocyte-tolerant Sprague-Dawley rats were injected with retrorsine, 2-acetaminofluorene or normal saline. L02 hepatocytes were then transplanted via the spleen. Human albumin and its mRNA, specific proliferating cell nuclear antigen (PCNA), L02 hepatocyte dynamic distribution, number density and area density of PCNA-positive cells in the liver were determined.. All the examined indicators were not significantly different between the rats treated with 2-acetaminofluorene and normal saline, which was not the case with rats treated with retrorsine. A dynamic distribution of L02 hepatocytes in the rat liver was detected from wk 1 to mo 6 after transplantation in the retrorsine group and from wk 1 to 10 in the 2-acetaminofluorene group. Human albumin and its mRNA were detected from wk 2 to mo 6 in the retrorsine group and from wk 1 to 8 in the 2-acetaminofluorene group. Specific human PCNA was detected in the rat liver from wk 2 to mo 6 in the retrorsine group and from wk 2 to 6 in the 2-acetaminofluorene group. Human albumin and its mRNA contents as well as the number of PCNA positive cells reached a peak at wk 4.. L02 human hepatocytes could not proliferate significiantly after transplantation to the normal, immunocompetent rats treated with 2-acetaminofluorene. L02 human hepatocytes can survive for 10 wk after transplantation and express human albumin for 8 wk. L02 human hepatocytes can proliferate and express human albumin for 6 mo after transplantation to the rats treated with retrorsine. The chimeric L02 human hepatocytes, which then underwent transplantation into tolerant rats, were normal in morphogenesis, biochemistry and function.

Topics: 2-Acetylaminofluorene; Animals; Carbocyanines; Cell Proliferation; Cell Survival; Female; Fluorescent Dyes; Gestational Age; Hepatectomy; Hepatocytes; Humans; Image Interpretation, Computer-Assisted; Immune Tolerance; Immunocompetence; Liver; Liver Regeneration; Microscopy, Fluorescence; Models, Animal; Pregnancy; Proliferating Cell Nuclear Antigen; Pyrrolizidine Alkaloids; Rats; Rats, Sprague-Dawley; Reverse Transcriptase Polymerase Chain Reaction; Serum Albumin; Time Factors; Transplantation Chimera

Activation of thin fibre muscle afferent nerves by metabolic by-products plays a critical role in the initiation and maintenance of the autonomic response to exercise and the metabolic profile of active muscle can influence the response. The purpose of this report was to determine the responsiveness of sensory neurones innervating muscles comprising predominantly glycolytic and oxidative fibres to protons and capsaicin using whole-cell patch clamp methods. Dorsal root ganglion (DRG) neurones from 4- to 6-week-old rats were labelled by injecting the fluorescence tracer DiI into the muscle 3-5 days prior to the recording experiments. The percentage of the DRG neurones innervating glycolytic and oxidative muscle was similar in response to both protons and capsaicin. However, the neurones innervating glycolytic muscle had greater inward current amplitude responses to protons and capsaicin as compared with oxidative muscle. The peak current amplitudes to pH 6.0 were 0.84 +/- 0.06 nA (oxidative muscle) versus 1.36 +/- 0.07 nA (glycolytic muscle, P < 0.05). The capsaicin-induced current amplitudes were 2.3 +/- 0.15 nA (oxidative muscle) versus 3.1 +/- 0.21 nA (glycolytic muscle, P < 0.05). Of neurones that responded to pH 6.0 with a sustained current, 88% also responded to capsaicin. Capsaicin exposure enhanced the proton responsiveness in the neurones innervating the muscle; and protons also increased the capsaicin response. These data suggest that (1) receptors mediating protons and capsaicin responses coexist in the DRG neurones innervating muscle; (2) the responsiveness of acidosis and capsaicin can be sensitized by each other; and (3) DRG neurones with nerve endings in a glycolytic muscle developed greater inward current responses to protons and capsaicin than did those with nerve endings in an oxidative muscle.

Topics: Action Potentials; Animals; Capsaicin; Carbocyanines; Electrophysiology; Hydrogen-Ion Concentration; Ion Transport; Male; Muscle Fibers, Skeletal; Muscle, Skeletal; Neurons, Afferent; Protons; Rats; Rats, Sprague-Dawley; Staining and Labeling

Cells are generally labeled during in vivo implantation studies enabling the cells to be traced. The relationship between the labeling efficiency and cellular proliferation after transplantation is critical for the interpretation of data obtained by detection of the signals on tissue sections. Here, we compare cellular labeling methods of rat marrow stromal cells that were labeled with 5-bromo-2-deoxyuridine (BrdU), 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI) and fluorescence in situ hybridization (FISH). Our data show that (i) BrdU uniformly labeled the nuclei, (ii) DiI-labeled cells had many dots or stained clear and uniform when a longer exposure time was used during detection and (iii) FISH labeled the cells with dots along the edges of the nuclei. The labeling efficiency was 94.1+/-8.6%, 97.6+/-3.4% and 90.5+/-3.0%, in BrdU, DiI- and FISH-labeled cells, respectively. After sub-culturing of labeled cells, the percentage of BrdU-positive cells was found to be 71.9+/-18.0% and 18.4+/-6.9%, after the first and second passages, respectively. The percentage of DiI-labeled cells detected depended on the exposure time: a long exposure time (>10 s) resulted in identification of 95.1+/-4.0% and 94.5+/-3.9% DiI-positive cells after the first and second sub-cultures, respectively. The percentage of FISH-positive cells was found to be 87.0+/-3.0% and 89.1+/-9.7%. The BrdU labeling signal quickly decreased over time. Thus, BrdU should only be used to temporarily label dividing cells. In contrast, our data indicate that DiI and FISH labeling may be used to steadily trace cells during in vivo experiments. To our knowledge, this is the first time that the effects of different labeling methods over time have been examined during a cell transplantation study.

Topics: Affinity Labels; Animals; Bone Marrow Cells; Bone Marrow Transplantation; Brain; Bromodeoxyuridine; Carbocyanines; Cell Count; Cell Division; Cell Movement; Cell Transplantation; Female; Fluorescent Dyes; In Situ Hybridization, Fluorescence; Male; Neurons; Rats; Rats, Sprague-Dawley; Sensitivity and Specificity; Staining and Labeling

Children exposed prenatally to alcohol can display a variety of neural deficits, including an altered development of the corpus callosum (CC), the largest interhemispheric axon pathway in the brain. Furthermore, these children show functional abnormalities that are related to brain regions with significant numbers of CC connections. Little is known about how alcohol imparts influence on CC development, but one possible mechanism is by affecting the corpus callosum projection neurons (CCpn) directly. The purpose of this study was to quantify the effects of prenatal alcohol exposure on the number, size, and distribution of CCpn within the visual cortex. The visual cortex was selected specifically due to the many vision-related deficits noted in fetal alcohol exposed children and because the critical role of the CC in visual cortex development is well documented. Sprague-Dawley rat pups received one of four alcohol dosages during gestational days (G) 1-20, or reared as nutritional or untreated control animals. Each litter was categorized according to the peak blood alcohol concentration experienced. Pups were removed from each litter on days equivalent to G29, G36, G43, and G50, for histology and measurement. Callosal axons were labeled retrogradely to their CCpn using 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI) and the CCpn were then examined using confocal laser scanning microscopy. Differences between alcohol-exposed and control animals were observed in CCpn cell body size, number, and location with the cortex. This was particularly true of animals exposed to high doses of alcohol. In addition, some trends of CCpn development were found to be unchanged as a result of prenatal alcohol exposure. The results demonstrate clear differences in the development of CCpn in the visual cortex between alcohol-exposed and control animals and suggest that this development is particularly affected in those animals exposed to high doses of alcohol.

Topics: Animals; Carbocyanines; Cell Count; Cell Size; Central Nervous System Depressants; Corpus Callosum; Dose-Response Relationship, Drug; Ethanol; Female; Fetus; Fluorescent Dyes; Gestational Age; Maternal-Fetal Exchange; Microscopy, Confocal; Neurons; Pregnancy; Rats; Rats, Sprague-Dawley; Visual Cortex

Normal human somatic cells in culture have a limited dividing potential. This is due to DNA end replication problem, whereby telomeres shorten with each subsequent cell division. When a critical telomere length is reached cells enter senescence. To overcome this problem, immortal HeLa cell line express telomerase, an enzyme that prevents telomere shortening. Although immortal, the existence of non-dividing cells that do not incorporate (3)H-thymidine over 24 h of growth has been well documented in this cell line. Using DiI labeling and high-speed cell sorting, we have separated and analyzed fractions of HeLa cells that divided vigorously as well as those that cease divisions over several days in culture. We also analyzed telomerase activity in separated fractions and surprisingly, found that the fraction of cells that divided 0-1 time over 6 days in culture have several times higher endogenous telomerase activity than the fastest dividing fraction. Additionally, the non-growing fraction regains an overall high labeling index and low SA-beta-Gal activity when subcultured again. This phenomenon should be considered if telomerase inhibition is to be used as an approach to cancer therapy. In this paper we also discuss possible molecular mechanisms that underlie the observed results.

Topics: beta-Galactosidase; Carbocyanines; Cell Proliferation; Cell Separation; Cellular Senescence; Female; Flow Cytometry; Fluorescent Dyes; HeLa Cells; Humans; Phenotype; Telomerase; Time Factors; Tritium; Uterine Cervical Neoplasms

Leishmania spp. are intracellular protozoa residing in mononuclear phagocytes. Leishmania organisms are susceptible to microbicidal responses generated in response to phagocytosis. Assuming that both phagocyte and parasite populations are heterogeneous, it is advantageous to examine the response of individual cells phagocytosing living parasites. Because Leishmania spp. lose virulence during the raising of transfectants, we developed a method to label live Leishmania chagasi short-term with fluorescent dyes. Up to six parasite divisions were detected by flow cytometry after labeling with carboxyfluorescein diacetate succinimidyl ester (CFSE), dioctadecyl-tetramethylindo carbocyanine perchlorate, or chloromethyl tetramethylrhodamine. Labeled parasites entered mononuclear phagocytes as determined by confocal and time-lapse microscopy. Dihydroethidium (DHE) was used to detect macrophage-derived oxidants generated during phagocytosis. Presumably Leishmania organisms are opsonized with host serum/tissue components such as complement prior to phagocytosis. Therefore, we investigated the effects of opsonization and found that this increased the efficiency of CFSE-labeled parasite entry into monocytes (84.6% +/- 8.8% versus 20.2% +/- 3.8% monocytes infected; P < 0.001). Opsonization also increased the percentage of phagocytes undergoing a respiratory burst (66.0% +/- 6.3% versus 41.0% +/- 8.3% of monocytes containing CFSE-labeled parasites; P < 0.001) and the magnitude of oxidant generation by each infected monocyte. Inhibitor data indicated that DHE was oxidized by products of the NADPH oxidase. These data suggest that opsonized serum components such as complement lead to more efficient entry of Leishmania into their target cells but at the same time activate the phagocyte oxidase to generate microbicidal products in infected cells. The parasite must balance these positive and negative survival effects in order to initiate a viable infection.

Topics: Animals; Carbocyanines; Cells, Cultured; Ethidium; Flow Cytometry; Fluoresceins; Fluorescent Dyes; Humans; Leishmania infantum; Microscopy, Confocal; Microscopy, Video; Monocytes; Opsonin Proteins; Oxidants; Phagocytes; Rhodamines; Staining and Labeling; Succinimides

Rather than being prepatterned, orientation of the embryonic-abembryonic (Em-Ab) axis of the mouse blastocyst has been claimed to depend on the conceptus being constrained by its zona pellucida (ZP) during blastulation. This hypothesis merited closer scrutiny, because it seemed at variance with observations on living conceptuses.. Two-cell conceptuses with an oil drop injected into the lesser diameter (LD) of the ZP at the first cleavage plane were cultured until shortly before blastulation when the blastomere underlying the drop was labelled with carbocyanine dye. After removing the ZP, conceptuses were re-cultured to the blastocyst stage for recording the position along the axis of the centres of the patches of labelled cells.. These centres showed significant bias towards the equatorial (Eq) region of the axis compared with those resulting from labelling a blastomere at random, even following softening of the ZP at the 2-cell stage. This was also true if conceptuses were denuded at the 2-cell stage and the blastomere underlying an intact second polar body (PB) labelled in morulae.. These findings further support the view that the Em-Ab axis of the mouse blastocyst is normally prepatterned and provide no evidence of a role for the ZP in its specification.

Topics: Animals; Blastocyst; Blastomeres; Body Patterning; Carbocyanines; Cell Polarity; Female; Mice; Pregnancy; Zona Pellucida; Zygote

Scavenger receptor class B type I (SR-BI) is the high-affinity high-density lipoprotein (HDL) receptor, and CLA-1 is the human homologue of the murine SR-BI. CLA-1/SR-BI receptor has been suggested as a new preventative and/or therapeutic target for atherosclerosis due to its pivotal role in overall HDL cholesterol (HDL-C) metabolism and its antiatherogenic activity in vivo. To search for active compounds that can increase CLA-1 transcription, a novel cell-based assay was developed for application in high-throughput screening (HTS). Human hepatoma HepG2 cells were transfected with a CLA-1-promoter-luciferase reporter gene construct, and the stable transfected cell line was selected and named CLAp-LUC HepG2. With rosiglitazone as a positive control, this stable cell line was used to establish a specific CLA-1 gene expression assay in a 96-well microplate format. The evaluating parameter Z' value of 0.64 showed that this cell-based HTS assay was robust and reliable. Screening of 6000 microbial secondary metabolite crude extracts identified 8 positive strains. Between 2 identified CLA-1 up-regulators produced by actinomycete strain 04-4776, 4776B may stimulate not only the expression of CLA-1 on the transcriptional and translational levels but also the activity of CLA-1 to uptake the HDL-C in HepG2 cells. The active compounds originated from this HTS assay may be developed to drug candidates or lead compounds for new antiatherosclerosis agents.

Topics: Actinomycetaceae; Biological Assay; Carbocyanines; Carcinoma, Hepatocellular; Cell Line, Tumor; Dose-Response Relationship, Drug; Drug Evaluation, Preclinical; Fermentation; Fluorescent Dyes; Gene Expression Regulation; Genes, Reporter; Humans; Hydroxyl Radical; Isoflavones; Lipoproteins, HDL; Liver Neoplasms; Luciferases; PPAR gamma; Receptors, Lipoprotein; Recombinant Fusion Proteins; Rosiglitazone; Scavenger Receptors, Class B; Thiazolidinediones; Transcription, Genetic; Up-Regulation

Recent molecular lineage analyses in mouse have demonstrated that the right ventricle is recruited from anterior mesoderm in later stages of cardiac development. This is in contrast to current views of development in the chicken heart, which suggest that the initial heart tube contains a subset of right ventricular precursors. We investigated the fate of the outflow tract myocardium using immunofluorescent staining of the myocardium, and lineage tracer, as well as cell death experiments. These analyses showed that the outflow tract is initially myocardial in its entirety, increasing in length up to HH24. The outflow tract myocardium, subsequently, shortens as a result of ventricularization, contributing to the trabeculated free wall, as well as the infundibulum, of the right ventricle. During this shortening, the overall length of the outflow tract is maintained because of the formation of a nonmyocardial portion between the distal myocardial border and the pericardial reflections. Cell death and transdifferentiation were found to play a more limited contribution to the initial shortening than is generally appreciated, if they play any part at all. Cell death, nonetheless, plays an important role in the disappearance of the myocardial collar that continues to invest the aorta and pulmonary trunk around HH30, and in the separation of the intrapericardial arterial vessels. Taken together, we show, as opposed to some current beliefs, the development of the arterial pole is similar in mammals and birds.

Topics: Animals; Aorta; Carbocyanines; Cell Death; Cell Lineage; Chick Embryo; Embryonic Development; Fluorescent Antibody Technique; Fluorescent Dyes; Heart; Heart Ventricles; Myocardium; Pulmonary Artery; Staining and Labeling

The mouse is an extremely valuable model for studying vagal development in relation to strain differences, genetic variation, gene manipulations or pharmacological manipulations. Therefore, a method using 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI) was developed for labeling vagal innervation of the gastrointestinal (GI) tract in embryonic and postnatal mice. DiI labeling was adapted and optimized for this purpose by varying several facets of the method. For example, insertion and crushing of DiI crystals into the nerve led to faster DiI diffusion along vagal axons and diffusion over longer distances as compared with piercing the nerve with a micropipette tip coated with dried DiI oil. Moreover, inclusion of EDTA in the fixative reduced leakage of DiI out of nerve fibers that occurred with long incubations. Also, mounting labeled tissue in PBS was superior to glycerol with n-propyl gallate, which resulted in reduced clarity of DiI labeling that may have been due to DiI leaking out of fibers. Optical sectioning of flattened wholemounts permitted examination of individual tissue layers of the GI tract wall. This procedure aided identification of nerve ending types because in most instances each type innervates a different tissue layer. Between embryonic day 12.5 and postnatal day 8, growth of axons into the GI tract, formation and patterning of fiber bundles in the myenteric plexus and early formation of putative afferent and efferent nerve terminals were observed. Thus, the DiI tracing method developed here has opened up a window for investigation during an important phase of vagal development.

Topics: Animals; Animals, Newborn; Axons; Carbocyanines; Diffusion; Drug Delivery Systems; Edetic Acid; Enteric Nervous System; Gastrointestinal Tract; Growth Cones; Image Cytometry; Mice; Mice, Inbred C57BL; Myenteric Plexus; Presynaptic Terminals; Sodium Chloride; Staining and Labeling; Vagus Nerve

To describe a previously uncharacterized structural specialization in the mouse lens fiber cell and to delineate its emergence relative to lens development and fiber cell differentiation.. Lens fixation efficiency was explored using (14)C-formaldehyde and autoradiography. Lens fiber cell architecture was examined by scanning electron microscopy and by DiI labeling of methacrylate sections in lenses ranging from 2 weeks to 8 months.. Scanning electron microscopy identified an elaborate structural specialization that emerges late in fiber cell differentiation, largely after the cell has lost its nucleus. These elaborations project from the short side of the cell, are regularly spaced throughout the central region of the cell and are aligned with similar structures in adjacent cells. The structures are not found in fiber cells of lenses younger than two weeks of age, nor in the fiber cells that initially differentiate before that time.. Fiber cells that arise later than 2 weeks of age undergo a structural differentiation program that is different from that of cells that arise earlier in development. This program includes the assembly of a series of regularly spaced, complex, lateral projections from the fiber cell that align themselves with similar structures in adjacent cells. Most if not all of the structural specialization occurs in cells that have lost their nuclei and organelles, suggesting that this component of fiber cell differentiation may not require ongoing transcription/translation.

Topics: Animals; Carbocyanines; Cell Differentiation; Fluorescent Dyes; Lens, Crystalline; Mice; Mice, Inbred C57BL; Microscopy, Electron, Scanning

Newly generated neurons are continuously added to the olfactory epithelium and olfactory bulbs of adult mammals. Studies also report newly generated neurons in the piriform cortex, the primary cortical projection site of the olfactory bulbs. The current study used BrdU-injection paradigms, and in vivo and in vitro DiI tracing methods to address three fundamental issues of these cells: their origin, migratory route and fate. The results show that 1 day after a BrdU-injection, BrdU/DCX double-labeled cells appear deep to the ventricular subependyma, within the white matter. Such cells appear further ventral and caudal in the ensuing days, first appearing in the rostral piriform cortex of mice at 2 days after the BrdU-injection, and at 4 days in the rat. In the caudal piriform cortex, BrdU/DCX labeled cells first appear at 4 days after the injection in mice and 7 days in rats. The time it takes for these cells to appear in the piriform cortex and the temporal distribution pattern suggest that they migrate from outside this region. DiI tracing methods confirmed a migratory route to the piriform cortex from the ventricular subependyma. The presence of BrdU/NeuN labeled cells as early as 7 days after a BrdU injection in mice and 10 days in the rat and lasting as long as 41 days indicates that some of these cells have extended survival durations in the adult piriform cortex.

Topics: Animals; Animals, Newborn; Bromodeoxyuridine; Carbocyanines; Cell Differentiation; Cell Lineage; Cell Movement; Cell Shape; Cell Survival; DNA-Binding Proteins; Doublecortin Domain Proteins; Doublecortin Protein; Fluorescent Dyes; Ki-67 Antigen; Male; Mice; Microscopy, Confocal; Microscopy, Immunoelectron; Microtubule-Associated Proteins; Nerve Tissue Proteins; Neurons; Neuropeptides; Nuclear Proteins; Olfactory Bulb; Olfactory Pathways; Rats; Rats, Sprague-Dawley; Staining and Labeling; Time Factors

The specific patterns of gastrointestinal motility in large herbivores may relate to differences in the organization of enteric nerve circuits, compared with other mammals. To investigate this possibility, we characterized the morphologies, chemical phenotypes, and projections of myenteric plexus (MP) neurons of the sheep ileum. Morphologies and projections were investigated after application of the carbocyanine dye (1,1', di-octadecyl-3,3,3',3',-tetramethylindo-carbocyanine perchlorate, DiI) to fixed tissues. To study chemical phenotypes, the fluorescent tracer Fast Blue (FB) was injected into the wall of the ileum, in vivo, 12-14 cm oral to the ileo-caecal junction. Over 80% of the descending and ascending DiI-labeled neurons had typical Dogiel type I morphology, whereas only a few Dogiel type II neurons were observed. Nevertheless, there were long projections (up to 10 cm) of Dogiel type II neurons in both directions. Both type II and type I neurons were neurofilament immunoreactive (IR). We observed long projections of descending (up to 18 cm) and ascending (up to 12-14 cm) FB-labeled MP neurons. Nitric oxide synthase (NOS)-IR, peripheral choline acetyltransferase (pChAT)-IR, and substance P (SP)-IR occurred in both descending and ascending myenteric neurons. NOS-IR was in approximately 60% of FB-labeled descending and ascending neurons, whereas those expressing pChAT-IR were 67 +/- 15% and 60 +/- 14%, respectively. Descending neurons expressing SP-IR were 48 +/- 15% and ascending were 56 +/- 12%. NOS-IR and pChAT-IR, and SP-IR and pChAT-IR were commonly colocalized in both ascending and descending pathways. In descending pathways, almost all SP-IR neurons were also pChAT-IR (98 +/- 3%) and NOS-IR (99 +/- 2 NOS(+)/SP(+)/pChAT(-)). Many FB-labeled descending neurons showed both NOS- and pChAT-IR. Descending neurons may represent inhibitory motor neurons (NOS(+)/SP(+)/pChAT(-)) and two classes of interneurons (pChAT(+)/NOS(-), and pChAT(+)/NOS(+)/SP(+)). In ascending pathways, most neurons are pChAT(+)/NOS(+)/SP(+). Thus, in sheep, ascending interneurons and ascending excitatory motor neurons both have the same phenotype, and other markers are needed to distinguish them.

Topics: Amidines; Animals; Carbocyanines; Choline O-Acetyltransferase; Gastrointestinal Motility; Ileum; Immunohistochemistry; Myenteric Plexus; Neurons; Nitric Oxide Synthase; Phenotype; Sheep; Substance P

In vertebrates, the endoderm is established during gastrulation and gradually becomes regionalized into domains destined for different organs. Here, we present precise fate maps of the gastrulation stage chick endoderm, using a method designed to label cells specifically in the lower layer. We show that the first population of endodermal cells to enter the lower layer contributes only to the midgut and hindgut; the next cells to ingress contribute to the dorsal foregut and followed finally by the presumptive ventral foregut endoderm. Grafting experiments show that some migrating endodermal cells, including the presumptive ventral foregut, ingress from Hensen's node, not directly into the lower layer but rather after migrating some distance within the middle layer. Cell transplantation reveals that cells in the middle layer are already committed to mesoderm or endoderm, whereas cells in the primitive streak are plastic. Based on these results, we present a revised fate map of the locations and movements of prospective definitive endoderm cells during gastrulation.

Topics: Animals; Body Patterning; Carbocyanines; Cell Movement; Chick Embryo; Endoderm; Fluorescent Dyes; Gastrula; In Situ Hybridization, Fluorescence; Intestinal Mucosa; Intestines; Mesoderm; Models, Biological

Gastrin-releasing peptide-preferring and neuromedin B-preferring receptors, members of the bombesin-like peptide receptor subfamily, are reported to regulate proliferation, migration and differentiation. Since they are expressed in developing brain, we postulated that the gastrin-releasing peptide-preferring and neuromedin B-preferring receptors might be involved in normal brain development. Here we examined the effects of the overexpressions of the gastrin-releasing peptide-preferring and neuromedin B-preferring receptors on chick brain development in vivo using a retrovirus. In the overexpressed exogenous gastrin-releasing peptide-preferring receptor brain, we found laminar disorganization in the telencephalon, tectum and particularly in the cerebellum with severe atrophy. Processes of the radial glial cells in the telencephalon and optic tectum, as well as the projections of the Bergmann glia in the cerebellum were distorted, which might disturb normal cell migration. Despite the atrophy of the cerebellum, densely-stained proliferating cell nuclear antigen- and phospho-histone H3-positive cells increased in number. Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling-positive cells also increased in the cerebellum, suggesting that the ectopically proliferating cells were subjected to apoptosis. Glial fibrillary acidic protein-positive cells also increased in the hyperpallium accessorium and in the outer layers of the tectum. We also found smaller and spindle-shaped cells which resembled undifferentiated embryonic tumor cells. On the other hand, the layer structures of the neuromedin B-preferring receptors overexpressed brain were well organized and developed, and the size of brain was generally enlarged. These results indicated that although the gastrin-releasing peptide-preferring and neuromedin B-preferring receptors are involved in normal brain development, both receptors contribute and exert their effects differently.

Topics: Animals; Apoptosis; Brain; Brain Chemistry; Calcium; Carbocyanines; Cattle; Chick Embryo; Coloring Agents; Immunohistochemistry; In Situ Hybridization; Mice; Microinjections; Ovum; Receptors, Bombesin; Retroviridae; Reverse Transcriptase Polymerase Chain Reaction; Transfection

We have characterized a method of labeling of axons in the post-mortem spinal cord using a silastic disc holding pins coated with DiI and DiO at the rostral and caudal ends of the cord. We optimized the DiI and DiO tracing techniques under different conditions of fixative concentration (1% versus 4% paraformaldehyde, PF), at room temperature (RT) versus 37 degrees C for up to 24 weeks. Crystal coated pins embedded in a silastic disc provided a novel method of dye application. Confocal microscopy of longitudinal sections showed DiI and DiO labeled both the axonal membrane and myelin sheath. DiI diffused significantly longer distances than DiO. Both dyes migrated greater distances at 37 degrees C compared with RT. No significant difference of dye labeling was found between 1% and 4% PF fixation. After prolonged incubation there was evidence that dye diffused through the aqueous medium and produced circumferential labeling of the cord. Placing a wax seal around the labeling site prevented this non-contiguous labeling. Labeling of myelin sheaths at extended distances into the cord suggested that dye could migrate between cells with prolonged incubation periods. Our data suggested that higher temperature facilitated dye diffusion along the axons, and demonstrated that with caution DiI and DiO could be used as specific tracers in the same spinal cords.

Topics: Animals; Axons; Carbocyanines; Coloring Agents; Diffusion; Female; Fixatives; Formaldehyde; Microscopy, Confocal; Nerve Regeneration; Rats; Rats, Sprague-Dawley; Spinal Cord; Temperature; Tissue Fixation

Hippocampal mossy fibers, which are the axons of dentate granule cells, are continuously generated owing to adult neurogenesis of granule cells. They extend exclusively into the stratum lucidum, a proximal layer of the CA3 pyramidal cells. We visualized the mossy fiber tracts by Timm histochemical staining and DiI labeling in the cultured hippocampal slices from newborn rats. The fibers were abnormally expanded when the slices were cultured in the presence of K252a, an inhibitor of the neurotrophin receptor Trk. Similar defasciculation was observed with an inhibitor of MEK, which is one of the signaling molecules downstream of Trk. This study suggests for the first time that Trk and the MEK pathway are required for mossy fiber pathfinding.

Topics: Animals; Brain-Derived Neurotrophic Factor; Carbazoles; Carbocyanines; Cell Death; Cells, Cultured; Enzyme Inhibitors; Fluorescent Dyes; Hippocampus; Histocytochemistry; Indole Alkaloids; MAP Kinase Kinase Kinases; Mossy Fibers, Hippocampal; Nerve Growth Factors; Nerve Regeneration; Organ Culture Techniques; Rats; Rats, Wistar; Receptor, trkA

To examine the morphology of the retinal ganglion cells (RGCs) in the lesser characterized area lying between the optic disk and the macula that consists of the central papillomacular area (PMA) and the arcuate papillomacular bundle (PMB).. Nineteen human and 10 monkey (Macaca fascicularis) retinas obtained after death were used in the study. Perikaryal, axonal, and dendritic silhouettes were examined by postvital application of the fluorescent dye DiI, which specifically labeled RGCs when placed onto the optic fiber layer. The retinas were freed from surrounding tissue, prepared as flat mounts on a nitrocellulose filter, and fixed overnight in 4% paraformaldehyde. DiI diffuses along the membranes of ganglion cell axons, thereby completely labeling them, their cell bodies, and dendrites, which enables the RGCs to be examined with fluorescence microscopy.. In both species, midget cells represented most of the RGCs within the PMA (96.15%) and possessed small, umbrella-like dendrites oriented toward the deeper retinal layers. Parasol cells were less abundant in both species and had small, typical symmetric dendrites. Also along the PMB, midget cells represented most cells (91.52%), whereas only 8.47% could be categorized as parasol cells. In both species, parasol cells of the PMB extended dendrites, which were oriented perpendicular to the axons.. The data show that the PMA and PMB mainly contain small midget cells of typical morphology and size but with atypically oriented dendrites, which are only characteristic for this retinal area.

Topics: Aged; Animals; Axons; Carbocyanines; Cell Count; Dendrites; Fluorescent Dyes; Humans; Macaca fascicularis; Macula Lutea; Middle Aged; Optic Disk; Retinal Ganglion Cells

The preparative method of a block copolymer of poly(dl-lactic acid) (PLA) and methoxypolyethylene glycol amine (MeO-PEG(N)), named PLA-(MeO-PEG), was refined. The degree of introduction of MeO-PEG(N) into PLA increased up to 55% (mol/mol) using a dichloromethane/methanol mixture (1:1, v/v) as a solvent at the reductive amination and taking all the fractions of the first peak in gel-chromatography. Plain and 1,1'-Dioctadecyl-3,3,3',3'-tetramethylindodicarbocyanine (DiD)-loaded nanoparticles prepared using the PLA/PLA-(MeO-PEG) mixture of 45:55 (mol/mol) showed a mean size of 113 and 154 nm, respectively, and a positive zeta potential in water. DiD solution, i.v. administered, showed a lower plasma level and high distribution in liver, though DiD was distributed into the blood cells to a fair extent. Nanoparticles exhibited a higher plasma concentration of DiD than the DiD solution at 1 and 8h, though DiD was distributed into the liver and spleen to a fair extent. Nanoparticles made of the PLA/PLA-(MeO-PEG) mixture of 44:55 (mol/mol) showed better plasma retention than those made of the PLA/PLA-(MeO-PEG) mixture of 64:36 (mol/mol). It is suggested that the PLA/PLA-(MeO-PEG) mixture nanoparticles with a higher PEG/PLA ratio should be useful as a carrier for the elevation of the plasma concentration of lipophilic drugs.

Topics: Animals; Blood Cells; Carbocyanines; Drug Carriers; Fluorescent Dyes; Lactic Acid; Male; Mice; Mice, Inbred Strains; Nanostructures; Plasma; Polyesters; Polyethylene Glycols; Polymers; Tissue Distribution

Unregulated uptake of oxidized low-density lipoproteins (ox-LDL) via macrophage scavenger receptors (SRs) such as lectin-like ox-LDL receptor-1 (LOX-1) is a key event in atherosclerosis. In this study, we examined the effects of five selected food phytochemicals on 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced LOX-1 mRNA expression in THP-1 human monocyte-like cells. Nobiletin, a citrus polymethoxylated flavone, markedly reduced it in dose- and time-dependent manners. It also suppressed the phosphorylation of extracellular signal-regulated protein kinase (ERK) 1/2, c-Jun NH2-terminal kinase (JNK) 1/2, and c-Jun (Ser-63), thereby inhibiting the transcriptional activity of activator protein-1. Further nobiletin attenuated expression of SR-A, SR-PSOX, CD36, and CD68, but not CLA-1, mRNA, leading to the blockade of DiI-acLDL uptake. Together, our results suggest that nobiletin is a promising phytochemical for regulating atherosclerosis with reasonable action mechanisms.

Topics: Carbocyanines; Cell Adhesion Molecules; Cells, Cultured; Citrus; Flavones; Flavonoids; Gene Expression; Humans; Hypolipidemic Agents; Lipoproteins, LDL; Mitogen-Activated Protein Kinases; Monocytes; Phorbol Esters; Phosphorylation; Receptors, Scavenger; RNA, Messenger; Scavenger Receptors, Class E; Tetradecanoylphorbol Acetate; Transcription Factor AP-1; Transcription, Genetic

We cultured bone marrow-derived endothelial progenitor cells by a simple ex vivo expansion method and observed the expansion efficacy. Bone marrow mononuclear cells from five mongrel adult dogs were cultured with EGM-2MV medium in culture flasks coated with fibronectin. Morphology was observed with phase contrast microscopy, and a growth curve was constructed to evaluate the efficacy of expansion. Incorporation of Dil-ac-LDL was tested to evaluate the function. At different time points, immunocytochemical staining for flk-1, CD133, and factor VIII-related antigens was done and compared to staining of endothelial cells and mesenchymal stem cells and percentages of CD133, vascular endothelial growth factor receptor 2 (VEGFR-2), and the double-positive cells measured with flow cytometry to identify the quality and efficacy of expansion. Cluster-like attached cells grew to confluence at an average time of 10 days, and the mean number of cells harvested from 1 mL of bone marrow was (1.3 +/- 0.3) x 10(6). The cells presented a cobblestone-like appearance and took up Dil-ac-LDL. Immunocytochemistry showed that flk-1, CD133, and factor VIII-related antigens were positive. Flow cytometry showed that VEGFR-2 and CD133 double-positive cells augmented 242-fold at the tenth day. Ex vivo expansion can effectively proliferate endothelial progenitor cells from bone marrow; the expansion efficacy could meet the requirements for tissue engineering of blood vessels.

Topics: AC133 Antigen; Animals; Antigens, CD; Bone Marrow Cells; Carbocyanines; Cell Differentiation; Cell Proliferation; Cell Shape; Cells, Cultured; Dogs; Endothelial Cells; Glycoproteins; Mesenchymal Stem Cells; Peptides; Time Factors; Tissue Engineering; von Willebrand Factor

The acid sensitive ion channels TRPV1 (transient receptor potential vanilloid receptor-1) and ASIC3 (acid sensing ion channel-3) respond to tissue acidification in the range that occurs during painful conditions such as inflammation and ischemia. Here, we investigated to which extent they are expressed by rat dorsal root ganglion neurons projecting to lung and pleura, respectively.. The tracer DiI was either injected into the left lung or applied to the costal pleura. Retrogradely labelled dorsal root ganglion neurons were subjected to triple-labelling immunohistochemistry using antisera against TRPV1, ASIC3 and neurofilament 68 (marker for myelinated neurons), and their soma diameter was measured.. Whereas 22% of pulmonary spinal afferents contained neither channel-immunoreactivity, at least one is expressed by 97% of pleural afferents. TRPV1+/ASIC3- neurons with probably slow conduction velocity (small soma, neurofilament 68-negative) were significantly more frequent among pleural (35%) than pulmonary afferents (20%). TRPV1+/ASIC3+ neurons amounted to 14 and 10% respectively. TRPV1-/ASIC3+ neurons made up between 44% (lung) and 48% (pleura) of neurons, and half of them presumably conducted in the A-fibre range (larger soma, neurofilament 68-positive).. Rat pleural and pulmonary spinal afferents express at least two different acid-sensitive channels that make them suitable to monitor tissue acidification. Patterns of co-expression and structural markers define neuronal subgroups that can be inferred to subserve different functions and may initiate specific reflex responses. The higher prevalence of TRPV1+/ASIC3- neurons among pleural afferents probably reflects the high sensitivity of the parietal pleura to painful stimuli.

Topics: Acid Sensing Ion Channels; Animals; Carbocyanines; Cell Size; Female; Fluorescent Dyes; Ganglia, Spinal; Immunohistochemistry; Lung; Membrane Proteins; Nerve Tissue Proteins; Neurofilament Proteins; Neurons, Afferent; Pleura; Rats; Rats, Wistar; Sodium Channels; TRPV Cation Channels

EphB receptors and their ephrin-B ligands are required for midline guidance decisions at several rostrocaudal levels of the developing CNS. In the embryonic vertebrate spinal cord, ephrin-B3 is localized to the floor plate (FP) at the ventral midline (VM), ephrin-B1 and ephrin-B2 are expressed in the dorsal spinal cord, and decussated EphB receptor-bearing commissural axons navigate between these ventral and dorsal ephrin-B domains. Despite these compelling expression patterns, the in vivo role(s) for EphB and ephrin-B proteins in regulating the guidance of spinal commissural axons has not been established. Here, we use DiI (1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate) labeling to assess the pathfinding of commissural axons in the spinal cords of ephrin-B and EphB mutant mouse embryos. In mice lacking ephrin-B3 or multiple EphB receptors, a significant number of axons followed aberrant trajectories in the immediate vicinity of the VM. Furthermore, forked transverse commissural (FTC) axons, a unique class of commissural axons that continues to project in the transverse plane on the contralateral side of the FP, were present at a markedly higher frequency in ephrin-B3 and EphB mutants, compared with wild-type embryos. Neither the midline guidance errors nor excessive numbers of FTC axons were observed in the spinal cords of ephrin-B3(lacz) mice that express a truncated form of ephrin-B3, which is capable of forward but not reverse signaling. In contrast to the midline guidance defects observed in EphB and ephrin-B3 mutant embryos, wild-type-like contralateral projections were observed in mice lacking ephrin-B1 and/or ephrin-B2.

Topics: Animals; Axons; Carbocyanines; Embryo, Mammalian; Ephrin-B3; Fluorescent Dyes; Mice; Mice, Knockout; Neural Pathways; Receptors, Eph Family; Spinal Cord; Synaptic Transmission

Muscarinic acetylcholine receptors (mAChRs) are known to mediate the acetylcholine inhibition of Ca(2+) channels in central and peripheral neurons. Stellate ganglion (SG) neurons provide the main sympathetic input to the heart and contribute to the regulation of heart rate and myocardial contractility. Little information is available regarding mAChR regulation of Ca(2+) channels in SG neurons. The purpose of this study was to identify the mAChR subtypes that modulate Ca(2+) channel currents in rat SG neurons innervating heart muscle. Accordingly, the modulation of Ca(2+) channel currents by the muscarinic cholinergic agonist, oxotremorine-methiodide (Oxo-M), and mAChR blockers was examined. Oxo-M-mediated mAChR stimulation led to inhibition of Ca(2+) currents through voltage-dependent (VD) and voltage-independent (VI) pathways. Pre-exposure of SG neurons to the M(1) receptor blocker, M(1)-toxin, resulted in VD inhibition of Ca(2+) currents after Oxo-M application. On the other hand, VI modulation of Ca(2+) currents was observed after pretreatment of cells with methoctramine (M(2) mAChR blocker). The Oxo-M-mediated inhibition was nearly eliminated in the presence of both M(1) and M(2) mAChR blockers but was unaltered when SG neurons were exposed to the M(4) mAChR toxin, M(4)-toxin. Finally, the results from single-cell RT-PCR and immunofluorescence assays indicated that M(1) and M(2) receptors are expressed and located on the surface of SG neurons. Overall, the results indicate that SG neurons that innervate cardiac muscle express M(1) and M(2) mAChR, and activation of these receptors leads to inhibition of Ca(2+) channel currents through VI and VD pathways, respectively.

Topics: Animals; Calcium Channel Blockers; Calcium Channels, N-Type; Carbocyanines; Data Interpretation, Statistical; Electrophysiology; Fluorescent Antibody Technique; Fluorescent Dyes; Ganglia, Sympathetic; Heart; Male; Microscopy, Fluorescence; Muscarinic Agonists; Neurons; omega-Conotoxin GVIA; Oxotremorine; Rats; Rats, Wistar; Receptor, Muscarinic M1; Receptor, Muscarinic M2; Reverse Transcriptase Polymerase Chain Reaction

The autosomal recessive hypercholesterolemia (ARH) protein plays a critical role in regulating plasma low density lipoprotein (LDL) levels. Inherited defects in ARH lead to a hypercholesterolemia that closely phenocopies that caused by a defective LDL receptor. The elevated serum LDL-cholesterol levels typical of ARH patients and the pronounced accumulation of the LDL receptor at the cell surface of hepatocytes in ARH-null mice argue that ARH operates by promoting the internalization of the LDL receptor within clathrin-coated vesicles. ARH contains an amino-terminal phosphotyrosine-binding domain that associates physically with the LDL receptor internalization sequence and with phosphoinositides. The carboxyl-terminal half of ARH contains a clathrin-binding sequence and a separate AP-2 adaptor binding region providing a plausible mechanism for how ARH can act as an endocytic adaptor or CLASP (clathrin-associated sorting protein) to couple LDL receptors with the clathrin machinery. Because the interaction with AP-2 is highly selective for the independently folded appendage domain of the beta2 subunit, we have characterized the ARH beta2 appendage-binding sequence in detail. Unlike the known alpha appendage-binding motifs, ARH requires an extensive sequence tract to bind the beta appendage with comparably high affinity. A minimal 16-residue sequence functions autonomously and depends upon ARH residues Asp253, Phe259, Leu262, and Arg266. We suggested that biased beta subunit engagement by ARH and the only other beta2 appendage selective adaptor, beta-arrestin, promotes efficient incorporation of this mechanistically distinct subset of CLASPs into clathrin-coated buds.

Topics: Adaptor Proteins, Signal Transducing; Alanine; Amino Acid Motifs; Amino Acid Sequence; Animals; Arginine; Arrestins; beta-Arrestins; Calorimetry; Carbocyanines; Cholesterol, LDL; Clathrin; Cytosol; DNA; DNA-Binding Proteins; Endocytosis; Fluorescent Dyes; Genes, Recessive; Glutathione Transferase; Green Fluorescent Proteins; Humans; Hypercholesterolemia; Kinetics; Lipoproteins, LDL; Mice; Microtubule-Associated Proteins; Models, Molecular; Molecular Sequence Data; Mutagenesis; Peptides; Protein Binding; Protein Conformation; Protein Structure, Secondary; Protein Structure, Tertiary; Receptors, LDL; Sequence Homology, Amino Acid; Transcription Factor AP-2; Transcription Factors; Two-Hybrid System Techniques

The zebrafish system is an excellent vertebrate genetic model to study hemostasis and thrombosis because saturation mutagenesis screens can identify novel genes that play a role in this vital physiologic pathway. To study hemostatic mutations, it is important to understand the physiology of zebrafish hemostasis and thrombosis. Previously, we identified zebrafish thrombocytes and have shown that they participate in arterial thrombus formation. Here, we recognized 2 populations of thrombocytes distinguishable by DiI-C18 (DiI) staining. DiI+ thrombocytes have a high density of adhesive receptors and are functionally more active than DiI- thrombocytes. We classified DiI+ thrombocytes as young and DiI- thrombocytes as mature thrombocytes. We found young and mature thrombocytes each formed independent clusters and that young thrombocytes clustered first. We have also shown that young thrombocytes initiate arterial thrombus formation. We propose that due to the increased adhesive receptor density on young thrombocytes, they adhere first to the subendothelial matrix, get activated rapidly, release agonists, and recruit more young thrombocytes, which further release more agonists. This increase in agonists activates the less active mature thrombocytes, drawing them to the growing thrombus. Since arterial thrombus formation is a fundamental hemostatic event, this mechanism may be conserved in mammals and may open new avenues for prevention of arterial thrombosis.

Topics: Animals; Arteries; Blood Platelets; Carbocyanines; Cellular Senescence; Fluorescent Dyes; Hemostasis; Humans; Thrombosis; Zebrafish

Self-organized lipid bilayers together with proteins are the essential building blocks of biological membranes. Membranes are associated with all living systems as they make up cell boundaries and provide basic barriers to cellular organelles. It is of interest to study the dynamics of individual molecules in cell membranes as the mechanism of how biological membranes function at the single molecule remains to be elucidated. In this letter we describe a study in which we incubate rat basophilic leukemia cells with a fluorescently labeled cell membrane component on a surface containing zero-mode waveguides (ZMWs). We used the ZMW to confine fluorescent excitation to an approximately 100-nm region of the membrane to monitor lipid diffusion along the cellular membrane. We showed that confinement with a ZMW largely reduced fluorescent contributions from the cytosolic pool that is present when using a more standard technique such as laser-induced confocal microscopy. We show that optical confinement with ZMWs is a facile way to probe dynamic processes on the membrane surface.

Topics: Animals; Biophysical Phenomena; Biophysics; Carbocyanines; Cell Line, Tumor; Cell Membrane; Fluorescent Dyes; Optics and Photonics; Rats; Spectrometry, Fluorescence

Recent studies in chick and mouse embryos have identified a previously unrecognized secondary heart field (SHF), located in the ventral midline splanchnic mesenchyme, which provides additional myocardial cells to the outflow tract as the heart tube lengthens during cardiac looping. In order to further delineate the contribution of this secondary myocardium to outflow development, we labeled the right SHF of Hamburger-Hamilton (HH) stage 14 chick embryos via microinjection of DiI/rhodamine and followed the fluorescently labeled cells over a 96-h time period. These experiments confirmed the movement of the SHF into the outflow and its spiraling migration distally, with the right side of the SHF contributing to the left side of the outflow. In contrast, when the right SHF was labeled at HH18, the fluorescence was limited to the caudal wall of the lengthening aortic sac. We then injected a combination of DiI and neutral red dye, and ablated the SHF in HH14 or 18 chick embryos. Embryos were allowed to develop until day 9, and harvested for assessment of outflow alignment. Of the embryos ablated at HH14, 76% demonstrated cardiac defects including overriding aorta and pulmonary atresia, while none of the sham-operated controls were affected. In addition, the more severely affected embryos demonstrated coronary artery anomalies. The embryos ablated at HH18 also manifested coronary artery anomalies but maintained normal outflow alignment. Therefore, the myocardium added to the outflow by the SHF at earlier stages is required for the elongation and appropriate alignment of the outflow tract. However, at later stages, the SHF contributes to the smooth muscle component of the outflow vessels above the pulmonary and aortic valves which is important for the development of the coronary artery stems. This work suggests a role for the SHF in a subset of congenital heart defects that have overriding aorta and coronary artery anomalies, such as tetralogy of Fallot and double outlet right ventricle.

Topics: Animals; Carbocyanines; Chick Embryo; Fluorescent Dyes; Heart; Immunohistochemistry; In Situ Hybridization; Models, Biological; Morphogenesis; Myocardium; Pulmonary Atresia; Tetralogy of Fallot

We have investigated the patterns of tissue flow underlying the formation of the primitive streak in the chick embryo. Analysis of time-lapse sequences of brightfield images to extract the tissue velocity field and of fluorescence images of small groups of DiI-labelled cells have shown that epiblast cells move in two large-scale counter-rotating streams, which merge at the site of streak formation. Despite the large-scale tissue flows, individual cells appear to move little relative to their neighbours. As the streak forms, it elongates in both the anterior and posterior directions. Inhibition of actin polymerisation via local application of the inhibitor latrunculin A immediately terminates anterior extension of the streak tip, but does not prevent posterior elongation. Inhibition of actin polymerisation at the base of the streak completely inhibits streak formation, implying that continuous movement of cells into the base of the forming streak is crucial for extension. Analysis of cycling cells in the early embryo shows that cell-cycle progression in the epiblast is quite uniform before the primitive streak forms then decreases in the central epiblast and incipient streak and increases at the boundary between the area pellucida and area opaca during elongation. The cell-cycle inhibitor aphidicolin, at concentrations that completely block cell-cycle progression, permits initial streak formation but arrests development during extension. Our analysis suggests that cell division maintains the cell-flow pattern that supplies the streak with cells from the lateral epiblast, which is critical for epiblast expansion in peripheral areas, but that division does not drive streak formation or the observed tissue flow.

Topics: Actins; Animals; Aphidicolin; Bridged Bicyclo Compounds, Heterocyclic; Bromodeoxyuridine; Carbocyanines; Cell Division; Cell Movement; Chick Embryo; Embryonic Development; Gastrula; Immunohistochemistry; Microscopy, Fluorescence; Thiazoles; Thiazolidines

A simple procedure to isolate neural stem cells would greatly facilitate direct studies of their properties. Here, we exploited the increase in EGF receptor (EGFR) levels, that occurs in late development stem cells or in younger precursors upon exposure to FGF-2, to isolate cells expressing high levels of EGFR (EGFR(high)) from the developing and the adult brain. Independently of age and region of isolation, EGFR(high) cells were highly enriched in multipotent precursors and displayed similar antigenic characteristics, with the exception of GFAP and Lex/SSEA-1 that were mainly expressed in adult EGFR(high) cells. EGFR levels did not correlate with neurogenic potential, indicating that the increase in EGFR expression does not directly affect differentiation. Instead, in the brain, many EGFR(high) precursors showed tangential orientation and, whether isolated from the cortex or striatum, EGFR(high) precursors displayed characteristics of cells originating from the ventral GZ such as expression Dlx and Mash-1 and the ability to generate GABAergic neurons and oligodendrocytes. Moreover, migration of EGFR(high) cells on telencephalic slices required EGFR activity. Thus, the developmentally regulated increase in EGFR levels may affect tangential migration of multipotent precursors. In addition, it can be used as a marker to effectively isolate telencephalic multipotent precursors from embryonic and adult tissue.

Topics: Animals; Blotting, Western; Brain; Carbocyanines; Cell Movement; ErbB Receptors; Female; Fibroblast Growth Factor 2; Flow Cytometry; Gene Expression Regulation, Developmental; Immunohistochemistry; Mice; Multipotent Stem Cells

Laser flow cytometric analysis was used in conjunction with in vivo labeling with the lipophilic fluorescent dye DiIC18(5)-DS to discriminate resident alveolar macrophages from newly infiltrating monocytes/macrophages in mice with and without pulmonary influenza A virus infection. Leukocytes in bronchoalveolar lavage (BAL) and peripheral blood were analyzed by 2-color flow cytometry as a function of time following intravenous injection of DiIC18(5)-DS. At 4 hours, dye-positive leukocytes were present in both BAL and blood of normal mice, indicating that DiIC18(5)-DS rapidly crossed the pulmonary endothelial-epithelial barrier. At 4 days after dye injection, 98% of BAL cells were DiIC18(5)-DS positive, and almost all of these were monocytes/macrophages based on labeling with fluorescein isothiocyanate (FITC)-conjugated antibody to the Mac-3 marker. Only 3.2% +/- 0.3% of peripheral blood monocytes (approximately 0.16% of total peripheral blood leukocytes) were DiIC18(5)-DS positive at 6 days after injection, whereas > 95% of BAL leukocytes were strongly dye-positive on days 6 to 28. When DiIC18(5)-DS was injected in mice 6 days prior to intranasal challenge with influenza A, flow cytometry indicated that 57.8% 5.6% and 60.7% +/- 8.5% of macrophages/monocytes in BAL were newly infiltrated (i.e., DiIC18(5)-DS negative, Mac-3 positive) at 4 and 7 days, respectively, post viral infection. The discrimination of subpopulations of resident and newly recruited macrophages in BAL should facilitate future mechanistic studies on pulmonary infection and inflammatory lung injury.

Topics: Animals; Antibodies, Monoclonal; Antigens, Differentiation; Blood Cells; Bronchoalveolar Lavage Fluid; Carbocyanines; Cell Movement; Cell Separation; Flow Cytometry; Fluorescein-5-isothiocyanate; Fluorescent Dyes; Influenza A virus; Macrophages, Alveolar; Male; Mice; Monocytes; Orthomyxoviridae Infections

Our purpose was to identify and localize intrinsic cardiac ganglia innervating distinct regions of the heart using postmortem tracing of nerve projections with DiI, a method not previously used to study the intrinsic cardiac nervous system. We also investigated the possibility of collateral innervation of myocardium and intrinsic ganglia. In isolated paraformaldehyde-fixed guinea pig hearts, crystals of DiI (1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate) were inserted into the posterior ventricular myocardium below the atrioventricular groove, the right atrium, or the left ventricular septum. Hearts were placed in the dark at 37 degrees C for 2-14 weeks to allow DiI diffusion within neuronal membranes. Labeled neurons were observed in intracardiac ganglia after at least 4 weeks of dye exposure. Labeling was restricted to the inferior-most ganglia (those near the atrioventricular groove) when DiI was inserted into the posterior ventricular myocardium and to ganglia near the sinus node after right atrial DiI placement. Application of DiI to the left ventricular septum resulted in neuron labeling in ganglia primarily in the interatrial septum near the atrioventricular node. After 8 weeks, DiI-labeled nerve fibers and varicosities were seen surrounding unlabeled neurons in some ganglia, suggesting that axons terminating in or passing through the DiI application site in posterior ventricular tissue had collateral branches innervating these ganglia. These results indicate that intrinsic innervation of major cardiac subdivisions is accomplished by regionally segregated cardiac ganglia. Also, tracing with DiI has provided evidence for collateral nerve projections that could be the substrate for novel intracardiac regulatory circuits.

Topics: Animals; Carbocyanines; Fluorescent Dyes; Ganglia; Guinea Pigs; Heart; Heart Atria; Heart Conduction System; Heart Septum; Heart Ventricles; Immunohistochemistry; In Vitro Techniques; Male; Myocardium; Neural Pathways; Neurons; Staining and Labeling

Neurabin and spinophilin are homologous protein phosphatase 1 and actin binding proteins that regulate dendritic spine function. A yeast two-hybrid analysis using the coiled-coil domain of neurabin revealed an interaction with Lfc, a Rho GEF. Lfc was highly expressed in brain, where it interacted with either neurabin or spinophilin. In neurons, Lfc was largely found in the shaft of dendrites in association with microtubules but translocated to spines upon neuronal stimulation. Moreover, expression of Lfc resulted in reduction in spine length and size. Both the translocation and the effect on spine morphology depended on the coiled-coil domain of Lfc. Coexpression of neurabin or spinophilin with Lfc resulted in their clustering together with F-actin, a process that depended on Rho activity. Thus, interaction between Lfc and neurabin/spinophilin selectively regulates Rho-dependent organization of F-actin in spines and is a link between the microtubule and F-actin cytoskeletons in dendrites.

Topics: Actins; Animals; Brain Chemistry; Carbocyanines; Coloring Agents; Dendrites; DNA, Complementary; Guanine Nucleotide Exchange Factors; Guanosine Diphosphate; Guanosine Triphosphate; Hippocampus; Immunohistochemistry; Microfilament Proteins; Microscopy, Confocal; Microtubules; Nerve Tissue Proteins; Phalloidine; Rats; Transfection

Relative sparing of the pupillary reflexes in patients with leber's hereditary optic neuropathy (LHON) has been observed clinically. This study sought to test histologically whether retino-pupillary fibers are spared in LHON.. Di-I, a fluorescein dye that allows anterograde labeling of axons, was injected into the brachium of the superior colliculus in post-mortem brain from a patient diagnosed with LHON (3460 mutation) and a normal control brain. After 4 weeks, serial fragmatome sections were obtained in the pretectal area and further stained with propidium iodide (PI stains DNA) to delineate the pretectal nuclei in the dorsal midbrain. Examination was performed under a confocal microscope. Optic nerves obtained from the above subjects were cut, mounted and stained with p-phenylenediamine (PPD) and trichrome stain for digital morphometry.. Di-I-labeled fibers were visible on all sections from the superior colliculus to the pretectum in the LHON and the control specimens, as were the nuclei in the cell bodies stained with PI. There was mild attenuation of the afferent pretectal fibers in LHON, but this was not as dramatic as the attenuation of the total population of fibers in the LHON optic nerve.. In our LHON patient, the preservation of retinofugal fibers to the pretectum lends support to the clinical observation of relatively preserved pupillary function in LHON.

Topics: Aged; Carbocyanines; DNA Mutational Analysis; DNA, Mitochondrial; Female; Fluorescent Dyes; Humans; Nerve Fibers; Neurons, Afferent; Optic Atrophy, Hereditary, Leber; Optic Nerve; Reflex, Pupillary

The third intracellular loop domain of G protein-coupled receptors regulates their desensitization, internalization, and resensitization. Colorectal and pancreatic cancers, but not the nonmalignant tissue, express a splice variant of the cholecystokinin 2 receptor (CCK2R) called CCK(2i4sv)R that, because of intron 4 retention, contains an additional 69 amino acids within its third intracellular loop domain. This structural alteration is associated with agonist-independent activation of Src kinase (Olszewska-Pazdrak, B., Townsend, C. M., Jr., and Hellmich, M. R. (2004) J. Biol. Chem. 279, 40400-40404). The purpose of the study was to determine the roles of intron 4 retention and Src kinase on CCK(2i4sv)R desensitization, internalization, and resensitization. Gastrin1-17 (G17) binds to both CCK2R and CCK(2i4sv)R and induces intracellular Ca2+ ([Ca2+]i) increases. Agonist-induced increases in [Ca2+]i were used to assess receptor activity. Src kinase activity was inhibited by transducing cells with a retrovirus containing a dominant-negative mutant Src (A430V). The subcellular location of enhanced green fluorescent protein-tagged receptors was monitored using laser scanning confocal microscopy. Both receptor variants desensitized at the same rate; however, CCK(2i4sv)R resensitized five times faster than CCK2R. Without agonist, 80% of CCK(2i4sv)R is located in an intracellular compartment. In contrast, 80% of CCK2R was located on the plasma membrane. Treatment with inverse agonist (YM022) or expression of dominant-negative Src blocked the constitutive internalization of CCK(2i4sv)R, resulting in its accumulation on the plasma membrane. Expression of dominant-negative Src slowed the rate of CCK(2i4sv)R resensitization. Inhibition of Src did not affect G17-induced internalization of either receptor variant. Constitutive internalization of CCK(2i4sv)R increases its rate of resensitization by creating an intracellular pool of receptors that can rapidly recycle back to the plasma membrane.

Topics: Alternative Splicing; Amino Acid Sequence; Benzodiazepines; Calcium; Carbocyanines; Cell Line; Cell Membrane; Clone Cells; Fluorescent Dyes; Gastrins; Genetic Variation; Green Fluorescent Proteins; Hormone Antagonists; Humans; Introns; Kinetics; Microscopy, Confocal; Models, Biological; Protein Structure, Tertiary; Receptor, Cholecystokinin B; Retroviridae; src-Family Kinases; Transduction, Genetic

The behavior of a fluid supported membrane during hydrolysis by phospholipase A(2) is for the first time visualized by time-resolved fluorescence imaging. After a lag phase, hydrolysis proceeds from the boundary of existing holes and via nucleation of new holes. During subsequent hydrolysis, the shape of the membrane boundary is determined both by hydrolysis and by shape relaxations due to the action of line tension. This is manifested by the appearance of Rayleigh instabilities in membrane rims and by an effect analogous to domain coarsening in phase transitions in which membrane holes decay when they are within a certain distance from larger and expanding holes.

Topics: Carbocyanines; Hydrolysis; Lipid Bilayers; Membrane Fluidity; Microscopy, Fluorescence; Phosphatidylcholines; Phospholipases A

The objective of this study was to assess the effect of hydrophilic/hydrophobic block chain lengths on the internalization of poly(ethylene oxide)-block-poly(epsilon-caprolactone) (PEO-b-PCL) micelles by cancer cells. PEO-b-PCL block copolymers with varied PEO and PCL chain lengths were synthesized, assembled to polymeric micelles and loaded with a hydrophobic fluorescent probe (DiI) through a co-solvent evaporation method of physical encapsulation. The slow release of the fluorescent probe from the micellar structure was evidenced following DiI transfer to lipid vesicles. The extent of micellar uptake by cancer cells was investigated through their incubation with MCF-7 cells followed by measurement of the fluorescent emission intensity of DiI (lambda=550 nm) in separated lysed cells. Cellular internalization of polymeric micelles was confirmed by laser scanning microscopy. The mechanism of micellar uptake was investigated by pretreatment of MCF-7 cells with chlorpromazine and cytochalasin B. Encapsulation of DiI in PEO-b-PCL micelles lowered the extent and rate of hydrophobic probe internalization by cancer cells. For polymeric micelles with 5000 gmol(-1) of PCL and varied PEO molecular weights of 2000, 5000 and 13,000 gmol(-1), maximum uptake was observed at a PEO molecular weight of 5000 gmol(-1). For polymeric micelles with 5000 gmol(-1) of PEO and varied PCL molecular weights of 5000, 13,000 and 24,000 gmol(-1), maximum uptake was observed at 13,000 gmol(-1) of PCL. Chlorpromazine reduced the cellular uptake of PEO-b-PCL micelles independent from the block copolymer structure, pointing to the involvement of clathrin mediated endocytosis mechanisms in the uptake of polymeric micelles by cancer cells. Inhibition of cellular uptake of PEO-b-PCL micelles by cytochalasin B, on the other hand, was found to be dependent on the chemical structure of the core/shell forming blocks.

Topics: Breast Neoplasms; Carbocyanines; Cell Line, Tumor; Cold Temperature; Drug Delivery Systems; Female; Fluorescent Dyes; Humans; Magnetic Resonance Spectroscopy; Micelles; Microscopy, Confocal; Polyesters; Staining and Labeling

The signaling of retinal ganglion cell (RGC) death after axotomy is partly dependent on the generation of reactive oxygen species. Shifting the RGC redox state toward reduction is protective in a dissociated mixed retinal culture model of axotomy. The hypothesis for the current study was that tris(2-carboxyethyl)phosphine (TCEP), a sulfhydryl reductant, would protect RGCs in a rat optic nerve crush model of axotomy.. RGCs of postnatal day 4 to 5 Long-Evans rats were retrogradely labeled with the fluorescent tracer DiI. At approximately 8 weeks of age, the left optic nerve of each rat was crushed with forceps and, immediately after, 4 muL of TCEP (or vehicle alone) was injected into the vitreous at the pars plana to a final concentration of 6 or 60 microM. The right eye served as the control. Eight or 14 days after the crush, the animals were killed, retinal wholemounts prepared, and DiI-labeled RGCs counted. Bandeiraea simplicifolia lectin (BSL-1) was used to identify microglia.. The mean number of surviving RGCs at 8 days in eyes treated with 60 microM TCEP was significantly greater than in the vehicle group (1250 +/- 156 vs. 669 +/- 109 cells/mm(2); P = 0.0082). Similar results were recorded at 14 days. Labeling was not a result of microglia phagocytosing dying RGCs. No toxic effect on RGC survival was observed with TCEP injection alone.. The sulfhydryl-reducing agent TCEP is neuroprotective of RGCs in an optic nerve crush model. Sulfhydryl oxidative modification may be a final common pathway for the signaling of RGC death by reactive oxygen species after axotomy.

Topics: Animals; Axotomy; Carbocyanines; Cell Count; Cell Survival; Disease Models, Animal; Fluorescent Dyes; Injections; Neuroglia; Neuroprotective Agents; Optic Nerve Injuries; Phosphines; Rats; Rats, Long-Evans; Retinal Ganglion Cells; Sulfhydryl Compounds; Vitreous Body

To develop a new high-throughput screening model for human high-density lipoprotein (HDL) receptor (CD36 and LIMPII analogous-1, CLA-1) agonists using CLA-1-expressing insect cells.. With the total RNA of human hepatoma cells BEL-7402 as template, the complementary DNA (cDNA) of CLA-1 was amplified by reverse transcription-polymerase chain reaction (RT-PCR). Bac-to-Bac baculovirus expression system was used to express CLA-1 in insect cells. CLA-1 cDNA was cloned downstream of polyhedrin promoter of Autographa californica nuclear polyhedrosis virus (AcNPV) into donor vector pFastBac1 and recombinant pFastBac1-CLA-1 was transformed into E. coli DH10Bac to transpose CLA-1 cDNA to bacmid DNA. Recombinant bacmid-CLA-1 was transfected into Spodoptera frugiperda Sf9 insect cells to produce recombinant baculovirus particles. Recombinant CLA-1 was expressed on the membrane of Sf9 cells infected with the recombinant baculoviruses. A series of parameters of DiI-lipoprotein binding assays of CLA-1-expressing Sf9 cells in 96-well plates were optimized.. Western blot analysis and DiI-lipoprotein binding assays confirmed that CLA-1 expressed in insect cells had similar immunoreactivity and ligand binding activity as its native counterpart. A reliable and sensitive in vitro cell-based assay was established to assess the activity of CLA-1 and used to screen agonists from different sample libraries.. Human HDL receptor CLA-1 was successfully expressed in Sf9 insect cells and a novel high-throughput screening model for CLA-1 agonists was developed. Utilization of this model allows us to identify potent and selective CLA-1 agonists which might possibly be used as therapeutics for atherosclerosis.

Topics: Animals; Baculoviridae; Biological Assay; Carbocyanines; Cell Line, Tumor; Cholesterol, HDL; DNA, Complementary; Fluorescent Dyes; Gene Expression; Humans; Lipoproteins, HDL; Lipoproteins, LDL; Receptors, Lipoprotein; Recombinant Proteins; Reverse Transcriptase Polymerase Chain Reaction; Scavenger Receptors, Class B; Spodoptera

Brain microglia are phagocytic cells that are the major inflammatory response cells of the central nervous system and widely held to play important pathophysiologic roles in Alzheimer's disease (AD) in both potentially neurotoxic responses and potentially beneficial phagocytic responses. In the study, we examined whether ginsonoside Rg3, a by-product of red ginseng, enhances the microglial phagocytosis of Abeta. We found that Rg3 promoted Abeta uptake, internalization, and digestion. Increased maximal Abeta uptake was observed at 4 and 8 h after Rg3 pre-treatment (25 microg/mL), and the internalized Abeta was almost completely digested from cells within 36 h when pretreated with Rg3 comparing with single non-Rg3-treated groups. The expression of MSRA (type A MSR) was also up-regulated by Rg3 treatment in a dose- and time-dependent manner which was coincidently identified in western blots for MSRA proteins in cytosol. These results indicate that microglial phagocytosis of Abeta may be enhanced by Rg3 and the effect of Rg3 on promoting clearance of Abeta may be related to the MSRA-associated action of Rg3. Thus, stimulation of the MSRA might contribute to the therapeutic potentials of Rg3 in microglial phagocytosis and digestion in the treatment of AD.

Topics: Amyloid beta-Peptides; Animals; Animals, Newborn; Blotting, Western; Carbocyanines; Cells, Cultured; Dose-Response Relationship, Drug; Fluorescent Dyes; Gene Expression; Ginsenosides; Lipoproteins, LDL; Mice; Microglia; Microscopy, Fluorescence; Peptide Fragments; Phagocytosis; Rats; Rats, Sprague-Dawley; Receptors, Scavenger; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Time Factors

Clinical application of the convection-enhanced delivery (CED) technique is currently limited by low infusion speed and reflux of the delivered agent. The authors developed and evaluated a new step-design cannula to overcome present limitations and to introduce a rapid, reflux-free CED method for future clinical trials.. The CED of 0.4% trypan blue dye was performed in agarose gel to test cannula needles for distribution and reflux. Infusion rates ranging from 0.5 to 50 microl/minute were used. Agarose gel findings were translated into a study in rats and then in cynomolgus monkeys (Macacafascicularis) by using trypan blue and liposomes to confirm the efficacy of the reflux-free step-design cannula in vivo. Results of agarose gel studies showed reflux-free infusion with high flow rates using the step-design cannula. Data from the study in rats confirmed the agarose gel findings and also revealed increasing tissue damage at a flow rate above 5-microl/minute. Robust reflux-free delivery and distribution of liposomes was achieved using the step-design cannula in brains in both rats and nonhuman primates.. The authors developed a new step-design cannula for CED that effectively prevents reflux in vivo and maximizes the distribution of agents delivered in the brain. Data in the present study show reflux-free infusion with a constant volume of distribution in the rat brain over a broad range of flow rates. Reflux-free delivery of liposomes into nonhuman primate brain was also established using the cannula. This step-design cannula may allow reflux-free distribution and shorten the duration of infusion in future clinical applications of CED in humans.

Topics: Animals; Brain; Carbocyanines; Catheterization; Coloring Agents; Convection; Drug Delivery Systems; Fluorescent Dyes; Gels; Liposomes; Macaca fascicularis; Male; Rats; Rats, Sprague-Dawley; Sepharose; Trypan Blue

Cell-to-cell communication is a crucial prerequisite for the development and maintenance of multicellular organisms. To date, diverse mechanisms of intercellular exchange of information have been documented, including chemical synapses, gap junctions, and plasmodesmata. Here, we describe highly sensitive nanotubular structures formed de novo between cells that create complex networks. These structures facilitate the selective transfer of membrane vesicles and organelles but seem to impede the flow of small molecules. Accordingly, we propose a novel biological principle of cell-to-cell interaction based on membrane continuity and intercellular transfer of organelles.

Topics: Actins; Animals; Biological Transport; Carbocyanines; Cell Communication; Cell Line; Cell Membrane; Cell Surface Extensions; Endocytosis; Endosomes; Fluorescent Dyes; Green Fluorescent Proteins; Luminescent Proteins; Membrane Proteins; Microscopy, Electron, Scanning; Microscopy, Fluorescence; Microscopy, Video; Organelles; PC12 Cells; Protein Prenylation; Protein Transport; Pseudopodia; Rats; Recombinant Fusion Proteins; Synaptophysin

Although liposomes have been used as a vehicle for delivery of therapeutic agents in oncology, their efficacy in targeting brain tumors has been limited due to poor penetration through the blood-brain barrier. Because convection-enhanced delivery (CED) of liposomes may improve the therapeutic index for targeting brain tumors, we conducted a three-stage study: stage 1 established the feasibility of using in vivo magnetic resonance imaging (MRI) to confirm adequate liposomal distribution within targeted regions in normal rat brain. Liposomes colabeled with gadolinium (Gd) and a fluorescent indicator, 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine-5,5'-disulfonic acid [DiI-DS; formally DiIC(18)(3)-DS], were administered by CED into striatal regions. The minimum concentration of Gd needed for monitoring, correlation of infused volume with distribution volume, clearance of infused liposome containing Gd and DiI-DS (Lip/Gd/DiI-DS), and potential local toxicity were evaluated. After determination of adequate conditions for MRI detection in normal brain, stage 2 evaluated the feasibility of in vivo MRI monitoring of liposomal distribution in C6 and 9L-2 rat glioma models. In both models, the distribution of Lip/Gd/DiI-DS covering the tumor mass was well defined and monitored with MRI. Stage 3 was designed to develop a clinically relevant treatment strategy in the 9L-2 model by infusing liposome containing Gd (Lip/Gd), prepared in the same size as Lip/Gd/DiI-DS, with Doxil, a liposomal drug of similar size used to treat several cancers. MRI detection of Lip/Gd coadministered with Doxil provided optimum CED parameters for complete coverage of 9L-2 tumors. By permitting in vivo monitoring of therapeutic distribution in brain tumors, this technique optimizes local drug delivery and may provide a basis for clinical applications in the treatment of malignant glioma.

Topics: Animals; Antibiotics, Antineoplastic; Brain; Brain Neoplasms; Carbocyanines; Convection; Doxorubicin; Fluorescent Dyes; Gadolinium; Glioma; Gliosarcoma; Liposomes; Magnetic Resonance Imaging; Male; Rats; Rats, Sprague-Dawley; Tissue and Organ Procurement

In adult mammals, the adipocyte-derived hormone leptin acts on the brain to reduce food intake by regulating the activity of neurons in the arcuate nucleus of the hypothalamus (ARH). Here, we report that neural projection pathways from the ARH are permanently disrupted in leptin-deficient (Lepob/Lepob) mice and leptin treatment in adulthood does not reverse these neuroanatomical defects. However, treatment of Lepob/Lepob neonates with exogenous leptin rescues the development of ARH projections, and leptin promotes neurite outgrowth from ARH neurons in vitro. These results suggest that leptin plays a neurotrophic role during the development of the hypothalamus and that this activity is restricted to a neonatal critical period that precedes leptin's acute regulation of food intake in adults.

Topics: Agouti-Related Protein; alpha-MSH; Animals; Animals, Newborn; Arcuate Nucleus of Hypothalamus; Axons; Carbocyanines; Culture Techniques; Dorsomedial Hypothalamic Nucleus; Eating; Feeding Behavior; Hypothalamic Area, Lateral; Hypothalamus; Intercellular Signaling Peptides and Proteins; Leptin; Mice; Mice, Inbred C57BL; Mice, Obese; Nerve Fibers; Neurites; Neurons; Paraventricular Hypothalamic Nucleus; Proteins; Recombinant Proteins; Signal Transduction

Neurophysiological studies have characterized the sensory responses of primary afferent nociceptors that innervate the intracranial dura. The present study used anatomical methods to examine in greater detail the axonal trajectories within the dura, as well as the axonal size distribution of the dural innervation. Immunostaining for CGRP in dural wholemounts revealed a network of fibers extending across the entire dura, with an especially dense plexus running along the borders of the transverse and superior sagittal sinuses. The plexus along the caudal border of the transverse sinus partially overlapped the dural area that shows the greatest density of mast cells. Visualization of axon bundles by DiI application in formalin-fixed tissue revealed two separate systems of fibers in the dura that could be distinguished by the orientation of their trajectories: one that runs parallel to the middle meningeal artery (MMA), and another with a more or less orthogonal orientation that runs rostromedially from the transverse sinus across the MMA. Axons traversed large distances across the dura, but the majority of the branching and arborization was usually concentrated in the distal part of the trajectory. In separate animals, measurement of myelinated axon diameters with electron microscopy showed that approximately one-third of the myelinated axons in the nerves supplying the dura (nervus spinosus and tentorial nerves) could be classified as A-beta, since they were comparable in size to the majority of axons in the trochlear nerve and the upper end of the size range in the trigeminal nerve (i.e., > 5 microm).

Topics: Animals; Axons; Calcitonin Gene-Related Peptide; Carbocyanines; Dura Mater; Fluorescent Dyes; Immunohistochemistry; Mast Cells; Microscopy, Electron; Neurons, Afferent; Rats

Despite the importance of cholesterol in the formation and function of caveolar microdomains in plasma membranes, almost nothing is known regarding the structural properties, cholesterol dynamics or intracellular factors affecting caveolar cholesterol dynamics. A non-detergent method was employed to isolate caveolae/raft domains from purified plasma membranes of murine fibroblasts. A series of fluorescent lipid probe molecules or a fluorescent cholesterol analogue, dehydroergosterol, were then incorporated into the caveolae/raft domains to show that: (i) fluorescence polarization of the multiple probe molecules [diphenylhexatriene analogues, DiI18 (1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate), parinaric acids and NBD-stearic acid [12-(N-methyl)-N-[(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-octadecanoic acid] indicated that acyl chains in caveolae/raft domains were significantly less 'fluid' (i.e. more rigid) and the transbilayer 'fluidity gradient' was 4.4-fold greater than in plasma membranes; (ii) although sterol was more ordered in caveolae/raft domains than plasma membranes, spontaneous sterol transfer from caveolae/raft domains was faster (initial rate, 32%; half-time, t(1/2), 57%) than from the plasma membrane; (iii) although kinetic analysis showed similar proportions of exchangeable and non-exchangeable sterol pools in caveolae/raft domains and plasma membranes, addition of SCP-2 (sterol carrier protein-2) 1.3-fold more selectively increased sterol transfer from caveolae/raft domains by decreasing the t(1/2) (50%) and increasing the initial rate (5-fold); (iv) SCP-2 was also 2-fold more selective in decreasing the amount of non-exchangeable sterol in caveolae/raft domains compared with plasma membranes, such that nearly 80% of caveolar/raft sterol became exchangeable. In summary, although caveolae/raft lipids were less fluid than those of plasma membranes, sterol domains in caveolae/rafts were more spontaneously exchangeable and more affected by SCP-2 than those of the bulk plasma membranes. Thus caveolae/raft domains isolated without the use of detergents display unique structure, cholesterol domain kinetics and responsiveness to SCP-2 as compared with the parent plasma membrane.

Topics: Animals; Carbocyanines; Carrier Proteins; Caveolae; Cell Line; Cell Membrane; Cholesterol; Diphenylhexatriene; Ergosterol; Fatty Acids, Unsaturated; Fibroblasts; Fluorescence Polarization; Fluorescent Dyes; Gels; Lipids; Membrane Microdomains; Methylamines; Mice; Molecular Probe Techniques; Molecular Probes; Peptides; Propionates; Protein Structure, Tertiary; Solutions; Sterols; Subcellular Fractions

We introduce a fast and simple one-step method, a variation of the methods of Barrett and Campbell and Bartlett, to synthesize monodisperse fluorescent particles that can be dispersed in organic solvents and have long excitation (649 nm) and emission wavelengths (679 nm). A lipophilic fluorescent dye, 1,1'-dioctadecyl-3,3,3',3'-tetramethylindodicarbocyanine perchlorate, is directly incorporated into PMMA particles through dispersion polymerization. A poly(hydroxystearic acid) graft (poly) methyl methacrylate (MM) and methacrylic acid (MA) copolymer is used as a stabilizer to prevent the particles from aggregating and flocculating in the nonaqueous solvents. The fluorescent PMMA particles are very uniform in size, bleach at very low rate, and behave like hard spheres in their ordering on substrates. One important achievement in our synthesis protocol is that we are able to produce particles of a desired size by choosing the composition of the reactants according to a predetermined relationship between particle size and composition of reactants. In addition, the effects of fluorescent dye and polar solvent (ethanol) on the formation and size of particles are discussed.

Topics: Biocompatible Materials; Carbocyanines; Excipients; Fluorescent Dyes; Materials Testing; Microscopy, Fluorescence; Organic Chemicals; Particle Size; Polymethacrylic Acids; Solubility; Solvents; Stearic Acids

Horizontal, bipolar, and amacrine cells in the zebrafish retina were morphologically characterized using DiOlistic techniques. In this method, 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI)-coated microcarriers are shot at high speed onto the surfaces of living retinal slices where the DiI then delineates axons, somata, and dendrites of isolated neurons. Zebrafish retinal somata were 5-10 microm in diameter. Three horizontal cell types (HA-1, HA-2, and HB) were identified; dendritic tree diameters averaged 25-40 microm. HA somata were round. Cells classified as HA-2 were larger than HA-1 cells and possessed an axon. HB somata were flattened, without an axon, although short fusiform structure(s) projected from the soma. Bipolar cells were separated into 17 morphological types. Dendritic trees ranged from 10 to 70 microM. There were six B(on) types with axon boutons only in the ON sublamina of the inner plexiform layer (IPL), and seven B(off) types with axon boutons or branches only in the OFF sublamina. Four types of bistratified bipolar cells displayed boutons in both ON and OFF layers. Amacrine cells occurred in seven types. A(off) cells (three types) were monostratified and ramified in the IPL OFF sublamina. Dendritic fields were 60-150 microM. A(on) pyriform cells (three types) branched in the ON sublamina. Dendritic fields were 50-170 microM. A(diffuse) cells articulated processes in all IPL strata. Dendritic fields were 15-90 microM. These findings are important for studies examining signal processing in zebrafish retina and for understanding changes in function resulting from mutations and perturbations of retinal organization.

Topics: Amacrine Cells; Animals; Biolistics; Carbocyanines; Diagnostic Imaging; Image Processing, Computer-Assisted; Neurons; Organ Culture Techniques; Zebrafish

The aims of this study were to obtain concentrated pinolenic acid (5,9,12-18:3) from dietary Korean pine (Pinus koraiensis) nut oil by urea complexation and to investigate its cholesterol-lowering effect on the LDL-receptor activity of human hepatoma HepG2 cells. Pine nut oil was hydrolyzed to provide a low-pinolenic acid-containing FA extract (LPAFAE), followed by crystallization with different ratios of urea in ethanol (EtOH) or methanol (MeOH) as a solvent to produce a high-pinolenic acid-containing FA extract (HPAFAE). The profiles of HPAFAE obtained by urea complexation showed different FA compositions compared with LPAFAE. The long-chain saturated FA palmitic acid (16:0) and stearic acid (18:0) were decreased with urea/FA ratios (UFR) of 1:1 (UFR1), 2:1 (UFR2), and 3:1 (UFR3). Linoleic acid (9,12-18:2) was increased 1.3 times with UFR2 in EtOH, and linolenic acid (9,12,15-18:3) was increased 1.5 times with UFR3 in MeOH after crystallization. The crystallization with UFR3 in EtOH provided the highest concentration of pinolenic acid, which was elevated by 3.2-fold from 14.1 to 45.1%, whereas that of linoleic acid (9,12-18:2) was not changed, and that of oleic acid (9-18:1) was decreased 7.2-fold. Treatment of HepG2 cells with HPAFE resulted in significantly higher internalization of 3,3'-dioctadecylindocarbocyanine-LDL (47.0 +/- 0.15) as compared with treatment with LPAFAE (25.6 +/- 0.36) (P< 0.05). Thus, we demonstrate a method for the concentration of pinolenic acid and suggest that this concentrate may have LDL-lowering properties by enhancing hepatic LDL uptake.

Topics: Animals; Carbocyanines; Cell Line, Tumor; Crystallization; Dietary Fats, Unsaturated; Fluorescent Dyes; Humans; Korea; Linolenic Acids; Nuts; Pinus; Plant Oils; Receptors, LDL; Urea

We previously characterized the patients with autosomal recessive hypercholesterolemia (ARH) as having severe hypercholesterolemia and retarded plasma low-density lipoprotein (LDL) clearance despite normal LDL receptor (LDLR) function in their cultured fibroblasts, and we identified a mutation in the ARH locus in these patients. ARH protein is an adaptor protein of the LDL and reportedly modulates its internalization. We developed ARH knockout mice (ARH-/-) to study the function of this protein. Plasma total cholesterol level was higher in ARH-/- mice than that in wild-type mice (ARH+/+), being attributed to a 6-fold increase of LDL, whereas plasma lipoprotein was normal in the heterozygotes (ARH+/-). Clearance of 125I-LDL from plasma was retarded in ARH-/- mice, as much as that found in LDLR-/- mice. Fluorescence activity of the intravenously injected 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI)-LDL was recovered in the cytosol of the hepatocytes of ARH+/+ mice, but not in those of ARH-/- or LDLR-/- mice. Also, less radioactivity was recovered in the liver of ARH-/- or LDLR-/- mice when [3H]cholesteryl oleyl ether (CE)-labeled LDL was injected. In contrast, uptakes of [3H]CE-labeled LDL, 125I-LDL, and DiI-LDL were all normal or slightly subnormal when the ARH-/- hepatocytes were cultured. We thus concluded that the function of the hepatic LDLR is impaired in the ARH-/- mice in vivo, despite its normal function in vitro. These findings were consistent with the observations with the ARH homozygous patients and suggested that certain cellular environmental factors modulate the requirement of ARH for the LDLR function.

Topics: Adaptor Proteins, Signal Transducing; Animals; Carbocyanines; Cells, Cultured; Cholesterol; Cholesterol, LDL; Female; Genes, Recessive; Genotype; Hepatocytes; Humans; Hyperlipoproteinemia Type II; Injections, Intravenous; Lipoproteins, LDL; Liver; Mice; Mice, Knockout; Mutagenesis, Insertional; Phenotype; Receptors, LDL

Erythropoiesis is characterized by 2 waves of production during mouse embryogenesis: a primitive one originating from the yolk sac (YS) and a definitive one produced from both the YS and the embryo proper. How the latter wave is generated remains unclear. To investigate our hypothesis that endothelial cells (ECs) could generate erythroid cells, we designed a method to label ECs at 10 days after coitus. This labeling method associates 2 techniques: an intracardiac inoculation that allows molecules to be delivered into the bloodstream followed by a whole-embryo culture period. DiI-conjugated acetylated low-density lipoproteins (Ac-LDL-DiI) were used to specifically tag ECs from the inside. One hour after inoculation, DiI staining was found along the entire endothelial tree. Fluorescence-activated cell sorter (FACS) analysis revealed that DiI+ cells were CD31+, CD34+, and CD45-, an antigen makeup characteristic of the endothelial lineage. Twelve hours after inoculation, 43% of DiI+ circulating cells belonged to the erythroid lineage. These cells expressed Ter119 and displayed an adult globin chain arrangement; thus they belonged to the definitive lineage as confirmed in erythroid colony formation. The remaining cells likely represent committed white blood cells or multipotent progenitors, as revealed by a mixed-colony formation. Beyond the 29-somite stage, the proportion of DiI+ erythroid cells gradually decreased. These results demonstrate the generation of hematopoietic cells from an endothelial intermediate, using in vivo tracing. We provide evidence for a release of these cells into the circulation and hypothesize that these cells are able to colonize the fetal liver and generate definitive erythrocytes in vivo.

Topics: Animals; Antigens, CD34; Carbocyanines; Cell Differentiation; Culture Techniques; Endothelium, Vascular; Erythroid Precursor Cells; Erythropoiesis; Flow Cytometry; Fluorescent Dyes; Gestational Age; Leukocyte Common Antigens; Lipoproteins, LDL; Liver; Mice; Mice, Inbred ICR; Platelet Endothelial Cell Adhesion Molecule-1; Reverse Transcriptase Polymerase Chain Reaction

The fluorescence lifetime and the fluorescence rate of single molecules are recorded as a function of the position of a Si3N4 atomic force microscopy tip with respect to the molecule. We observe a decrease of the excited state lifetime and the fluorescence rate when the tip apex is in close proximity to the molecule. These effects are attributed to the fact that the dielectric tip converts non-propagating near-fields to propagating fields within the dielectric tip effectively quenching the fluorescence. The spatial extension of the quenching area is of subwavelength dimensions. The results are discussed in terms of molecular fluorescence in a system of stratified media. The experiment provides surprising new insights into the interactions between a fluorescent molecule and a dielectric tip. The methodology holds promise for applications in ultra high-resolution near-field optical imaging at the level of single fluorophores.

Topics: Carbocyanines; Electricity; Energy Transfer; Microscopy, Scanning Probe; Models, Structural; Nanotechnology; Optics and Photonics; Polymethyl Methacrylate; Silicon Compounds; Spectrometry, Fluorescence

Patterning of the ventral head has been attributed to various cell populations, including endoderm, mesoderm, and neural crest. Here, we provide evidence that head and heart development may be influenced by a ventral midline endodermal cell population. We show that the ventral midline endoderm of the foregut is generated directly from the extreme rostral portion of Hensen's node, the avian equivalent of the Spemann organizer. The endodermal cells extend caudally in the ventral midline from the prechordal plate during development of the foregut pocket. Thus, the prechordal plate appears as a mesendodermal pivot between the notochord and the ventral foregut midline. The elongating ventral midline endoderm delimits the right and left sides of the ventral foregut endoderm. Cells derived from the midline endoderm are incorporated into the endocardium and myocardium during closure of the foregut pocket and fusion of the bilateral heart primordia. Bilateral ablation of the endoderm flanking the midline at the level of the anterior intestinal portal leads to randomization of heart looping, suggesting that this endoderm is partitioned into right and left domains by the midline endoderm, thus performing a function similar to that of the notochord in maintaining left-right asymmetry. Because of its derivation from the dorsal organizer, its extent from the forebrain through the midline of the developing face and pharynx, and its participation in formation of a single midline heart tube, we propose that the ventral midline endoderm is ideally situated to function as a ventral organizer of the head and heart.

Topics: Animals; Body Patterning; Carbocyanines; Chick Embryo; Chimera; Coturnix; Digestive System; Endoderm; Gene Expression Regulation, Developmental; Genes, Homeobox; Head; Heart; Homeodomain Proteins; Models, Biological; Organizers, Embryonic; Rhodamines

The migratory response of surface fibroblasts to flexor tendon injury was studied by their selective labeling with a vital dye.. The surfaces of 30 rat deep flexor tendons were bathed in 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine percholate (DiI), a vital dye for 5 minutes. The residual dye was removed by thorough irrigation. A partial tenotomy was made in the stained section by cutting out a central window. Semiquantitative cell counts and position of stained fibroblasts were noted by examination under fluorescent light at 0, 1, 3, 5, and 7 days.. The surface fibroblasts readily took up the vital dye at day 0. By day 1 dyed cells had moved into the cut. By day 3 they had migrated laterally into the core substance of the tendon. Core dyed cell counts at days 1, 3, 5, and 7 were significantly different compared with day 0 core dyed cell counts.. This cell migration from the surface of the cut to the tendon core is likely to be vital in the early stages of tendon healing.

Topics: Animals; Carbocyanines; Cell Count; Fluorescent Dyes; Hindlimb; Histological Techniques; Male; Rats; Rats, Sprague-Dawley; Statistics, Nonparametric; Tendon Injuries; Tendons; Wound Healing

The connections of the dorsal telencephalon (pallium) of the Senegal bichir (Polypterus senegalus) were investigated in order to test the hypothesis that the simple everted pallium of polypterid fishes is directly comparable to the evaginated pallium of most vertebrates. Neuroanatomical tracers (DiI or biotinylinated dextran amines) were injected into the three traditionally recognized divisions of the bichir pallium to determine their afferent and efferent connections. The connections were corroborated with injections into regions identified as projecting to, or receiving input from, one or more pallial divisions. The results suggest that the bichir pallium consists not of three, but only two primary divisions: a dorsomedial one and a dorsolateral one. This reinterpretation refutes the hypothesis of a direct one-to-one comparison with the tripartite pallium of most vertebrates. Homologues of the bichir dorsomedial and dorsolateral pallium are recognized in the lateral and medial pallium of anuran amphibians, respectively. The pallium of bichirs is compared to that of derived ray-finned fishes as a link between derived ray-finned fishes and other vertebrates. The available information on the connections of the pallium of teleosts suggests that only the olfactory recipient (pars posterior) of the pallium can be directly compared to bichirs and amphibians and that the remaining divisions of the pallium in teleosts are uniquely derived features of the teleost telencephalon.

Topics: Animals; Carbocyanines; Coloring Agents; Diencephalon; Fishes; Globus Pallidus; Nerve Fibers; Neural Pathways; Telencephalon

The neural crest is a vertebrate-specific cell population that contributes to the facial skeleton and other derivatives. We have performed focal DiI injection into the cranial neural tube of the developing lamprey in order to follow the migratory pathways of discrete groups of cells from origin to destination and to compare neural crest migratory pathways in a basal vertebrate to those of gnathostomes. The results show that the general pathways of cranial neural crest migration are conserved throughout the vertebrates, with cells migrating in streams analogous to the mandibular and hyoid streams. Caudal branchial neural crest cells migrate ventrally as a sheet of cells from the hindbrain and super-pharyngeal region of the neural tube and form a cylinder surrounding a core of mesoderm in each pharyngeal arch, similar to that seen in zebrafish and axolotl. In addition to these similarities, we also uncovered important differences. Migration into the presumptive caudal branchial arches of the lamprey involves both rostral and caudal movements of neural crest cells that have not been described in gnathostomes, suggesting that barriers that constrain rostrocaudal movement of cranial neural crest cells may have arisen after the agnathan/gnathostome split. Accordingly, neural crest cells from a single axial level contributed to multiple arches and there was extensive mixing between populations. There was no apparent filling of neural crest derivatives in a ventral-to-dorsal order, as has been observed in higher vertebrates, nor did we find evidence of a neural crest contribution to cranial sensory ganglia. These results suggest that migratory constraints and additional neural crest derivatives arose later in gnathostome evolution.

Topics: Animals; Branchial Region; Carbocyanines; Cartilage; Cell Movement; Fluorescent Dyes; Ganglia; Lampreys; Melanocytes; Mesencephalon; Neural Crest; Pharynx; Rhombencephalon; Skull; Species Specificity

The olfactory placodes generate the primary sensory neurons of the olfactory sensory system. Additionally, the olfactory placodes have been proposed to generate a class of neuroendocrine cells containing gonadotropin-releasing hormone (GnRH). GnRH is a multifunctional decapeptide essential for the development of secondary sex characteristics in vertebrates as well as a neuromodulator within the central nervous system. Here, we show that endocrine and neuromodulatory GnRH cells arise from two separate, nonolfactory regions in the developing neural plate. Specifically, the neuromodulatory GnRH cells of the terminal nerve arise from the cranial neural crest, and the endocrine GnRH cells of the hypothalamus arise from the adenohypophyseal region of the developing anterior neural plate. Our findings are consistent with cell types generated by the adenohypophysis, a source of endocrine tissue in vertebrate animals, and by neural crest, a source of cells contributing to the cranial nerves. The adenohypophysis arises from a region of the anterior neural plate flanked by the olfactory placode fields at early stages of development, and premigratory cranial neural crest lies adjacent to the caudal edge of the olfactory placode domain [Development 127 (2000), 3645]. Thus, the GnRH cells arise from tissue closely associated with the developing olfactory placode, and their different developmental origins reflect their different functional roles in the adult animal.

Topics: Animals; Carbocyanines; Gonadotropin-Releasing Hormone; Head; Immunohistochemistry; Mutation; Neural Crest; Pituitary Gland, Anterior; Staining and Labeling; Transcription Factors; Zebrafish; Zebrafish Proteins; Zinc Finger Protein Gli2

Confocal fluorescence microscopy and fluorescence correlation spectroscopy (FCS) have been employed to investigate the lipid spatial and dynamic organization in giant unilamellar vesicles (GUVs) prepared from ternary mixtures of dioleoyl-phosphatidylcholine/sphingomyelin/cholesterol. For a certain range of cholesterol concentration, formation of domains with raft-like properties was observed. Strikingly, the lipophilic probe 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI-C18) was excluded from sphingomyelin-enriched regions, where the raft marker ganglioside GM1 was localized. Cholesterol was shown to promote lipid segregation in dioleoyl-phosphatidylcholine-enriched, liquid-disordered, and sphingomyelin-enriched, liquid-ordered phases. Most importantly, the lipid mobility in sphingomyelin-enriched regions significantly increased by increasing the cholesterol concentration. These results pinpoint the key role, played by cholesterol in tuning lipid dynamics in membranes. At cholesterol concentrations >50 mol%, domains vanished and the lipid diffusion slowed down upon further addition of cholesterol. By taking the molecular diffusion coefficients as a fingerprint of membrane phase compositions, FCS is proven to evaluate domain lipid compositions. Moreover, FCS data from ternary and binary mixtures have been used to build a ternary phase diagram, which shows areas of phase coexistence, transition points, and, importantly, how lipid dynamics varies between and within phase regions.

Topics: Carbocyanines; Cell Membrane; Cholesterol; Fluorescent Dyes; Lipid Metabolism; Membrane Microdomains; Microscopy, Confocal; Microscopy, Fluorescence; Models, Statistical; Spectrometry, Fluorescence; Sphingomyelins; Temperature

In the present study, the influence of vesicle size on the penetration of two fluorescently labeled substances into the human skin was investigated. For the measurements either a hydrophilic fluorescent compound [carboxyfluorescein (CF)] or a lipophilic one [1,1'-dioctadecyl-3,3,3',3'-tertramethylindocarbo-cyanine perchlorate (DiI)] were encapsulated into vesicles. Liposomal formulations were prepared by extruding the vesicles through polycarbonate membrane filters with pores of different sizes. In vitro penetration studies into human abdominal skin were performed by using the Franz diffusion cell and a standardized skin stripping technique in attempt to find an optimum size for topical drug delivery by liposomes. Confocal laser scanning microscopy (CLSM) was used to visualize the effect of penetration ability of liposomal DiI. The maximum DiI fluorescence in the skin was observed with smaller liposomes of 71 nm diameter. The liposomes with a size of 120 nm diameter showed statistically enhanced penetration of CF into the skin as compared to larger ones. The results indicated that the CF penetration was inversely related to the size of the liposomes, which was confirmed by the data of the confocal laser scanning microscopy studies.

Topics: Administration, Topical; Carbocyanines; Diffusion; Female; Fluoresceins; Fluorescent Dyes; Humans; In Vitro Techniques; Liposomes; Microscopy, Confocal; Particle Size; Skin; Skin Absorption

Virtually all cell types in the inner ear develop from the cells of the otic vesicle. The otic vesicle is formed by the invagination of non-neural ectodermal cells known as the otic placode. We investigated whether a recently described cell population, originating from the ventral part of the hindbrain neural tube known as the ventrally emigrating neural tube (VENT) cells, also contributes cells to the otic vesicle. The ventral hindbrain neural tube cells were labeled with the fluorescent vital dye DiI or replication-deficient retroviruses containing the LacZ gene in chick embryos on embryonic day 2, after the emigration of neural crest from this region. One day later, the labeled cells were detected only in the hindbrain neural tube. Shortly thereafter, the labeled cells began to appear in the eighth (vestibulocochlear) cranial nerve and otic vesicle. From embryonic day 3.5-5, the labeled cells were detected in the major derivatives of the otic vesicle, i.e. the endolymphatic duct, semicircular canals, utricle, saccule, cochlea, and vestibulocochlear ganglion. That the emigrated cells originated from the ventral part of the hindbrain neural tube was confirmed by focal application of DiI impregnated filter paper and with quail chimeras. It is concluded that, in addition to the otic placode cells, the otic vesicle also contains the ventrally emigrating neural tube cells, and that both cell populations contribute to the structures and cell types in the inner ear. It is well known that inductive signals from the hindbrain are required for the morphogenesis of the inner ear. The migration of the hindbrain neural tube cells into the otic vesicle raises the possibility that the inductive effect of the hindbrain might be mediated, at least in part, by the ventrally emigrating neural tube cells and that, therefore, a mechanism exists that involves cells rather than diffusible molecules only.

Topics: Animals; Carbocyanines; Cell Differentiation; Cell Movement; Chick Embryo; Ear, Inner; Lac Operon; Morphogenesis; Quail; Rhombencephalon; Stem Cells; Vestibulocochlear Nerve

Gastrulation is characterized by the extensive movements of cells. Fate mapping is used to follow such cell movements as they occur over time, and prospective fate maps have been constructed for several stages of the model organisms used in modern studies in developmental biology. In chick embryos, detailed fate maps have been constructed for both prospective mesodermal and ectodermal cells. However, the origin and displacement of the prospective endodermal cells during crucial periods in gastrulation remain unclear. This study had three aims. First, we determined the primitive-streak origin of the endoderm using supravital fluorescent markers, and followed the movement of the prospective endodermal cells as they dispersed to generate the definitive endodermal layer. We show that between stages 3a/b and 4, the intraembryonic definitive endoderm receives contributions mainly from the rostral half of the primitive streak, and that endodermal movements parallel those of ingressing adjacent mesodermal subdivisions. Second, the question of the epiblast origin of the endodermal layer was addressed by precisely labeling epiblast cells in a region known to give rise to prospective somitic cells, and following their movement as they underwent ingression through the primitive streak. We show that the epiblast clearly contributes prospective endodermal cells to the primitive streak, and subsequently to definitive endoderm of the area pellucida. Finally, the relationship between the hypoblast and the definitive endoderm was defined by following labeled rostral primitive-streak cells over a short period of time as they contributed to the definitive endoderm, and combining this with in situ hybridization with a riboprobe for Crescent, a marker of the hypoblast. We show that as the definitive endodermal layer is laid down, there is cell-cell intercalation at its interface with the displaced hypoblast cells. These data were used to construct detailed prospective fate maps of the endoderm in the chick embryo, delineating the origins and migrations of endodermal cells in various rostrocaudal levels of the primitive streak during key periods in early development.

Topics: Animals; Carbocyanines; Chick Embryo; Endoderm; Fluoresceins; Fluorescent Dyes; Gastrula; Succinimides

Neurons generated early in development of the ventral diencephalon have been shown to play a key role in defining the midline and the caudal boundary of the optic chiasm in the mouse retinofugal pathway. These functions have been attributed to a surface bound adhesion molecule, CD44 that is expressed in these chiasmatic neurons. In this study, we investigated the effects of perturbing normal CD44 functions on axon routing in brain slice preparations of the mouse retinofugal pathway. Two CD44 antibodies (Hermes-1 and IM7) were used that bind to distinct epitopes on the extracellular domain of the molecule. We found that both antibodies produced dramatic defects in routing of the retinal axons that arrive early in the chiasm. In preparations of embryonic day 13 (E13) and E14 pathways, the crossed component in the chiasm was significantly reduced after antibody treatment. However, such reduction in axon crossing was not observed in E15 chiasm, indicating that the lately generated crossed axons lost their responses to CD44. Furthermore, the anti-CD44 treatment produced a reduction in the uncrossed component in the E15 but not in younger pathways, suggesting a selective response of the lately generated axons, mostly from ventral temporal retina, but not those generated earlier, to the CD44 at the chiasmatic midline in order to make their turn for the uncrossed pathway. These findings provide evidence that a normal function of CD44 molecules in the chiasmatic neurons is essential for axon crossing and axon divergence at the mouse optic chiasm.

Topics: Animals; Antibodies; Axons; Carbocyanines; Diencephalon; Embryo, Mammalian; Embryonic and Fetal Development; Epitopes; Female; Hyaluronan Receptors; Immunohistochemistry; In Vitro Techniques; Mice; Mice, Inbred C57BL; Microscopy, Confocal; Neural Pathways; Optic Chiasm; Pregnancy; Retina; Time Factors

To study morphologically the relationship between climbing fibre and Purkinje cell in the developing mouse cerebellum, we established a novel tract tracing using injection of 1,1'-dioctodecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI) into the inferior cerebellar peduncle, the half point of olivocerebellar projection. In this tracing method, only a certain number of climbing fibres were labelled with DiI and they revealed the single-fibre resolution, individually. These technical advantages enabled us to follow the projection of a climbing fibre to a Purkinje cell at the light microscopic level. To further investigate how a single labelled olivocerebellar axon interacts with a Purkinje cell, we introduced a photoconversion method into this tracing method and successfully observed the photo-oxidized climbing fibre terminals at the electron microscopic level. At postnatal days 7 and 9, a single DiI-labelled climbing fibre arborized around some adjacent Purkinje cell bodies in a distinguishable nest. At this pericellular nest stage, we first demonstrated that the terminal arborization stemmed from a single climbing fibre formed synapses simultaneously on both a soma and dendrites of a Purkinje cell. This finding suggests that the pericellular nest may be such an efficient form that a single climbing fibre innervates a Purkinje cell at both perisomatic and peridendritic sites. Thus, we succeeded in establishing an effective tracing method to investigate a single climbing fibre synaptogenesis with a Purkinje cell both at the light and electron microscopic levels.

Topics: Animals; Animals, Newborn; Carbocyanines; Cerebellum; Fluorescent Dyes; Mice; Microscopy, Electron; Microscopy, Fluorescence; Nerve Fibers; Purkinje Cells; Synapses

The aim of this study was to investigate the effect of dietary oxidized fats on the lipoprotein profile and the atherogenicity of LDL. Two experiments with male Sprague-Dawley rats were conducted. In Experiment 1, diets with either fresh fat or oxidized fat, prepared by heating at temperatures of 50, 105 or 190 degrees C, containing either 25 or 250 mg alpha-tocopherol equivalents/kg were used. In Experiment 2, diets with fresh or oxidized fat, heated at a temperature of 55 degrees C, containing 25 mg alpha-tocopherol equivalents/kg, were used. In Experiment 1, rats fed all types of oxidized fats had higher concentrations of HDL cholesterol and lower ratios between plasma and HDL cholesterol than rats fed the diet containing the fresh fat. As determined from the lag time, the susceptibility of LDL to copper-induced lipid peroxidation was higher in rats fed oxidized fats heated at 105 or 190 degrees C than in rats fed the diets containing the fresh fat or the oxidized fat treated at 50 degrees C, irrespective of the dietary vitamin E concentration. However, in Experiment 2, the composition of LDL apolipoproteins and uptake of LDL by macrophages were not different between rats fed the fresh fat diet and those fed the oxidized fat diet. We conclude that ingestion of oxidized fats does not adversely affect the lipoprotein profile in the rat model used, and does not cause modifications of apolipoproteins that would lead to enhanced uptake of LDL via macrophage scavenger receptors.

Topics: alpha-Tocopherol; Animals; Apolipoproteins; Body Weight; Carbocyanines; Cholesterol, HDL; Cholesterol, LDL; Copper; Diet; Dietary Fats; Fatty Acids; Feeding Behavior; Hot Temperature; Lipid Peroxidation; Lipoproteins, LDL; Macrophages; Male; Osmolar Concentration; Oxidation-Reduction; Rats; Rats, Sprague-Dawley

Recently, increasing attention has been paid to the study of intermediate targets and their relay guidance role in long-range pathfinding. In the present study, mechanisms of corticothalamic and thalamocortical pathfinding were investigated in C57BL/6 mice using in vitro DiI labeling and in vivo cholera toxin labeling. Specifically, three important intermediate targets, the subplate, ganglionic eminence, and reticular thalamic nucleus, were studied for their role in corticothalamic and thalamocortical pathfinding. The results show that the neuroepithelium of the ganglionic eminence is a source of pioneer neurons and pioneer fibers. Through radial and tangential migration, these pioneer neurons and fibers can approach the differentiating field of the ganglionic eminence, the subplate and thalamic reticular nucleus to participate in the formation of these three intermediate targets. Furthermore, the subplate, ganglionic eminence and thalamic reticular nucleus are linked by pioneer neurons and fibers to form a guidance axis. The guidance axis and the three important intermediate targets provide an ideal environment of contact guidance and chemical guidance for the corticothalamic and thalamocortical pathfinding. The concept of a "waiting time" in the subplate and the thalamic reticular nucleus is likely due to the expression of a guidance effect, so that the thalamocortical and corticothalamic projections can be deployed spatially and temporally to the subplate and thalamic reticular nucleus before these projections enter their final destinations, the neocortex and thalamus.

Topics: Animals; Animals, Newborn; Brain; Carbocyanines; Fluorescent Dyes; Ganglia; Intralaminar Thalamic Nuclei; Mice; Mice, Inbred C57BL; Microscopy, Confocal; Neocortex; Neural Pathways; Neurons; Subthalamic Nucleus; Thalamus

The septal organ represents one of the three chemosensory subsystems found in most vertebrate species. Analyzing the projection pattern of septal organ neurons using the OMP-GFP transgenic mouse line revealed that axons navigate in highly variable fiber tracks across the main olfactory epithelium toward the main olfactory bulb. All septal organ axons cross through the cribriform plate at a spatially defined site and terminate exclusively in the posterior, ventromedial aspect of the bulb. Here, one portion of axons forms a dense network on the medial side where they apparently enter glomeruli which are mainly innervated by axons of olfactory sensory neurons from the main olfactory epithelium. Another significant portion of the axons targets a few glomeruli which appear to receive input exclusively from the septal organ neurons.

Topics: Animals; Axons; Carbocyanines; Green Fluorescent Proteins; Luminescent Proteins; Mice; Mice, Transgenic; Microscopy, Confocal; Nerve Tissue Proteins; Olfactory Marker Protein; Olfactory Mucosa

Choroidal melanoma is the most common primary ocular cancer among the adult population. To avoid enucleation, there has been a concerted effort to develop therapies that spare the affected eye and the patient's vision. Blood flow helps shape the tumor's microenvironment, plays a key role in the tumor's response to many different types of therapy, and is necessary for delivery of chemotherapeutic agents. To rationally design new therapies and optimize existing treatments, it is essential to learn as much as possible about blood flow and the microcirculation in this tumor. In recent years, much has been discovered about the anatomy of the microvasculature and the dynamics of overall blood flow in choroidal melanoma, but little is known about the factors that determine microvascular blood flow. In this study hemodynamic parameters in individual microvessels of a human choroidal melanoma xenograft were compared with those same parameters in a normal rat choroid.. Nude, athymic WAG/RijHs-rnu rats were used in this study. The human choroidal melanoma cell line OCM-1 was used to grow solid tumors subcutaneously in the flanks of donor rats. Small pieces of these tumors were then implanted into the choroidal space of recipient rats. After 6 to 8 weeks, the rats were anesthetized with a subcutaneous injection of urethane, and the sclera was exposed. Rhodamine-labeled liposomes and red blood cells (RBCs) labeled with 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI) were injected intravenously. Epifluorescent, intravital microscopy was used to visualize the flow of fluorescent RBCs through individual vessels in the choroid or tumor. Flow through multiple vessels was recorded on videotape for later analysis. From the recordings, RBC flux, RBC velocity (V(c)), and microvascular hematocrit (HCT(m)) were determined. Similar experiments were performed in rats with no tumor growth, and these same parameters were calculated in normal choroidal vessels. RBC flow was characterized in 55 vessels in six OCM-1 tumors and in 153 choroidal vessels in five non-tumor-bearing rats.. RBC flux was higher in larger tumor vessels (>30 micro m in diameter) compared with similarly sized choroidal vessels. There were no differences in the velocities of RBCs through the two types of vessels. HCT(m) was significantly higher in medium-sized (>20 micro m in diameter) and larger tumor vessels compared with normal choroidal vessels.. These experiments demonstrate differences between hemodynamic parameters in normal choroidal microvessels and microvessels in choroidal melanoma in this animal model. Because HCT(m) is a key determinant of apparent viscosity, abnormally high HCT(m) in the tumor vessels would increase vascular resistance and decrease flow. This could have a negative impact on the tumor oxygen levels and on the ability to deliver drugs effectively. On the contrary, higher local HCT(m) has also been shown to increase oxygen delivery. The impact and interplay of these two effects on tumor oxygenation remain to be determined.

Topics: Animals; Blood Flow Velocity; Carbocyanines; Choroid; Choroid Neoplasms; Erythrocytes; Fluorescent Dyes; Hematocrit; Hemodynamics; Melanoma; Microcirculation; Microscopy, Fluorescence; Models, Animal; Neovascularization, Pathologic; Rats; Rats, Mutant Strains; Rhodamines; Transplantation, Heterologous; Video Recording

Mediators of inflammation, such as PGE(2), are known to sensitize the airways to inhaled irritants and circulating autacoids. Evidence from in vivo studies has shown the involvement of vagal pulmonary C-fiber afferents in the PGE(2)-elicited airway hypersensitivity. However, whether PGE(2) acts directly on these sensory nerves is unclear. The present study aimed to investigate whether PGE(2) has direct potentiating effects on nodose and jugular pulmonary C neurons cultured from adult Sprague-Dawley rats and, if so, determine whether the EP(2) prostanoid receptor is involved. Pulmonary neurons were identified by retrograde labeling with a fluorescent tracer 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate. Using perforated patch-clamp technique, our results showed that 1) PGE(2) pretreatment (1 microM) increased the whole cell current density elicited by capsaicin and phenylbiguanide, chemical agents known to stimulate pulmonary C fibers; 2) selective activation of the EP(2) prostanoid receptor by butaprost (3-10 microM) increased the whole cell current density elicited by capsaicin; and 3) PGE(2), as well as butaprost, increased the number of action potentials evoked by current injection. Therefore, we conclude that PGE(2) directly sensitizes vagal pulmonary C neurons to chemical and electrical stimulation. Furthermore, butaprost modulates the neurons in a manner similar to that of PGE(2), suggesting that the effects of PGE(2) are mediated, at least in part, through the EP(2) prostanoid receptor.

Topics: Alprostadil; Animals; Bronchi; Carbocyanines; Cells, Cultured; Dinoprostone; Electric Stimulation; Fluorescent Dyes; Lung; Male; Neurons, Afferent; Patch-Clamp Techniques; Prostaglandins E, Synthetic; Rats; Rats, Sprague-Dawley; Stimulation, Chemical; Vagus Nerve

Alveolar macrophages (AMs) from immunocompetent animals were isolated from bronchoalveolar lavage and labeled with the fluorescent marker 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI). These AMs were administered intratracheally into mechanically ventilated SCID mice. From 1 to 28 days later, the recipient mice underwent bronchoalveolar lavage to isolate their AMs. To determine whether reconstituted AMs were still immunocompetent, the recovered AMs were assayed for their ability to phagocytose fluorescein-labeled zymosan-coated beads. After incubation with the beads, samples were assayed using a fluorescent-activated cell sorter to identify DiI-labeled reconstituted AMs, unlabeled resident AMs, and the proportion of these two groups undergoing phagocytosis. DiI-labeled AMs accounted for approximately 50% of all returned AMs. Additionally, the reconstituted AMs from normal BALB/c mice retained phagocytic activity compared with AMs from immunodeficient SCID mice. Reconstituted AMs demonstrated enhanced phagocytic activity compared with resident SCID AMs for up to 28 days following reconstitution. These results indicate that immunocompetent AMs can be successfully reconstituted into an immunodeficient host to partially restore alveolar host defense.

Topics: Animals; Bronchoalveolar Lavage Fluid; Carbocyanines; Flow Cytometry; Fluorescein; Fluorescent Dyes; Intubation, Intratracheal; Lung; Macrophages, Alveolar; Mice; Mice, Inbred BALB C; Mice, SCID; Microscopy, Fluorescence; Microscopy, Phase-Contrast; Phagocytosis; Severe Combined Immunodeficiency; Zymosan

To reveal the morphological features and dynamic processes of the development of inter-connections in the retina, lateral geniculate nucleus (LGN), superior colliculus (SC) and visual cortex (VC) in human fetal life by using a fluorescent tracer, 1, 1'-dioctadecyl-3, 3, 3', 3'-tetramethylin-docarbocyanine perchlorate (DiI).. DiI was embedded into the optic tract, brachium of superior colliculus and subplate of visual cortex in fixed postmortem human tissues of 7 fetuses. The tissue was incubated at 37 degrees C for 4 to 10 weeks (ws). After DiI had satisfactorily diffused via axons of the visual system, the tissue was sectioned, mounted and observed under a confocal laser scanning microscope.. In 12 week-fetus, retinogeniculate axon has already reached LGN, but there was no cellular lamination. After embedment, axons from retina arrived at SC, and the fibers were distributed along the dorsal part of the SC. At 12 and 22 ws, there were subplates under visual cortex.. The retinogeniculate axon reaches LGN before 12 ws and forms cellular lamination after 12 ws. The axon from retina reaches SC before 12 ws. The subplate under visual cortex forms before 12 ws and disappears after 22 ws. DiI can be easily and effectively used to label the axon of visual system of human fetus to study the prenatal development of human visual pathway.

Topics: Axons; Carbocyanines; Fetus; Fluorescent Dyes; Geniculate Bodies; Humans; Microscopy, Confocal; Optic Nerve; Retina; Visual Cortex; Visual Pathways

We follow somite segmentation in living chick embryos and find that the shaping process is not a simple periodic slicing of tissue blocks but a much more carefully choreographed separation in which the somite pulls apart from the segmental plate. Cells move across the presumptive somite boundary and violate gene expression boundaries thought to correlate with the site of the somite boundary. Similarly, cells do not appear to be preassigned to a given somite as they leave the node. The results offer a detailed picture of somite shaping and provide a spatiotemporal framework for linking gene expression with cell movements.

Topics: Animals; Boron Compounds; Carbocyanines; Cell Adhesion; Cell Movement; Cell Size; Central Nervous System; Ceramides; Chick Embryo; Epithelial Cells; Gene Expression; Gene Expression Profiling; Mesoderm; Microscopy, Confocal; Models, Biological; Receptor, EphA4; Somites; Time Factors

The high-density lipoprotein (HDL) receptor, scavenger receptor, class B, type I (SR-BI), mediates both the selective uptake of lipids, mainly cholesterol esters, from HDL to cells and the efflux of cholesterol from cells to lipoproteins. The mechanism underlying these lipid transfers is distinct from classic receptor-mediated endocytosis, but it remains poorly understood. To investigate SR-BI's mechanism of action and in vivo function, we developed a high-throughput screen to identify small molecule inhibitors of SR-BI-mediated lipid transfer in intact cells. We identified five compounds that in the low nanomolar to micromolar range block lipid transport (BLTs), both selective uptake and efflux. The effects of these compounds were highly specific to the SR-BI pathway, because they didn't interfere with receptor-mediated endocytosis or with other forms of intracellular vesicular traffic. Surprisingly, all five BLTs enhanced, rather than inhibited, HDL binding by increasing SR-BI's binding affinity for HDL (decreased dissociation rates). Thus, the BLTs provide strong evidence for a mechanistic coupling between HDL binding and lipid transport and may serve as a starting point for the development of pharmacologically useful modifiers of SR-BI activity and, thus, HDL metabolism.

Topics: Adrenal Cortex; Adrenocorticotropic Hormone; Animals; Biological Transport; Carbocyanines; CD36 Antigens; Cell Line; Chlorocebus aethiops; CHO Cells; Clathrin; Cricetinae; Cricetulus; Drug Antagonism; Endocytosis; Fibroblasts; Fluorescent Dyes; HeLa Cells; Humans; Lipoproteins, HDL; Membrane Proteins; Mice; Molecular Weight; Protein Binding; Receptors, Immunologic; Receptors, Lipoprotein; Receptors, Scavenger; Recombinant Fusion Proteins; Scavenger Receptors, Class B; Substrate Specificity

Liver-derived apolipoprotein E (apoE) decreases atherosclerosis without altering the circulating concentrations of plasma lipoproteins. We evaluated the effects of apoE and lipoprotein lipase (LpL) on the interactions of triglyceride-rich particles (TGRPs) in the arterial wall.. Quantitative fluorescence microscopy was used to study the interactions of TGRPs (25- to 35-nm diameter) in the arterial wall. Carotid arteries were harvested from rats, placed in a perfusion chamber, and perfused with fluorescently labeled TGRPs. In the absence of apoE or LpL, 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine-TGRP (100 microg neutral lipid/mL) was poorly retained in the arterial wall. The addition of either apoE (10 microg/mL) or LpL (10 microg/mL) increased TGRP accumulation 220% and 100%, respectively. This effect was attenuated by heparin (10.0 IU/mL). Histological analyses of cross sections from these vessels demonstrate that in the absence of apoE or LpL, there is deep penetration of lipid into the arterial wall. With the addition of either apoE or LpL, arterial wall penetration of TGRP is blocked.. These results demonstrate that although apoE and LpL increase arterial wall accumulation of TGRPs, these proteins also reduce the penetration of TGRPs into the arterial wall. We postulate that this may represent a novel antiatherogenic property of apoE and LpL.

Topics: Animals; Apolipoproteins E; Carbocyanines; Carotid Arteries; Diffusion Chambers, Culture; Fluorescent Dyes; Heparan Sulfate Proteoglycans; In Vitro Techniques; Lipoprotein Lipase; Lipoproteins; Male; Microscopy, Fluorescence; Perfusion; Protein Binding; Rats; Rats, Sprague-Dawley; Triglycerides

The apical dendrites of pyramidal neurons in the cerebral cortex form vertical bundles whose distribution and density vary across species and areas. To understand their relationships with cortical columns, we labeled retrogradely neurons from the white matter underlying the visual cortex with 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI) at P3 and P10 and with biotinylated dextran amine at P30. We also mapped the distribution of apical dendrites in tangential sections, immunostained for microtubule-associated proteins (MAP2). Their composition and distribution were studied with Neurolucida and NeuroExplorer software. The apical dendrites of pyramidal neurons formed different bundle types: at P3 we found bundles formed (a) by neurons located in cortical plate; (b) by layer V neurons; and (c) by upper layer V neurons and cortical plate neurons. At P10, the amount of supragranular neurons participating in the bundles increased. The inter-dendritic and inter-bundle distances increased with age. These findings confirm that dendritic bundles are present in the rat visual cortex early in development and are formed by neurons belonging to different cortical layers. The existence of different types of bundles relative to the layer of location of their parent neurons suggests that they are heterogeneous from each other in nature and in the pattern of connectivity.

Topics: Animals; Biotin; Carbocyanines; Dendritic Cells; Dextrans; Fluorescent Dyes; Microtomy; Microtubule-Associated Proteins; Pyramidal Cells; Rats; Visual Cortex

We have investigated raft formation in human platelets in response to cell activation. Lipid phase separation and domain formation were detected using the fluorescent dye 1,1'-dioctadecyl-3,3,3',3'-tetramethyl-indocarbocyanine perchlorate (diI-C(18)) that preferentially partitions into gel-like lipid domains. We showed that when human platelets are activated by cold and physiological agonists, rafts coalesce into visible aggregates. These events were disrupted by depletion of membrane cholesterol. Using Fourier transform infrared spectroscopy (FTIR), we measured a thermal phase transition at around 30 degrees C in intact platelets, which we have assigned as the liquid-ordered to the liquid-disordered phase transition of rafts. Phase separation of the phospholipid and the sphingomyelin-enriched rafts could be observed as two phase transitions at around 15 and 30 degrees C, respectively. The higher transition, assigned to the rafts, was greatly enhanced with removal of membrane cholesterol. Detergent-resistant membranes (DRMs) were enriched in cholesterol (50%) and sphingomyelin (20%). The multi-functional platelet receptor CD36 selectively partitioned into DRMs, whereas the GPI-linked protein CD55 and the major platelet integrin alpha(IIb)beta(3a) did not, which suggests that the clustering of proteins within rafts is a regulated process dependent on specific lipid protein interactions. We suggest that raft aggregation is a dynamic, reversible physiological event triggered by cell activation.

Topics: beta-Cyclodextrins; Blood Platelets; Blotting, Western; Carbocyanines; CD36 Antigens; CD55 Antigens; Cell Separation; Cholesterol; Cyclodextrins; Fluorescent Dyes; Humans; Membrane Microdomains; Membranes, Artificial; Microscopy, Fluorescence; Platelet Activation; Platelet Glycoprotein GPIIb-IIIa Complex; Spectroscopy, Fourier Transform Infrared; Sphingomyelins; Temperature

The expression of the cellular retinoic acid binding protein type I (CRABP I) was examined in the early phase of cerebellar development in the mouse. The CRABP I was expressed from embryonic day (E) 10.5 to E15.5 in the cerebellar plate. The expression was diffused at E10.5-E11.5 and thereafter localized in a small rostrodorsal area of the cerebellar territory of both sides. By using in situ hybridization and both immunohistochemistry and carbocyanine tracing procedures, we identified the fastigial cells as the population that expresses CRABP I in the cerebellum. The results suggest that these cells play a critical role in the early development of the cerebellum.

Topics: Animals; Carbocyanines; Cerebellum; Immunohistochemistry; In Situ Hybridization; Mice; Neural Pathways; Receptors, Retinoic Acid; RNA, Messenger

General mechanisms initiating the gastrulation process in early animal development are still elusive, not least because embryonic morphology differs widely among species. The rabbit embryo is revived here as a model to study vertebrate gastrulation, because its relatively simple morphology at the appropriate stages makes interspecific differences and similarities particularly obvious between mammals and birds. Three approaches that centre on mesoderm specification as a key event at the start of gastrulation were chosen. (1) A cDNA fragment encoding 212 amino acids of the rabbit Brachyury gene was cloned by RT-PCR and used as a molecular marker for mesoderm progenitors. Whole-mount in situ hybridisation revealed single Brachyury-expressing cells in the epiblast at 6.2 days post conception, i.e. several hours before the first ingressing mesoderm cells can be detected histologically. With the anterior marginal crescent as a landmark, these mesoderm progenitors are shown to lie in a posterior quadrant of the embryonic disc, which we call the posterior gastrula extension (PGE), for reasons established during the following functional analysis. (2) Vital dye (DiI) labelling in vitro suggests that epiblast cells arrive in the PGE from anterior parts of the embryonic disc and then move within this area in a complex pattern of posterior, centripetal and anterior directions to form the primitive streak. (3) BrdU labelling shows that proliferation is reduced in the PGE, while the remaining anterior part of the embryonic disc contains several areas of increased proliferation. These results reveal similarities with the chick with respect to Brachyury expression and cellular migration. They differ, however, in that local differences in proliferation are not seen in the pre-streak avian embryo. Rather, rabbit epiblast cells start mesoderm differentiation in a way similar to Drosophila, where a transient downregulation of proliferation initiates mesoderm differentiation and, hence, gastrulation.

Topics: Amino Acid Sequence; Animals; Carbocyanines; Cell Division; Cell Movement; Cloning, Molecular; Female; Fetal Proteins; Fluorescent Dyes; Gastrula; Gene Expression Regulation, Developmental; Mesoderm; Molecular Sequence Data; Rabbits; Sequence Homology, Amino Acid; Stem Cells; T-Box Domain Proteins

In the present study, we applied a lipophilic tracer, Dil (1,1-dilinoleyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate), to the synaptic region of the medial olfactory bulb in formaldehyde-fixed preparations from the crucian carp. We observed staining both in the axons of secondary neurons leading to the brain and in the olfactory receptor neurons (ORNs) of the olfactory epithelium. In those preparations, where staining of the tract was restricted to axons of the medial part of the medial olfactory tract, the majority (86-98%) of the somata of the sensory neurons were found in the deep layers of olfactory epithelium. Since the medial bundle of the medial olfactory tract mediates alarm behaviour in the crucian carp, we conclude that the sensory neurons with long dendrites participate in the reception of alarm pheromones.

Topics: Animals; Axons; Carbocyanines; Carps; Coloring Agents; DNA Probes; Epithelium; Fluorescent Dyes; Histocytochemistry; Neurites; Olfactory Bulb; Olfactory Pathways; Olfactory Receptor Neurons

After their final mitosis, cerebellar granule cells remain in the external granular layer (EGL) for 20-48 hr before initiating their radial migration across the molecular layer (ML), but the significance of this latent period is not well understood. In the present study, we used a confocal microscope to examine morphogenetic changes and behavior of postmitotic granule cells restricted to the EGL in slice preparations of the postnatal mouse cerebellum. We found that, coincident with the extension of two uneven horizontal processes oriented parallel to the longitudinal axis of the folium, postmitotic granule cells start to migrate tangentially in the direction of the larger process. Interestingly, their morphology and the speed of cell movement change systematically with their position within the EGL. The rate of tangential cell movement is fastest (approximately 14.8 micrometer/hr) in the middle of the EGL, when cells have two short horizontal processes. As granule cells elongate their somata and extend longer horizontal processes at the bottom of the EGL, they move at a reduced rate (approximately 12.6 micrometer/hr). At the interface of the EGL and ML where cells migrate tangentially at the slowest rate (approximately 4.1 micrometer/hr), their somata round and then begin to extend couples of the descending processes into the ML. After the stationary period, granule cells abruptly extend a single vertical process and initiate the transition from tangential to radial migration, reshaping their rounded somata into a vertically elongated spindle. These observations suggest that tangential migration of granule cells within the EGL may provide the developmental mechanisms for their appropriate allocation across parasagittal compartments of the expanding cerebellar cortex.

Topics: Animals; Bromodeoxyuridine; Carbocyanines; Cell Division; Cell Movement; Cerebellum; Cytoplasmic Granules; Fluorescent Dyes; In Vitro Techniques; Mice; Microscopy, Confocal; Microscopy, Video; Time Factors

Transient brain ischemia induces significant alterations in lipid structures of neuronal membranes, which are believed to result from lipid peroxidation and free radical attack. Such a membrane structural change may serve as an important histological marker of cell injury. In the present study, we examined how the dynamics of DiI/membrane incorporation may reflect early membrane metabolism and dynamic changes following sodium-potassium pump inhibition. Ouabain (1mM) was stereotactically co-administered with either 1,1'-dioctadecyl-3,3,3',3'-tetramethyl-indocarbocyanine perchlorate DiI (50 microg/ml) or ethidium homodimer (4 microM) into the granule cell layer of the adult rat hippocampus. Tissue was cryosectioned and examined with epifluorescence microscopy at 1, 2, 3, 4, 6, 8 and 72h post-injection. Alternate sections were stained with thionine or haematoxylin and eosin to evaluate morphological changes. Ouabain-induced pump inhibition resulted in a dramatic increase in DiI fluorescence in granule cell layer neurons as early as 4h post-injection. This increase in DiI incorporation coincided both spatially and temporally with the appearance of reactive changes characterizing early neuronal injury. However, the fluorescence increase was not a result of membrane breakdown because ethidium homodimer, a membrane-impermeable nucleic acid probe used for labeling cells with compromised membranes, when applied in a similar fashion, failed to show any fluorescence changes. The results of this study suggest that pump inhibition results in a specific increase in membrane lipophilicity possibly due to altered lipid structure.

Topics: Animals; Carbocyanines; Cell Count; Cell Death; Cell Membrane; Cell Size; Ethidium; Fluorescent Dyes; Hippocampus; Male; Microinjections; Microscopy, Fluorescence; Ouabain; Rats; Rats, Long-Evans; Sodium-Potassium-Exchanging ATPase

A study was done to determine how lipid microspheres (LM) containing prostaglandin E1 (lipo PGE1) accumulate in injured arterial tissue. After administration of lipo PGE1 labeled with a fluorescence probe, 1,1'-dioctadecyl-3, 3, 3',3'-tetramethyl-indocarbocyanine perchlorate (DiI-lipo PGE1) to rats 14 of days after balloon injury of the carotid artery, localization into the injured site was examined using a fluorescence confocal laser scanning microscope (CLSM). In contrast with the normal carotid artery, DiI-lipo PGE1 accumulated remarkably in neointima of the injured site which was occupied mainly by the migration of the proliferating and quiescent vascular smooth muscle cells (SMC). In vitro cellular uptake of DiI-lipo PGE1 was semi-quantitatively measured using CLSM, regarding differentiated and proliferative phenotypes of human vascular SMC, compared with the human endothelial cells (EC) and mouse fibroblasts. The differentiated SMC incorporated DiI-lipo PGE1 to equal or a higher level of the proliferative phenotype, and was significantly higher than EC and fibroblasts. The uptake of DiI-lipo PGE1 by both SMC and EC was inhibited at 4 degrees C, by dansylcadaverine and excessive LM, but was unaffected by cytochalasin B. These results suggest that the uptake of DiI-lipo PGE1 by SMC plays an important role in localization of DiI-lipo PGE1 at the injured site, and that the uptake seems to be a receptor mediated endocytosis.

Topics: 3T3 Cells; Alprostadil; Animals; Cadaverine; Carbocyanines; Carotid Artery Injuries; Catheterization; Cytochalasin B; Fluorescent Dyes; Humans; Liposomes; Male; Mice; Microspheres; Muscle, Smooth, Vascular; Rats; Rats, Wistar; Tunica Intima

The absorption and fluorescence spectra of 3,3'-dioctadecyloxacarbocyanine [DiOC18(3)], a cationic oxacarbocyanine dye have been studied in aqueous and nonaqueous media containing egg phosphatidylcholine (PC) as well as in different solvents of diverse nature. The results show the evidence of complex formation of the dye in the ground and in the excited states with PC. The excited state interaction of the dye with PC suggests the electron transfer from PC to dye and this is supported by photovoltage generation in a photoelectrochemical cell consisting of dye and PC in aqueous medium. An attempt has been made to determine the polarity of the microenvironment of the dye in PC liposome or PC reverse micelle from the spectral studies of the dye in different solvents of known polarity.

Topics: Carbocyanines; Fluorescent Dyes; Membranes, Artificial; Micelles; Models, Biological; Solvents

Cholesterol-laden macrophages are the hallmark of atherogenesis. The class B scavenger receptor, CD36, binds oxidized low density lipoprotein (OxLDL), is found in atherosclerotic lesions, and is upregulated by OxLDL. We tested the effects of alpha-tocopherol (AT) enrichment of human monocyte-derived macrophages on CD36 expression and cholesteryl ester accumulation. Monocytes isolated from normal volunteers were cultured into macrophages. Macrophages were enriched overnight with various doses of AT (25, 50, and 100 microM). LDL from normal volunteers was oxidized or acetylated (AcLDL) and incubated with macrophages for 48 h at a concentration of 50 or 100 microg/ml. CD36 expression was assessed by flow cytometry. Quantitative analysis of scavenger receptor class A (SR-A) activity was performed with 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanide perchlorate (DiI)-labeled LDL. CD36 expression was maximal after 8;-10 days of culture. AT (> or =50 microM) significantly decreased CD36 expression upregulated by OxLDL and AcLDL (P < 0.01). Other antioxidants (beta- or gamma-tocopherol) or protein kinase C inhibitors failed to decrease CD36 expression. Concomitantly, DiI-AcLDL and DiI-OxLDL uptake was significantly decreased after AT treatment (P < 0.001). Cholesteryl ester accumulation was significantly decreased after AT enrichment (AcLDL + AT, 77% inhibition; OxLDL + AT, 42% inhibition). In conclusion, AT decreases both CD36 and SR-A expression and cholesteryl ester accumulation in human macrophages. This provides additional scientific support for the antiatherogenic properties of AT.

Topics: Antioxidants; Carbocyanines; CD36 Antigens; Cells, Cultured; Cholesterol Esters; Enzyme Inhibitors; Flow Cytometry; Fluorescent Dyes; Humans; Lipoproteins, LDL; Macrophages; Membrane Proteins; Monocytes; Receptors, Immunologic; Receptors, Lipoprotein; Receptors, Scavenger; RNA, Messenger; Scavenger Receptors, Class A; Scavenger Receptors, Class B; Time Factors; Vitamin E

Translational and rotational diffusion of fluorescent molecules on the surface of small biological systems such as vesicles, proteins and micelles depolarize the fluorescence. A recent study has treated the case of the translational dynamics of surface probes (M.M.G. Krishna, R. Das, N. Periasamy and R. Nityananda, J. Chem. Phys., 112 (2000) 8502-8514) using Monte Carlo and theoretical methods. Here we extend the application of the methodologies to apply the case of rotational dynamics of surface probes. The corresponding fluorescence anisotropy decays were obtained using the Monte Carlo simulation methods for the two cases: surface probes undergoing rotational dynamics on a plane and on a sphere. The results were consistent with the theoretical equations which show that Monte Carlo methods can be used to simulate the surface diffusion problems. The anisotropy decay for the rotational diffusion of a molecule on a planar surface is single exponential and the residual anisotropy is zero. However, residual anisotropy is finite for the case of rotational diffusion on a sphere because of the spatial averaging of the anisotropy function. The rotational correlation time in both the cases is (4Drot)(-1) with Drot being the rotational diffusion coefficient. Rotational dynamics of a surface bound dye in a single giant liposome and in sonicated vesicles were studied and the results were explained according to the theoretical equations. A fast component of fluorescence depolarization was also observed for sonicated vesicles which was interpreted as wobbling-in-cylinder dynamics of the surface-bound dye.

Topics: Carbocyanines; Caveolae; Fluorescence Polarization; Fluorescent Dyes; Membrane Lipids; Monte Carlo Method; Rotation; Surface Properties

Although Na+/H+ exchange (NHE) has been implicated in myocardial reperfusion injury, participation of coronary microvascular endothelial cells (CMECs) in this pathogenesis has been poorly understood. NHE-induced intracellular Ca2+ concentration ([Ca2+]i) overload in CMECs may increase the synthesis of intercellular adhesion molecules (ICAM), which is potentially involved in myocardial reperfusion injury. The present study tested the hypothesis that NHE plays a crucial role in [Ca2+]i overload and ICAM-1 synthesis in CMECs. Primary cultures of CMECs isolated from adult rat hearts were subjected to acidic hypoxia for 30 min followed by reoxygenation. Two structurally distinct NHE inhibitors, cariporide and 5-(N-N-dimethyl)-amiloride (DMA), had no significant effect on the acidic hypoxia-induced decrease in intracellular pH (pH(i)) of CMECs but significantly retarded pH(i) recovery after reoxygenation. These NHE inhibitors abolished the hypoxia- and reoxygenation-induced increase in [Ca2+]i. Expression of ICAM-1 mRNA was markedly increased in the vehicle-treated CMECs 3 h after reoxygenation, and this was significantly inhibited by treatment with cariporide, DMA, or Ca2+-free buffer. In addition, enhanced ICAM-I protein expression on the cell surface of CMECs 8 h after reoxygenation was attenuated by treatment with cariporide, DMA, or Ca2+-free buffer. These results suggest that NHE plays a crucial role in the rise of [Ca2+]i and ICAM-1 expression during acidic hypoxia/reoxygenation in CMECs. We propose that inhibition of ICAM-1 expression in CMECs may represent a novel mechanism of action of NHE inhibitors against ischemia-reperfusion injury.

Topics: Amiloride; Animals; Anti-Arrhythmia Agents; Calcium; Carbocyanines; Cell Hypoxia; Cells, Cultured; Coronary Vessels; Endothelium, Vascular; Flow Cytometry; Guanidines; Hydrogen-Ion Concentration; Intercellular Adhesion Molecule-1; Lipoproteins, LDL; Male; Microcirculation; Myocardial Reperfusion Injury; Oxygen; Rats; Rats, Sprague-Dawley; RNA, Messenger; Sodium-Calcium Exchanger; Sodium-Hydrogen Exchangers; Sulfones

Enrichment of chemoattractant receptors on the neutrophil surface has been difficult to assess, primarily because of limitations in sensitivity of visualization. Using an ultrasensitive, cooled charge-coupled device camera, we investigated spatial-temporal relationships between N-formyl peptide receptor distribution and directional motility of human neutrophils. Live cells were labeled with fluorescent receptor ligands, i.e., fluoresceinated tert-butyl-oxycarbonyl-Phe-(D)-Leu-Phe-(D)-Leu-Phe-OH (Boc-FLFLF) and formyl-Nle-Leu-Phe-Nle-Tyr-Lys (fnLLFnLYK), while fixed cells were labeled with either fluorescent peptides or monoclonal antibodies. Double labeling of receptors and filamentous actin (F-actin) was done to investigate possible colocalization. N-Formyl peptide receptors on unstimulated cells were randomly distributed. However, on polarized neutrophils, the receptors accumulated toward regions involved in motility and distributed nonuniformly. In fixed neutrophils, antibody-labeled receptors colocalized with the F-actin-rich leading edge whereas peptide-labeled receptors lagged behind this region. We suggest that neutrophils use an asymmetric receptor distribution for directional sensing and sustained migration. A separation between receptors labeled with peptides and those labeled with antibodies reflects two functionally distinct receptor populations at the membrane of motile neutrophils.

Topics: Actins; Adult; Antibodies, Monoclonal; Carbocyanines; Cells, Cultured; Chemotactic Factors; Fluorescent Dyes; Humans; Ligands; Neutrophils; Oligopeptides; Receptors, Formyl Peptide; Receptors, Immunologic; Receptors, Peptide

Heterogeneity of peripheral blood monocytes is characterized by specific patterns in the membrane expression of Fc gamma-receptor III (FcgammaRIII/CD16) and the lipopolysaccharide receptor (LPS receptor CD14), allowing discrimination of distinct subpopulations. The aim was to analyze the correlation of these phenotypic differences to the early interaction of freshly isolated monocytes with modified lipoproteins by the use of either enzymatically degraded low density lipoprotein (E-LDL), acetylated low density lipoprotein (ac-LDL), oxidized low density lipoprotein (ox-LDL), or native low density lipoprotein. Highest E-LDL binding was observed on CD14(high) CD16(+) monocytes as determined by flow cytometry, suggesting a selective interaction of E-LDL with distinct subpopulations of monocytes. E-LDL induced rapid foam cell formation both in predifferentiated monocyte-derived macrophages and, in contrast to ac-LDL or ox-LDL, also in freshly isolated peripheral blood monocytes. This was accompanied by upregulation of the 2 class B scavenger receptors CLA-1/SR-BI (CD36 and LIMPII Analogous-1/scavenger receptor type B class I) and CD36. Cellular binding and uptake of E-LDL was neither competed by ac-LDL nor the class A scavenger-receptor inhibitor polyinosinic acid but was partially inhibited by an excess of ox-LDL. In predifferentiated monocyte-derived macrophages, an anti-CD36 antibody inhibited cellular binding and uptake of E-LDL by approximately 20%, suggesting that recognition of these hydrolase-modified low density lipoprotein particles is mediated only in part by the class B scavenger receptor CD36.

Topics: Affinity Labels; Antibodies, Monoclonal; Binding, Competitive; Carbocyanines; CD36 Antigens; Foam Cells; Humans; Lipids; Lipopolysaccharide Receptors; Lipoproteins, LDL; Membrane Proteins; Monocytes; Poly I; Receptors, IgG; Receptors, Immunologic; Receptors, LDL; Receptors, Lipoprotein; Receptors, Scavenger; RNA, Messenger; Scavenger Receptors, Class A; Scavenger Receptors, Class B; Up-Regulation

Dorsal root ganglion (DRG) neurons that have dichotomizing axons to the lumbar facet joint and to the sciatic nerve were investigated in rats using a double fluorescent labeling technique.. To clarify the existence of DRG neurons with dichotomizing axons projecting to the lumbar facet joint and to the sciatic nerve in rats.. DRG neurons having dichotomizing axons have been reported in several species and are considered to be related to referred pain. However, such DRG neurons have not been investigated in the lumbar spine. Clinically, pain from the lumbar facet joint is sometimes referred to the lower extremities innervated by the sciatic nerve.. Two kinds of neurotracers (DiI and FG) were used in the present double-labeling study. DiI crystals were placed in the left L5-L6 facet joint, and FG was applied to the ipsilateral sciatic nerve or along the midline of the L5 dermatome. Bilateral DRGs T13-S1 were observed by fluorescence microscope.. DRG neurons double labeled with DiI and FG were recognized only in the ipsilateral DRGs from L3 to L6 levels. Approximately 3% of DRG neurons innervating the L5-L6 facet joint had other axons to the sciatic nerve. By contrast, no double-labeled neurons were observed after FG was applied to the L5 dermatome.. In rats approximately 3% of DRG neurons innervating the lumbar facet joints have dichotomized axons projecting to the sciatic nerve.

Topics: Animals; Axons; Carbocyanines; Fluorescent Dyes; Ganglia, Spinal; Hindlimb; Lumbar Vertebrae; Male; Neurons, Afferent; Rats; Rats, Sprague-Dawley; Sciatic Nerve; Skin; Stilbamidines; Synaptic Transmission

The nucleus paragigantocellularis lateralis (PGL) is located in the rostral ventrolateral medulla (RVLM), a brainstem region that regulates homeostatic functions, such as blood pressure and cardiovascular reflexes, respiration. central chemosensitivity and pain. In the present study, we examined anatomic relationships of the human nucleus paragigantocellularis lateralis using a bidirectional lipophilic fluorescent tracer, 1,1'-dioctadecyl-3,3.3',3'-tetramethylindocarbocyanine perchlorate (DiI), in nine postmortem human fetal midgestational brainstems. The areas which were labeled by diffusion of DiI from the nucleus paragigantocellularis lateralis included the arcuate nucleus (ARC) of the medulla, caudal raphe (nucleus raphe obscurus and pallidus), hilum and amiculum of the inferior olive, bilateral "reticular formation" (including the nucleus paragigantocellularis lateralis, nucleus gigantocellular-is and the intermediate reticular zone (IRZ)). vestibular and cochlear nuclei, cells and fibers at the floor of the fourth ventricle with morphologic features of tanycytes, parabrachial nuclei (PBN), medial lemniscus, lateral lemniscus, inferior cerebellar peduncle and cerebellar white matter, central tegmental tract, and the capsule of the red nucleus. This pattern of DiI labeling bears many similarities with the pattern of connections of the nucleus paragigantocellularis lateralis previously demonstrated by tract-tracing methods in experimental animals, and is consistent with the role of the nucleus paragigantocellularis lateralis in central regulation of homeostatic functions. In contrast to the animal studies, however, we did not demonstrate connections of the nucleus paragigantocellularis lateralis with the nucleus of the tractus solitarius (nTS) (only connections with the rostral subdivision were examined), locus coeruleus, or the periaqueductal gray (PAG) in the human midgestational brainstem. In our previous studies, six medullary areas showed reduced serotonin receptor binding in a subset of victims of sudden infant death syndrome (SIDS). The present study demonstrated DiI labeling in all of these six areas, suggesting that they are interconnected.

Topics: Autonomic Nervous System; Carbocyanines; Fetus; Fluorescent Dyes; Humans; Infant, Newborn; Medulla Oblongata; Neural Pathways; Olivary Nucleus; Raphe Nuclei; Respiratory Center; Sudden Infant Death

Little is known about the molecular mechanisms that cause excitation of neurons which innervate the teeth. We investigated whether rat dental sensory neurons express the vanilloid (capsaicin) receptor (VR1). Dental sensory neurons were identified by retrograde transport of the fluorescent dye DiIC18 placed in maxillary molars. Patch-clamp recordings in culture showed that 65% of DiIC18-labeled rat trigeminal ganglion neurons are excited by capsaicin. Responders covered the entire range of cell sizes examined (soma diameter, 24 to 48 microm). All non-responders had a soma diameter > 33 microm. Capsazepine (1 microM) reduced the capsaicin-evoked membrane current (6/6) and depolarization (7/7 responders). RT-PCR amplified a 375-bp product from DiIC18-labeled neurons which was identical to that expected for VR1. Thus, many rat dental primary afferent neurons are excited by capsaicin, and the response appears to be mediated by VR1. These results suggest that pharmacological blockers of VR1 may provide significant relief of dental pain.

Topics: Animals; Capsaicin; Carbocyanines; Cells, Cultured; Dental Pulp; Electrophysiology; Fluorescent Dyes; Male; Neurons, Afferent; Nociceptors; Patch-Clamp Techniques; Rats; Rats, Sprague-Dawley; Receptors, Drug; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Trigeminal Ganglion

Cell proliferation and cell movement during early regeneration of zebrafish caudal fins were examined by injecting BrdU and Di-I, respectively. In normal fins of adult fish, a small number of proliferating cells are observed in the epidermis only. Shortly following amputation, epithelial cells covered the wound to form the epidermal cap but did not proliferate. However, by 24 hr, epithelial cells proximal to the level of amputation were strongly labeled with BrdU. Label incorporation was also detected in a few mesenchymal cells. Proliferating cells in the basal epithelial layer were first observed at 48 hr at the level of the newly formed lepidotrichia. At 72 hr, proliferating mesenchymal cells were found distal to the plane of amputation whereas more proximal labeled cells included mainly those located between the lepidotrichia and the basal membrane. When BrdU-injected fins were allowed to regenerate for longer periods, labeled cells were observed in the apical epidermal cap, a location where cells are not thought to proliferate. This result is suggestive of cell migration. Epithelial cells, peripheral to the rays or in the tissue between adjacent rays, were labeled with Di-I and were shown to quickly migrate towards the site of amputation, the cells closer to the wound migrating faster. Amputation also triggered migration of cells of the connective tissue located between the hemirays. Although cell movement was induced up to seven segments proximal from the level of amputation, cells located within two segments from the wound provided the main contribution to the blastema. Thus, cell proliferation and migration contribute to the early regeneration of zebrafish fins.

Topics: Animals; Bromodeoxyuridine; Carbocyanines; Cell Division; Cell Movement; Epidermis; Extremities; Fluorescent Dyes; Regeneration; Time Factors; Zebrafish

We examined the pharmacology of dendritic morphologic changes in cultured cortical neurons exposed to sublethal oxygen-glucose deprivation (OGD). Confocal analysis of DiI-labeled neurons demonstrated transient dendritic swelling and spine loss after OGD. These morphological changes were reproduced by direct application of NMDA, kainate, veratridine, ionomycyin, and gramicidin, but not KCl. Blockade of voltage-gated sodium or calcium channels did not prevent OGD-induced dendritic spine loss. In contrast, the NMDA receptor antagonist, MK-801, fully prevented these changes. An AMPA/kainate receptor antagonist, NBQX, had no effect by itself but reduced spine loss when added to MK-801. While alterations in dendrite morphology may be triggered by activation of disparate ion channels, rapid spine loss in hypoxic cortical neurons is mediated preferentially through activation of the NMDA subtype glutamate receptor.

Topics: Animals; Anti-Bacterial Agents; Astrocytes; Calcium Channel Blockers; Carbocyanines; Cell Hypoxia; Cell Surface Extensions; Cells, Cultured; Coculture Techniques; Dose-Response Relationship, Drug; Excitatory Amino Acid Agonists; Excitatory Amino Acid Antagonists; Fluorescent Dyes; Glucose; Hypoxia, Brain; Ionophores; Kainic Acid; Mice; N-Methylaspartate; Neurons; Oxygen; Peptides; Potassium Chloride; Tetrodotoxin; Veratridine

Up to now, single molecule fluorescence experiments were performed by dividing the time into a set of intervals and to observe the number of fluorescence photons arriving in each interval. It is obvious that the detected photons carry less information than the arrival times of the photons themselves. From the arrival times, one can still calculate the number of photons in any user-defined interval; whereas, when only the number of photons in an interval are recorded, information about their positions in time is lost. Therefore, we present a new analysis method of single molecule fluorescence data based on the positions in time of the detected fluorescence photons. We derive mathematically different statistical characteristics describing the single molecule fluorescence experiment assuming an immobilized molecule. The theory of point processes using the generating functionals formalism is ideally suited for a consistent description, linking the statistical characteristics of the excitation and detected photons to the statistical characteristics of the single motionless molecule. We then use computer-generated data sets mimicking the single molecule fluorescence experiment to explore the parametric estimation of mono- and bi-exponential single molecule impulse response functions (SMIRFs) via the following statistical characteristics: the probability density distributions (pdd) of the single and first photocount time positions in a user-defined detection interval, the probability distribution of the number of photocounts per user-defined detection interval, the time correlation function and the pdd of the time interval between two consecutive photocounts. It is shown that all of the above characteristics ensure a satisfactory recovery of the decay time of mono-exponential SMIRFs for a broad range of excitation intensities and widths of user-defined detection intervals. For bi-exponential SMIRFs, the selection of the experimental conditions is more critical and dependent on the detection procedure. At lower excitation intensities it is advantageous to use the pdds of the single and first photocount time occurrences in the user-defined detection interval. To show the practical usefulness of the new analysis method, series of photon arrival times from immobilized single molecules of DiI and rhodamine 6G were analyzed to estimate triplet lifetimes and intersystem crossing yields.

Topics: Biophysical Phenomena; Biophysics; Carbocyanines; Fluorescent Dyes; Models, Statistical; Photons; Rhodamines; Spectrometry, Fluorescence; Time Factors

Based on a study using a retrograde neurotracer, we have previously found that the dorsal portion of the L5/6 disc in the rat is multisegmentally innervated by dorsal root ganglia (DRG) from the level of T13 to L6, and that sensory nerve fibres from DRG of T13, L1 and L2 pass through the paravertebral sympathetic trunks. In this study in newborn rats, we injected crystals of 1,1'-dioctadecyl-3,3,3',3'-tetramethylinedocarbocyanine perchlorate (DiI) into the DRG of T13, L1 and L2 and showed DiI-labelled sensory nerve fibres in the dorsal portion of the discs from the level of T13/L1 to L5/6. Our results show that the dorsal portion of the lumbar discs is innervated by the DRG from levels T13 to L2.

Topics: Animals; Animals, Newborn; Carbocyanines; Fluorescent Dyes; Ganglia, Spinal; Lumbar Vertebrae; Rats; Rats, Sprague-Dawley; Thoracic Vertebrae

To isolate retinal endothelial cells (RECs) from Lewis rats using magnetic beads coated with antibodies to rat platelet-endothelial cell adhesion molecule-1 (PECAM-1) and to characterize the cultured RECs.. Magnetic beads were coated with anti-rat PECAM-1 antibodies. Retinas were obtained from Lewis rat eyes. After the retinas were digested with collagenase, they were incubated with the antibody-coated beads with agitation. RECs that stuck to the beads were collected with a magnetic particle concentrator and cultured in fibronectin coated wells. The characteristics of the RECs were examined by immunohistochemical study utilizing von Willebrand's Factor, acetylated low-density lipoprotein uptake and transmission electron microscopy.. The cells isolated using the PECAM-1-coated magnetic-bead technique formed a contact-inhibited cobblestone monolayer that stained positive for von Willebrand's Factor. These cells revealed low-density lipoprotein uptake. Ultrastructurally, the isolated cells exhibited pinocytic vesicles and a high density of intercellular junctions without fenestrations.. These results indicate that the isolated cells were vascular endothelial cells showing both morphologic and functional characteristics of retinal vascular endothelium. The magnetic-bead technique was useful for isolating high purity RECs that can be cultured to study the physiological, immunological and biochemical role of the endothelium in various ocular diseases.

Topics: Animals; Antibodies; Carbocyanines; Cell Separation; Cells, Cultured; Endothelium, Vascular; Fluorescent Dyes; Lipoproteins, LDL; Magnetics; Microspheres; Platelet Endothelial Cell Adhesion Molecule-1; Rats; Rats, Inbred Lew; Retinal Vessels; von Willebrand Factor

The potential biomedical utility of the photoinduced destabilization of liposomes depends in part on the use of green to near infrared light with its inherent therapeutic advantages. The polymerization of bilayers can be sensitized to green light by associating selected amphiphilic cyanine dyes, i.e. the cationic 1,1'-dioctadecyl-3,3,3', 3'-tetramethylindocarbocyanine (DiI), or the corresponding anionic disulfonated DiI (DiI-DS), with the lipid bilayer. The DiI sensitization of the polymerization of 1, 2-dioleoyl-sn-glycero-3-phosphoethanolamine/1,2-bis[10-(2', 4'-hexadienoyloxy)-decanoyl]-sn-glycero-3-phosphocholine liposomes caused liposome destabilization with release of encapsulated aqueous markers. In separate experiments, similar photosensitive liposomes were endocytosed by cultured HeLa cells. Exposure of the cells and liposomes to 550 nm light caused a net movement of the liposome-encapsulated 8-hydroxypyrene-1,3,6-trisulfonic acid (HPTS) from low pH compartment(s) to higher pH compartment(s). This suggests that photolysis of DiI-labelled liposomes results in delivery of the contents of the endocytosed liposomes to the cytoplasm. The release of HPTS into the cytoplasm appears to require the photoactivated fusion of the labelled liposomes with the endosomal membrane. These studies aid in the design of visible light sensitive liposomes for the delivery of liposome-encapsulated reagents to the cytoplasm.

Topics: Arylsulfonates; Carbocyanines; Color; Cytoplasm; Drug Carriers; Endocytosis; Endosomes; Fluorescent Dyes; HeLa Cells; Humans; Hydrogen-Ion Concentration; Light; Liposomes; Membrane Fusion; Naphthalenes; Phosphatidylcholines; Phosphatidylethanolamines; Photolysis; Pyridinium Compounds; Sulfonic Acids; Temperature; Ultraviolet Rays

A retroendocytotic pathway for high-density lipoprotein 3 (HDL(3)) in cultured intestinal epithelial cell lines has been described. In small intestinal crypt cells and Caco-2, HDL(3) is internalized, transported to lipid droplets and, after solubilization of these lipid droplets, resecreted. In the present study we examined the mechanisms of intracellular transport of HDL(3) in the Caco-2 cell line.. Apolipoprotein E free HDL(3 )was gold-labeled for transmission electron microscopy and 1, 1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine iodide [DiI(3)] labeled for fluorescence and confocal laser scanning microscopy. For tubulin desintegration Caco-2 cells were incubated with taxol, colchicine and beta- and gamma-lumicolchicine. Tubulin staining was performed using a FITC labeled antibody. Uptake of HDL(3) was quantified by FACS analysis.. HDL(3) was rapidly internalized and found to be in contact with lipid droplets in the perinuclear region after 10 min. By transmission electron microscopy a frequent colocalization of HDL(3)-containing vesicles and tubular structures was demonstrated. The close association of HDL(3)-containing vesicles with fluorescence stained tubulin could be confirmed by confocal laser scanning microscopy. Preincubation of the cells with taxol and colchicine did not completely prevent internalization but reduced it during a 2-hour incubation period to less than 50% of the control cells. The transport of DiI(3)-labeled HDL(3) to the lipid droplets in the perinuclear region was almost completely blocked by taxol and colchicine.. Internalization and intracellular transport of HDL(3) in intestinal epithelial cells (Caco-2) is dependent on a tubulin-mediated mechanism.

Topics: Biological Transport; Caco-2 Cells; Carbocyanines; Cell Adhesion; Endocytosis; Epithelial Cells; Flow Cytometry; Fluorescent Dyes; Humans; Intestinal Mucosa; Lipoproteins, HDL; Lipoproteins, HDL3; Microscopy, Confocal; Tubulin

Specialization of the ganglion cell layer (GCL) was studied by Nissl-staining and axonal tract-tracing methods in chicks and chick embryos. The changes in the retinal area and the cell number in the GCL produced a disparity in the cell density that occurred through the two different processes, cell generation (before embryonic days 10-14, E10-14) and cell loss (after E10-14). One high-density area was found in the retinal fundus on E8 (presumptive central area, pCA) and its density decreased toward the peripheral retina. Another high-density area was found in the dorsal retina on E11 (presumptive dorsal area, pDA). Cell densities of the pCA and the pDA on E11 decreased gradually to 25-30% by P1, and after that they further decreased to 40-60% by P30. The pCA was still identified on P30, but the pDA became very obscure by this age. In contrast, ganglion cell sizes increased 5-7 times in the pCA and pDA from E8 to P30, and increased 12 times in the temporal periphery. The present study suggests that the center-peripheral gradient of cell density results from lager scale of cell genesis in the pCA, but not from lager scale of cell loss in the peripheral retina. However, obscuration of the pDA results from equalization of cell density in cellular degeneration processes.

Topics: Animals; Benzoxazines; Carbocyanines; Chick Embryo; Chickens; Coloring Agents; Horseradish Peroxidase; Image Processing, Computer-Assisted; Oxazines; Retinal Ganglion Cells

Xenopus laevis has three distinctive olfactory neuroepithelia. We examined the axonal projection from each of these epithelia to the olfactory bulb by Di-I labeling, and confirmed that the Xenopus primary olfactory pathways involve the dorsal pathway from the olfactory epithelium to the dorsal region of the main olfactory bulb, the ventral pathway from the middle chamber epithelium to the ventral region of the main olfactory bulb, and the vomeronasal pathway from the vomeronasal epithelium to the accessory olfactory bulb. We next examined expression patterns of glycoconjugates in the three olfactory pathways by lectin-histochemistry using 21 biotinylated lectins. Fourteen out of 21 lectins stained the Xenopus primary olfactory system. RCA-I stained the three olfactory pathways uniformly. PHA-E stained only the dorsal pathway. LEL, STL, PNA, ECL and UEA-I stained the dorsal pathway more intensely than the ventral pathway, and among them, only UEA-I stained the vomeronasal pathway. In contrast, s-WGA, DBA, SBA, BSL-I VVA, SJA and PHA-L showed intense stainings in the ventral pathway and moderate stainings in the vomeronasal pathway, but faint or weak stainings in the dorsal pathway. These observations suggest that the ventral pathway expresses glycoconjugates shared commonly with either the dorsal or the vomeronasal pathway. In addition, from the binding patterns of the lectins with a binding specificity for N-acetylgalactosamine, glycoconjugates containing this saccharide seem to play an important role for the organization of the olfactory pathways.

Topics: Affinity Labels; Animals; Biotinylation; Carbocyanines; Female; Fluorescent Dyes; Glycoconjugates; Histocytochemistry; Lectins; Male; Olfactory Bulb; Xenopus laevis

The major goal of this study was to determine the interactions of VLDL surface and core lipids with the artery wall. We first demonstrated in vitro that surface lipid in VLDL could be traced using the phospholipid-like fluorescent probe 1,1'-dioctadecyl-3,3, 3',3'-tetramethyl-indocarbocyanine (DiI). The core of VLDL particles was traced by fluorescently labeling apolipoprotein B with TRITC. The labeled VLDLs were perfused through rat carotid arteries, and accumulation of the fluorescently labeled VLDL components in the arterial walls was determined by quantitative fluorescence microscopy. Addition of lipoprotein lipase increased the accumulation of both DiI and TRITC by >2.3-fold. Histological examination showed that DiI and TRITC were primarily localized to the endothelial layer; however, DiI also accumulated as small "lakes" deeper in the artery, in a subendothelial position. Addition of HDL to the perfusion decreased the accumulation of surface lipid and apolipoprotein B-containing particles and eliminated the DiI lakes. Moreover, the increase in endothelial layer permeability associated with lipolysis was attenuated 21% by HDL. If VLDL surface lipid first was allowed to accumulate in the arterial wall, its subsequent rate of loss was more than twice as fast if HDL was included in the perfusate. These studies directly demonstrate atherogenic effects of VLDL lipolysis and their inhibition by HDL.

Topics: Animals; Apolipoproteins B; Carbocyanines; Carotid Arteries; Endothelium, Vascular; Fluorescent Dyes; In Vitro Techniques; Lipolysis; Lipoproteins, HDL; Lipoproteins, VLDL; Muscle, Smooth, Vascular; Perfusion; Permeability; Rats; Rhodamines

The hypothalamus and perhaps its function appear to be similar among vertebrates. Thus, studying the teleostean hypothalamus could be a good model for understanding common neural circuits and mechanisms retained through the vertebrates. However, connections of the inferior lobe, which is considered the hypothalamus in teleosts, is poorly known. The corpus mamillare (CM) is a nucleus of the inferior lobe named after the mammalian mamillary body based on similarities in external morphology. Afferent connections of the CM have been reported only in cypriniform teleosts. These include projections from the nucleus pretectalis superficialis pars magnocellularis, a nucleus lacking in percomorph teleosts, and projections from the secondary gustatory nucleus. Efferent connections of the CM have not been reported in teleosts. In the present study, the CM and its subdivisions and the connections of these subnuclei were identified in isolated and maintained brains of tilapia Oreochromis niloticus by local DiI and biocytin injection. Afferent connections confirmed by reciprocal injections were from the nucleus diffusus lobi inferioris (NDLI) and the nucleus diffusus tori lateralis (NDTL). Efferent connections of each CM subnuclei were also reciprocally confirmed. These connections were to the area dorsalis pars medialis of the telencephalon, the nucleus ventromedialis (NVM) of the thalamus, the tectum opticum (TO), and the nucleus posterioris periventricularis. Because the NDLI is known to receive gustatory information in tilapia, the CM could relay gustatory inputs to multisensory areas, the TO and NVM, for which there are no current reports regarding gustatory inputs.

Topics: Afferent Pathways; Animals; Biotin; Carbocyanines; Dextrans; Female; Fluorescent Dyes; Histocytochemistry; Hypothalamus; Lysine; Male; Mammillary Bodies; Nerve Fibers; Tilapia

Foam cell formation is a key event in the onset and progression of atherosclerotic lesions. We have previously reported that internalization of aggregated low density lipoproteins (agLDLs) by vascular smooth muscle cells (VSMCs) produces cholesteryl ester (CE) accumulation in these cells. The aim of this study was to analyze whether the low density lipoprotein receptor-related protein (LRP) mediates the uptake of agLDL by VSMCs. First, immunocytochemistry and fluorescence microscopic analysis with the use of anti-LRP antibodies indicated that there was a high expression of LRP in VSMCs. Confocal microscopic analysis with the use of agLDLs labeled with fluorochrome 1,1'-dioctadecyl-3,3,3', 3'-tetramethylindocarbocyanine and anti-LRP antibodies showed the colocalization of agLDL and LRP. The second approach was to analyze the effect of LRP ligands on agLDL internalization; lactoferrin strongly inhibited CE accumulation from agLDLs (85.0+/-5.7% at 25 microg/mL) by impairing agLDL binding. Coincubation of agLDL with anti-LRP antibodies decreased in a dose-dependent manner agLDL-derived CE accumulation (from 20% at 12.5 microg/mL to 80% at 50 microg/mL). The third approach was to evaluate whether antisense LRP oligodeoxynucleotides were able to block agLDL internalization. Treatment of VSMCs with 5 micromol/L antisense LRP oligodeoxynucleotides reduced agLDL-derived CE accumulation by 84+/-2%. In conclusion, these results from immunologic, biochemical, and molecular interventions demonstrate that LRP mediates the binding and internalization of agLDL in human VSMCs. Because LRP is highly expressed in VSMCs and the uptake of 1 LDL aggregate amounts to the deposition of several hundreds of LDL particles, the uptake of agLDL through LRP could have a crucial role for lipid deposition in VSMCs.

Topics: Antibodies; Aorta; Carbocyanines; Cells, Cultured; Cholesterol Esters; Fluorescent Dyes; Humans; Immunoglobulin G; Immunohistochemistry; Lactoferrin; Lipoproteins, LDL; Low Density Lipoprotein Receptor-Related Protein-1; Microscopy, Confocal; Muscle, Smooth, Vascular; Oligonucleotides, Antisense; Poly I; Receptors, Immunologic; Receptors, LDL

The objective of this study was to determine the schedule of the arrival of the axons from the hypothalamus to the posterior lobe of the pituitary (PL) in rats during ontogenesis by using the fluorescent lipophilic carbocyanine dye 1,1'-dioctadecyl-3,3,3', 3'-tetramethylindocarbocyanine perchlorate (DiI) as a retrograde tracer. After preliminary fixation of the brain, DiI crystals were implanted in the PL on embryonic day 15 (E15), E16, E17, and E19 as well as on postnatal day 2 (P2) and P9. This was followed by a DiI retrograde diffusion along the plasma membrane and subsequent staining of hypothalamic neuronal cell bodies. The supraoptic nucleus (SO) contained an accumulation of fluorescent cells that extended toward the diamond-like swelling of the third ventricle as early as E15. These data suggest that the magnocellular neurons of the SO send their axons to the PL at the very beginning of differentiation, perhaps even before reaching their final position. The initial axons of the neurons of the paraventricular nucleus proper (PV) appeared to reach the PL significantly later, at E17. In addition to the SO and the PV, accessory magnocellular nuclei contributed to the innervation of the PL in perinatal rats. The neurons of the retrochiasmatic accessory nucleus first sent their axons to the PL on E16-E17. Axons that originated from other accessory hypothalamic nuclei reached the PL after birth, suggesting a delay in their involvement in the regulation of visceral functions compared with other magnocellular nuclei. Thus, the axons of magnocellular neurons reach the PL unexpectedly early in embryogenesis, raising the possibility of the functional significance of vasopressin and oxytocin as fetal neurohormones.

Topics: Aging; Animals; Animals, Newborn; Carbocyanines; Embryo, Mammalian; Embryonic and Fetal Development; Fluorescent Dyes; Hypothalamus; Pituitary Gland, Posterior; Rats; Rats, Wistar; Synaptic Transmission

Animal studies have shown that connections between the retina, lateral geniculate nucleus (LGN), and visual cortex begin to develop prenatally. To study the development of these connections in humans, regions of fixed brain from fetuses of 20-22 gestational weeks (GW) were injected with the fluorescent tracer DiI. Placement of DiI in the optic nerve or tract labeled retinogeniculate projections. In the LGN, these projections were already segregated into eye-specific layers by 20 GW. Retinogeniculate segregation thus preceded cellular lamination of the LGN, which did not commence until 22 GW. Thalamocortical axons, labeled from DiI injections into the optic radiations, densely innervated the subplate, but did not significantly innervate the cortical plate. This pattern was consistent with observations of a "waiting period" in animals, when thalamocortical axons synapse in the subplate for days or weeks before entering the cortical plate. Cortical efferent neurons (labeled retrogradely from the optic radiations) were located in the subplate and deep layers of the cortical plate. In summary, human visual connections are partially formed by mid-gestation, and undergo further refinement during and after this period. The program for prenatal development of visual pathways appears remarkably similar between humans and other primates.

Topics: Carbocyanines; Efferent Pathways; Embryonic and Fetal Development; Fetus; Fluorescent Dyes; Geniculate Bodies; Gestational Age; Humans; Retina; Synaptic Transmission; Visual Cortex; Visual Pathways

Mammalian skeletal muscles with long fascicle lengths are predominantly composed of short muscle fibers that terminate midbelly with no direct connection to the muscle origin or insertion. The manner in which these short fibers terminate and transmit tension through the muscle to their tendons is poorly understood. We made an extensive morphological study of a series-fibered muscle, the guinea pig sternomastoid, in order to define the full range of structural specializations for tension transmission from short fibers within this muscle. Terminations were examined in single fibers, teased small bundles of fibers, and in sections at both the light and electron microscopic level. In many cases, sites of fiber termination were defined by reactivity for the enzyme acetylcholinesterase, which also marks myotendinous junctions. Additionally, transport of the lipophilic fluorescent dye, DiI, or injection of Lucifer Yellow were used to visualize undisturbed fiber terminations in whole muscles using confocal and fluorescence microscopy. At the light microscopic level, we find that intrafascicularly terminating fibers end about equally often in either a long progressive taper, or in a series of small or larger blunt steps. Combinations of these two morphologies are also seen. However, when analyzed at higher resolution with confocal or electron microscopy, the apparently smooth progressive tapers appear also to be predominantly composed of a series of fine stepped terminations. Stepwise terminations in most cases join face-to-face with complementary endings of neighboring muscle fibers, some via an extended collagenous bridge and others at close interdigitating myomyonal junctions. These muscle-to-muscle junctions show many of the features of myotendinous junctions, including dense subsarcolemmal plaques in regions of myofibrillar termination and we suggest that they serve to pass tension from fiber to fiber along the longitudinal axis of the muscle. In addition, we observe regions of apparent side-to-side adhesion between neighboring fibers at sites where there is no apparent fiber tapering or structural specialization typical of myofibril termination. These sites show acetylcholinesterase reactivity, and large numbers of collagen fibers passing laterally from fiber to fiber. These latter connections seem most likely to be involved in lateral transmission of tension, either from fiber to fiber, or from fiber to endomysium. Overall, our results suggest that tension

Topics: Acetylcholinesterase; Animals; Carbocyanines; Collagen; Female; Fluorescent Dyes; Guinea Pigs; Hydrogen-Ion Concentration; Isoquinolines; Microscopy, Confocal; Microscopy, Electron; Microscopy, Electron, Scanning; Muscle Contraction; Muscle Fibers, Skeletal; Muscle Proteins; Neck Muscles; Tendons

A non-ideal lipid binary mixture (dilauroylphosphatidylcholine/distearoylphosphatidylcholine), which exhibits gel/fluid phase coexistence for wide temperature and composition ranges, was studied using photophysical techniques, namely fluorescence anisotropy, lifetime and resonance energy transfer (FRET) measurements. The FRET donor, N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-dilauroylphosphatidylethanol amine, and a short-tailed FRET acceptor, 1,1'-didodecil-3,3,3',3'-tetramethylindocarbocyanine (DiIC12(3)), were shown to prefer the fluid phase by both intrinsic anisotropy, lifetime and FRET measurements, in agreement with published reports. The other studied FRET acceptor, long-tailed probe 1,1'-dioctadecil-3,3,3',3'-tetramethylindocarbocyanine (DiIC18(3)), is usually reported in the literature as partitioning mainly to the gel. While intrinsic lifetime studies indeed indicated preferential partition of DiIC18(3) into a rigidified environment, FRET analysis pointed to an increased donor-acceptor proximity as a consequence of phase separation. These apparently conflicting results were rationalized on the basis of segregation of DiIC18(3) to the gel/fluid interphase. In order to fluid-located donors sense these interphase-located acceptors, fluid domains should be small (not exceed approximately 10-15 nm). It is concluded that membrane probes which apparently prefer the gel phase may indeed show a non-random distribution in this medium, and tend to locate in an environment which simultaneously leads to less strict packing constraints and to favorable hydrophobic matching interactions.

Topics: 4-Chloro-7-nitrobenzofurazan; Carbocyanines; Fluorescent Dyes; Phosphatidylcholines; Phosphatidylethanolamines; Spectrometry, Fluorescence; Temperature

Retinal light exposure is a source of oxidative stress, and retinal cells contain molecules that scavenge or inactivate reactive oxygen species (ROS). Yet, ROS also play a role in signal transduction, and some retinal cells (e.g., neurotrophin-dependent retinal ganglion cells, RGCs) may use ROS as part of the signaling process for cell death. RGCs might therefore have specialized mechanisms for regulating ROS levels. The hypothesis that RGCs might regulate ROS differently from other retinal cells was tested by studying their differential response to oxidative stress in vitro.. RGCs were retrogradely labeled by injecting the fluorescent tracer DiI into the superior colliculi of postnatal day 2 through 4 Long-Evans rats. At postnatal days 7 through 9 the retinas were dissociated with papain and cultured with and without specific ROS-generating systems and/or scavengers. RGCs were identified by their DiI positivity using rhodamine filters. Living cells, determined by metabolism of calcein-AM viewed with fluorescein filters, were counted in triplicate. Degenerate reverse transcription-polymerase chain reaction (RT-PCR) using primers specific to peroxidase homology regions was used to survey for novel peroxidases expressed within normal retinas.. Compared with other retinal cells, RGCs were remarkably resistant to cell death induced by superoxide anion, hydrogen peroxide, or hydroxyl radical. Catalase counteracted the effect of each ROS-generating system on retinal cells, consistent with damage occurring via a hydrogen peroxide intermediate. Aminotriazole, L-buthionine sulfoximine, and sodium azide partly abrogated the RGC resistance to oxidative stress, suggesting that this resistance may be mediated by catalase and/or glutathione peroxidase. A limited expression survey within the retina using degenerate RT-PCR did not demonstrate novel peroxidases.. These data suggest a role for one or more endogenous peroxidases within RGCs, which could possibly be protective under conditions of axonal damage. Exploration of the unique characteristics of RGC resistance and susceptibility to injury may help in better understanding the pathophysiology of diseases associated with primary axonal damage.

Topics: Animals; Carbocyanines; Catalase; Cell Count; Cell Survival; Cells, Cultured; DNA Primers; Fluoresceins; Fluorescent Dyes; Hydrogen Peroxide; Neoplasm Proteins; Oxidative Stress; Peroxidases; Peroxiredoxins; Rats; Rats, Long-Evans; Reactive Oxygen Species; Retinal Ganglion Cells; Reverse Transcriptase Polymerase Chain Reaction; Vitamin K; Xanthine Oxidase

Approximately 50% of New Zealand Black mice (NZB/BINJ) and 80% of NXSM-D/EiJ mice prenatally develop neocortical layer I ectopias, mostly in somatosensory cortices. These cortical anomalies are similar to those seen in the brains of individuals with dyslexia. Neurofilament staining revealed a radial column of tightly packed fiber bundles in the layers underlying ectopias. This suggested that the connectivity of the ectopic neurons was aberrant. The present study used the tracers 1,1'-dioctadecyl- 3,3,3',3'-tetramethylindo- carbocyanine perchlorate (DiI) and biotinylated dextran amine (BDA) to more thoroughly explore the cortical and thalamic connectivity of the ectopias. DiI placement into ectopias again revealed a distinct bundle of fibers extending from the ectopic neurons to the deep cortical layers. This bundle split in the white matter with some fibers traveling to the corpus callosum and others to the internal capsule. Thalamic connections were concentrated in the ventrobasal com- plex (VB) and posterior thalamic nucleus group (Po). Injections of BDA into VB revealed reciprocal connections between VB and the ectopic cortical neurons. Ipsilateral corticocortical projections were seen between ectopias in primary somatosensory and motor and secondary somatosensory cortices, but no contralateral connections of the ectopic neurons were seen. These findings confirm the notion that layer I ectopias are anomalously connected by comparison to neurons in homologous cortex, which may underlie widespread dysfunction of brains containing ectopias.

Topics: Animals; Autoimmunity; Biotin; Brain Diseases; Carbocyanines; Choristoma; Dextrans; Female; Fluorescent Dyes; Male; Mice; Mice, Inbred Strains; Motor Cortex; Neural Pathways; Neurons; Somatosensory Cortex; Thalamic Nuclei

The ability of liposomes bearing anti-HLA-DR Fab' fragments at the end termini of polyethyleneglycol chains (sterically stabilized immunoliposomes) to target HLA-DR expressing cells and increase the accumulation of liposomes into lymphoid organs has been evaluated and compared to that of conventional liposomes, sterically stabilized liposomes and conventional immunoliposomes after a single subcutaneous injection to mice. The accumulation of sterically stabilized liposomes in lymph nodes was higher than that of conventional liposomes. Sterically stabilized immunoliposomes accumulated much better than conventional immunoliposomes in all tissues indicating that the presence of PEG has an important effect on the uptake of immunoliposomes by the lymphatic system. Fluorescence microscopy studies showed that sterically stabilized liposomes are mainly localized in macrophage-rich areas such as the subcapsular region of lymph nodes and in the red pulp and marginal zone of the spleen. In contrast, sterically stabilized immunoliposomes mostly accumulated in the cortex in which follicles are located and in the white pulp of the spleen. As the human HLA-DR determinant of the major histocompatibility complex class II is expressed on activated CD4+ T lymphocytes and antigen presenting cells such as monocyte/macrophages and dendritic cells, known as the cellular reservoirs of HIV-1, liposomes bearing anti-HLA-DR antibodies constitute an attractive approach to concentrate drugs in HIV-1 reservoirs and improve their therapeutic effect.

Topics: Animals; Antibodies; Carbocyanines; Drug Delivery Systems; Female; Flow Cytometry; Fluorescent Dyes; HIV-1; HLA-DR Antigens; Immunoglobulin Fab Fragments; In Vitro Techniques; Liposomes; Lymph Nodes; Lymphoid Tissue; Mice; Mice, Inbred C3H; Microscopy, Fluorescence; Polyethylene Glycols; Spleen; Tissue Distribution

A cell-line was established from bovine placental cotyledon. When cultured in M199 with 10% fetal bovine serum, this cell-line had a doubling time of about 18 h. With immunohistochemistry, it was demonstrated that this cell-line expressed vimentin and angiotensin-converting enzyme (ACE). While both molecules are expressed in endothelial cells, ACE is usually considered to be a specific marker for endothelial cells. Furthermore, cells were shown to take up Dil-Ac-LDL (acetylated low-density lipoprotein labeled with 1,1'-dioctadecyl-3,3,3'-tetramethylindo-carbocyanine perchlorate). This characteristic feature has been used to identify endothelial cells. Finally, when cultured on matrigel, this cell-line formed tube-like structures similar to those formed by endothelial cells. Tube-formation on matrigel is a physiological property specific to endothelial cells. In conclusion, these three lines of evidence strongly suggest that this cell-line is endothelial cell in nature. Further studies using an endothelial cell-line from bovine placenta may help to elucidate the cause of bovine placental retention, a major cause for economic loss in bovine industry. Furthermore, an endothelial cell-line could be an important tool in research areas such as tissue remodeling, angiogenesis, and cancer.

Topics: Animals; Carbocyanines; Cattle; Cell Culture Techniques; Cells, Cultured; Collagen; Drug Combinations; Endothelium; Fluorescent Dyes; Humans; Laminin; Lipoproteins, LDL; Peptidyl-Dipeptidase A; Placenta; Proteoglycans; Vimentin

This study reports the detection of single mammalian cells, specifically T cells (T lymphocytes) labeled with dextran-coated superparamagnetic iron oxide particles, using magnetic resonance microscopy. Size amplification due to sequestration of the superparamagnetic particles in vacuoles enhances contrast in localized areas in high-resolution magnetic resonance imaging. Magnetic resonance images of samples containing differing concentrations of T cells embedded in 3% gelatin show a number of dark regions due to the superparamagnetic iron oxide particles, consistent with the number predicted by transmission electron microscopy. Colabeling of T cell samples with a fluorescent dye leads to strong correlations between magnetic resonance and fluorescence microscopic images, showing the presence of the superparamagnetic iron oxide particles at the cell site. This result lays the foundation for our approach to tracking the movement of a specific cell type in live animals and humans.

Topics: Animals; Biophysical Phenomena; Biophysics; Carbocyanines; Cell Movement; Dextrans; Ferrosoferric Oxide; Fluorescent Dyes; Humans; Iron; Magnetic Resonance Imaging; Magnetics; Magnetite Nanoparticles; Microscopy, Electron; Microscopy, Fluorescence; Oxides; Rats; Rats, Inbred F344; T-Lymphocytes

Neurons with axons that extend to the contralateral side of the spinal cord--commissural interneurons (CINs)--coordinate left/right alternation during locomotion. Little is known about the organization of CINs in the mammalian spinal cord. To determine the numbers, distribution, dendritic morphologies, axonal trajectories, and termination patterns of CINs located in the lumbar spinal cord of the neonatal rat, several different retrograde and anterograde axonal tracing paradigms were performed with fluorescent dextran amines and the lipophilic tracer 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI). CINs with ascending (aCINs) and descending (dCINs) axons were labeled independently. The aCINs and dCINs occupied different but overlapping domains within the transverse plane. The aCINs were clustered into four recognizable groups, and the dCINs were clustered into two recognizable groups. All dCINs and most aCINs were located within the gray matter, with somata ranging from 10-30 microm in diameter and with large, multipolar dendritic trees. One group of aCINs was located outside the gray matter along the dorsal and dorsolateral margin and had dendrites that were nearly confined to the dorsolateral surface. All CIN axons traversed the ventral commissure at right angles to the midline. CIN axons coursed up to six or seven segments rostrally and/or caudally in the ventral and ventrolateral white matter and gave off collaterals over a shorter range, predominantly to the ventral gray matter. These findings show that the lumbar spinal cord of the neonatal rat contains substantial numbers of CINs with axon projections and collateral ranges spanning several segments and that CINs projecting rostrally vs. caudally have different distributions in the transverse plane. The study provides an anatomical framework for future electrophysiological studies of the spinal neuronal circuits underlying locomotion in mammals.

Topics: Afferent Pathways; Animals; Animals, Newborn; Axons; Carbocyanines; Dendrites; Efferent Pathways; Fluorescent Dyes; Interneurons; Lumbosacral Region; Motor Activity; Rats; Rats, Wistar; Signal Transduction; Spinal Cord

The neural connections of the parapineal organ of two species of lampreys were studied with the fluorescent dye 1,1'-dioctadecyl-3,3,3'3'-tetramethylindocarbocyanine perchlorate (DiI) and with immunocytochemistry. The lamprey parapineal organ consists of a vesicle and a ganglion that are connected to the left habenula. Labeling experiments included the application of DiI to the parapineal organ, left and right fasciculus retroflexus, left habenula, and the left pretectal region. Afferent parapineal fibers run in the left fasciculus retroflexus to the interpeduncular nucleus. The parapineal fibers of this fascicle arose from parapineal ganglion cells, whereas DiI application to the left habenula labeled both neurons of this ganglion and bipolar cells in the parapineal vesicle. Efferent neurons were observed in the left habenula, and bilaterally in the subhippocampal nucleus and the dorsal pretectum. Labeling with DiI also revealed a hippocampal projection. Immunocytochemical study of the parapineal vesicle revealed serotonin-immunoreactive cells in both species of lamprey, as well as substance P-immunoreactive (SP-ir) cells in sea lamprey and choline acetyltransferase-immunoreactive (ChAT-ir) cells in the river lamprey. The SP-ir cells and ChAT-ir cells formed a rich neuropil in the parapineal ganglion. Calretinin-ir cells were numerous in the ganglion. Neuropeptide Y-immunoreactive and gamma-aminobutyric acid-immunoreactive efferent fibers were observed in the parapineal organ. Neuropeptide Y-immunoreactive fibers originate in the subhippocampal nucleus, whereas gamma-aminobutyric acid-immunoreactive fibers might also arise in the pretectal nucleus. A few galanin-ir fibers were observed. These results indicate that the parapineal connections are completely different from those of the pineal organ. The possible homology between parapineal organs of vertebrates is discussed.

Topics: Afferent Pathways; Animals; Carbocyanines; Efferent Pathways; Fluorescent Dyes; Immunohistochemistry; Lampreys; Neurons; Photoreceptor Cells; Pineal Gland; Species Specificity

To investigate the mechanisms underlying the reorganization of barrels in the whisker-tobarrel pathway, the facial vibrissae of mice were damaged by electrocauterization at alternate positions on either postnatal day 0 (P0) or P3, before or after the onset of cell aggregation in the thalamus and cortex. Animals were subsequently killed on P8, topographical changes were examined by cytochrome C oxidase histochemistry, and afferent connections were identified using DiI tracer. The cytoarchitecture was characterized with bisbenzimide counterstain. Regardless of when damage was done, the reorganized barreloids and barrels in the thalamus and cortex, respectively, were integrated in an array that represented the topography of undamaged vibrissae. In the brainstem, although the original framework of the array was preserved, defective cell aggregates remained, possibly still in contact with damaged vibrissae. During normal development, on P0 cell aggregates are formed only in the brainstem, and begin to be organized at the other levels of the pathway on P3. Therefore, when damage is induced on P3, the primary cell aggregates are replaced by new, possibly recombined, cell aggregates in the thalamus and cortex to represent the new peripheral topography. The presumably recombined aggregates indicate that cell reassemblage occurred between neighboring cell aggregates. Concomitant with these changes, afferent fibers originating in the brainstem and thalamus extended their terminal arborizations to delineate the new cell aggregates in the thalamus and cortex, respectively. These findings indicate that activity-dependent competitive interactions of afferents may play a crucial role in organizing the topography of cell aggregation and reassemblage in response to vibrissal damage at each level of the pathway.

Topics: Afferent Pathways; Animals; Animals, Newborn; Bisbenzimidazole; Carbocyanines; Cerebral Cortex; Coloring Agents; Face; Fluorescent Dyes; Mice; Mice, Inbred ICR; Neuronal Plasticity; Neurons; Thalamic Nuclei; Vibrissae

Vibrating blade microtomes are used extensively in biological research to section non-frozen tissue. There are a wide variety of commercial instruments available for this purpose, however, they are designed to cut thin sections primarily from a tissue block less than one centimeter in height. Herein is described a simple modification of a microscope frame that creates a vibrating blade microtome capable of producing a sequential series of sections through three centimeters of tissue. We illustrate the use of this device to identify and reconstruct a column of rat spinal motor neurons retrogradely labeled from a peripheral muscle.

Topics: Animals; Axonal Transport; Carbocyanines; Equipment Design; Fluorescent Dyes; Forelimb; Microtomy; Motor Neurons; Muscle, Skeletal; Rats; Specimen Handling; Spinal Cord; Vibration

Local field potentials evoked either by auditory or by mechanosensory (water displacement) lateral line stimuli were recorded in sensory subregions of the telencephalic nucleus dorsalis pars medialis (Dm) in the weakly electric fish Gnathonemus petersii. The neural tracer Neurobiotin was injected into these two physiologically defined subregions. A reciprocal connection between the two subregions of Dm, as well as cell bodies and terminals in other telencephalic regions, whose distribution was somewhat different for the two injection types, were found. The course of labeled fibers outside the telencephalon was similar after injections in both Dm regions. Fibers were seen running through the lateral forebrain bundle (lfb) to the ventral surface area of the brain within the diencephalic preglomerular region (PGv). Within a narrow streak along the ventral side of the brain densely arranged cell bodies were labeled. The locations of labeled cells within PGv were indistinguishable after tracer was injected into either acoustical or lateral line areas of Dm. Only after injection into the mechanosensory Dm region labeled cell bodies were found in the anterior preglomerular nucleus (PGa), in addition. When crystals of the fluorescent tracer DiI were inserted in the ventral part of PGv, a path of labeled fibers similar to that after telencephalic injections was found. Labeled terminals, but no cell bodies, were located both in the acoustical and in the mechanosensory regions of Dm as well as in several other telencephalic areas. Even though sensory regions in Dm that process acoustical and mechanical stimuli are segregated and unimodal, they both receive input from neurons of PGv. The specificity of the mechanosensory region of Dm might originate from the additional input from PGa and from other endbrain areas.

Topics: Animals; Auditory Pathways; Biotin; Brain Mapping; Carbocyanines; Evoked Potentials, Auditory; Fishes; Fluorescent Dyes; Functional Laterality; Microinjections; Stress, Mechanical; Telencephalon

To understand the mechanisms for endocytic sorting of lipids, we investigated the trafficking of three lipid-mimetic dialkylindocarbocyanine (DiI) derivatives, DiIC16(3) (1,1'-dihexadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate), DiIC12(3) (1,1'- didodecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate), and FAST DiI (1,1'-dilinoleyl-3,3,3', 3'-tetramethylindocarbocyanine perchlorate), in CHO cells by quantitative fluorescence microscopy. All three DiIs have the same head group, but differ in their alkyl tail length or unsaturation; these differences are expected to affect their distribution in membrane domains of varying fluidity or curvature. All three DiIs initially enter sorting endosomes containing endocytosed transferrin. DiIC16(3), with two long 16-carbon saturated tails is then delivered to late endosomes, whereas FAST DiI, with two cis double bonds in each tail, and DiIC12(3), with saturated but shorter (12-carbon) tails, are mainly found in the endocytic recycling compartment. We also find that DiOC16(3) (3,3'- dihexadecyloxacarbocyanine perchlorate) and FAST DiO (3, 3'-dilinoleyloxacarbocyanine perchlorate) behave similarly to their DiI counterparts. Furthermore, whereas a phosphatidylcholine analogue with a BODIPY (4,4-difluoro-4-bora-3a,4a-diaza-s-indacene) fluorophore attached at the end of a 5-carbon acyl chain is delivered efficiently to the endocytic recycling compartment, a significant fraction of another derivative with BODIPY attached to a 12-carbon acyl chain entered late endosomes. Our results thus suggest that endocytic organelles can sort membrane components efficiently based on their preference for association with domains of varying characteristics.

Topics: Animals; Boron Compounds; Carbocyanines; Cell Membrane; CHO Cells; Cricetinae; Endocytosis; Endosomes; Fluorescent Dyes; Humans; Lipid Metabolism; Lipids; Lysosomes; Models, Biological; Receptors, Transferrin

The aim of this work was to examine the influence of subcortical afferents on the development of corticocortical projections in the cat's visual cortex. In the adult, corticocortical axons project with precision to link retinotopically corresponding points in visual areas 17 and 18. In the newborn kitten, an excess of corticocortical connections is generated, leading to a degree of imprecision in the early pathways. During the first postnatal month, the loss of some of these early connections lowers their densities and increases the accuracy with which they project. These processes occur in an environment already influenced by afferents from the lateral geniculate nucleus and we tested the extent to which these existing inputs are required for corticocortical development. We lesioned the lateral geniculate nucleus with ibotenic acid in newborn kittens and studied connections from area 17 to area 18, and vice versa, after 1 month. In lesioned kittens, there were fewer corticocortical projections than normal in these reciprocal pathways and those that were present retained an immature, widespread pattern of projection. These results suggest that geniculate afferents are crucial for generating sufficient numbers of corticocortical projections and for creating the precision in their mapping.

Topics: Animals; Brain Mapping; Carbocyanines; Cats; Excitatory Amino Acid Agonists; Geniculate Bodies; Ibotenic Acid; Visual Cortex; Visual Pathways

Classification of retinal ganglion cells (RGCs) in the chick central retina was studied by retrograde labeling of carbocyanine dye (DiI) and intracellular filling with Lucifer Yellow. Ganglion cells were divided into 4 groups, Group Ic/Is, Group IIc/IIs, Group IIIs, Group IVc, according to sizes of somal area and dendritic field and dendritic branching pattern. Group I cells had small somal area and small dendritic field. They were further divided into 2 subgroups by complexity (subgroup Ic) and simplicity (subgroup Is) of the dendritic arborization. Group II cells had medium-sized soma and dendritic field. They were also divided into subgroup IIc and IIs by the same definitions as those of subgroup Ic and Is. Group IIIs had medium-sized soma, large and simple dendritic arborization. Group IVc in which all cells had large soma, showed large and complex dendritic arborization. Cell populations of each group were 51.8% (subgroup Ic), 21.1% (subgroup Is), 6.2% (subgroup IIc), 14.6% (subgroup IIs), 4.2% (Group IIIs), and 2.1% (Group IVc). Subgroup Ic cells, which were very similar to beta-cells in the mammalian central area, represented about a half of the ganglion cell population. Cells in subgroup Is and IIs, which were not reported in the mammalian retina, were found in the chick central retina in relatively high population (35.7%). Morphological features of chick RGCs in the central retina were considered in comparison with those of other vertebrates.

Topics: Amidines; Animals; Axonal Transport; Carbocyanines; Chickens; Dendrites; Fluorescent Dyes; Isoquinolines; Mammals; Retina; Retinal Ganglion Cells

Permanent, novel retinal projections to the principal thalamic somatosensory (ventrobasal) or auditory (medial geniculate) nuclei can be produced in adult hamsters if the superior colliculus is ablated bilaterally and the somatosensory and auditory lemniscal axons are transected unilaterally on the day of birth. We studied the development of those novel projections by labeling retinal axons with the fluorescent tracer 1,1'-dioctadecyl-3,3,3', 3'-tetramethylindocarbocyanine perchlorate to examine the relative roles of intrinsic factors and axon-target interactions in the specification of retinal axon connections. Our principal findings are as follows: (1) In hamsters operated on the day of birth to produce the novel retinal projections, retinal ganglion cell axons projecting to the ventrobasal or medial geniculate nuclei develop in three morphologically distinct stages, i.e., elongation, collateralization, and arborization, as do retinal axons projecting to the dorsal lateral geniculate nucleus, the principal thalamic visual nucleus, in normal hamsters. (2) In both the ventrobasal and medial geniculate nuclei of operated hamsters, as in the dorsal lateral geniculate nucleus of normal hamsters, collateral branches were initially formed by retinal ganglion cell axons in both the superficial and internal components of the optic tract and only collaterals from the superficial component formed permanent projections. (3) The retinofugal axon terminal arbors in the ventrobasal and medial geniculate nuclei of mature, operated hamsters resemble the same three morphologic classes that are observed in the lateral geniculate nucleus of normal hamsters, although their absolute size appears to be altered. These data suggest that both superficial and internal optic tract axons can produce thalamic collaterals during development but that only superficial optic tract axons can permanently retain thalamic collaterals. Furthermore, the same morphologic types of retinofugal axons appear to contribute to normal and surgically induced retinal projections.

Topics: Animals; Axons; Carbocyanines; Cricetinae; Fluorescent Dyes; Geniculate Bodies; Mesocricetus; Nerve Transfer; Retinal Ganglion Cells; Thalamic Nuclei; Visual Pathways

In the rat dentate gyrus, the lateral perforant path, the medial perforant path, and the major part of the hilar projection to the molecular layer share the lamination domain, mainly in the outer one-third of the molecular layer, the middle one-third, and the inner one-third, respectively. To reveal the order of the afferent fiber lamination and to have an indication of how the synaptic sites on dendrites are determined, we investigated the ontogeny of afferent fiber lamination in the dorsal hippocampus by injecting 1, 1'-dioctodecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI) into the entorhinal cortex and hippocampus in vivo. Fibers from the contralateral hilar region were found under the pia mater of the infrapyramidal blade at postnatal day 3 (P3), whereas the entorhinal afferent fibers were absent in the infrapyramidal blade. Then the medial and the lateral perforant path appeared under the pia mater in the infrapyramidal blade as riding on top of the preexisting laminae by P7 and by P11, respectively. Based on the established knowledge that most entorhinal layer II neurons simultaneously innervate both the suprapyramidal blade and infrapyramidal blade by branching, it is assumed that the medial and lateral perforant path in the suprapyramidal blade await an appropriate timing for sprouting of interstitial branches into the infrapyramidal blade. The granule cells in the infrapyramidal blade had dendritic growth cones by P11. Calretinin immunohistochemistry revealed Cajal-Retzius cells in the infrapyramidal blade even at P14. Under the pia mater, axon growth cones of ingrowing afferent fibers may interact with the dendritic growth cones or the Cajal-Retzius cells, and determines the synaptic sites on the granule cell dendrites.

Topics: Afferent Pathways; Aging; Animals; Animals, Newborn; Carbocyanines; Dendrites; Dentate Gyrus; Entorhinal Cortex; Fluorescent Dyes; Nerve Fibers; Perforant Pathway; Rats; Rats, Sprague-Dawley; Synapses

The present study identified and characterised myenteric neurones involved in the innervation of the gastric mucosa. We applied retrograde neuronal tracing methods by using the dye DiI (1,1'-didodecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorat) in combination with the immunohistochemical demonstration of choline acetyltransferase (ChAT), enkephalin (ENK), neuropeptide Y (NPY), nitric oxide synthase (NOS), substance P (SP), and vasoactive intestinal peptide (VIP). This method showed distinct neurochemical coding of DiI-labelled neurones with projections to the mucosa (mucosa neurones): ChAT/- (indicating the presence of ChAT only, 32%), ChAT/NPY/ +/- VIP (22%), NOS/NPY/ +/- VIP (19%), ChAT/SP/ +/- ENK (12%), NOS/- (indicating the presence of NOS only, 8%), or ChAT/ENK (4.6%). DiI-labelled mucosa neurones did not contain calretinin, serotonin, or somatostatin. All ChAT population had primarily ascending projections, whereas the NOS populations had mainly descending projections. Both were further classified as longitudinally and circumferentially projecting neurones, the latter having projection preferences towards the lesser or greater curvature. All subpopulations exhibited projection preferences. Nitrergic projections primarily arose from cell bodies located at the lesser curvature. ChAT/- projections, which dominated the cholinergic pathway, mainly arose from cell bodies located at the greater curvature. The other major cholinergic pathway with the code ChAT/NPY/ +/- VIP consisted of neurones located mainly at the lesser curvature. The results suggest specific coding of gastric myenteric neurones with projections to the mucosa. Polarised projections consisted of ascending cholinergic and descending nitrergic neurones; the additional presence of NPY/VIP was a prominent feature in both pathways. Chemical coding, polarity, and projection preferences of enteric pathways to the gastric mucosa are remarkably different from those of other regions in the gut.

Topics: Animals; Axonal Transport; Axons; Carbocyanines; Choline O-Acetyltransferase; Enkephalins; Fluorescent Dyes; Gastric Mucosa; Guinea Pigs; Immunohistochemistry; Myenteric Plexus; Neurons; Neuropeptide Y; Nitric Oxide Synthase; Substance P; Vasoactive Intestinal Peptide

To examine the time course of plasticity of the cranial nucleus during axonal regeneration, we followed the topographical reorganization of the cat facial nucleus (FN) up to 24 months after facio-facial nerve suture using retrograde labeling methods. The trunk of the temporal-zygomatico-orbital and both superior and inferior buccolabial branches (defined as main branch) of the facial nerve was cut and sutured again under ketamine hydrochloride anesthesia. At 11-722 days after nerve suture, Fast Blue (FB) and 1,1'-dioctadecyl-3, 3, 3', 3'-tetramethylindocarbocyanine perchlorate (Dil) or horseradish peroxidase (HRP) were injected into the distal part of the sutured main branch and the unoperated posterior auricular branch, respectively. Until about 3 months after suture, the topographical pattern in FN was similar to that observed in normal cats. At about 4 months after suture, FB-labeled motoneurons were distributed not only in the lateral part (including intermediate, dorsal and ventrolateral divisions) but also in the medial subdivision of FN. After a survival period of 18-24 months, FB-labeled neurons were found all over the FN, and their number increased significantly. Interestingly, in the longer survival cases, we noticed that the Dil- or HRP-labeled posterior auricular branch motoneurons also showed a tendency to distribute outside the medial region. The present study showed that somatotopic disorganization starts at around 4 months after suture, which seems to be somewhat slower than that in rats, and continues until a much later postoperative period. Furthermore, we suggested a possibility that the regeneration of one branch may affect the somatotopy of the unoperated nerve branch. These phenomena may contribute to aberrant facial nerve functions such as abnormal associated movement and facial spasm observed after nerve injury.

Topics: Amidines; Animals; Carbocyanines; Cats; Facial Nerve; Facial Nerve Injuries; Histocytochemistry; Horseradish Peroxidase; Motor Neurons; Nerve Regeneration; Time Factors

There is a long-standing controversy regarding the mechanisms that generate the functional subdivisions of the cerebral neocortex. One model proposes that thalamic axonal input specifies these subdivisions; the competing model postulates that patterning mechanisms intrinsic to the dorsal telencephalon generate neocortical regions. Gbx-2 mutant mice, whose thalamic differentiation is disrupted, were investigated. Despite the lack of cortical innervation by thalamic axons, neocortical region-specific gene expression (Cadherin-6, EphA-7, Id-2, and RZR-beta) developed normally. This provides evidence that patterning mechanisms intrinsic to the neocortex specify the basic organization of its functional subdivisions.

Topics: Animals; Axons; Cadherins; Calbindin 2; Carbocyanines; DNA-Binding Proteins; Gene Expression; Homeodomain Proteins; Immunohistochemistry; In Situ Hybridization; Inhibitor of Differentiation Proteins; Lymphoid Enhancer-Binding Factor 1; Mice; Mutation; Neocortex; Nerve Fibers; Proteins; Receptors, Cell Surface; Receptors, Cytoplasmic and Nuclear; Receptors, Melatonin; S100 Calcium Binding Protein G; Steroid 17-alpha-Hydroxylase; Telencephalon; Thalamus; Transcription Factors

Expression of the class A macrophage scavenger receptor (MSR) contributes to the uptake of modified low density lipoproteins (LDL) by macrophages and transformation of these cells into lipid-laden foam cells, which characterize atherosclerosis. Many environmental factors, in particular, proinflammatory cytokines and growth factors, can exert regulatory effects on MSR expression, whereas intracellular accumulation of cholesterol itself does not influence MSR levels to any considerable extent. In the present study, by using an in vitro model, we examined whether stimulation with interleukin-6 (IL-6), an immunoregulatory, multipotential cytokine, modulates the expression and activities of the MSR in macrophages. When treated with IL-6, macrophages derived from peripheral monocytes and phorbol 12-myristate 13-acetate (PMA)-differentiated THP-1 monocytic cells showed significantly reduced uptake and/or binding of the MSR ligand, acetylated LDL. This effect was paralleled by a reduction in the expression of MSR protein and mRNA. Analysis of MSR promoter activity in THP-1 cells transfected with an MSR promoter-reporter gene construct demonstrated decreased activity of the MSR promoter in IL-6-treated THP-1 macrophages. Electrophoretic mobility gel shift assay also showed a reduction in the binding of a transcription factor to the MSR promoter AP-1/ets elements in IL-6-treated cells. Thus, exposure to IL-6 may inhibit expression of the class A MSR in differentiated macrophages at transcriptional levels. This result suggests that this cytokine may modulate foam cell formation during atherogenesis.

Topics: Carbocyanines; Gene Expression; Humans; Interleukin-6; Leukocytes, Mononuclear; Lipoproteins, LDL; Macrophages; Promoter Regions, Genetic; Receptors, Immunologic; Receptors, Scavenger; Scavenger Receptors, Class A; Transcription Factors; Tumor Cells, Cultured

The functional organization of the trigeminal nuclei during embryogenesis was investigated using multiple-site optical recording with a fast voltage-sensitive dye. Brainstem preparations with three classified trigeminal nerve afferents, the ophthalmic, maxillary and mandibular nerves, together with motor nerve fibers, were dissected from five- to eight-day-old chick embryos. Electrical responses evoked by trigeminal nerve stimulations were optically recorded simultaneously from many loci of the stained preparations. We identified three response areas related to the trigeminal nerve: area I, located cephalic to the level of the trigeminal ganglion; area II, located caudal to the level of the trigeminal ganglion; and area III, located at the level of the trigeminal root. The neural responses in areas I and II were evoked by ophthalmic, maxillary or mandibular nerve stimulation, while the responses in area III were detected when the stimulation was applied to the trigeminal motor nerve. In comparison with the morphology indicated by DiI labeling, the results suggest that areas I, II and III correspond to the principal sensory nucleus of the trigeminal nerve, the spinal sensory nucleus of the trigeminal nerve and the trigeminal motor nucleus, respectively. We identified two components of the optical response: a fast and a slow signal. In five-day-old preparations, fast spike-like signals related to action potentials were recorded from the three response areas. In six-day-old preparations, slow optical signals which reflect glutamate-mediated excitatory postsynaptic potentials were detected from area II only when the ophthalmic nerve was stimulated: no slow signal was evoked by maxillary or mandibular nerve stimulation. In seven- and eight-day-old preparations, slow signals were detected from both areas I and II with every nerve stimulation. These results suggest that synaptic function is first generated in the spinal trigeminal nucleus by the six-day embryonic stage, and the developmental organization of synaptic function is not the same in the three trigeminal nerves or in the two sensory nuclei. Contour line maps of the signal amplitude revealed that the size and the area of the neural responses within the trigeminal nuclei changed dramatically with development. We compared the spatial distribution and temporal dynamics of the optical signals between the ophthalmic, maxillary and mandibular nerve stimulations, and we found that somatotopic organization is l

Topics: 6-Cyano-7-nitroquinoxaline-2,3-dione; Animals; Brain Mapping; Cadmium; Calcium; Carbocyanines; Chick Embryo; Coloring Agents; Electric Stimulation; Electrophysiology; Excitatory Amino Acid Antagonists; Motor Neurons; Neurons; Ophthalmic Nerve; Rhodanine; Synaptic Transmission; Thiazolidines; Trigeminal Nuclei; Valine

Peripheral projections of neurones whose cell bodies lie in the mesencephalic nucleus of the fifth cranial nerve, situated between the central grey and mesencephalic reticular formation, were studied in mouse embryos aged between day 9 and 15 and in postnatal day 1 mice. Nonspecific neural antibody staining allowed visualisation of the developing cranial nerves, in particular the descending mesencephalic tract. This facilitated successful dissection of the descending mesencephalic tract and trigeminal ganglion in the heads of fresh mouse embryos and postnatal mice. The fluorescent dye, 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (Dil), was injected into the descending mesencephalic tract in mouse embryos aged 12.5, 13.5 and 15 days of gestation and also into postnatal day 1 mice. Following a period of incubation, 100 microm sections were viewed under visible light and episcopic fluorescence. Mesencephalic neurones were observed to pass superiorly over the trigeminal ganglion and enter the maxillary division to innervate vibrissal follicle-sinus complexes, whilst none was observed innervating mandibular and maxillary intraoral structures. There was no fluorescent labelling in non-Dil injected control specimens. Using a highly specific neuronal tracer, this study shows that mesencephalic neurones in the periphery project exclusively to follicle sinus complexes in the developing mouse embryo and remain at least until postnatal day 1. These observations, contrary to those made in other animals, indicate a species specificity of mesencephalic peripheral projections.

Topics: Animals; Animals, Newborn; Carbocyanines; Efferent Pathways; Embryo, Mammalian; Embryonic and Fetal Development; Fluorescent Dyes; Immunohistochemistry; Mesencephalon; Mice; Paranasal Sinuses; Trigeminal Nerve; Vibrissae

During development of the neocortex, the marginal zone (layer I) and the subplate (layer VII) are the first layers to form from a primordial plexiform neoropil. The cortical plate (layers II-VI) is subsequently established between these superficial and deep components of the primordial plexiform neuropil. Neurons in the early zones are thought to play important roles in the formation of the cortex: the Cajal-Retzius cells of the marginal zone are instrumental in neuronal migration and laminar formation, and cells of the subplate are involved in the formation of cortical connections. Using the fluorescent tracer 1,1'-dioctodecyl-3,3,3', 3'-tetramethylindocarbocyanine (DiI), we have shown here that a substantial proportion of neurons of the marginal zone, including cells with features of Cajal-Retzius cells, and of the subplate and lower intermediate zone are not born in the ventricular neuroepithelium but instead originate in the medial ganglionic eminence (MGE), the pallidal primordium. These neurons follow a tangential migratory route to their positions in the developing cortex. They express the neurotransmitter GABA but seem to lack the calcium binding protein calretinin; some migrating cells found in the marginal zone express reelin. In addition, migrating cells express the LIM-homeobox gene Lhx6, a characteristic marker of the MGE. It is suggested that this gene uniquely or in combination with other transcription factors may be involved in the decision of MGE cells to differentiate in situ or migrate to the neocortex.

Topics: Animals; Brain; Carbocyanines; Cell Movement; Cerebral Cortex; Embryonic and Fetal Development; Fluorescent Dyes; Gene Expression Regulation, Developmental; Homeodomain Proteins; Morphogenesis; Nerve Tissue Proteins; Neurons; Organ Culture Techniques; Rats; Rats, Sprague-Dawley; Reelin Protein; Zinc Fingers

A mitotic wave before an increase in juvenile hormone (JH) synthesis was observed in the corpora allata during each ovarian cycle in the cockroach Diploptera punctata and was shown subsequently to be inhibited by the brain until adult females mated. Each corpus allatum (CA) was innervated by groups of neurons in the contralateral pars intercerebralis (PI) and the ipsilateral pars lateralis (PL). In this article, a third set of neurons is identified that innervates the CA located extralaterally in the contralateral PL. The topography of the brain neurons innervating the CA was reconstructed from confocal optical sections after vital staining with two fluorescent carbocyanine dyes, 1,1'-dihexadecyl-3, 3,3',3'-tetramethylindocarbocyanine perchlorate [DiIC(16) (3)] and 3, 3'-dioctadecyloxacarbocyanine perchlorate [DiOC(18) (3)]. This provided information necessary to disconnect specific pathways within the brain of virgin females and to determine the effect on mitosis in the corpora allata and on the growth of oocytes. Disconnection of the CA from the PI neurons was followed by a prolonged period of moderate mitotic activity in the glands. Disconnection of the CA from PI and contralateral PL neurons was followed by a rapid wave of high mitotic activity in the CA similar to that observed after mating. The ipsilateral PL neurons did not appear to influence CA cell proliferation, because mitotic activity in glands was similar whether or not glands were connected to these neurons. Disconnection of the contralateral PI and/or ipsilateral PL neurons, but not the contralateral PL neurons, from the CA resulted in oocyte growth indicative of increased JH synthesis. The authors conclude that, whereas contralateral PI neurons inhibit both CA cell proliferation and JH synthesis, ipsilateral PL neurons affect only JH synthesis, and contralateral PL neurons modulate cell proliferation slightly only.

Topics: Animals; Carbocyanines; Cell Division; Cockroaches; Corpora Allata; Fluorescent Dyes; Ganglia, Invertebrate; Juvenile Hormones; Microscopy, Confocal; Neural Inhibition; Neurons; Neurosecretory Systems; Oocytes

Stimulatory effects of a novel isobenzofranone, MD-700, on low density lipoprotein (LDL) receptor activity were investigated in vitro and in vivo. MD-700 at 0.03 microg/ml elevated the expression of LDL receptor in HepG2 cells within 4 h. Corresponding to this, uptake of fluorescent labeled-LDL (3,3'-dioctadecylindocarbocyanine-LDL) by the cells increased linearly in time- and dose-dependent manner by MD-700 for up to 12 h. In the experiment using HepG2 cells transiently transfected with promoter-luciferase gene constructs, MD-700 increased luciferase activity in a dose-dependent manner from 0.03 to 0.1 microg/ml. In contrast, luciferase activity was not stimulated by MD-700 in construct with a deleted sterol regulatory element (SRE)-1, suggesting importance of SRE-1 in stimulation of the LDL receptor gene promoter by MD-700. Binding experiments on liver membranes from MD-700-treated hamsters showed about a 60% increase in 125I-labeled LDL binding. A Scatchard plot revealed that MD-700 increased the maximal binding without affecting binding affinity. In contrast to findings with an inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A reductase, pravastatin, MD-700 had no effect on the sterol synthesis in hamster liver homogenates. These results suggest that MD-700 stimulates the expression of LDL receptor, presumably in a manner independent of change in sterol metabolism, and thereby promotes LDL clearance. Hypocholesterolemic actions of MD-700 in hamsters were then examined. MD-700 lowered serum cholesterol levels in hamsters fed normal chow or a high-fat diet. Fractionation of serum lipoproteins demonstrated that MD-700 selectively decreased LDL and very low density lipoprotein cholesterol. Dose-dependent decrease in serum cholesterol was also seen in hypercholesterolemic rats. Thus, the hypocholesterolemic action of MD-700 may be attributed to up-regulation of the LDL receptor, based on stimulation of the transcription of the LDL receptor gene. Although pravastatin stimulates LDL uptake and lowers serum cholesterol in a manner similar to that seen with MD-700, the mechanism responsible for hypocholesterolemic action appears to differ.

Topics: Animals; Benzofurans; Blotting, Northern; Carbocyanines; Carcinoma, Hepatocellular; Cell Membrane; Cholesterol; Cricetinae; Disease Models, Animal; DNA Primers; Fluorescent Dyes; Humans; Hypercholesterolemia; Lipoproteins, LDL; Lipoproteins, VLDL; Liver Neoplasms; Male; Promoter Regions, Genetic; Rats; Rats, Wistar; Receptors, LDL; RNA, Messenger; RNA, Neoplasm; Sterols; Transcription, Genetic; Tumor Cells, Cultured; Up-Regulation

A signature feature of mature ferret retinal ganglion cells (RGCs) is the stratification of their dendrites within either ON or OFF sublayers of the retinal inner plexiform layer (IPL). Dendritic stratification is achieved through the gradual restriction of RGC dendrites which initially ramify throughout the IPL. We examined the time course of stratification by retrogradely labeling ferret retinas with DiI at various postnatal ages. Stratification of beta and alpha RGC dendrites into either the ON or OFF sublayers of the IPL begins around postnatal day 5, when class-specific morphologies begin to emerge, and is largely completed by eye opening, at the end of the first postnatal month. Our results imply that dendritic stratification of ferret ON and OFF RGCs, as in other mammals, occurs independently of visually driven activity.

Topics: Aging; Animals; Carbocyanines; Cats; Dendrites; Female; Ferrets; Fluorescent Dyes; Pregnancy; Retina; Retinal Ganglion Cells

Toxoplasma gondii is a member of the phylum Apicomplexa, a diverse group of intracellular parasites that share a unique form of gliding motility. Gliding is substrate dependent and occurs without apparent changes in cell shape and in the absence of traditional locomotory organelles. Here, we demonstrate that gliding is characterized by three distinct forms of motility: circular gliding, upright twirling, and helical rotation. Circular gliding commences while the crescent-shaped parasite lies on its right side, from where it moves in a counterclockwise manner at a rate of approximately 1.5 microm/s. Twirling occurs when the parasite rights itself vertically, remaining attached to the substrate by its posterior end and spinning clockwise. Helical gliding is similar to twirling except that it occurs while the parasite is positioned horizontally, resulting in forward movement that follows the path of a corkscrew. The parasite begins lying on its left side (where the convex side is defined as dorsal) and initiates a clockwise revolution along the long axis of the crescent-shaped body. Time-lapse video analyses indicated that helical gliding is a biphasic process. During the first 180(o) of the turn, the parasite moves forward one body length at a rate of approximately 1-3 microm/s. In the second phase, the parasite flips onto its left side, in the process undergoing little net forward motion. All three forms of motility were disrupted by inhibitors of actin filaments (cytochalasin D) and myosin ATPase (butanedione monoxime), indicating that they rely on an actinomyosin motor in the parasite. Gliding motility likely provides the force for active penetration of the host cell and may participate in dissemination within the host and thus is of both fundamental and practical interest.

Topics: Animals; Carbocyanines; Cell Movement; Cytochalasin D; Diacetyl; Fibroblasts; Fluorescent Antibody Technique; Fluorescent Dyes; Humans; Kinetics; Microscopy, Electron; Microscopy, Fluorescence; Microscopy, Video; Toxoplasma

The chronological relation between the establishment of lipid continuity and fusion pore formation has been investigated for fusion of cells expressing hemagglutinin (HA) of influenza virus to planar bilayer membranes. Self-quenching concentrations of lipid dye were placed in the planar membrane to monitor lipid mixing, and time-resolved admittance measurements were used to measure fusion pores. For rhodamine-PE, fusion pores always occurred before a detectable amount of dye moved into an HA-expressing cell. However, with DiI in the planar membrane, the relationship was reversed: the spread of dye preceded formation of small pores. In other words, by using DiI as probe, hemifusion was clearly observed to occur before pore formation. For hemifused cells, a small pore could form and subsequently fully enlarge. In contrast, for cells that express a glycosylphosphatidylinositol-anchored ectodomain of HA, hemifusion occurred, but no fully enlarged pores were observed. Therefore, the transmembrane domain of HA is required for the formation of fully enlarging pores. Thus, with the planar bilayer membranes as target, hemifusion can precede pore formation, and the occurrence of lipid dye spread does not preclude formation of pores that can enlarge fully.

Topics: 3T3 Cells; Animals; Biophysical Phenomena; Biophysics; Carbocyanines; Fluorescent Dyes; Hemagglutinin Glycoproteins, Influenza Virus; Lipid Bilayers; Membrane Fusion; Mice; Microscopy, Fluorescence

Retinal microglial cells of newborn Lewis rats were isolated and cultured, and the effect of macrophage colony-stimulating factor (M-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), and interferon-gamma (IFN-gamma) on microglial expression of the accessory molecules required for antigen presentation were studied.. Retinal microglia were isolated from newborn Lewis rats and cultured in media supplemented with either M-CSF or GM-CSF. Immunohistochemical tests using anti-macrophage complement receptor 3 (OX42) or anti-monocyte-macrophage (ED1) and DiI-ac-low-density lipoprotein (LDL) uptake were used to identify microglia. The effect on accessory molecule expression of microglial cells cultured under varying conditions (M-CSF, GM-CSF, and M-CSF plus IFN-gamma) was analyzed by fluorescence-activated cell sorter, using one of the following antibodies: anti-OX3, anti-OX6, anti-rat intercellular adhesion molecule (ICAM)-1, anti-rat B7-1, or anti-rat B7-2.. The cultured retinal microglia were positive for macrophage-related antigens (ED1 and OX42) and also showed uptake of LDL. Furthermore, ICAM-1 and B7-2 were expressed constitutively on these cells, and MHC class II and B7-1 were also expressed after IFN-gamma stimulation.. In vitro, the retinal microglia express the molecules required for effective antigen presentation to CD4-positive T cells. These findings suggest that microglia may play a role in local antigen presentation, especially when they are exposed to IFN-gamma.

Topics: Animals; Animals, Newborn; Antigen-Presenting Cells; Antigens, Surface; B7-1 Antigen; Biomarkers; Carbocyanines; Cell Separation; Cells, Cultured; Flow Cytometry; Fluorescent Dyes; Granulocyte-Macrophage Colony-Stimulating Factor; Histocompatibility Antigens Class II; Immunoenzyme Techniques; Immunophenotyping; Intercellular Adhesion Molecule-1; Interferon-gamma; Lipoproteins, LDL; Macrophage Colony-Stimulating Factor; Microglia; Rats; Rats, Inbred Lew; Retina

Cajal-Retzius (CR) cells are characteristic horizontally orientated, early-generated transient neurons in the marginal zones of the neocortex and hippocampus that synthesize the extracellular matrix protein reelin. They have been implicated in the pathfinding of entorhino-hippocampal axons, but their role in this process remained unclear. Here we have studied the axonal projection of hippocampal CR cells. Following injection of the carbocyanine dye DiI into the entorhinal cortex of aldehyde-fixed rat embryos and young postnatal rats, neurons in the outer molecular layer of the dentate gyrus and stratum lacunosum-moleculare of the hippocampus proper with morphological characteristics of CR cells were retrogradely labelled. In a time course analysis, the first retrogradely labelled CR cells were observed on embryonic day 17. This projection of hippocampal CR cells to the entorhinal cortex was confirmed by retrograde tracing with Fast Blue in new-born rats and by intracellular biocytin filling of CR cells in acute slices from young postnatal rat hippocampus/entorhinal cortex and in entorhino-hippocampal slice cocultures using infrared videomicroscopy in combination with the patch-clamp technique. In double-labelling experiments CR cells were identified by their immunocytochemical staining for reelin or calretinin, and their interaction with entorhino-hippocampal axons labelled by anterograde tracers was analysed. Future studies need to investigate whether this early transient projection of hippocampal CR cells to the entorhinal cortex is used as a template by the entorhinal axons growing to their target layers in the hippocampus.

Topics: Amidines; Animals; Animals, Newborn; Axonal Transport; Calbindin 2; Carbocyanines; Cell Adhesion Molecules, Neuronal; Cells, Cultured; Coculture Techniques; Entorhinal Cortex; Extracellular Matrix Proteins; Fluorescent Dyes; Hippocampus; Lysine; Microscopy, Video; Nerve Tissue Proteins; Neural Pathways; Neurons; Rats; Rats, Sprague-Dawley; Rats, Wistar; Reelin Protein; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; S100 Calcium Binding Protein G; Serine Endopeptidases

Chicken ovalbumin upstream promotor-transcription factor I (COUP-TFI), an orphan member of the nuclear receptor superfamily, is highly expressed in the developing nervous systems. In the cerebral cortex of Coup-tfl mutants, cortical layer IV was absent due to excessive cell death, a consequence of the failure of thalamocortical projections. Moreover, subplate neurons underwent improper differentiation and premature cell death during corticogenesis. Our results indicate that the subplate neuron defects lead to the failure of guidance and innervation of thalamocortical projections. Thus, our findings demonstrate a critical role of the subplate in early corticothalamic connectivity and confirm the importance of afferent innervation for the survival of layer IV neurons. These results also substantiate COUP-TFI as an important regulator of neuronal development and differentiation.

Topics: Animals; Antimetabolites; Axons; Bromodeoxyuridine; Carbocyanines; Cell Death; Cell Differentiation; Cerebral Cortex; COUP Transcription Factor I; DNA-Binding Proteins; Fluorescent Dyes; Immunohistochemistry; In Situ Hybridization; Male; Mice; Mutation; Neural Pathways; Neurons; Receptors, Glucocorticoid; Thalamus; Transcription Factors

The endorgan-specific distribution of vestibular ganglion cells was studied in neonatal and postnatal rats and mice using indocarbocyanine dye (DiI) and dextran amines for retrograde and anterograde labeling. Retrograde DiI tracing from the anterior vertical canal labeled neurons scattered throughout the whole superior vestibular ganglion, with denser labeling at the dorsal and central regions. Horizontal canal neurons were scattered along the dorsoventral axis with more clustering toward the dorsal and ventral poles of this axis. Utricular ganglion cells occupied predominantly the central region of the superior vestibular ganglion. This utricular population overlapped with both the anterior vertical and horizontal canals' ganglion cells. Posterior vertical canal neurons were clustered in the posterior part of the inferior vestibular ganglion. The saccular neurons were distributed in the two parts of the vestibular ganglion, the superior and inferior ganglia. Within the inferior ganglion, the saccular neurons were clustered in the anterior part. In the superior ganglion, the saccular neurons were widely scattered throughout the whole ganglion with more numerous neurons at the posterior half. Small and large neurons were labeled from all endorgans. Examination of the fiber trajectory within the superior division of the vestibular nerve showed no clear lamination of the fibers innervating the different endorgans. These results demonstrate an overlapping pattern between the different populations within the superior ganglion, while in the inferior ganglion, the posterior canal and saccular neurons show tighter clustering but incomplete segregation. This distribution implies that the ganglion cells are assigned for their target during development in a stochastic rather than topographical fashion.

Topics: Animals; Animals, Newborn; Axonal Transport; Axons; Carbocyanines; Coloring Agents; Dextrans; Fluoresceins; Ganglia, Sensory; Horseradish Peroxidase; Mice; Neurons, Afferent; Rats; Rats, Sprague-Dawley; Saccule and Utricle; Spiral Ganglion; Vestibular Nerve; Xanthenes

Developmental fates of cells emigrating from the primitive streak were traced by a fluorescent dye Dil both in chick and in quail embryos from the fully grown streak stage to 12-somite stage, focusing on the development of mesoderm and especially on the timing of ingression of each level of somitic mesoderm. The fate maps of the chick and quail streak were alike, although the chick streak was longer at all stages examined. The anterior part of the primitive streak predominantly produced somites. The thoracic and the lumbar somites were shown to begin to ingress at the 5 somite-stage and 10 somite-stage in a chick embryo, and 6 somite-stage and 9 somite-stage in a quail embryo, respectively. The posterior part of the streak served mainly as the origin of more lateral or extra embryonic mesoderm. As development proceeded, the fate of the posterior part of the streak changed from the lateral plate mesoderm to the tail bud mesoderm and then to extra embryonic, allantois mesoderm. The fate map of the primitive streak in chick and quail embryo presented here will serve as basic data for studies on mesoderm development with embryo manipulation, especially for transplantation experiments between chick and quail embryos.

Topics: Animals; Body Patterning; Carbocyanines; Chick Embryo; Coturnix; Fluorescent Dyes; Mesoderm; Notochord; Somites; Stem Cells

Developing axons reach their final targets as a result of a series of axonal projections to successive intermediate targets. Long-range chemoattraction by intermediate targets plays a key role in this process. Growing axons, however, do not stall at the intermediate targets, where the chemoattractant concentration is expected to be maximal. Commissural axons in the metencephalon, initially attracted by a chemoattractant released from the floor plate, were shown to lose responsiveness to the chemoattractant when they crossed the floor plate in vitro. Such changes in axon responsiveness to chemoattractants may enable developing axons to continue to navigate toward their final destinations.

Topics: Animals; Axons; Carbocyanines; Cell Adhesion Molecules, Neuronal; Chemotaxis; Contactin 2; Culture Techniques; Fluorescent Dyes; Membrane Glycoproteins; Nerve Growth Factors; Netrin-1; Pons; Rats; Rats, Wistar; Rhombencephalon; Tumor Suppressor Proteins

Calcium-calmodulin-dependent protein kinase II (CaMKII) promotes the maturation of retinotectal glutamatergic synapses in Xenopus. Whether CaMKII activity also controls morphological maturation of optic tectal neurons was tested using in vivo time-lapse imaging of single neurons over periods of up to 5 days. Dendritic arbor elaboration slows with maturation, in correlation with the onset of CaMKII expression. Elevating CaMKII activity in young neurons by viral expression of constitutively active CaMKII slowed dendritic growth to a rate comparable to that of mature neurons. CaMKII overexpression stabilized dendritic structure in more mature neurons, whereas CaMKII inhibition increased their dendritic growth. Thus, endogenous CaMKII activity limits dendritic growth and stabilizes dendrites, and it may act as an activity-dependent mediator of neuronal maturation.

Topics: Animals; Axons; Benzylamines; Calcium-Calmodulin-Dependent Protein Kinase Type 2; Calcium-Calmodulin-Dependent Protein Kinases; Carbocyanines; Dendrites; Fluorescent Dyes; Microscopy, Confocal; Neurons; Receptors, AMPA; Receptors, N-Methyl-D-Aspartate; Recombinant Proteins; Sulfonamides; Superior Colliculi; Synapses; Synaptic Transmission; Time Factors; Vaccinia virus; Xenopus laevis

Retrograde tracing, combined with immunohistochemistry, was used to study the projections of 5-hydroxytryptamine (5-HT)-accumulating neurones within the ileum of the guinea-pig, with confocal microscopy being used to characterise further their morphology. Two classes of neurones in the myenteric plexus, capable of taking up 5-HT or analogues, were distinguished. One class had Dogiel type I morphology with lamellar dendrites, was located on the edge or in the middle of ganglia and lacked immunoreactivity for somatostatin (SOM). The other class had smooth ovoid cell bodies with multiple filamentous dendrites and a single axon and represented a subset of the SOM-immunoreactive interneurones in the myenteric plexus. Varicosities immunoreactive for 5-HT alone, 5-HT/SOM or SOM alone were present in the myenteric ganglia. Both classes of 5-HT-accumulating neurones had long aboral projections within the myenteric plexus (up to 100 mm long) and to the submucous plexus and probably function as descending interneurones.

Topics: Animals; Axonal Transport; Carbocyanines; Dendrites; Female; Fluorescent Dyes; Guinea Pigs; Immunohistochemistry; Male; Microscopy, Confocal; Myenteric Plexus; Neurons; Serotonin

The corticopontine projection develops exclusively by collateral branches that form along the length of corticospinal axons days after they have passed their hindbrain target, the basilar pons. In vitro evidence suggests that the basilar pons releases a diffusible activity that initiates and directs the growth of collateral branches. This study investigates whether contact-dependent mechanisms may also influence the formation of collateral branches. By using immunocytochemistry, electron microscopy, and neuronal tracing techniques, we examined the region of the axon tract, the cerebral peduncle, overlying the basilar pons for cellular structures that correlate spatially and temporally with collateral branch formation. We found that radial glia are excluded from the tract. Oligodendrocyte precursors are found only at low density. Although mature astrocytes are absent, immature astrocytes are present throughout the tract. However, our evidence does not suggest a direct role for glial cell types in collateral branch formation. In contrast, dendrites of basilar pontine neurons are transiently present in the tract during the time of collateral branch formation. Although collateral branches are observed in regions of the tract devoid of dendrites, the orientation and location of most collateral branches correlates at the light microscopic level with dendrites. Electron microscopy reveals sites of increased collateral branch formation near neuronal cell bodies or dendrites. However, cell processes, whether dendritic or otherwise, are rarely found in direct contact with collateral branch points. A common and unexpected feature is the bundles of corticopontine collateral branches, oriented transversely to their parent corticospinal axons and directed across the tract to the basilar pons. Dendrites were often apposed to or embedded within the transverse bundles. These findings suggest that dendrites are not essential for collateral branch formation but that they may enhance this process and define discrete preferred locations for collateral branch initiation and elongation within the cerebral peduncle.

Topics: Animals; Astrocytes; Axons; Carbocyanines; Cerebral Cortex; Dendrites; Female; Lysine; Microscopy, Electron; Neural Pathways; Neuroglia; Pons; Pregnancy; Rats; Rats, Sprague-Dawley

To assess the degree of order exhibited during development by crossed and uncrossed retinocollicular pathways, focal deposits of 1,1'-dioctodecyl-3,3,3'3'-tetramethylinodocarbocyanine perchlorate (DiI) were made into the temporal or nasal retina of prenatal and postnatal ferrets. This procedure revealed that the first retinal fibers (from the ipsilateral temporal retina) grow into the superior colliculus at embryonic (E) day 30. Both crossed and uncrossed fibers innervate the colliculus by E34. At this age, terminal arbors were lacking, and there was no evidence of extensive axonal branching. Retinocollicular arbors first appeared at E38, with both the crossed and uncrossed projections forming well-defined terminal zones that appeared to be localized to topographically appropriate regions. At E38, the ipsilateral terminal zone was significantly larger but notably less dense than the contralateral zone. At this and later ages (postnatal day [P] 0 and P7), a few crossed and uncrossed fibers extended beyond the terminal zone. Four days later, at P0, the terminal zone of the uncrossed projection was reduced in size in comparison with that of earlier ages, whereas the crossed projection became substantially larger. By P7, the few misprojecting fibers seen in younger ferrets had been virtually eliminated. When focal retinal deposits of tracer were made into the nasal retina of E36 and E40 ferrets, crossed fibers were found to innervate the caudal segment of the superior colliculus. These crossed nasal cells appear to project to the topographically appropriate region of the superior colliculus (caudal segment) but on the wrong side of the brain. Collectively, the present findings indicate that throughout development the ferret retinocollicular pathway is characterized by a remarkable degree of topographic precision as evident by the paucity of axonal branches and the low number of grossly misprojecting axons.

Topics: Animals; Axons; Carbocyanines; Female; Ferrets; Fluorescent Dyes; Immunohistochemistry; Microscopy, Confocal; Pregnancy; Retina; Superior Colliculi; Visual Pathways

1. Neuronal retrograde tracing with the dye DiI (1,1'-didodecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate), in combination with immunohistochemical detection of choline acetyltransferase (ChAT) and vasoactive intestinal peptide (VIP), were used to identify the innervation of the mucosa of the guinea-pig proximal colon by submucosal neurones. Ussing chamber experiments were performed to measure changes in short circuit current (delta Isc) evoked by electrical stimulation of the oral or anal end of the preparation. 2. The tracing studies revealed that the mucosa was primarily innervated by descending neurones (78%); the vast majority of these were VIP positive (85%). The numerically smaller ascending pathway (13%) was predominantly ChAT positive (69%). A small population (9%) of DiI-labelled neurones projected circumferentially. 3. Ussing chamber experiments revealed that oral electrical stimulation induced a significantly larger delta Isc than anal stimulation. The VIP antagonist VIP(6-28) significantly reduced only orally induced delta Isc. Anally induced delta Isc were significantly more atropine sensitive that orally induced delta Isc. Tissue incubation with carbachol or VIP significantly potentiated delta Isc induced by VIP and carbachol, respectively, indicating cross-potentiation. 4. This study provides the first functional demonstration of polarized innervation patterns from submucosal neurones to enteric mucosa. The ascending ChAT and descending VIP pathways suggest the existence of reflexes resulting in preferential release of VIP or acetylcholine. The distinct pathways might favour the observed cross-potentiation of cholinergic and VIPergic mediated secretion.

Topics: Action Potentials; Affinity Labels; Animals; Carbocyanines; Choline O-Acetyltransferase; Colon; Diffusion Chambers, Culture; Dose-Response Relationship, Drug; Electric Stimulation; Evoked Potentials; Guinea Pigs; Hormone Antagonists; Immunohistochemistry; Intestinal Mucosa; Neurons; Sensitivity and Specificity; Vasoactive Intestinal Peptide

The receptiveness of the brain to monocyte infiltration was studied in rats that had been injected intracerebrally with Corynebacterium parvum. At 0-17 days after intracerebral injection and 18 h after intravenous injection of diI-labeled isogenous mononuclear cells, host rats were sacrificed and cells from the vicinity of the injection site and from the contralateral cerebral hemisphere were dissociated and analyzed by flow cytometry. In rats sacrificed 4-11 days postinjection of C. parvum, diI-labeled mononuclear cells were detected in cell preparations from the hemisphere ipsilateral and, to a lesser extent, contralateral to the injection site. No extravasation of cells from the blood to the brain was detected in rats injected intracerebrally with saline. By immunohistochemistry, many macrophages were detected in the hemisphere ipsilateral to injection of C. parvum. In additional experiments, the dissociated CNS cell population was labeled with OX-42 antibodies to the type 3 complement receptor, which is present on monocytes but not lymphocytes. Some cells in the brain were labeled with both diI and OX-42 and therefore were identified as monocytes that had entered the brain from the blood. In conclusion, monocytes can home to both sides of the brain after unilateral injection of a strong inflammatory agent but monocyte infiltration into the brain is delayed in comparison to monocyte inflammatory responses that have been reported in nonneural tissues.

Topics: Adoptive Transfer; Animals; Antigens, CD; Antigens, Neoplasm; Antigens, Surface; Avian Proteins; Basigin; Blood Proteins; Brain; Carbocyanines; Flow Cytometry; Fluorescent Dyes; Gram-Positive Bacterial Infections; Inflammation; Leukocytes, Mononuclear; Male; Membrane Glycoproteins; Monocytes; Propionibacterium acnes; Rats; Rats, Inbred Lew; Reproducibility of Results; Time Factors

The carbocyanine dyes DiI, DiA and DiO were microinjected into the cerebral ganglion of intact Mesocestoides corti tetrathyridia to determine the spatial organisation and connectivity patterns of the CNS. Of the dyes tested, DiI proved to be the most effective, giving highly fluorescent and persistent staining of even very fine calibre afferent and efferent nerve fibres. DiI labelling, in conjunction with transmission electron microscopy, revealed the nervous system to consist of sensory endings, directly connected to the cerebral ganglion by elongated cellular tracts, efferent nerve fibres which innervated the suckers, and longitudinal nerve cords which travelled along the remainder of the body.

Topics: Animals; Carbocyanines; Fluorescent Dyes; Ganglia, Invertebrate; Mesocestoides; Microinjections; Nervous System; Neural Pathways; Sensory Receptor Cells

Single blastomeres of the sixth-cleavage veg1 and veg2 tiers of Strongylocentrotus purpuratus embryos were labeled with DiI lineage tracer, and the disposition of the progeny was followed through the blastula and gastrula stages in order to determine their respective endodermal and ectodermal contributions. In the endoderm of postgastrula embryos, veg1-derived cells constituted nearly all of the prospective hindgut and about half of the prospective midgut, while veg2-derived cells made up the prospective foregut and half the midgut. Oral veg1 clones consistently contributed more cells to endoderm than aboral veg1 clones. Oral veg1 clones extended along the archenteron up to the foregut region, while aboral veg1 clones contributed only small numbers of hindgut cells but large patches of ectoderm cells that extended out to the prospective larval vertex. The oral/aboral asymmetry in veg1 allocations was also demonstrated using chimeric embryos, the animal halves of which were labeled with a rhodamine-dextran. Lineages expressing the vegetal plate marker Endo16 were more precisely determined by combining lineage tracer injection with whole-mount in situ hybridization. Endo16 expression was found in all cells that are going to participate in gastrulation. Recruitment of new cells to the Endo16 domain occurs in advance of the actual invagination of those cells. During the blastula stages Endo16 expression expands radially until all cells in the veg2 lineages express this gene. The first phase of gastrulation, including the normal buckling of the vegetal plate and primary invagination of the archenteron, involves only the Endo16-expressing cells of the veg2 lineages. As the archenteron begins to elongate, marking the onset of the second phase of gastrulation, there is an asymmetric expansion of Endo16 into the veg1-derived cells that will contribute to the hindgut and midgut in accordance with lineage tracing observations. The results indicate a relatively late specification of veg1-derived cells, resulting in late recruitment to the periphery of the vegetal plate territory as gastrulation proceeds. Differential recruitment of veg1-derived cells on the oral side of the embryo introduces an oral bias to gastrulation by disproportionately increasing the number of cells on the oral side that are competent to participate in gastrulation.

Topics: Animals; Biomarkers; Blastomeres; Carbocyanines; Cell Adhesion Molecules; Cell Lineage; Endoderm; Fluorescent Dyes; Gastrula; Image Processing, Computer-Assisted; In Situ Hybridization; Mesoderm; Morphogenesis; Proteins; Sea Urchins; Staining and Labeling

During rodent fetal development, maternal lipoproteins can be sources of cholesterol for the membrane synthesis required for tissue growth in the developing embryo and for steroid hormone production in the extraembryonic tissues. Although the mechanisms underlying the maternal-fetal lipoprotein cholesterol transport system are not well defined, the placenta and yolk sac seem to play major roles in this process, serving as functionally active interfaces between maternal circulation and the embryo. In rodents, the principal cholesterol transporter in the plasma is HDL, and the HDL receptor SR-BI is a physiologically important mediator of cholesterol uptake in adult liver and steroidogenic tissues. To begin to investigate SR-BI's role in maternal cholesterol uptake by the fetus, we used immunofluorescence microscopy to determine the pattern of SR-BI expression during murine embryogenesis. At day E7.5 in gestation, there was significant SR-BI expression in endothelial cells of the decidua, but little in intraembryonic and extraembryonic tissues. By day E8.5, there was a dramatic increase in SR-BI expression in the trophoblast cells which surround the developing embryo. Beginning at day E10, SR-BI was expressed in both the placenta and yolk sac. The expression in these extraembryonic tissues was correlated with significant uptake of fluorescent dye by the yolk sac visceral endodermal cells from DiI-labeled HDL injected into pregnant mice. Within the embryo proper, SR-BI expression appeared by day E14.5 at high levels in the adrenal gland. SR-BI expression was not detected in the embryonic liver through day E17.5 of gestation; however, it could be observed in neonatal livers. These findings suggest that SR-BI may play a role in the rodent maternal-fetal lipoprotein cholesterol transport system, supplying HDL cholesterol for either membrane or steroid hormone synthesis, or both.

Topics: Animals; Biological Transport; Carbocyanines; CD36 Antigens; Cholesterol; Embryonic and Fetal Development; Female; Fluorescent Dyes; Gene Expression; Membrane Proteins; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Microscopy, Fluorescence; Placenta; Placentation; Pregnancy; Receptors, Immunologic; Receptors, Lipoprotein; Receptors, Scavenger; Scavenger Receptors, Class B; Time Factors; Yolk Sac

The class A macrophage scavenger receptors (SR-A) are macrophage-specific trimeric integral membrane glycoproteins that have been implicated in many macrophage-associated physiological and pathological processes including atherosclerosis, Alzheimer's disease, and host defense. There are two forms of the receptor that have been previously cloned, and both are generated by alternative splicing of a single gene. Here we report the cloning of a third, alternatively spliced isoform of the human SR-A gene (type III hSR-A). The novel isoform is expressed in the human monocytic leukemia cell line THP-1 and also in primary human monocyte derived macrophages. When expressed in CHO-K1 cells, type III hSR-A does not internalize AcLDL despite having the domain shown to mediate this function in type I and II hSR-A. We show that type III protein has altered intracellular processing and is trapped within the endoplasmic reticulum, making it unable to perform endocytosis. Type III protein acts as a dominant negative isoform by reducing modified LDL uptake in CHO cells stably expressing either type I or type II SR-A. The demonstration that a naturally occurring splice variant of SR-A mRNA can act as a dominant negative isoform suggests a novel mechanism for regulation of scavenger receptor activity in macrophages.

Topics: Alternative Splicing; Amino Acid Sequence; Animals; Base Sequence; Carbocyanines; Cell Line; CHO Cells; Cloning, Molecular; Cricetinae; Fluorescent Dyes; Gene Expression; Humans; Leukemia, Monocytic, Acute; Lipoproteins, LDL; Macrophages; Molecular Sequence Data; Polymerase Chain Reaction; Receptors, Immunologic; Receptors, Scavenger; Scavenger Receptors, Class A; Transfection; Tumor Cells, Cultured

The neuroanatomical connections of the diencephalic torus lateralis and inferior lobe of the goldfish (Carassius auratus) were studied by retrograde and anterograde labeling with the carbocyanine dye DiI. Both structures have afferents originating in the central zone of the dorsal telencephalic area as well as in the supracommissural nucleus of the ventral telencephalic area, and in the secondary gustatory, tertiary gustatory, and posterior thalamic nuclei. Both structures investigated have efferents to the tertiary gustatory and posterior thalamic nuclei, as well as to the dorsal hypothalamus (dorsal hypothalamic neuropil) and superior reticular formation. The torus lateralis receives additional afferents from the secondary general visceral nucleus and, sparsely, from the dorsal tegmental nucleus. The inferior lobe receives additional afferents from the medial zone of the dorsal telencephalic area, as well as from the suprachiasmatic, posterior pretectal, central posterior thalamic, caudal preglomerular, two tegmental nuclei (T1 and T2), corpus mamillare, and, sparsely, from the cerebellar valvula. The inferior lobe has additional efferents to the dorsal and ventral thalamus and subglomerular nucleus. The lateral torus and inferior lobe are also mutually interconnected. The lateral torus and inferior lobe map topographically onto the vagal-related (intraoral) or onto the facial-related (extraoral) portions, respectively, of both the secondary and tertiary gustatory nuclei. Because the posterior thalamic nucleus is reciprocally connected with the lateral torus and inferior lobe and is further known to project in turn to the area doralis telencephali, it likely represents a quaternary gustatory projection nucleus to the telencephalon in cyprinids. Whereas the lateral torus seems to be exclusively involved with gustatory and general visceral systems, the inferior lobe has inputs from additional sensory (e.g., octavolateralis, visual) systems, and, thus, likely represents a multisensory integration center.

Topics: Animals; Brain Mapping; Carbocyanines; Diencephalon; Fluorescent Dyes; Goldfish; Hypothalamus; Neural Pathways; Prosencephalon; Taste

Nucleus praeeminentialis (nPr) is an isthmic nucleus that has been described in the brains of electrosensory teleost fishes and a single non-electrosensory species. The nucleus receives axon collaterals of ascending medullary sensory lemniscal neurons. Axons of nPr neurons project in turn back down onto those same populations of medullary projection neurons via a descending parallel fiber system (the molecular layer or cerebellar crest). Thus nPr forms a link in a sensory feedback loop that modulates the activity of neurons that relay information from medulla to midbrain. The purpose of this study is to investigate the nature of the afferent and efferent connections of the nPr with the specific aim of investigating other sources of input into this modulatory circuit. Transport of neuronal tracers (horseradish peroxidase, DiI and dextran amines) revealed that nPr has extensive interconnections with nuclei in the basal metencephalon, cerebellum, octavolateralis column and basal medulla. A previously described source of afference, the torus semicircularis in the mesencephalon, was not indicated by our studies. Our studies suggest that in addition to regulating the sensitivity and resolution of electrosensory and mechanosensory lateral line systems, the nPr may play a role in the resolution of signal ambiguities posed by auditory or vestibular stimulation of the saccular endorgan of the inner ear.

Topics: Afferent Pathways; Animals; Axons; Brain; Carbocyanines; Efferent Pathways; Electrophysiology; Fluorescent Dyes; Histocytochemistry; Horseradish Peroxidase; Ictaluridae; Nerve Fibers; Sensory Receptor Cells

Many studies demonstrate that differentiation of certain sensory receptors during development is induced by their nerve supply. Thus the navigational accuracy of pioneering fibres to their targets is crucial to this process. The special gustatory elements of the facial and glossopharyngeal nerves are used extensively as model systems in this field. We examined the chorda tympani, the gustatory component of the facial nerve, to determine the precise time course of its development in mice. The transganglionic fluorescent tracer DiI was injected into the anterior aspect of the mandibular arch of fixed embryos aged between 30 and 50 somites (E10-E12). It was allowed to diffuse retrogradely via the geniculate ganglion to the brainstem for 4 wk, before the distribution of DiI was determined using confocal laser scanning microscopy. Geniculate ganglion cells were first labelled at the 34 somite stage (E10). Pioneering chorda tympani fibres that arise from these cells passed peripherally and followed an oblique course as they grew towards the mandibular arch. At the 36 somite stage (E10.5), the peripheral component followed an intricate postspiracular course and passed anteriorly to arch over the primitive tympanic cavity, en route to the lingual epithelium. From the 36 to 50 somite stages (E10.5-E12), it consistently traced in the fashion of a 'U' bend. The central fascicle also traced at the 36 somite stage (E10.5) and just made contact with the brainstem. At the 40 somite stage (E11), the central fibres clearly chose a route of descent into the spinal trigeminal tract and branched into the solitary tract. Pioneering chorda tympani fibres contact the lingual epithelium when the target is primordial. The lingual epithelium may be a source of a neurotropic factor that attracts peripheral chorda tympani fibres to the sites of putative papillae. However, the chorda tympani is probably not a vital influence on the subsequent differentiation of gustatory papillae, since the papillae are elaborated 5 d later at E15 in murine embryos. The early morphology of the nerve is true to the amniote vertebrate phenotype.

Topics: Animals; Carbocyanines; Chorda Tympani Nerve; Fluorescent Dyes; Geniculate Ganglion; Gestational Age; Mice; Mice, Inbred Strains; Microscopy, Confocal; Morphogenesis; Trigeminal Ganglion

The pallium of hagfishes (myxinoids) is unique: It consists of a superficial "cortical" mantle of gray matter which is subdivided into several layers and fields, but it is not clear whether or how these subdivisions can be compared to those of other craniates, i.e., lampreys and gnathostomes. The pallium of hagfishes receives extensive secondary olfactory projections (Wicht and Northcutt [1993] J. Comp. Neurol. 337:529-542), but there are no experimental data on its nonolfactory connections. We therefore investigated the pallial and dorsal thalamic connections of the Pacific hagfish. Injections of tracers into the pallium labeled many cells bilaterally in the olfactory bulbs. Other pallial afferents arise from the contralateral pallium, the dorsal thalamic nuclei, the preoptic region, and the posterior tubercular nuclei. Descending pallial efferents reach the preoptic region, the dorsal thalamus, and the mesencephalic tectum but not the motor or premotor centers of the brainstem. Injections of tracers into the dorsal thalamus confirmed the presence of reciprocal thalamopallial connections. In addition, these injections revealed that there is no "preferred" pallial target for the ascending thalamic fibers; instead, ascending thalamic and secondary olfactory projections overlap throughout the pallium. The mesencephalic tectum and tegmentum, which receive afferents from a variety of sensory sources, are interconnected with the dorsal thalamus; thus, ascending nonolfactory sensory information may reach myxinoid pallia via a tectal-thalamic-telencephalic route. A comparative analysis of pallial organization reveals that the subdivisions of the pallium in gnathostomes (i.e., medial, dorsal, and lateral pallia) cannot be recognized with certainty in hagfishes.

Topics: Animals; Carbocyanines; Diencephalon; Fluorescent Dyes; Functional Laterality; Hagfishes; Histocytochemistry; Mesencephalon; Neostriatum; Neural Pathways; Prosencephalon; Telencephalon; Thalamus

Clonal analysis of the chick embryo hindbrain has shown that during the first 48 hr of neurogenesis the large majority of neural progenitor cells generate clones of neurons of only a single major phenotype or of only closely related phenotypes. This is despite considerable spatial intermixing of diverse neuronal phenotypes at these stages of development and suggests that phenotype may be decided early in mitotic precursors and remembered through several subsequent rounds of division and dispersal (Lumsden et al. [1994] Development 120:1581-1589). Here we have used fate-mapping and clonal analysis to study neuroepithelial cell dispersal and mixing in the early hindbrain and discuss this data in relation to the generation of single phenotype neuronal clones. We find that dispersal is not uniform throughout the dorsoventral axis of the neural tube, but is highly dependent on position along that axis. Neuronal identity is related to the spatial origin and, hence, environment of the cell, and the spatial intermixing of diverse neuronal phenotypes at HH stage 20 is largely the result of circumferential neuronal migration as medially born branchial motor neurons migrate laterally while the more laterally born mlf neurons migrate medially. Constraints on the dispersal of clonally related progenitors, in particular those that lie adjacent to the floor plate, may serve to restrict the fate of these cells to the generation of only one major neuronal phenotype, i.e., motor neurons.

Topics: Animals; Carbocyanines; Cell Differentiation; Cell Movement; Chick Embryo; Dextrans; Epithelial Cells; Fluoresceins; Fluorescent Dyes; Intracellular Fluid; Iontophoresis; Neurons; Phenotype; Rhodamines; Rhombencephalon; Stem Cells

Developing facial primordia change shape substantially in stages leading up to primary palate formation. We investigated expansion of cell populations within each of the four facial primordia of chick embryos between HH-stages 20 and 28, by using DiI labelling. Populations of cells centred around the nasal pits in the upper face, the midline of the paired mandibular primordia in the lower face, and at sites of fusion contribute most to overall expansion. Abundant Msx-1 transcripts are found in regions of high expansion, and Fgf-8 transcripts are seen in ectoderm associated with some of these regions. Many cell populations display preferential expansion along one axis. Maxillary and mandibular primordia cell populations expand along the proximodistal axis, whereas at the distal tip of the frontonasal mass, cell populations expand mediolaterally. Thus outgrowth occurs at the tips of mandibular and maxillary primordia, but at the base of the frontonasal mass. At regions where adjacent primordia abut each other, we found bidirectional movement of cells between primordia, unidirectional movement or could detect no movement at all. Regions of highest expansion in each primordium have the highest percentage of S phase labelled cells. Cell death occurs in some regions of low expansion but it seems likely that cell rearrangements and intercalations also contribute to shaping. These rearrangements could be associated with stretching of the primordia by neighbouring tissues. Treatment of chick embryos with retinoic acid causes clefts of the primary palate (Tamarin et al. [1984] J. Embryol. Exp. Morphol. 84:105-123). We found a decrease in expansion of cell populations that normally contribute to primary palate formation but surprisingly little ectopic cell death. Expansion of other cell populations in the treated upper face was more even rather than directed. This further supports the idea that tension exerted by neighbouring tissues plays a major role in global shaping of the upper face.

Topics: Animals; Carbocyanines; Cell Death; Cell Division; Chick Embryo; Facial Bones; Fibroblast Growth Factor 8; Fibroblast Growth Factors; Fluorescent Dyes; Gene Expression; Homeodomain Proteins; Mandible; Maxilla; Maxillofacial Development; MSX1 Transcription Factor; Palate; Transcription Factors; Tretinoin

Langmuir-Blodgett (LB) monolayers and bilayers of L-alpha-dipalmitoylphosphatidylcholine (DPPC), fluorescently doped with 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (diIC18), are studied by confocal microscopy, atomic force microscopy (AFM), and near-field scanning optical microscopy (NSOM). Beyond the resolution limit of confocal microscopy, both AFM and NSOM measurements of mica-supported lipid monolayers reveal small domains on the submicron scale. In the NSOM studies, simultaneous high-resolution fluorescence and topography measurements of these structures confirm that they arise from coexisting liquid condensed (LC) and liquid expanded (LE) lipid phases, and not defects in the monolayer. AFM studies of bilayers formed by a combination of LB dipping and Langmuir-Schaefer monolayer transfer exhibit complex surface topographies that reflect a convolution of the phase structure present in each of the individual monolayers. NSOM fluorescence measurements, however, are able to resolve the underlying lipid domains from each side of the bilayer and show that they are qualitatively similar to those observed in the monolayers. The observation of the small lipid domains in these bilayers is beyond the spatial resolving power of confocal microscopy and is complicated in the topography measurements taken with AFM, illustrating the utility of NSOM for these types of studies. The data suggest that the small LC and LE lipid domains are formed after lipid transfer to the substrate through a dewetting mechanism. The possible extension of these measurements to probing for lipid phase domains in natural biomembranes is discussed.

Topics: 1,2-Dipalmitoylphosphatidylcholine; Biophysical Phenomena; Biophysics; Carbocyanines; Fluorescent Dyes; In Vitro Techniques; Lipid Bilayers; Microscopy, Atomic Force; Microscopy, Confocal; Molecular Structure

The ontogeny of the myotome was investigated using [3H]thymidine or Brdu treatment in conjunction with 1,1', di-octadecyl-3, 3, 3', 3',-tetramethylindo-carbocyanine perchlorate (DiI) labeling and expression of specific markers. We have identified a subset of early post-mitotic cells that is present in the dorsomedial aspect of epithelial somites and is homogeneously distributed along their entire rostrocaudal extent. The post-mitotic quality of this cell subset enabled us to trace their fate in time-course experiments. Following initial somite dissociation, this epithelial post-mitotic layer bends underneath the medial portion of the nascent dermomyotome. Then, these cells progressively lose epithelial arrangement and migrate in a rostral direction where they accumulate temporarily. Subsequently, these early post-mitotic precursors extend processes that reach both rostral and caudal edges of each segment. Medial somite-derived myofibers also fill the entire mediolateral extent of the segment and reach the dorsomedial lip of the dermomyotome, thus forming the primary myotome. During this process, their large nuclei localize to a narrow stripe in the middle of the nascent myotome. Consistent with the proliferation studies, DiI labeling of the medial epithelial somite cells gave rise to a primary myotomal structure, and continuous pulsing of the DiI-injected embryos with radioactive thymidine revealed that these fibers indeed developed from post-mitotic progenitors. As these early post-mitotic cells that arise prior to somite dissociation are the first wave of progenitors that constitutes the myotome, we have termed them avian muscle pioneers. We propose that the primary myotome formed by the muscle pioneers constitutes a longitudinal scaffold that serves as a substrate for the addition of subsequent waves of myotomal cells.

Topics: Animals; Biomarkers; Body Patterning; Carbocyanines; Cell Lineage; Coturnix; DNA Replication; Epithelium; Fluorescent Dyes; MyoD Protein; Somites; Time Factors

The molecular layer of the cerebellum contains parallel fibers, the axons of granule neurons. We have examined the morphology and behavior of parallel fiber growth cones in the early postnatal rat cerebellum using the fluorescent tracer DiI. Parallel fiber growth cones distributed into three categories based on size and shape: short torpedo-like, long torpedo-like, and lamellopodial in form. The torpedo-like growth cones were modified by the addition of lamellopodia and/or filopodia, and the lamellopodial growth cones were often decorated with a filopodium. These three different growth cone morphologies were found throughout the growing region of the molecular layer. The nascent axons elaborated by premigratory granule neurons differed form the longer axons of more developed neurons in that they often had forked growth cones and extensive lamellopodial decoration along the axon shaft. Growth cones in living slices closely resembled those observed in the fixed preparations. The living growth cones exhibited frequent lamellopodial rearrangement and a side-to-side headwaving movement. The axon proximal to the growth cone was also dynamic. The axons curved and undulated, and mobile swellings formed along the axon shaft. These observations show that the growth cones of parallel fibers are similar to growth cones described for axons in other developing systems in terms of size, morphological characteristics, and dynamic behavior.

Topics: Aging; Animals; Animals, Newborn; Axons; Carbocyanines; Cerebellum; Fluorescent Dyes; Nerve Fibers; Rats; Rats, Sprague-Dawley; Time Factors

Using DiI fluorescent dextrans, we have created fate maps of the neural plate and early neural tube describing the extent of progenitor cell dispersal and the spatial origin of morphologically distinct neuronal cell types along the dorsoventral axis of the developing chick spinal cord. Nonuniform dispersal and mixing of progenitors occur within the early neuroepithelium, with the degree of dispersal being determined by the initial position of the cells along the mediolateral axis of the neural plate. Dispersal is greatest in the midregions of the ventricular epithelium and decreases toward the dorsal and ventral midlines. Phenotypically diverse classes of neurons are born at specific dorsoventral locations in the neural tube. Motor neurons are the most ventral cell type generated followed, at progressively more dorsal positions, by distinct classes of interneurons. Several genes show dorsoventrally restricted patterns of expression within the neural tube and the fate maps were used to investigate the relationship between one of these genes, Pax3, and progenitor cell dispersal and fate. The results indicate that the dorsoventral pattern of Pax3 expression is not maintained by restrictions to cell mixing and are consistent with a role for this transcription factor in specifying the identity of neurons with contralateral descending axons.

Topics: Animals; Body Patterning; Carbocyanines; Cell Differentiation; Cell Lineage; Cell Movement; Chick Embryo; DNA-Binding Proteins; Fluorescent Dyes; Neurons; Paired Box Transcription Factors; PAX3 Transcription Factor; Phenotype; Spinal Cord; Stem Cells; Time Factors; Transcription Factors

Connections in the developing nervous system are thought to be formed initially by an activity-independent process of axon pathfinding and target selection and subsequently refined by neural activity. Blockade of sodium action potentials by intracranial infusion of tetrodotoxin in cats during the early period when axons from the lateral geniculate nucleus (LGN) were in the process of selecting visual cortex as their target altered the pattern and precision of this thalamocortical projection. The majority of LGN neurons, rather than projecting to visual cortex, elaborated a significant projection within the subplate of cortical areas normally bypassed. Those axons that did project to their correct target were topographically disorganized. Thus, neural activity is required for initial targeting decisions made by thalamic axons as they traverse the subplate.

Topics: Action Potentials; Animals; Auditory Cortex; Axons; Carbocyanines; Cats; Dendrites; Geniculate Bodies; Neural Pathways; Tetrodotoxin; Visual Cortex

Anterograde tracing with the carbocyanine tracer DiI and the aminostyrol derivative DiA was used to selectively label fibers from the nucleus ambiguus, dorsal motor nucleus and nodose ganglion, respectively, terminating in the rat esophagus, and to compare them with the innervation of the gastric fundus in the same animals. Ambiguus neurons terminated on motor endplates distributed mainly to the ipsilateral half of the esophagus. There was no evidence of preganglionic innervation of myenteric ganglia from ambiguus neurons. Neurons of the dorsal motor nucleus supplied sparse fibers to only about 10% of enteric ganglia in the esophagus while they innervated up to 100% of myenteric ganglia in the stomach. Neurons of the nodose ganglion terminated profusely on more than 90% of myenteric ganglia of the esophagus and on about 50% of ganglia in the stomach. Afferent vagal fibers were also frequently found in smooth muscle layers starting at the esophago-gastric junction. In contrast, they were extremely rare in the striated muscle part of the esophagus. These morphological data suggest a minor influence of neurons of the dorsal motor nucleus and a prominent influence of vagal afferent terminals onto myenteric neurons in the rat esophagus.

Topics: Animals; Carbocyanines; Esophagus; Fluorescent Dyes; Ganglia, Autonomic; Histocytochemistry; Male; Medulla Oblongata; Motor Neurons; Myenteric Plexus; Neurons, Afferent; Neurons, Efferent; Nodose Ganglion; Pyridinium Compounds; Rats; Spinal Nerve Roots; Vagus Nerve

The retrograde tracer 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI) was used to label sympathetic preganglionic neurons (SPN) and motoneurons (MN) in postmortem human spinal cord. Seven months after microinjection of DiI into the ventral part of spinal thoracic segments T4 and T8, DiI-labelled neurons were identified and analyzed. Cryostat sections of spinal cord were prepared for light microscopy, while vibratome sections were analyzed using confocal microscopy. The majority of retrogradely labelled SPNs were located within the intermediolateral nucleus, with a few labelled dendrites having a mediolateral orientation. SPNs were also located within the nucleus intercalatus, around the central canal and in the lateral funiculus. Cell bodies of retrogradely labelled IML neurons were oval, kite- or spindle-shaped. The soma area of SPNs in T4 was approximately 422.9 +/- 20.9 microm2 with a median diameter of 14 +/- 0.6 microm. MNs in the ventral horn were round or oval in shape and often appeared with a few labelled neurites. The soma area of the MNs in T4 was approximately 842.3 +/- 35.1 microm2, with a median diameter of 18.3 +/- 0.1 microm. The mean values for MN soma area and diameter measurements were significantly greater compared to SPNs. However, no difference was observed between MNs in different segments or between SPNs in the same segments. No retrogradely labelled cells were observed within the dorsal horn. These findings indicate that DiI is a useful method for studying fixed human central nervous system tissue.

Topics: Adult; Carbocyanines; Fluorescent Dyes; Ganglia, Sympathetic; Histocytochemistry; Humans; Male; Microscopy, Confocal; Motor Neurons; Spinal Cord; Sympathetic Nervous System

The axons of the optic nerve layer are known to be myelinated by oligodendrocytes in the chick retina. The development of the retinal oligodendrocytes has been studied immunohistochemically with antibodies against oligodendrocyte lineage: monoclonal antibodies O4 and O1, and an antibody against myelin basic protein. O4 positive (O4+) cells were first detected in the retina on the tenth day of incubation (embryonic day (E)10, stage 36). The labeled cells were located in the optic nerve layer close to the optic fissure. Most were unipolar in shape, extending a leading process with a growth cone toward the periphery of the retina. By E12, unipolar O4+ cells had spread to the middle of the retina. Many O4+ cells close to the optic fissure showed radial arrangement with extension of processes toward the inner limiting membrane. O1+ oligodendrocytes were first observed in the E14 retina positioned just above (interiorly to) retinal ganglion cells. These labeled cells extended fine processes in the optic nerve layer. Limited numbers of myelin basic protein-positive cells were present by E16 and located interiorly to the retinal ganglion cells. In addition to the oligodendrocyte in the optic nerve layer, a limited number of O4+ cells were observed in the inner nuclear layer by E14, and they became O1+ by E18. Furthermore, explant culture experiments showed E10 to be the youngest stage at which the retina contained oligodendrocyte precursors. An intraventricular inj ection of fluorescent dye 1,1',dioctadecyl-3,3,3',3-tetramethylindocarbocyanine perchlorate (DiI) at E6 yielded O4+/DiI+ cells in the retina at E10, which provided direct evidence to support migration of oligodendrocyte precursor into the retina. The present results demonstrated the sequential appearance of the cells of oligodendrocyte lineage and the detailed morphology of the developing oligodendrocytes in the retina. These morphologic features strongly suggested that retinal oligodendrocytes were derived from the optic nerve and spread by migration through the optic nerve layer.

Topics: Animals; Antibodies, Monoclonal; Carbocyanines; Chick Embryo; Fluorescent Dyes; Immunohistochemistry; Injections, Intraventricular; Oligodendroglia; Optic Nerve; Retina

Our present understanding of the morphology of neuroepithelial bodies (NEBs) in mammalian lungs is comprehensive. Several hypotheses have been put forward regarding their function but none has been proven conclusively. Microscopic data on the innervation that appears to affect the reaction of NEBs to stimuli have given rise to conflicting interpretations. The aim of this study has been to check the validity of the hypothesis that pulmonary NEBs receive an extensive vagal sensory innervation. The fluorescent neuronal tracer DiI was injected into the vagal sensory nodose ganglion and NEBs were visualized in toto by using immunocytochemistry and confocal microscopy on 100-micrometer-thick frozen sections of the lungs of adult rats. The most striking finding was the extensive intraepithelial terminal arborizations of DiI-labelled vagal afferents in intrapulmonary airways, apparently always co-appearing with calcitonin gene-related peptide (CGRP)-immunoreactive NEBs. Not all NEBs received a traced nerve fibre. Intrapulmonary CGRP-containing nerve fibres, including those innervating NEBs, always appeared to belong to a nerve fibre population different from the DiI-traced fibres and hence did not arise from the nodose ganglion. Therefore, at least some of the pulmonary NEBs in adult rats are supplied with sensory nerve fibres that originate from the vagal nodose ganglion and form beaded ramifications between the NEB cells, thus providing support for the hypothesis of a receptor function for NEBs.

Topics: Animals; Calcitonin Gene-Related Peptide; Carbocyanines; Epithelial Cells; Fluorescent Dyes; Immunohistochemistry; Lung; Microscopy, Confocal; Nerve Endings; Neurons, Afferent; Nodose Ganglion; Rats; Rats, Wistar

Angiogenesis and coronary artery collateral formation can improve blood flow and thereby prevent myocardial ischemia. The role of perivascular fibroblasts in neovascularization remains incompletely understood. Here we investigated the effects of epicardial and myocardial fibroblasts on angiogenesis in vitro by using a serum-free microcarrier-based fibrin gel angiogenesis system. To clearly distinguish between different cell types, we either stained endothelial cells or fibroblasts in the living with 1, 1'-dioctadecyl-3,3,3',3'-tetramethyl-indocarbocyanine-perchlorate (DiI). In cocultures, low numbers of heart fibroblasts stimulated endothelial sprouting, and capillary growth was also induced by fibroblast-conditioned media, indicating a paracrine mechanism. Capillary formation was decreased by increasing the density of fibroblasts in the cocultures, indicating contact-dependent inhibition. Using time-lapse studies, it turned out that close contacts between fibroblasts and endothelial cells resulted in rapid retraction of endothelial cells or, rarely, in cell death. Depending on the local ratio of fibroblasts to endothelial cell numbers, fibroblasts determined the location of capillary growth and the size of developing capillaries and thereby contributed to capillary network remodeling. In contrast to primary heart fibroblasts, NIH 3T3 fibroblasts did not display contact-dependent inhibition of endothelial sprouts. NIH fibroblasts were frequently seen in close association with endothelial capillaries, resembling pericytes. Contact-dependent inhibition of angiogenesis by epicardial fibroblasts could not be reversed by addition of neutralizing anti-TGF-beta1 antibodies, by addition of serum, of medium conditioned by hypoxic tumor cells or myocardium, by various cytokines or by growing cocultures under hypoxic conditions. Our results implicate a pivotal role of periendothelial mesenchymal cells for the regulation of microvascular network remodeling and collateral formation.

Topics: Animals; Carbocyanines; Cell Communication; Coculture Techniques; Collateral Circulation; Coronary Circulation; Coronary Vessels; Culture Media, Conditioned; Culture Media, Serum-Free; Endothelium, Vascular; Fibrin; Fibroblasts; Gels; Humans; Hypoxia; Myocardium; Neovascularization, Physiologic; Swine

The primary olfactory projections of channel catfish Ictalurus punctatus have been examined with postmortem tracing by using either 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate or 1,1-dilinoleyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI). Following DiI deposition into small areas in different parts of the posterior olfactory bulb, olfactory sensory neurons always were labeled throughout the olfactory epithelium. No obvious topographical mapping exists between the epithelium and olfactory bulb. The different dye placements, however, did result in labeling of different morphologies of receptor cells, depending on the site of injection. Retrogradely labeled neurons in the olfactory epithelium were classified into three types on the basis of their height: tall, intermediate, and short. Tall olfactory sensory neurons had perikarya at the bottom one-fourth of the epithelium, extended slender dendrites to the epithelial surface, and possessed numerous cilia on the apical dendritic tips. These tall olfactory sensory neurons were labeled predominantly following DiI applications to the ventral part of the posterior olfactory bulb. In contrast, the short olfactory sensory neurons had perikarya situated within the superficial half of the epithelium and with short apical dendrites bearing microvilli. These short olfactory sensory neurons projected predominantly to the dorsal, posterior olfactory bulb. Thus, short microvillous receptor cells and tall ciliated receptor cells connect to different parts of the olfactory bulb, although the receptor cells are intermingled within the olfactory epithelium. Because different parts of the olfactory bulb are thought to respond preferentially to different classes of odorants, these results suggest that receptor cell morphology may be related to odorant quality detection. In addition, to compare this study with previous in vivo studies, Fluoro-Gold was injected in vivo into either the olfactory bulb or intraperitoneally. These in vivo studies show that so-called "type II ciliar receptor cells" of the nonsensory epithelium are labeled nonselectively by blood-borne substances, but they are not labeled by postmortem injections of DiI anywhere in the olfactory bulb.

Topics: Animals; Brain Mapping; Carbocyanines; Cilia; Dendrites; Fluorescent Dyes; Ictaluridae; Microvilli; Olfactory Bulb; Olfactory Mucosa; Olfactory Pathways; Olfactory Receptor Neurons; Stilbamidines

We have used carbocyanine dye tracing techniques in conjunction with photoconversion and electronmicroscopy to examine the prenatal development of the central and peripheral processes of those vestibular ganglion cells projecting to the cerebellum. Developmental changes in the number of vestibular ganglion cells were assessed in paraffin-embedded material by nucleolar counting. In agreement with the results of parvalbumin staining, afferents to the cerebellum from the vestibular ganglion pursued a superficial course during early fetal life (E13 to E15). From E16 to E19, this superficial position was progressively lost and vestibulocerebellar fibres were seen to be directed towards the ventricular surface (prospective posterior/inferior vermis). The change in the course of vestibular afferents to the cerebellum coincided with a profound reduction in the number of ganglion cells which could be retrogradely 1a-belled from the cerebellar anlage (mean+/-SD: E16-2040+/-1130; E19-510+/-440). During that same period the total number of vestibular ganglion cells rose to peak at a mean of 9200 at E19, although there was a subsequent decline to an average of 4660 at P0. This population size was maintained through to adult life (4600). We also examined the development of connections between the vestibular ganglion and the vestibular apparatus. Peripheral processes of vestibular ganglion cells invaded the macula utricle and saccule and cristae of the semicircular canals from E13. We found that the peripheral vestibular ganglion cell processes themselves did not show any significant morphological changes from E16 to E21, but the sensory epithelium itself adopts a mature pseudostratified appearance by E21. This suggests that the loss of vestibular ganglion cells from E19 to birth is not related to major morphological changes in the peripheral axons, at least as revealed by carbocyanine dye labelling of these from the cerebellum, but may be associated with differentiation of the sensory epithelium to the mature pseudostratified form. Electronmicroscopy of photoconverted vestibulocerebellar fibres showed that at E14 these afferents were grouped in tight bundles of up to 20 axons. No particular association with the superficially placed external granular layer cells was found at that age. By E16 photoconverted vestibulocerebellar axons were no longer as tightly bundled and could be seen coursing more ventrally through the cerebellar anlage. The findings indicate that vestib

Topics: Afferent Pathways; Animals; Animals, Newborn; Axons; Carbocyanines; Cell Count; Cerebellum; Efferent Pathways; Embryonic and Fetal Development; Female; Fluorescent Dyes; Ganglia, Sensory; Male; Morphogenesis; Neurons; Parvalbumins; Pregnancy; Rats; Rats, Wistar; Synapses; Vestibular Nerve

The Brn-3 class of POU-domain transcription factors includes three genes in mammals which have key roles in the development of specific groups of sensory neurons. Here, we have identified three avian genes which correspond to the murine genes Brn-3.0, Brn-3.1, and Brn-3.2. Using an in situ hybridization probe generic for this gene class, the earliest detectable expression of Brn-3 in the chick is at stage 15, in placodal and migrating precursors of the trigeminal ganglion. By stage 19, Brn-3.0 protein is detectable in the trigeminal and vestibulocochlear ganglia with Brn-3.0-specific antisera, and Brn-3 message expression has extended to the dorsal root ganglia. At later stages, when condensation of the trigeminal ganglion is complete, Brn-3.0-immunoreactive neurons are concentrated in the portion of the ganglion distal to the brain stem. To examine the developmental origin of the Brn-3 expressing cells, we combined lipophilic dye (DiI) labeling with in situ hybridization. DiI labeling of the placodal surface ectoderm and of premigratory neural crest cells in the neural tube reveals that all, or nearly all, of the Brn3-expressing neurons in the trigeminal ganglia are derived from the sensory placodes and not from the neural crest, and thus, that Brn-3 is an early marker of the placode-derived sensory neural lineage.

Topics: Amino Acid Sequence; Animals; Brain; Brain Chemistry; Caenorhabditis elegans; Carbocyanines; Chick Embryo; Cloning, Molecular; Coloring Agents; DNA-Binding Proteins; Drosophila; Immunohistochemistry; In Situ Hybridization; Mice; Molecular Sequence Data; Neurons, Afferent; Reverse Transcriptase Polymerase Chain Reaction; Transcription Factor Brn-3; Transcription Factor Brn-3A; Transcription Factors

The binding and internalization of a circulating insect lipoprotein, high density lipophorin (HDLp), by insect fat body cells was studied at the electron-microscopic level using ultrasmall gold-labeled HDLp and DiI-labeled HDLp, which were visualized by silver enhancement and diaminobenzidine photoconversion, respectively. Internalization of HDLp seems to conflict with the selective process by which the lipids are transported between HDLp and fat body cells. The pathway followed by the internalized lipoproteins was investigated. In addition, the localizations of HDLp in fat body cells of young and older adult locusts were compared because of the previously reported age-related differences in distribution of cell-associated and internalized HDLp. In the present study, internalized labeled HDLp was observed in early endosomes, late endosomes, and putative lysosomes. In older adults, these labeled structures were much less abundant than in young adults. Moreover, in these animals, the labeled endosomal/lysosomal vesicles were located close to the plasma membranes. A more intense labeling was observed in the extracellular matrix in older adults compared to young adults. In both developmental stages, an apparent accumulation of labeled HDLp was found in extracellular spaces. We propose that this entrapment of HDLp may be essential for selective lipid transport between HDLp and fat body cells.

Topics: Affinity Labels; Aging; Animals; Carbocyanines; Carrier Proteins; Cell Membrane; Endocytosis; Endosomes; Extracellular Space; Fat Body; Gold; Grasshoppers; Lipoproteins; Lysosomes; Microscopy, Electron

A precise knowledge of the timing and origin of the first cortical axons to project through the corpus callosum (CC) and of the subsequent emergence of callosal projection cells is essential for understanding the early ontogeny of this commissure. By using a series of mouse embryos and fetuses of the hybrid cross B6D2F2/J weighing from 0.36 g to 1.0 g (embryonic day E15.75-E17.25), we examined the spatial and temporal distribution of callosal projection cells by inserting crystals of the lipophilic dye (DiI: 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate) into the contralateral white matter just lateral to the midsagittal plane. Around 0.4 g or E15.8, retrogradely labeled cells were found restricted to a discrete cluster continuously distributed from the most ventral part of presumptive cingulate cortex to the hippocampus. During subsequent development, however, the tangential distribution of these labeled cells in ventromedial cortex did not extend further dorsally, and in fetuses where the CC became distinct from the hippocampal commissure (HC), labeled axons of cells in the ventral cingulate cortex were observed to intersect the callosal pathway and merge with labeled axons of the HC derived from cells in the hippocampus. The first cortical axons through the CC crossed the midline at about 0.64 g or E16.4, and these axons originated from a scattered neuronal population in the dorsal to lateral part of the presumptive frontal cortex. The earliest callosal cells were consistently located in the cortical plate and showed an immature bipolar appearance, displaying an ovoid- or pearl-shaped perikaryon with an apical dendrite coursing in a zig-zagging manner toward the pial surface and a slender axon directed toward the underlying white matter. Callosal projection cells spread progressively with development across the tangential extent of the cerebral cortex in both lateral-to-medial and rostral-to-caudal directions. In any cortical region, the first labeled cells appeared in the cortical plate and their number in the subplate was insignificant compared to that in the cortical plate. Thus, these results clarify that the CC is pioneered by frontal cortical plate cells, and the subsequent ontogeny of callosal projection cells proceeds according to the gradient of cortical maturation.

Topics: Animals; Axons; Brain Mapping; Carbocyanines; Cerebral Cortex; Corpus Callosum; Female; Fluorescent Dyes; Male; Mice; Mice, Inbred C57BL; Mice, Inbred DBA; Neural Pathways; Neurons; Time Factors

Topics: Animals; Carbocyanines; Chick Embryo; Fluorescent Dyes; Lipids; Microscopy, Confocal; Microscopy, Fluorescence; Vestibulocochlear Nerve

We present a fast and simple method for a general, fluorescent double stain that differentially labels various cellular components and visualizes all cells in confocal laser scanning microscopy. The technique is useful for two- and three-dimensional visualization of neural tissue and facilitates quantification of a variety of neuroanatomical parameters. Examples from cerebellum and retina are shown to demonstrate the broad applicability.

Topics: Animals; Carbocyanines; Cell Nucleolus; Cerebellum; Cytosol; Fluorescent Dyes; Isoquinolines; Microscopy, Confocal; Neurons; Perches; Rats; Retina

Tendon cells have complex shapes, with many cell processes and an intimate association with collagen fibre bundles in their extracellular matrix. Where cells and their processes contact one another, they form gap junctions. In the present study, we have examined the distribution of gap junction components in phenotypically different regions of rat Achilles tendon. This tendon contains a prominent enthesial fibrocartilage at its calcaneal attachment and a sesamoid fibrocartilage where it is pressed against the calcaneus just proximal to the attachment. Studies using DiI staining demonstrated typical stellate cell shape in transverse sections of pure tendon, with cells withdrawing their cell processes and rounding up in the fibrocartilaginous zones. Coincident with change in shape, cells stopped expressing the gap junction proteins connexins 32 and 43, with connexin 43 disappearing earlier in the transition than connexin 32. Thus, there are major differences in the ability of cells to communicate with one another in the phenotypically distinct regions of tendon. Individual fibrocartilage cells must sense alterations in the extracellular matrix by cell/matrix interactions, but can only coordinate their behaviour via indirect cytokine and growth factor signalling. The tendon cells have additional possibilities--in addition to the above, they have the potential to communicate direct cytoplasmic signals via gap junctions. The formation of fibrocartilage in tendons occurs because of the presence of compressive as well as tensile forces. It may be that different systems are used to sense and respond to such forces in fibrous and cartilaginous tissues.

Topics: Achilles Tendon; Animals; Biomechanical Phenomena; Calcaneus; Carbocyanines; Cartilage; Cell Communication; Cell Differentiation; Cell Size; Connexin 43; Connexins; Female; Fluorescent Antibody Technique, Indirect; Fluorescent Dyes; Gap Junction beta-1 Protein; Gap Junctions; Hindlimb; Male; Microscopy, Confocal; Rats; Rats, Wistar

The dorsal cochlear nucleus (DCN) of mammals displays a cortical structure containing a number of cell types organized into distinct layers. In the present study, the migratory mode of large multipolar cells and granule cells as well as the morphological differentiation of the projection neurons were investigated in the development of the mouse DCN. The classification of the DCN neurons followed that of Ryugo and Willard. The mode of neuronal migration was examined by immunohistochemical bromodeoxyuridine labeling. Large multipolar neurons originated from the primary rhombic lip and small granule cells from the secondary rhombic lip. Large multipolar neurons migrated radially from the ventricular zone into the forming DCN. Granule cells were generated later than the large multipolar neurons and migrated via the subependymal and subpial routes. Large multipolar neurons and small granule cells were thus segregated early in the DCN development and intermixed later during perinatal maturation. Projection neurons retrogradely labeled by DiI application to the contralateral inferior colliculus showed neurite extension between the pial surface and the ventricular zone during migration in the DCN primordium. The retrogradely labeled projection neurons showed a well-differentiated morphology of the large multipolar neurons as early as the late embryonic stage. The arrangement of the radial glial processes coincided with that of the migratory projection neurons. The migratory immature neurons showed close apposition with the radial glial processes, suggesting that glial scaffolds are involved in the migration and settlement of the large multipolar neurons. Thus, it is suggested that the mode of migration and settlement of large multipolar neurons and granule cells in the developing DCN is highly similar to that of Purkinje and granule cell migration in the cerebellar development, based on the findings of this study and the structural similarity between the cerebellum and DCN.

Topics: Animals; Bromodeoxyuridine; Carbocyanines; Cell Differentiation; Cell Movement; Cochlear Nucleus; Embryonic and Fetal Development; Fluorescent Dyes; Immunohistochemistry; Mice; Mice, Inbred ICR; Neural Pathways; Neuroglia; Neurons; Pyramidal Cells; Staining and Labeling

Topics: Carbocyanines; Cell Adhesion; Cell Membrane; Cytosol; Diffusion; Fluorescent Dyes; Humans; Lipid Bilayers; Membrane Fusion; Microinjections; Neutrophils; Phosphatidylcholines; Pressure

Fetal hippocampal cells grafted into the excitotoxically lesioned hippocampus of adult rats are capable of extending axonal projections into the host brain. We hypothesize that the axonal growth of grafted fetal cells into specific host targets, and the establishment of robust long-distance efferent graft projections, require placement of fetal cells in close proximity to appropriate axon guidance pathways. Intracerebroventricular administration of kainic acid in adult rats leads to a specific loss of hippocampal CA3 pyramidal neurons. We grafted 5'-bromodeoxyuridine-labeled embryonic day 19 hippocampal cells into adult hippocampus at four days post-kainic acid lesion, and quantitatively measured the projection of grafted cells into the contralateral hippocampus and the septum after three to four months survival using Fluoro-Gold and 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine (Dil) tracing. Grafts located in or near the degenerated CA3 cell layer exhibited numerous neurons which established commissural projections with the contralateral hippocampus. However, such projection did not occur in intrahippocampal grafts located away from the CA3 cell layer. In contrast, neurons in all grafts established robust projections into the septum regardless of location within hippocampus although grafts located near the degenerated CA3 cell layer displayed more neurons with such projections. Location of grafted cells clearly influences the development of efferent graft projections into distant targets in the adult host brain, particularly access to axon guidance pathways to facilitate the formation of projections. The establishment of robust long-distance commissural projections of fetal hippocampal grafts is clearly dependent on their placement in or near the degenerated CA3 cell layer, suggesting that appropriate axon guidance pathways for commissural pathways are tightly focussed near this cell layer. However, the establishment of septal projections of these grafts was not dependent on specific location within the CA3 cell layer, suggesting that axonal guidance mechanisms to the septum are more diffuse and not limited to the CA3 dendritic layers. The results underscore that fetal hippocampal grafts are capable of partly restoring lesioned hippocampal circuitry in adult animals when appropriately placed in the host hippocampus.

Topics: Animals; Antimetabolites; Brain Tissue Transplantation; Bromodeoxyuridine; Carbocyanines; Cell Survival; Excitatory Amino Acid Agonists; Fetal Tissue Transplantation; Fluorescent Dyes; Hippocampus; Kainic Acid; Neural Pathways; Neurons, Efferent; Rats; Rats, Inbred F344; Stilbamidines

The induction of postsynaptic structures by presynaptic terminals is suggested in a teleost brain. Neurons in the nucleus pretectalis superficialis pars magnocellularis (PSm) in the common carp are known to send fibers to the corpus mamillare (CM) and the nucleus lateralis valvulae (NLV). Individual axons of PSm neurons bifurcate (or give off an axon collateral), both of which reach the target areas in the CM and NLV. The morphology of horseradish peroxidase-labeled terminals in the CM and NLV appears quite different in light microscopy. Terminals in the CM appear as a fine network of beaded (2-4 microns in diameter) fibers, while those in the NLV are larger (8-12 microns in transverse diameter) and cup-shaped, partially enveloping the soma of individual NLV neurons. In electron microscopy, however, these synapses in the CM and NLV share several ultrastructural similarities. Small (0.2 to 0.4-micron thick, 0.4 to 0.7-micron long) spine-like protrusions arising from dendrites in the CM, and from cell bodies in the NLV, invaginate into the axon terminals, and the synaptic junctions are always formed at the base of the protrusion in both areas. Development of this unusual morphology is inferred to be directed from the presynaptic side. The morphological similarity of the spine-like protrusions to the "spinule," which is thought to be formed in response to synaptic activation, is discussed.

Topics: Animals; Axons; Carbocyanines; Carps; Fluorescent Dyes; Histocytochemistry; Horseradish Peroxidase; Microscopy, Electron; Neurons; Presynaptic Terminals; Superior Colliculi

The distribution of commissural neurons and terminals was revealed by DiI and Fluorogold injections in gerbils. The distribution of corpus callosum projections in visual cortical areas changes significantly during postnatal development of the gerbil. On postnatal days 5, 8 and 10, the animals do not show callosal projections. Widespread callosal endings are developed by postnatal day 15. This diffuse projection concentrates in strips at the border of area 17/18A in adult animals (postnatal day 20 and older).

Topics: Aging; Animals; Axons; Carbocyanines; Corpus Callosum; Fluorescent Dyes; Gerbillinae; Mice; Nerve Endings; Species Specificity; Stilbamidines; Visual Cortex

Tumour cell attachment to the endothelial cell lining of the circulatory system is of utmost importance in the process of cancer spread. We describe here a method of quantifying tumour cell attachment to an endothelial cell layer in vitro, using the fluorescent carbocyanine dye, 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine (DiI). We show that by incubation of human tumour cells with this fluorochrome, a high degree of fluorescent label can be incorporated into the cells without cytotoxic effects. These labelled tumour cells can then be used in subsequent attachment assays involving confluent human endothelial cell layers and subsequently quantified by using a fluorescent plate reader. Monitoring of this assay by fluorescent microscopy showed no transfer of the dye between tumour and attached endothelial cells. The labelled cells remained fluorescent for more than 3 days with no observable cytotoxicity. We suggest that DiI is of use in an assay system such as this to determine the effects of various factors on tumour cell-endothelial cell attachment.

Topics: Bisbenzimidazole; Carbocyanines; Cell Adhesion; Cell Communication; Endothelium, Vascular; Fluorescent Dyes; Humans; Neoplasms; Sensitivity and Specificity; Tetrazolium Salts; Thiazoles

We have devised a simple method that combines retrograde labeling of projecting neurons and in situ hybridization histochemistry to examine mRNA expression in the retrogradely labeled neurons. First, projecting neurons were retrogradely labeled in vivo by injection of the lipophilic neuronal tracer Dil. The fluorescence of the labeled neurons in the brain slices was photoconverted into stable DAB precipitate by green light illumination. The slices were cut into thinner sections and processed for detection of specific mRNA by in situ hybridization. Using this highly sensitive method, we demonstrate here that the corticospinal tract neurons in newborn rats express mRNA for the cell adhesion molecule L1. TAG-1 mRNA was not detected in these neurons. Therefore, the present method provides an important tool to study the molecular expression of projection neurons during the development of neuronal circuitry.

Topics: Affinity Labels; Animals; Carbocyanines; Cell Adhesion Molecules; Cell Adhesion Molecules, Neuronal; Contactin 2; Fluorescent Dyes; In Situ Hybridization, Fluorescence; Leukocyte L1 Antigen Complex; Membrane Glycoproteins; Neural Cell Adhesion Molecules; Pyramidal Cells; Pyramidal Tracts; Rats; Rats, Sprague-Dawley; RNA, Messenger

The sensory apparatus of the extraocular muscles attains special interest because of the great variation among different species with respect to the proprioceptors. The sensory innervation of the guinea pig extraocular muscles, lacking both muscle spindles and tendon organs, was investigated with a fluorescence double-labelling method. Primary sensory perikarya were assessed by postmortem application of 1,1'-dioctadecyl-3,3,3'3'-tetramethylindocarbocyanine perchlorate (Di-I) to the extraocular muscle nerves. Traced neurons were found in the ipsilateral ophthalmic part of the trigeminal ganglion. This is in line with findings in other species. Calcitonin gene-related peptide (CGRP) was detected immunohistochemically within the trigeminal ganglion. No somatotopic organization was observed for CGRP-like immunoreactive perikarya. Small (maximal diameter below 30 microm), medium (maximal diameter between 30 and 50 microm), and large (maximal diameter larger than 50 microm) trigeminal ganglion cells were found among the primary afferent perikarya from extraocular muscles. Among CGRP-like immunoreactive cells, only small and medium cells were observed. Double-labelling experiments indicated the CGRP content of primary afferents of the guinea pig extraocular muscles. The relationship to former morphological categories of ganglion cells is discussed. Primary afferent neurons with CGRP-like immunoreactivity might have efferent functions and might also be involved in inflammatory processes of extraocular muscles.

Topics: Animals; Calcitonin Gene-Related Peptide; Carbocyanines; Cell Size; Fluorescent Dyes; Guinea Pigs; Immunohistochemistry; Male; Neurons, Afferent; Oculomotor Muscles; Proprioception; Trigeminal Ganglion

It is assumed that pioneer neurons will predominantly show path finding ability, and that other neurons only follow the pioneer fibers. Here, tracers were injected into the newborn rat spinal cord followed by a search for labeled cells other than those in layer V. In all cases, neurons in the lower intermediate zone (IZ) of the dorsomedial cortex were retrogradely labeled. The IZ neurons have several characteristics required for pioneer neurons and project to the spinal cord first. Thus, the IZ neurons may function as pioneer neurons of the corticospinal projection and help in the formation of the permanent tract in rats.

Topics: Animals; Animals, Newborn; Carbocyanines; Cerebral Cortex; Fluorescent Dyes; gamma-Aminobutyric Acid; Immunohistochemistry; Microtubule-Associated Proteins; Neural Pathways; Neurons; Rats; Rats, Sprague-Dawley; Spinal Cord; Synaptic Transmission

The preotic neural tube has a variable ability for regeneration of neural crest depending on the neuraxial level. There is robust regeneration of neural crest in the caudal midbrain/rostral hindbrain. In contrast, removal of the cardiac neural crest results in cardiovascular abnormalities suggesting the lack of regeneration in this area, although the regenerative capacity of the cardiac crest region has never been tested directly. Premigratory cardiac neural crest was ablated bilaterally using laser irradiation or extirpation by tungsten needle, and the remaining ventral neural tube was labeled with DiI to examine any neural crest regeneration from the neural tube. The results indicate that there is very little regeneration of crest cells from the cardiac region of the neural tube if the ablation is done prior to the 5-somite stage and no regeneration after the 6-somite stage with either ablation procedure. Furthermore no compensatory response occurs from the adjacent regions of the neural crest. By contrast, we were able to confirm that regeneration of neural crest occurs in the preotic rhombencephalic neural tube even after laser irradiation. An analysis in the trunk region suggests that the trunk neural tube is similar to the cardiac region in that it does not regenerate crest cells in the ventral migratory pathway after ablation. However, melanocytes generated cranial and caudal to the ablated region migrate radially and fill in the ablated region so that there is no interruption of the normal pigment pattern. This study indicates that even though there is a variable capacity for crest regeneration in the preotic neural tube, the postotic neural tube does not have such regenerative ability.

Topics: Animals; Carbocyanines; Cell Differentiation; Cell Movement; Central Nervous System; Chick Embryo; Fluorescent Dyes; Heart; Histocytochemistry; Melanocytes; Nerve Regeneration; Neural Crest

In the embryonic CNS, preformed pathways precede the growth of axonal fasciculi [Katz M. J. and Lasek R. J. (1980) Cell Motil. 1, 141-157; Katz M. J. et al. (1980) Neuroscience 5, 821-833]. What are the developmental events that lead to the elaboration of these preformed pathways? To answer this question, we investigated the organization of the primitive neural tube and more particularly the arrangement of the early-generated cells using [3H]thymidine autoradiography or bromodeoxyuridine. Our data suggest that the position of early-generated cells might be involved in the setting of such pathways. In the brain stem of E12(0) (12 days and 0 h) and E12(15) rat embryos, the first-generated cells were organized into three longitudinal columns associated with glycoconjugate-rich extracellular spaces in the adjacent primitive marginal layer. Also, axons traced with 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI) were contiguous to the early-generated cellular columns and represented the primordium of the medial longitudinal fasciculus, the lateral longitudinal tract and the mesencephalic trigeminal tract. Our results show a correlation between the organization of early-generated cells, likely neurons, and the pattern of extracellular spaces in the marginal layer where axons grow. It has been reported in the literature that neurons produce elements of the extracellular matrix such as growth-modulating molecules or space-creating molecules. We therefore suggest that the position of early-generated neurons could be involved in the elaboration of a template for the setting of some major longitudinal tracts during embryonic development of the brainstem.

Topics: Animals; Antimetabolites; Axons; Brain Stem; Bromodeoxyuridine; Carbocyanines; Female; Fluorescent Dyes; Histocytochemistry; Neural Pathways; Neurons; Pregnancy; Rats; Rats, Sprague-Dawley; Thymidine

The arcuate nucleus (ARC) at the ventral surface of the human medulla has been historically considered a precerebellar nucleus. More recently, it has been implicated in central chemoreception, cardiopulmonary coupling and blood pressure responses. A deficiency of the ARC has been reported in a subset of putative human developmental disorders of ventilatory function. To investigate anatomic relationships of the ARC with brainstem regions involved in cardiorespiratory control, we applied crystals of DiI, a lipophilic dye which labels cells and cell processes by lateral diffusion along cell membranes, to 23 paraformaldehyde-fixed human fetal brainstems at 19 to 22 weeks postconceptional age. After 7 to 15.5 months diffusion, serial frozen sections were examined by florescence microscopy. DiI diffusion from the ARC labeled fibers and cell bodies in the medullary raphé, and the external arcuate fibers. Diffusion from the medullary raphé [corrected] labeled the reticular formation, medullary raphé, and the ARC. Diffusion from the pyramid and the basis pontis (negative control) labeled the corticospinal tract, with no labeling of the medullary raphé or ARC. The results suggest the existence of cellular connections between the ARC and the caudal raphé, a region implicated in cardiorespiratory control.

Topics: Arcuate Nucleus of Hypothalamus; Carbocyanines; Fetus; Fluorescent Dyes; Humans; Medulla Oblongata; Microscopy, Fluorescence; Neural Pathways; Pyramidal Tracts; Raphe Nuclei; Reticular Formation

Fat body cells of insects exhibit a high-affinity lipoprotein binding site at their cell surfaces. In the present study, the lipoprotein binding site was identified as an endocytotic receptor involved in receptor-mediated uptake of its lipoprotein ligand, high density lipophorin. After an initial period of high endocytotic uptake of high density lipophorin in the adult stage, this process strongly diminished. In the same period, a dramatic increase in cell surface-associated lipoproteins was observed. When animals were starved, however, internalization of lipoproteins was maintained. The pathway followed by the internalized lipoproteins appears to be different from the endosomal/lysosomal pathway, as the vast majority of apolipoproteins seemed to escape from lysosomal hydrolysis. In addition, no substantial intracellular accumulation of apolipoproteins was observed, suggesting that internalized lipoproteins were resecreted. It is unlikely that internalization is required for transport of the two major lipid components of insect lipoproteins, diacylglycerol and cholesterol, as inhibition of endocytosis neither affected the exchange of these lipids between lipoproteins and fat body cells nor influenced the loading of diacylglycerol onto lipoproteins in response to adipokinetic hormone. We postulate that the endosomal environment may facilitate transport of components which, unlike diacylglycerol and cholesterol, cannot be transported by simple aqueous diffusion.

Topics: Ammonium Chloride; Animals; Binding Sites; Carbocyanines; Carrier Proteins; Chloroquine; Down-Regulation; Endocytosis; Fat Body; Fluorescent Dyes; Food Deprivation; Grasshoppers; Hemolymph; Indicators and Reagents; Lipid Mobilization; Lipoproteins; Lipoproteins, HDL; Male; Molting; Receptors, Lipoprotein; Succinimides; Time Factors; Tritium

The initial pain from tissue damage may result from the release of cytoplasmic components that act upon nociceptors, the sensors for pain. ATP was proposed to fill this role because it elicits pain when applied intradermally and may be the active compound in cytoplasmic fractions that cause pain. Moreover, ATP opens ligand-gated ion channels (P2X receptors) in sensory neurons and only sensory neurons express messenger RNA for the P2X3 receptor. To test whether ATP contributes to nociception, we developed a tissue culture system that allows comparison of nociceptive (tooth-pulp afferent) and non-nociceptive (muscle-stretch receptor) rat sensory neurons. Low concentrations of ATP evoked action potentials and large inward currents in both types of neuron. Nociceptors had currents that were similar to those of heterologously expressed channels containing P2X3 subunits, and had P2X3 immunoreactivity in their sensory endings and cell bodies. Stretch receptors had currents that differed from those of P2X3 channels, and had no P2X3 immunoreactivity. These results support the theory that P2X3 receptors mediate a form of nociception, but also suggest non-nociceptive roles for ATP in sensory neurons.

Topics: Action Potentials; Adenosine Triphosphate; Animals; Carbocyanines; Cell Line; Culture Techniques; Dental Pulp; Fluorescent Dyes; Humans; Male; Mechanoreceptors; Membrane Potentials; Muscles; Neurons; Neurons, Afferent; Nociceptors; Rats; Rats, Sprague-Dawley; Receptors, Purinergic P2; Receptors, Purinergic P2X3; Recombinant Proteins

The mechanisms underlying mechanotransduction in baroreceptor neurons (BRNs) are undefined. In this study, we specifically identified aortic baroreceptor neurons in primary neuronal cell cultures from nodose ganglia of rats. Aortic baroreceptor neurons were identified by labeling their soma with the fluorescent dye 1,1'-dioleyl-3,3,3',3'-tetramethylin-docarbocyanine (DiI) applied to the aortic arch. Using Ca2+ imaging with fura 2, we examined these BRNs for evidence of Ca2+ influx and determined its mechanosensitivity and voltage dependence. Mechanical stimuli were produced by ejecting buffer from a micropipette onto the cell surface with a pneumatic picopump, producing a shift in the center of mass of the cell that was related to intensity of stimulation. Ninety-three percent of DiI-labeled neurons responded to mechanical stimulation with an increase in [Ca2+]i. The magnitude of the increases in [Ca2+]i was directly related to the intensity of the stimulus and required the presence of external Ca2+. The trivalent cations Gd3+ or La3+ in equimolar concentrations (20 mumol/L) eliminated the K(+)-induced rises in [Ca2+]i, demonstrating that both trivalent cations are equally effective at blocking voltage-gated Ca2+ channels in these baroreceptor neurons. In contrast, the mechanically induced increases in [Ca2+]i were blocked by Gd3+ (20 mumol/L) only and not by La3+ (20 mumol/L). Stretch-activated channels (SACs) have been shown in other preparations to be blocked by Gd3+ specifically. Our data demonstrate that (1) BRNs, specifically identified as projecting to the aortic arch, have ion channels that are sensitive to mechanical stimuli; (2) mechanically induced Ca2+ influx in these cells is mediated by a Gd(3+)-sensitive ion channel and not by voltage-gated Ca2+ channels; (3) the magnitude of the Ca2+ influx is dependent on the intensity of the stimulus and the degree and duration of deformation; and (4) repeated stimuli of the same intensity result in comparable increases in [Ca2+]i. We conclude that mechanical stimulation increases Ca2+ influx into aortic BRNs independent of voltage-gated Ca2+ channels. The results suggest that Gd(3+)-sensitive SACs are the mechanoelectrical transducers in baroreceptors.

Topics: Animals; Aorta; Calcium; Carbocyanines; Cells, Cultured; Extracellular Space; Fluorescent Dyes; Ion Channel Gating; Ion Channels; Male; Mechanoreceptors; Neurons; Physical Stimulation; Pressoreceptors; Rats; Rats, Sprague-Dawley

We have produced detailed fate maps for mesenchyme and apical ridge of a stage 20 chick wing bud. The fate maps of the mesenchyme show that most of the wing arises from the posterior half of the bud. Subapical mesenchyme gives rise to digits. Cell populations beneath the ridge in the mid apical region fan out into the anterior tip of the handplate, while posterior cell populations extend right along the posterior margin. Subapical mesenchyme of the leg bud behaves similarly. The absence of anterior bending of posterior cell populations has implications when considering models of vertebrate limb evolution. The fatemaps of the apical ridge show that there is also a marked anterior expansion and cells that were in anterior apical ridge later become incorporated into non-ridge ectoderm along the margin of the bud. Mesenchyme and apical ridge do not expand in concert--the apical ridge extends more anteriorly. We used the fatemaps to investigate the relationship between cell lineage and elaboration of Hoxd-13 and Fgf-4 domains. Hoxd-13 and Fgf-4 are initially expressed posteriorly until about the mid-point of the early wing bud in mesenchyme and apical ridge respectively. Later in development, the genes come to be expressed throughout most of the handplate and apical ridge respectively. We found that at the proximal edge of the Hoxd-13 domain, cell populations stopped expressing the gene as development proceeded and found no evidence that the changes in extent of the domains were due to initiation of gene expression in anterior cells. Instead the changes in extent of expression fit with the fate maps and can be attributed to expansion and fanning out of cell populations initially expressing the genes.

Topics: Animals; Body Patterning; Carbocyanines; Chick Embryo; Ectoderm; Fibroblast Growth Factor 4; Fibroblast Growth Factors; Fluorescent Dyes; Gene Expression Regulation, Developmental; Homeodomain Proteins; Limb Buds; Mesoderm; Proto-Oncogene Proteins; Pyridinium Compounds; Transcription Factors; Wings, Animal

We examined the effects of Lovastatin on LDL receptor (LDL-R) expression and rate of internalization in interleukin-2 (IL-2) expanded phytohemagglutinin-stimulated lymphocytes. Lovastatin increased the surface LDL-R expression, but not DiI-LDL uptake, by up to 30% regardless of whether cell proliferation was affected. It caused a dose-dependent reduction in the LDL-R internalization rate as determined with monensin. Lovastatin had no effect on IL-2 receptor internalization. Inhibition of DNA synthesis by hydroxyurea or protein tyrosine kinase activity by genistein failed to affect the LDL-R internalization rate. Co-incubation of cells with Lovastatin and mevalonate or LDL completely restored the rate of LDL-R internalization. We conclude that Lovastatin increases the apparent surface LDL-R expression by retarding the rate of LDL-R internalization. The effect is mediated through the mevalonate pathway but not the anti-mitogenic property of Lovastatin.

Topics: Carbocyanines; Cell Division; Endocytosis; Flow Cytometry; Fluorescent Dyes; Genistein; Hydroxyurea; Interleukin-2; Isoflavones; Lipoproteins, LDL; Lovastatin; Lymphocyte Activation; Lymphocytes; Mevalonic Acid; Monensin; Phytohemagglutinins; Receptors, Interleukin-2; Receptors, LDL

We investigated the responses of putative aortic baroreceptor neurons to mechanical stimulation of their processes. Putative aortic baroreceptor neurons were identified by applying the carbocyanine dye DiI to the adventitia of the aortic arch of anesthetized rats. After at least 1 week, the nodose ganglia were removed and the neurons were cultured. Within 2-3 days, neurite outgrowth was evident on many neurons. The soma was voltage-clamped using whole cell patch clamp techniques while the neurites were deformed with pneumatic ejection of bath solution at 5-15 psi using a glass pipette (7-15 microm) positioned at least 50 microm from the neurite. Mechanical stimulation induced an inward current in 15 out of 17 putative aortic baroreceptor neurons. The magnitude of the current was related to the intensity of stimulation. The current was blocked by 20 microM gadolinium (n = 11), a reported blocker of mechanically sensitive ion channels, or by incubating the cells overnight in 10 microM phalloidin, which binds to actin filaments (n = 5). We conclude that mechanical deformation of neurites of putative baroreceptor neurons activates a mechanosensitive inward current in the soma and that the cytoskeletal actin filaments are involved in the generation of this current.

Topics: Animals; Aorta, Thoracic; Carbocyanines; Cells, Cultured; Fluorescent Dyes; Muscle, Smooth, Vascular; Neurites; Neurons; Nodose Ganglion; Patch-Clamp Techniques; Physical Stimulation; Pressoreceptors; Rats

The dendritic distribution of mitral/tufted (M/T) cells in the opossum accessory olfactory bulb (AOB) was investigated using intracellular injection of Lucifer yellow and DiI labeling. Lucifer yellow labeling demonstrated that the primary dendrites of M/T cells are restricted to one of the two subregions (anterior or posterior) of the glomerular layer. When DiI was placed in the anterior or posterior subregion of the glomerular layer, virtually all of the labeled cell bodies in the AOB were located in the anterior or posterior part of the M/T cell layer, respectively. These results demonstrate that the anterior and posterior subregions of the AOB glomerular layer are termination sites for dendrites belonging to distinct populations of M/T cells.

Topics: Animals; Carbocyanines; Dendrites; Female; Isoquinolines; Male; Neurons; Olfactory Bulb; Olfactory Pathways; Opossums; Synapses

Nucleus rostrolateralis is a distinctive diencephalic nucleus in some ray-finned fishes. In the osteoglossomorph Pantodon, it is a large ovoid nucleus with visual system connections. A topologically and cytoarchitectonically similar nucleus has been found in six species of non-osteoglossomorph fishes, two species of gars and four euteleosts. Of the latter, two are ostariophysans of the genus Danio, one an atherinomorph, and one a notothenioid percomorph. A variety of characteristic similarities can be found in nucleus rostrolateralis among all these species. The present study reports on these similarities in Danio and in the euteleost Xiphophorus as compared with the nucleus in Pantodon. While this nucleus has a phylogenetic distribution that might imply convergent evolution, the high degree of similarity in its features across species strongly suggests that its genetic basis may be the same despite the lack of phenotypic homology.

Topics: Animals; Biological Evolution; Carbocyanines; Diencephalon; Fishes; Fluorescent Dyes; Species Specificity; Superior Colliculi; Visual Pathways

The adult facial nerve contains the axons from two populations of efferent neurons. First, the branchiomotor efferent neurons that innervate the muscles of the second arch. These neurons project out of the hindbrain in the motor root and form the facial motor nuclei. Second, the preganglionic efferent neurons that innervate the submandibular and pterygopalatine ganglia. These neurons project from the hindbrain via the intermediate nerve and form the superior salivatory nucleus. The motor neurons of the facial nerve are known to originate within rhombomeres 4 and 5. In the kreisler mouse mutant there is a specific disruption of the hindbrain rhombomeres 5 and 6 appear to be absent. To investigate changes in the organization of the facial motor neurons in this mutant, we have used lipophilic dyes to trace the facial motor components both retrogradely and anterogradely. As expected, facial motor neurons are missing from rhombomere 5 in this mutant. In addition, the loss of these neurons correlates with the specific loss of the superior salivatory nucleus. In contrast, the branchiomeric neurons, that originate in rhombomere 4, appear to develop normally. This includes the caudal migration of their cell bodies forming the genu of the facial nerve. Our studies confirm that rhombomeres are critical to hindbrain development and that they are the fundamental unit at which motor neurons are specified.

Topics: Animals; Carbocyanines; Facial Nerve; Fluorescent Dyes; Mice; Mice, Mutant Strains; Motor Neurons

The availability of a vehicle to deliver lipid soluble agents to a brain injury site may be of potential value in management of brain injury. This work describes the aggregation of intravenously administered Lipid-Coated Microbubbles (LCM) in the injury site following an experimental radiofrequency rat brain lesion. The bubbles can be identified around the region of the injury after the lesion has matured at least 48 h. The greatest bubbles density is evident after the lesion has matured for 10 days. This bubble density, reflecting "affinity," decreases to a plateau level from the second to the third week after injury. In order to investigate the potential relationship of bubble influx to posttraumatic astrocytosis and to cell turnover in the region, we utilized dual-channel laser-scanning confocal microscopy to track both bubble influx into the region and concomitant Glial Fibrillary Acidic Protein (GFAP) expressing astroctyte cell distribution. Cell turnover was assayed in separate sections using immunohistochemical staining of Proliferating Cell Nuclear Antigen (PCNA). We suggest a relationship between the LCM affinity and reactive astrocytes, but found no affinity of LCM for cells which stained positive with PCNA.

Topics: Animals; Brain Injuries; Carbocyanines; Cell Adhesion; Cell Division; Feasibility Studies; Fluorescent Dyes; Glial Fibrillary Acidic Protein; Immunohistochemistry; Lipids; Male; Microspheres; Proliferating Cell Nuclear Antigen; Rats; Rats, Sprague-Dawley; Surface Properties

The octavolateral area of lampreys, which receives primary fibers from the octaval and lateral line nerves, is involved in the premotor organization of body movements through secondary projections to the reticular formation. Here, the typology of neurons of the three octavolateral nuclei (ventral, medial, and dorsal) that putatively project to the middle and posterior rhombencephalic reticular nuclei were studied by retrograde transport of horseradish peroxidase (HRP) applied to these reticular nuclei. Several types of neurons were labeled in the ventral nucleus, both ipsilateral and contralateral to the site of HRP application. Some of these neurons showed a rather simple morphology (octavomotor neurons, monopolar cells), but most had more- or less-branched dendrites that were associated with one, or several, fields of terminal fibers in the octavolateral area. Unlike those of the ventral nucleus, labeled neurons of the medial nucleus were homogeneous in appearance (mostly pear-shaped). The dorsal nucleus was scarcely developed in larvae, as judged from the very simple and small labeled cells. The presence of terminal or "en-passant" boutons of secondary octavolateral fibers in the reticular area and the commissural nature of these fibers were also investigated by means of application of HRP or indocarbocyanine dye to the octavolateral nuclei. In addition, neurons of other alar plate nuclei that were labeled by the HRP application to the reticular nuclei (trigeminal descending root nucleus and solitary nucleus) were also characterized. The functional significance of these results is discussed.

Topics: Animals; Brain Mapping; Carbocyanines; Horseradish Peroxidase; Lampreys; Larva; Medulla Oblongata; Movement; Neural Pathways; Neurons; Reticular Formation; Rhombencephalon; Solitary Nucleus; Trigeminal Nuclei

We have reinvestigated the embryonic development of the vestibulocochlear system in mice using anterograde and retrograde tracing techniques. Our studies reveal that rhombomeres 4 and 5 include five motor neuron populations. One of these, the abducens nucleus, will not be dealt with here. Rhombomere 4 gives rise to three of the remaining populations: the facial branchial motor neurons; the vestibular efferents; and the cochlear efferents. The migration of the facial branchial motor neurons away from the otic efferents is completed by 13.5 days post coitum (dpc). Subsequently the otic efferents separate into the vestibular and cochlear efferents, and complete their migration by 14.5 dpc. In addition to their common origin, all three populations have perikarya that migrate via translocation through secondary processes, form a continuous column upon completion of their migrations, and form axonal tracts that run in the internal facial genu. Some otic efferent axons travel with the facial branchial motor nerve from the internal facial genu and exit the brain with that nerve. These data suggest that facial branchial motor neurons and otic efferents are derived from a common precursor population and use similar cues for pathway recognition within the brain. In contrast, rhombomere 5 gives rise to the fourth population to be considered here, the superior salivatory nucleus, a visceral motor neuron group. Other differences between this group and those derived from rhombomere 4 include perikaryal migration as a result of translocation first through primary processes and only then through secondary processes, a final location lateral to the branchial motor/otic efferent column, and axonal tracts that are completely segregated from those of the facial branchial and otic efferents throughout their course inside the brain. Analysis of the peripheral distribution of the cochlear efferents and afferents show that efferents reach the spiral ganglion at 12.5 dpc when postmitotic ganglion cells are migrating away from the cochlear anlage. The efferents begin to form the intraganglionic spiral bundle by 14.5 dpc and the inner spiral bundle by 16.5 dpc in the basal turn. They have extensive collaterals among supporting cells of the greater epithelial ridge from 16.5 dpc onwards. Afferents and efferents in the basal turn of the cochlea extend through all three rows of outer hair cells by 18.5 dpc. Selective labeling of afferent fibers at 20.5 dpc (postnatal day 1) shows that

Topics: Animals; Axons; Brachial Plexus; Carbocyanines; Cell Differentiation; Cochlea; Dextrans; Facial Nerve; Female; Mice; Motor Neurons; Neurons, Afferent; Neurons, Efferent; Pregnancy; Rhombencephalon; Vestibule, Labyrinth

The use of cyclosporine-A (CsA) immunosuppression for neural transplantation has become the treatment of choice for ameliorating host-graft rejection responses in human and animal transplant studies. However, the cytotoxic effects of CsA have warranted a search for alternative methods of protecting neural transplants. Sertoli cells produce an immunosuppressant factor, Fas ligand (Fas-L), that may provide the testis with its immunoprivileged status. Therefore, it has recently been suggested that these cells may be useful in producing localized immunosuppression for transplants. If Sertoli cells do produce localized immunosuppression, then it should be possible to successfully transplant Sertoli cells without additional immunosuppression following transplant surgery. The present study was undertaken to determine whether rat or porcine Sertoli cells transplanted into rat brain would survive for an extended period of time without CsA immunosuppression. Isolated rat or porcine Sertoli cells prelabeled with DiI were transplanted into normal rat brain. We report that both rat Sertoli cell allografts and porcine Sertoli cell xenografts survived for at least 2 months posttransplantation into the rat brain without CsA immunosuppression, indicating that these grafts were capable of producing sufficient localized immunosuppression to survive at the site of transplant without additional systemic immunosuppression.

Topics: Animals; Carbocyanines; Cell Survival; Corpus Striatum; Cyclosporine; Fluorescent Dyes; Immunosuppressive Agents; Male; Microscopy, Fluorescence; Rats; Rats, Sprague-Dawley; Sertoli Cells; Swine; Transplantation, Heterologous; Transplantation, Homologous

Because of their many inputs and bilateral projections, interneurons surrounding the trigeminal motor nucleus (MotV) are thought to be very important in control of jaw movements and reflexes. However, their interactions with the trigeminal motoneurons are almost unknown. In the present study an in vitro slice preparation was used to investigate this relationship in rat. The zone bordering MotV has been subdivided into four regions: the supra-, juxta-, and intertrigeminal areas (SupV, JuxtV, and IntV, respectively) and the parvocellular reticular formation ventral and caudal to MotV. Stimulation of all areas evoked short-latency excitatory postsynaptic potentials (EPSPs) in masseteric motoneurons. Frequently the EPSPs masked inhibitory postsynaptic potentials (IPSPs) or were followed by long-lasting inhibitory potentials. Only responses obtained from stimulation of JuxtV and IntV seemed devoid of inhibitory components. The EPSPs were mediated through kainate/alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors, whereas the IPSPs appear to be due to gamma-aminobutyric acid and glycine. EPSPs and IPSPs were also recorded in SupV premotor interneurons after stimulation of IntV and MotV, respectively, thus suggesting that reciprocal connections exist between premotor areas and also between premotor interneurons of SupV and inhibitory interneurons located within MotV. It is concluded that the preparation used here will doubtless prove useful for further investigation of the circuitry involved in the bilateral coordination of the jaw.

Topics: Animals; Bicuculline; Carbocyanines; Central Nervous System Stimulants; Electric Stimulation; Excitatory Amino Acid Antagonists; Excitatory Postsynaptic Potentials; GABA Antagonists; Histocytochemistry; In Vitro Techniques; Interneurons; Masseter Muscle; Motor Neurons; Quinoxalines; Rats; Strychnine; Synapses; Trigeminal Nuclei

The development of trigeminal projections between the thalamus and cortex has been investigated in the marsupial mammal, the wallaby, by using a carbocyanine dye, horseradish peroxidase conjugated to wheat germ agglutinin (WGA-HRP), Neurobiotin, and biocytin as pathway tracers. The appearance of whisker-related patterns in the cortex in relation to their appearance in the brainstem and thalamus was examined, as was the presence or absence of a waiting period for thalamocortical afferents and the identity of the first cortical cells to project to the thalamus. Thalamic afferents first reached the cortex at postnatal day (P) 15 and were distributed up to the deep edge of the compact cell zone in the superficial cortical plate throughout development, in both dye and WGA-HRP labelled material, with no evidence of a waiting period. The initial corticothalamic projection, detected by retrograde transport of WGA-HRP from the thalamus, occurred at P60 from layer 5 cells. This was confirmed by labelling of corticothalamic axons after cortical injections of Neurobiotin and biocytin. Scattered, labelled cells seen before P60 after dye labelling from the thalamus presumably resulted from transcellular labelling via thalamic afferents. Clustering of afferents in layer 4 and cell bodies and their dendrites in layers 5 and 6 first occurred simultaneously at P76. There is a sequential onset of pattern formation, first in brainstem, then in thalamus, and finally in cortex, with a long delay between afferent arrival and pattern formation at each level. Independent regulation at each level, likely depending on target maturation, is suggested.

Topics: Animals; Axons; Biotin; Brain Mapping; Carbocyanines; Cerebral Cortex; Fluorescent Dyes; Lysine; Macropodidae; Neural Pathways; Thalamus; Time Factors; Trigeminal Nerve; Wheat Germ Agglutinin-Horseradish Peroxidase Conjugate

We previously reported that retrovirally marked clones in the mature chick diencephalon were widely dispersed in the mediolateral, dorsoventral and rostrocaudal planes. The current study was undertaken to define the migration routes that led to the dispersion. Embryos were infected between stages 10 and 14 with a retroviral stock encoding alkaline phosphatase and a library of molecular tags. Embryos were harvested 2.5-5.5 days later and the brains were fixed and serially sectioned. Sibling relationships were determined following PCR amplification and sequencing of the molecular tag. On embryonic day 4, all clones were organized in radial columns spanning the neuroepithelium, which was composed primarily of a ventricular zone at this age. No tangential migration was seen in the ventricular zone. On embryonic day 5, most clones remained radial with many cells located in the ventricular zone; however, a few clones had cells migrating perpendicular to the radial column, in either a rostrocaudal or dorsoventral direction. The tangential migration began just beyond the basal limit of the ventricular zone. On embryonic days 6 and 7, many clones had cells migrating perpendicular to the radial column, which spanned from the ventricular to the pial surface. The migrating cells appeared to be aligned along axes that were perpendicular to the radial column. Using a combination of DiI tracing, immunohistochemistry and electron microscopy, we have determined that axonal tracts are present and are aligned with the migrating cells, suggesting that they support the non-radial cell migration. These data indicate that migration along pathways independent of radial glia occur outside of the ventricular zone in more than 50% of the clones in the chick diencephalon.

Topics: Animals; Axons; Carbocyanines; Cell Movement; Chick Embryo; Clone Cells; Diencephalon; Fluorescent Dyes; Immunohistochemistry; Microscopy, Electron; Neuroglia; Neurons

It has been suggested that the entorhino-hippocampal circuit is involved in memory formation. To investigate the way that associative memory is elaborated in the circuit, the entorhino-dentate projection was studied with the fluorescent lipophilic tracer Dil. We investigated the projection originating in the dorsal part of the entorhinal cortex by injecting Dil along the rhinal sulcus. Anterograde fluorescent labeling allowed us to examine sections of the sample with a confocal microscope or in wholemount preparations with a fluorescence microscope. Quantitative analysis of the distribution of the Dil-labeled perforant path by confocal microscopy was performed in the septal one third level of the hippocampus. The analysis confirmed that the topographical map along the mediolateral dimension of the entorhinal cortex was transferred to the proximodistal level (from the inner one third to the edge of the molecular layer) of the granule cell dendrites in a gradually shifting manner. The fiber profile observed after lateral entorhinal injection was thick in the suprapyramidal blade and thin in the infrapyramidal blade. The fiber profile observed after medial entorhinal injection was thin in the suprapyramidal blade and thick in the infrapyramidal blade. Fluorescence microscopic observation of wholemount preparations showed that projections from the Dil injection site were distributed wider than half the dentate gyrus in the longitudinal direction. In transverse sections, the range of the labeled fiber distribution was confirmed to be more than two thirds of the dentate gyrus in the same direction regardless of the mediolateral level of the injection site. It has been suggested that the dorsoventral axis of the entorhinal cortex is represented in the septotemporal levels of the dentate gyrus, but that the topographical correspondence might be weak and vague. Although our investigation was limited to the projection from the dorsal entorhinal cortex to the dorsal part of the dentate gyrus, we conclude that the widely distributed projection covers the dentate gyrus in a nontopographic manner.

Topics: Animals; Brain Mapping; Carbocyanines; Dendrites; Dentate Gyrus; Entorhinal Cortex; Fluorescent Dyes; Male; Memory; Microscopy, Confocal; Microscopy, Fluorescence; Neural Pathways; Rats; Rats, Sprague-Dawley

During telencephalic development, a boundary develops that restricts cell movement between the dorsal cortical and basal striatal proliferative zones. In this study, the appearance of this boundary and the mechanism by which cell movement is restricted were examined through a number of approaches. The general pattern of neuronal dispersion was examined both with an early neuronal marker and through the focal application of DiI to telencephalic explants. Both methods revealed that, although tangential neuronal dispersion is present throughout much of the telencephalon, it is restricted within the boundary region separating dorsal and ventral telencephalic proliferative zones. To examine the cellular mechanism underlying this boundary restriction, dissociated cells from the striatum were placed within both areas of the boundary, where dispersion is limited, and areas within the cortex, where significant cellular dispersion occurs. Cells placed within the boundary region remain round and extend only thin processes, whereas progenitors placed onto the cortical ventricular zone away from this boundary are able to migrate extensively. This suggests that the boundary inhibits directly the migration of cells. To examine whether the signal inhibiting dispersion within the boundary region acts as a long- or short-range cue, we apposed explants of boundary and nonboundary regions in vitro. Within these explants we found that migration was neither inhibited in nonboundary regions nor induced in boundary regions. This suggests that the boundary between dorsal and ventral telencephalon isolates these respective environments through either a contact-dependent or a short-range diffusible mechanism.

Topics: Acridine Orange; Animals; Biomarkers; Carbocyanines; Cell Communication; Cell Division; Cell Movement; Cerebral Cortex; Corpus Striatum; Diffusion; Embryonic Induction; Fluorescent Dyes; Microscopy, Fluorescence; Morphogenesis; Rats; Telencephalon

We have examined the development of the laminar and areal distribution of cortical commissural neurons in a marsupial mammal, the wallaby Macropus eugenii. In this species, commissural axons approach the major cerebral commissure, the anterior commissure, via either the internal capsule or the external capsule and first cross the midline at postnatal day 14 (P14). By retrogradely labelling these axons with 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine (DiI) at P15, we show here that the cell bodies of these neurons are restricted to a region of cortex adjacent to the rhinal fissure. Most of these labelled neurons are located in the compact cell zone of the cortical plate, with only a few labelled cells found in the zone of loosely packed cells deep to this layer. Over the subsequent 66 days, commissural neurons are found progressively more dorsally, rostrally, and caudally, so that, by P80, they are present throughout the extent of the neocortex. At this age, they are mainly pyramidal in morphology and form a single band within the deeper part of layer 5 of the developing cortex. From P80 to adulthood, the distribution of commissural neurons has been assessed in the visual cortex by using retrograde transport of horseradish peroxidase. At P80, labelled neurons with immature pyramidal morphology are present throughout the occipital cortex; as in DiI material, somata are located in deep layer 5. At P165, previously shown to be the age when commissural axon numbers peak, widespread labelling is present in the occipital region, with labelled cells now found in two bands corresponding to layers 3 and 5. After this age, neurons become more restricted in distribution, so that, by adulthood, commissural neurons are no longer apparent throughout area 17 but are restricted to a localised region around the area 17/18 boundary. Within this region, labelling is still present in layers 3 and 5 but is more dense in layer 3. The gradual restriction of commissural fields seen here in the wallaby is similar to that reported in the neocortex in many eutherians. These findings also support studies in eutheria, suggesting that subplate neurons do not appear to play a major role in commissural development.

Topics: Aging; Animals; Brain; Brain Mapping; Carbocyanines; Fluorescent Dyes; Horseradish Peroxidase; Macropodidae; Neural Pathways; Neurons; Visual Cortex

The details of the morphology of vertically migrating granule cells were examined semiquantitatively in the postnatal mouse cerebellum by a Golgi method, with special reference to the growth cone-related structures such as filopodia and lamellipodia. The first sign of inward migration was extension of short, vertical filopodium-like processes from the sides of the perikarya of tangentially oriented granule cells, followed by a change of orientation of cell bodies to the vertical axis showing a T-shaped morphology. The T-shaped migratory cells formed sprouted filopodia (side spikes) from their vertical leading processes and perikarya at right angles to the vertical axis. More than three-quarters of the migratory cells extended the side spikes. The presence of such side spikes was confirmed with laser scanning confocal microscopy of granule cells labeled with 1,1', dioctadecyl-3,3,3',3-tetramethylindocarbocyanine perchlorate and also with transmission electron microscopy (TEM). In addition, about one-fourth of migratory cells extended lamellipodia of web-like forms along the stem or at the tip of the leading process, some of which showed a typical growth cone. Several morphological variations of vertical granule cells were also observed. Furthermore, TEM observation confirmed that side spikes from migratory cells made direct contact with parallel fibers. The present results suggest that, during vertical migration, growth cone-related structures of the leading processes of granule cells adhere to and probably recognize tangentially oriented parallel fibers. Therefore, the mechanisms of the vertical guidance and migration of granule cells in the cerebellar cortex seem to be multiple, involving not only parallel contact guidance by the Bergmann glia fibers but also perpendicular contact guidance by the parallel fibers. These parallel and perpendicular geometric cues surrounding the granule cells seem to have produced the varying morphology of vertically migrating granule cells.

Topics: Animals; Carbocyanines; Cell Movement; Cerebellum; Cytoplasmic Granules; Fluorescent Dyes; Mice; Mice, Inbred C3H; Microscopy, Confocal; Microscopy, Electron

Urodele amphibians are the only vertebrates that can regenerate amputated limbs, even as adults. However, we have previously shown that amputated chick wing bud stumps can be induced to ((regenerate)) and to form a complete set of correctly-patterned skeletal elements, following implantation of beads soaked in fibroblast growth factor-4 (FGF-4). We have now performed Dil injection experiments to determine which cells contribute to FGF-4-induced chick wing bud ((regenerates)). We show that the FGF-4-induced outgrowth of the regenerating wing bud stump is comprised of mesenchyme cells that originate from a region within 200 microm of the FGF-4 bead, and that cells proximal to the bead move distally.

Topics: Animals; Carbocyanines; Chick Embryo; Fibroblast Growth Factor 4; Fibroblast Growth Factors; Fluorescent Dyes; Limb Buds; Mesoderm; Microinjections; Proto-Oncogene Proteins; Regeneration; Wings, Animal

To visualize the structure and determine the continuity of lipid membranes in lens fiber cells (LFCs) from human aged normal and cataractous lenses.. Thick sections from human nuclear cataracts and aged normal lenses were stained with the lipophilic probe DiI, and then analyzed by confocal microscopy. Staining patterns of membranes were observed in individual optical sections or three-dimensional projections of z-series taken in longitudinal section and cross-section of LFCs from different regions within the lens nucleus.. DiI bound to and delineated the plasma membrane of LFCs from all regions of the lens nucleus. Three-dimensional projections of z-series from aged normal and cataractous lenses suggested that some of the stained lipid membranes were not continuous with LFC plasma membrane of cataractous lenses.. The results obtained using these methods demonstrated that lipid membranes, discontinuous with the plasma membrane of LFCs, were indicative of a novel process occurring predominately in cataractous human lenses.

Topics: Aged; Aged, 80 and over; Carbocyanines; Cataract; Cell Membrane; Fluorescent Dyes; Humans; Lens Nucleus, Crystalline; Lens, Crystalline; Membrane Lipids; Microscopy, Confocal; Middle Aged

The growth of cerebellar granule cell axons was examined by placing focal implants of 1,1',dioctadecyl-3,3,3',3'-tetramethyl-indocarbocyanine perchlorate (DiI) in the cerebella of normal and staggerer mutant mice at a series of developmental ages between postnatal day 2 (P2) and P30. Parallel fibers contacting the implant site were brightly labeled by the fluorescent dye, as were the associated granule cell bodies located principally in the internal granule layer. The extent of parallel fiber labeling in the molecular layer and the distance from the implant to the most extreme labeled granule cells were measured in sectioned material. Two additional measures describing the distribution of labeled granule cells about the implant site suggest length bounds for most parallel fibers. Parallel fiber growth is surprisingly rapid; all measures approached peak values at P3-P5, only a few days after the earliest postmitotic granule cells differentiate and migrate. At intermediate ages (P8 and P10), parallel fiber lengths appeared to decrease transiently. At later ages (P15 and beyond), the measures of fiber length increased to their mature values. These values differed little from lengths measured at P3-P5, suggesting that most parallel fiber growth occurs within a few days of cell birth. At early and intermediate ages, parallel fiber lengths in staggerer mice were comparable to controls, suggesting that an interaction with normal healthy Purkinje cells is not essential for parallel fiber outgrowth.

Topics: Aging; Animals; Animals, Newborn; Carbocyanines; Cerebellum; Fluorescent Dyes; Mice; Mice, Neurologic Mutants; Nerve Fibers; Reference Values

Topics: Affinity Labels; Animals; Carbocyanines; Chick Embryo; Dissection; Fluorescent Dyes; Microscopy, Fluorescence; Neurons; Spinal Cord

Although the aortic nerves contain vagal afferents that terminate in both the wall of the aortic arch (putative baroreceptors) and its associated glomus tissue (putative chemoreceptors) in most mammalian species, the aortic nerves of the rat have been widely assumed to contain only baro- or pressor afferents. The present study reconsidered this anomaly by characterizing vagal afferent endings and their targets in the aortic arch region of the rat, both qualitatively and quantitatively. Eight Sprague-Dawley rats received intracranial vagal motor rhizotomy unilaterally to eliminate efferents in the nerve and then, two weeks later, injections of the tracer DiI (1,1'-dioleyl-3,3,3',3'-tetramethylindocarbocyanine methanesulfonate) into the ipsilateral nodose ganglion. The aortic arch and its surrounding tissue, with the common carotid and subclavian arteries attached, were examined with both conventional epifluorescence and confocal microscopes. Consistent with earlier observations, vagal afferents formed both flower-spray and end-net terminals rather diffusely within the wall of the aortic arch. More interestingly, vagal afferents also innervated glomus or SIF (i.e., small intensely fluorescent) cell bodies at the junction areas of the common carotid and subclavian arteries. To identify the course of these fibers, six additional animals received DiI injection into the nodose unilaterally after a complete cervical vagotomy caudal to the nodose; in these animals, the aortic nerve had been separated from the vagal trunk and kept intact. There were no marked differences in innervation patterns between the nonvagotomized and the cervically vagotomized animals, indicating that the vagal axons innervating the walls of the blood vessels and the SIF cells in the aortic arch region travel through the aortic nerves. Using a stereological method, we estimated the relative number of chemo- and baroreceptor afferents innervating the aortic arch. About 16.4% (left) and 13.1% (right) of fibers in the aortic nerves innervate SIF cells. These findings challenge the general consensus that the aortic nerves of rats contain exclusively baroreceptor fibers.

Topics: Animals; Aorta, Thoracic; Carbocyanines; Cell Count; Chemoreceptor Cells; Fluorescent Dyes; Functional Laterality; Male; Microscopy, Confocal; Nerve Fibers; Neurons, Afferent; Pressoreceptors; Rats; Rats, Sprague-Dawley; Rhizotomy; Vagus Nerve

We have recently published papers in which sensory neurons that innervate either the tooth pulp or masseter muscle spindles were labelled in vivo and later identified and studied in primary tissue culture (Taddese et al., 1995; Cook et al., 1997). Here, we provide detailed descriptions of cell labelling and tissue culture methods that we used. The purpose of the preparations is to compare nociceptive and non-nociceptive sensory neurons in vitro. The spindles in mastication muscles are the only muscle afferents whose cell bodies reside in the mesencephalic nucleus (MeN5) of the fifth nerve (Corbin and Harrison, J Neurophysiol, 1940; Cody et al., J Physiol, 1972). Thus, labelling neurons projecting to the masseter muscle and dissecting the MeN5 isolates muscle spindle afferents. Pain is the only conscious sensation elicited by physiological stimulus of tooth pulp (Anderson and Matthews, 1967; Edwall and Olgart, 1977; Ahlquist et al., 1984; Narhi et al., 1994); there may be unconscious sensations that arise from the pulp, but these have never been demonstrated. Thus, tooth pulp afferents represent at least a highly enriched, and possibly a pure, population of nociceptors. In broad outline, the methods of labelling and tissue culture are standard, but we have honed many details in order to obtain practical yields.

Topics: Affinity Labels; Animals; Carbocyanines; Cell Culture Techniques; Cell Separation; Cells, Cultured; Centrifugation, Density Gradient; Dissection; Fluorescent Dyes; Injections, Intramuscular; Masseter Muscle; Mesencephalon; Neuroglia; Neurons, Afferent; Rats; Rats, Sprague-Dawley; Solutions; Surface Properties; Tooth Germ; Trigeminal Ganglion

Previous lineage tracing experiments have shown that the vegetal blastomers of cleavage stage embryos give rise to all the mesoderm and endoderm of the sea urchin larva. In these studies, vegetal blastomers were labeled no later than the sixth cleavage division (60-64 cell stage). In an earlier study we showed that single cells in the vegetal plate of the blastula stage Lytechinus variegatus embryo could be labeled in situ with the fluorescent, lipophilic dye, DiI(C18), and that cells labeled in the central region of the vegetal plate of the mesenchyme blastula primarily gave rise to homogeneous clones consisting of a single secondary mesenchyme cell (SMC) type (Ruffins and Ettensohn (1993) Dev. Biol. 160, 285-288). Our clonal labeling showed that a detailed fate map could be generated using the DiI(C18) labeling technique. Such a fate map could provide information about the spatial relationships between the precursors of specific mesodermal and endodermal cell types and information concerning the movements of these cells during gastrulation and later embryogenesis. We have used this method to construct the first detailed fate map of the vegetal plate of the sea urchin embryo. Ours is a latitudinal map; mapping from the plate center, where the mesodermal precursors reside, through the region which contains the endodermal precursors and across the ectodermal boundary. We found that the precursors of certain SMC types are segregated in the mesenchyme blastula stage vegetal plate and that prospective germ layers reside within specific boundaries. To determine whether the vegetal plate is radially symmetrical with respect to mesodermal cell fates, single blastomeres of four cell stage embryos were injected with lysyl-rhodamine dextran (LRD). The resulting ectodermal labeling patterns were classified and correlated with the SMC types labeled. This analysis indicates that the dorsal and ventral blastomers do not contribute equally to SMC derivatives in L. variegatus.

Topics: Animals; Blastocyst; Blastomeres; Carbocyanines; Cell Count; Fluorescent Dyes; Mesoderm; Microscopy, Electron, Scanning; Sea Urchins; Stem Cells; Time Factors

We investigated the potential role of rostral-caudal and dorsal-ventral subdivisions of the early rostral brain by relating these subdivisions to the early patterning of neuron cell bodies and their axon projections. The earliest neurons were mapped using the lipophilic axon tracers diI and diO on embryos fixed on embryonic days 9.5-10.5 (E9.5-E10.5); neuromeric boundaries were marked by diO. The tracts were small in number, were organized orthogonally (2 dorsal-ventral and 4 rostral-caudal), and originated from groups of cell bodies which we term "sources." Two parallel longitudinal axon systems, one dorsal (the tract of the postoptic commissure and the mesencephalic tract of the trigeminal nerve) and one ventral (the mammillotegmental tract and the medial longitudinal fasciculus), projected caudally from the prosencephalon into the rhombencephalon. We argue that the dorsal longitudinal pathway marked the boundary between the alar and basal plates along the entire neuraxis. The dorsal-ventral axons coursed circumferentially and either crossed the midline (forming the posterior and ventral tegmental commissures) or turned caudally without crossing the midline. The dorsal-ventral axons were not generally restricted to the interneuromeric boundaries, as others have suggested. Earlier, all neighboring neurons projected their axons together; later, nearby neurons projected into different pathways. Some tracts originated in single neuromeres, while other tracts had origins in two or more neuromeres. The dorsal longitudinal axons altered course at several of the borders, but the ventral longitudinal axons did not. In summary, the early subdivisions appeared to influence some, but not all, aspects of tract formation.

Topics: Animals; Carbocyanines; Immunohistochemistry; Mesencephalon; Mice; Molecular Probes; Neural Pathways; Neurons; Oculomotor Nerve; Prosencephalon; Trigeminal Nerve; Trochlear Nerve

The recent establishment of an immortalized clonal cell line of rat parotid acinar cells (2RSG) by transfecting isoproterenol-stimulated parotid cells with a plasmid vector, pSV3neo which carries the large T-antigen gene from SV40 virus, afforded the opportunity to develop a model for parotid acinar cell transplantation. Single cell suspensions of 2RSG cells labeled with a fluorescent tracer, DiI, were injected into the parotid gland or oral submucosa of allogeneic adult rats. The grafted cells survived and were functionally viable for at least 30 days. Histological sections revealed no evidence of infiltration of leukocytes or lymphocytes. Grafted cells did not form tumors. Results suggest that allogeneic parotid acinar cell transplantation is a feasible technique in the animal model.

Topics: Animals; Antigens, Polyomavirus Transforming; Carbocyanines; Cell Line, Transformed; Cell Transformation, Viral; Cell Transplantation; Clone Cells; Feasibility Studies; Fluorescent Dyes; Graft Survival; Male; Mouth Mucosa; Parotid Gland; Rats; Rats, Sprague-Dawley; Simian virus 40; Transplantation, Heterotopic; Xerostomia

Teeth are formed by reciprocal interactions between the epithelium and mesenchyme in the first pharyngeal arch. Although the contribution of midbrain and hindbrain crest cells to the first pharyngeal arch has been previously examined in rodent embryos, no direct evidence exists that these cells are actually involved in the dental mesenchyme. In order to elucidate the contribution of the cranial neural crest cells in tooth formation, we first identified the emigration sites and stages providing the crest cells that migrate to the presumed tooth-forming region of the mandibular prominence. Focal labeling with DiI was performed at the midbrain and anterior hindbrain crests in rat embryos, and the labeled embryos were cultured for 30 or 60 hr. The resultant migration patterns indicated that posterior midbrain crest cells emigrating by the end of the 4-somite stage predominantly migrated to the region where tooth buds normally develop. Second, we established a new type of long-term culture system in which whole embryo culture is followed by a mandibular organ culture. Using this system, rat embryos were maintained from the early-somite stage and the molars in the explants were able to reach the bud stage within 8 days. Finally, to ascertain if posterior midbrain crest cells emigrating by the end of the 4-somite stage were involved in the dental mesenchyme, these cells were labeled with DiI and processed for the long-term culture. Labeled crest cells were clearly detectable in the dental mesenchyme. These findings indicate that the early-emigrating posterior midbrain crest cells contribute to mandibular molar tooth development in rat embryos.

Topics: Animals; Carbocyanines; Cell Movement; Female; Fluorescent Dyes; Gestational Age; Mandible; Mesencephalon; Mesoderm; Molar; Neural Crest; Odontogenesis; Organ Culture Techniques; Pregnancy; Rats; Rats, Sprague-Dawley; Rhombencephalon

We have recently characterized a strain of rabbits that shows a low atherosclerotic response (LAR) to dietary hypercholesterolemia in contrast to the usual high atherosclerotic response (HAR) of rabbits [1]. Presently, we have focused on three well established and important stages of atherogenesis, i.e., monocyte adhesion to endothelium, cell mediated peroxidative modification of lipoproteins and induction of a receptor that recognizes modified low density lipoprotein (LDL). The results obtained show that (1) beta-very low density lipoprotein (beta-VLDL) from LAR and HAR rabbits enhanced monocyte adhesion to endothelial cells to the same extent; (2) Cell mediated peroxidation of LDL and beta-VLDL, tested by loss of alpha-tocopherol and formation of thiobarbituric acid reacting substances (TBARS), was compared using macrophages, fibroblasts and smooth muscle cells (SMC) of LAR and HAR rabbits and no significant differences were found; (3) Induction of scavenger receptor by phorbol ester (phorbol 12-myristate 13-acetate (PMA)) and platelet-derived growth factor-BB (PDGF-BB) was determined in SMC or fibroblasts from LAR and HAR rabbits using 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate-acetylated LDL (DiL-acLDL). We found a significantly higher uptake of DiI-acLDL in SMC and fibroblasts derived from HAR rabbits as compared with cells from LAR rabbits. Similar results were also obtained with [125I]-acLDL in fibroblasts from LAR and HAR rabbits with respect to cellular lipoprotein degradation after PMA pretreatment. Even though the attenuated atherosclerotic response to hypercholesterolemia of LAR rabbits may have multiple underlying causes, the most prominent so far is an apparent difference in inducibility of scavenger receptor in SMC and fibroblasts.

Topics: Animals; Arteriosclerosis; Carbocyanines; Cell Adhesion; Cells, Cultured; Diet, Atherogenic; Endothelium, Vascular; Fibroblasts; Gene Expression Regulation; Humans; Hypercholesterolemia; Lipid Peroxidation; Lipoproteins; Lymphoma, Large B-Cell, Diffuse; Macrophages; Membrane Proteins; Monocytes; Muscle, Smooth, Vascular; Platelet-Derived Growth Factor; Rabbits; Receptors, Immunologic; Receptors, LDL; Receptors, Lipoprotein; Receptors, Scavenger; Scavenger Receptors, Class B; Tetradecanoylphorbol Acetate; Thiobarbituric Acid Reactive Substances; Tumor Cells, Cultured; Vitamin E

Resection and re-anastomosis of the bowel interrupts enteric neuronal pathways. The reestablishment of neuronal connections across a colonic anastomosis was studied using immunohistochemical, retrograde tracing and physiological techniques. In control guinea-pig proximal colon, retrograde labelling with 1,1'-didodecyl-3,3,3,3'-tetramethylindocarbocyanine perchlorate (DiI) revealed that enteric neurons with anally-directed projections are more numerous and have longer axons than orally-projecting neurons. In resected bowel, up to 26 weeks after re-anastomosis, descending neuronal pathways were substantially interrupted. Immunohistochemical labelling of nerve fibres revealed that some enteric nerve fibres did regenerate across narrow regions of the anastomosis, growing preferentially in the oral to anal direction. However, nerve fibres immunoreactive for neurofilament protein triplet were substantially depleted in myenteric ganglia anal to the anastomosis, even after the longest recovery periods, demonstrating that axonal regrowth was limited. This was confirmed in retrograde tracing studies, as no nerve cell bodies oral to an anastomosis were labelled when DiI was placed on myenteric ganglia just anal to the anastomosis. Physiological studies confirmed that regrowth of nerve fibres across the anastomosis occurred and that it was asymmetric, as electrical stimulation led to aboral conduction across the anastomosis more reliably than oral conduction, as measured by circular muscle contraction. After resection and re-anastomosis of the colon, the disruption of neuronal pathways in the enteric nervous system was observed, with limited and preferential re-establishment of aborally-directed long connections.

Topics: Anastomosis, Surgical; Animals; Biological Transport; Carbocyanines; Colon; Electric Stimulation; Female; Fluorescent Dyes; Guinea Pigs; Immunohistochemistry; Male; Muscle Contraction; Muscle, Smooth; Nerve Fibers; Nerve Regeneration

The use of traditional neuroanatomic tracing methods with tracers such as horseradish peroxidase requires living systems to take up and transport the label. These tracers have limited application in the study of prenatal systems. The advent of the carbocyanine dye DiI provided a tool by which neuronal pathways may be traced in postmortem fixed tissue. This dye allows careful dissection of prenatal organisms and specific application of the tracer to the neuroanatomic structure under investigation. Although DiI has become increasingly popular, it is limited by the difficulty in applying crystals to peripheral nerves and in the lengthy trace times, particularly in fixed tissue. A chemically modified version of DiI has been introduced that may overcome some of the limitations in using this tracer. The newer dye, fast-DiI, is easy to apply and can trace neural pathways in a shorter period. We describe our experience with the use of fast-DiI in the prenatal rat for the investigation of motoneurons that subserve upper respiratory tract structures. We have determined protocols for fixation, application of dye, processing of tissue, and visualization of traces. The entire protocol can be completed within 1 week, and the use of fast-DiI is easy to learn and apply. The resultant labeling of traced nerves is specific and clearly demonstrates respective motor nuclei and individual motoneurons.

Topics: Animals; Carbocyanines; Cell Movement; Cranial Nerves; Female; Fluorescent Dyes; Motor Neurons; Pregnancy; Rats; Rats, Sprague-Dawley; Respiratory System

Mitral cells are the primary output neurons of the vertebrate olfactory bulb and are major recipients of sensory input from the periphery. The morphogenesis of mitral cell dendrites was followed to elucidate their early spatial and temporal interactions with olfactory receptor neurons and glia during the construction of olfactory glomeruli. Monodelphis domestica, a marsupial born at an extremely immature stage, and rats were examined. Mitral cells were retrogradely labeled by application of the lipophilic dye 1,1' dihexadecyl-3,3,3'3'-tetramethylin-docarbocyanine perchlorate (DiI) to the lateral olfactory tract. In double-labeling experiments, olfactory receptor neurons were stained with 3,3' dihexadecyloxacarbocyanine perchlorate (DiO), or olfactory nerve Schwann cells were visualized using S-100 protein immunohistochemistry. Tissue was examined with a confocal laser scanning microscope. Some preparations were subsequently investigated with an electron microscope. In Monodelphis, differentiation of mitral cells starts with an outgrowth of numerous, uniform, and widespread dendrites. As soon as terminals of olfactory receptor axons coalesce into glomerular knots within the presumptive glomerular layer, dendrites of individual mitral cells innervate several adjacent glomeruli where they receive sensory synaptic input. With maturation, supernumerary mitral cell dendrites retract, leaving one primary dendrite bearing a terminal glomerular tuft. Simultaneously, secondary dendrites begin to arise. The formation of glomeruli begins earlier and progresses faster in the rat compared to Monodelphis. Nevertheless, mitral cell differentiation in both species follows a common sequence: overproduction of dendrites, selection of usually one primary apical dendrite, and elimination of supernumerary processes. Since olfactory receptor neurons form synaptic contacts with the widespread mitral cell dendrites, considerable synaptic rearrangement must occur within the olfactory glomeruli during maturation.

Topics: Animals; Axons; Carbocyanines; Coloring Agents; Dendrites; Fluorescent Dyes; Immunohistochemistry; Lasers; Microscopy, Confocal; Microscopy, Electron; Olfactory Bulb; Olfactory Pathways; Olfactory Receptor Neurons; Opossums; Rats; Schwann Cells

A prospective fate map of the late gastrulation mouse primitive streak has been charted in 8.5 dpc mouse embryos developed in culture, using the lineage marker DiI to label groups of cells. As at earlier stages, the fate of cells in the 8.5 dpc primitive streak is regionalised such that successively more caudal regions of the streak give rise to more lateral mesoderm. While most labelled cells over a 24 or 48 h culture period exit from the primitive streak, some are consistently found to remain within it. The most conspicuous resident population is present in the node. To determine when ingression of ectoderm through the streak ceases, ectoderm cells of the streak and posterior neuropore of 8.5-10.0 dpc embryos were labelled. Involution of surface cells to form mesoderm continues until closure of the posterior neuropore but is not seen thereafter.

Topics: Affinity Labels; Animals; Carbocyanines; Cell Lineage; Ectoderm; Endoderm; Fluorescent Dyes; Gastrula; Mice; Morphogenesis

Neural connections of the small parapineal organ of the adult rainbow trout were experimentally investigated by using a lipophilic carbocyanine dye as a tracer. The dye was applied to the parapineal organ, to the pineal organ, or to the left or right habenular ganglion. The parapineal organ mainly projected via a coarse parapineal tract to a conspicuous neuropil in the rostrodorsal part of the left habenular ganglion. A small accessory parapineal tract projecting to the right habenular ganglion was also found in some animals. No pineal afferents were observed in the parapineal, nor was any neuron in the brain seen to project to this organ. These results suggest a functional relationship of the parapineal organ to the limbic system.

Topics: Animals; Carbocyanines; Fluorescent Dyes; Ganglia; Neural Pathways; Oncorhynchus mykiss; Pineal Gland; Thalamus

The projections of myenteric neurons within the myenteric plexus of the guinea-pig small intestine were established using retrograde tracing in organotypic culture. Three days after applying the fluorescent dye DiI to a single internodal strand in the myenteric plexus, 500-1000 nerve cell bodies were labelled. Of these, 77% were located oral to the application site, 15% were located anally and 7% were located within 1 mm of this site. Three major morphological types of neurons could be distinguished. Dogiel type I neurons had lamellar dendrites and single axons, Dogiel type II neurons had large smooth cell bodies and several long processes, and filamentous neurons had smooth ovoid cell bodies, single axons and several filamentous dendrites. Dogiel type I, II and filamentous neurons accounted for 54.6%, 38% and 7.4% of all filled cells, respectively. Labelled nerve cell bodies were present up to 13 mm aboral to the DiI application site; all neurons more than 2 mm aboral had Dogiel type I features. On the oral side, Dogiel type I neurons were found up to 110 mm, Dogiel type II neurons up to 100 mm and filamentous neurons up to 80 mm. Neurons with 2 mm oral or aboral to the DiI application site were located up to 7 mm circumferentially and were mainly Dogiel type II cells. This work revealed remarkable polarity within the myenteric plexus, with a significant prevalence of myenteric neurons projecting anally for longer distances than those projecting orally. These long pathways are probably involved in the coordination of intestinal motility.

Topics: Animals; Carbocyanines; Female; Fluorescent Dyes; Guinea Pigs; Intestine, Small; Male; Microscopy, Confocal; Microscopy, Fluorescence; Myenteric Plexus; Nerve Fibers; Neural Pathways; Neurons

Immunological studies of human endothelial cells have been performed almost entirely on endothelial cells (EC) derived from foetal (umbilical vein) or adult (aorta) large vessels. Immunologically these EC have in common the fact that they are negative for major histocompatibility complex (MHC) class II antigens (HLA-DR), in contrast to microvascular cells within the heart which are constitutively positive for HLA-DR antigens. Here we describe a simple method for isolating microvascular EC from chunks of adult human ventricle using antibody (anti-HLA-DR) or Ulex lectin-coated magnetic beads. Cytospins of primary isolates reveal the majority of cells binding the beads are CD31+ve, CD36+ve, and HLA-DR+ve. After 14 weeks in culture, 19% of cells remained HLA-DR+ve but CD36 expression had disappeared. Cultures after 4-7 passages were analysed by flow cytometry and immunocytochemical staining of cytospins with mAbs against conventional endothelial antigens (EN4, PECAM-1, vWF, uptake of acetylated LDL) and nonendothelial antigens (CD45, cytokeratin, alpha-actin, desmin, MHC class I and class II). The observation that all the cells remained positive for CD31 and did stain strongly for cytokeratin confirmed their endothelial origin. There was some persistent, albeit low level, expression of HLA-DR which was upregulated by interferon-gamma, showing kinetics of induction similar to that found on large vessel EC. These microvascular cells should be very useful for those wishing to compare microvascular with macrovascular EC and for immunological studies of heart endothelial cells.

Topics: Adult; Antigens, Differentiation, Myelomonocytic; Carbocyanines; CD36 Antigens; Cell Adhesion Molecules; Cell Culture Techniques; Cell Separation; Cells, Cultured; Endothelium, Vascular; Fluorescent Dyes; HLA-DR Antigens; Humans; Interferon-gamma; Keratins; Microcirculation; Myocardium; Platelet Endothelial Cell Adhesion Molecule-1; Recombinant Proteins; Time Factors

Previous studies in the rat femoral nerve have shown that regenerating motor neurons preferentially reinnervate a terminal nerve branch to muscle as opposed to skin, a process that has been called preferential motor reinnervation. However, the ability of sensory afferent neurons to accurately reinnervate terminal nerve pathways has been controversial. Within the dorsal root ganglia, sensory neurons projecting to muscle are interspersed with sensory neurons projecting to skin. Thus, anatomical studies assessing the accuracy of sensory neuron regeneration have been hampered by the inability to reliably determine their original innervation status. A sensory neuron that regenerated an axon into a terminal nerve branch to muscle might represent either an appropriate return of an original sensory afferent to muscle stretch receptors or the inappropriate recruitment of a cutaneous sensory afferent that originally innervated skin. The current experiments used a labeling strategy that effectively labels motor and sensory neurons projecting to a terminal nerve branch before experimental manipulation of the parent mixed nerve. Our results confirm previous observations concerning preferential motor reinnervation for motor neurons, and show for the first time anatomical evidence of specificity during regeneration of sensory afferent projections to muscle. In addition, the accuracy of sensory afferent regeneration was highly correlated with the accuracy of motor regeneration. This suggests that these two distinct neuronal populations that project to muscle respond in parallel to specific guidance factors during the regeneration process.

Topics: Animals; Carbocyanines; Femoral Nerve; Fluorescent Dyes; Hindlimb; Motor Neurons; Muscle, Skeletal; Nerve Regeneration; Neurons, Afferent; Rats; Rats, Sprague-Dawley; Stilbamidines; Synaptic Transmission

Both antennal receptor cell axons and uniglomerular projection neurons of the antennal lobe were specifically labeled, and their synaptic relationship was studied at the fine structural level. The labelings were applied in different combinations: i) Experimentally induced anterograde degeneration of sensory-afferent axons was combined with injection of horseradish peroxidase into uniglomerular projection neurons. ii) Lucifer Yellow was injected into uniglomerular projection neurons, and receptor cell axons were anterogradely labeled with the lipophilic dye DiI. The fluorescent dyes were transformed by immuno- or photochemical treatment into electron-dense markers. In both types of preparations, a considerable number of monosynaptic output synapses from antennal receptor neurons onto processes of uniglomerular projection neurons were identified within the glomeruli of the lobe. In most cases, the receptor axon was connected in a dyadic fashion firstly to a process of a projection neuron and secondly to a nonlabeled process. The results clearly demonstrate a direct connection between receptor cells and output neurons of the cockroach antennal lobe which exists in parallel to the already proposed and demonstrated polysynaptic connection via inhibitory local interneurons.

Topics: Animals; Carbocyanines; Fluorescent Dyes; Male; Microscopy, Electron; Nerve Degeneration; Neural Pathways; Neurons; Olfactory Receptor Neurons; Periplaneta; Sense Organs; Signal Transduction; Synapses

To determine how target-derived nerve growth factor (NGF) affects sensory neuronal survival and the development of topographic nerve projections in the spinal cord, anatomical studies were performed on transgenic mice that overexpress NGF in skin and other keratinized epithelial structures. Transgenic animals showed a 100% increase in the number of sensory neurons in specific dorsal root ganglia and exhibited significantly more fibers immunoreactive for calcitonin gene-related peptide in the dorsal horn compared to control animals. This confirms earlier studies which suggested that naturally occurring sensory neuronal death is decreased, or eliminated, in the transgenic mice. Nerve labeling studies showed that the somatotopic organization of cutaneous nerve projections was not altered in the transgenic animals. These data suggest that neuronal death does not act to remove sensory neurons that project to inappropriate regions of the spinal cord.

Topics: Animals; Carbocyanines; Cell Death; Epidermis; Fluorescent Dyes; Ganglia, Spinal; Immunohistochemistry; Mice; Mice, Transgenic; Nerve Fibers; Nerve Growth Factors; Neural Pathways; Neurons, Afferent

Numerous studies have reported successful allotransplantation of hepatocytes. However, none have shown long-term correction of a liver-related metabolic defect. In this study, we used a method of regional hepatocyte transplantation and subsequent induction of transplanted cell proliferation by regeneration response in the transplant-bearing liver lobes. New Zealand White rabbits were used as cell donors and Watanabe heritable hyperlipidemic (WHHL) rabbits were used as cell recipients (2 x 10(8) cells/rabbit). All recipient rabbits were maintained on daily cyclosporine. Two weeks after baseline serum cholesterol determination, group I WHHL rabbits (n = 7) received an infusion of cells into the right lateral liver lobe, and a loose ligature was placed around the portal venous branch supplying the anterior lobe. After 1 week, to allow engraftment, the portal venous branch was ligated, which resulted in the atrophy of the affected liver parenchyma and induction of hyperplasia in the transplant-bearing liver tissue. Group II rabbits (n = 6) were transplanted with New Zealand White hepatocytes without portal branch ligation (PBL) and group III rabbits (n = 4) were subjected to sham transplantation (saline) and PBL. The experimental period extended to 150 days after transplantation. All WHHL rabbits transplanted with normal hepatocytes showed reduction in serum cholesterol and low-density lipoprotein (LDL) levels. Group I (PBL-stimulated) recipients demonstrated a more pronounced and sustained effect than group II animals (P < 0.05). Group III controls showed only a slight, typical for aging decrease in serum cholesterol. Group I recipient livers perfused with LDL labeled with 1,1'-dioctadecyl-3,3,3',3'-tetramethyl indocarbocyanine perchlorate (DiI) showed much higher numbers of DiI-LDL-positive hepatocytes than those of group II recipients. In conclusion, a liver regeneration stimulus enhanced the population of transplanted hepatocytes and their functional effect in a large animal model of inborn error of liver metabolism.

Topics: Alanine Transaminase; Animals; Carbocyanines; Cell Transplantation; Cholesterol; Fluorescent Dyes; Hypercholesterolemia; Lipoproteins, LDL; Liver; Liver Regeneration; Organ Size; Rabbits

We have studied the intrinsic connections of the anterior dorsal ventricular ridge (ADVR) in the lacertid lizard Psammodromus algirus by means of retrograde transport of horseradish peroxidase (HRP) and fluorescent labeling with the lipophilic carbocyanine dye DiI. We injected HRP into different regions in the ADVR arrayed in a medial-to-lateral sequence, with each consisting of three distinct superficial-to-deep zones. When HRP was injected into a given region, many labeled neurons (always located ipsilateral to the injection site) were found at all mediolateral regions of ADVR in locations rostrally distant from the injection site. DiI crystals were applied on different superficial-to-deep zones within each region. Two patterns could be recognized: DiI crystals applied on the periventricular (most superficial) zone resulted in a labeling of cells widely distributed throughout the ADVR independently of the mediolateral region of the application site, whereas DiI crystals applied on deeper zones resulted in a staining of cells mostly restricted to a narrow radial area. Results from both types of labeling confirm that the ADVR has a prominent radial component in its intrinsic organization, but they also demonstrate that some areas of the ADVR receive projections from distant, rostrally located neurons in every ipsilateral region of the ridge itself, which establishes a clear non-radial component. This organization may have important functional properties with regard to a putative integration of different sensory modalities conveyed by thalamic afferent fibers to the ADVR. Last, we analyzed some evolutionary implications of our results.

Topics: Animals; Carbocyanines; Fluorescent Dyes; Horseradish Peroxidase; Lizards; Telencephalon

The fluorescent carbocyanine dye Dil has been used to retrogradely label the neuronal connections between the forebrain septal area and the interpeduncular nucleus. Previous works based on retrograde horseradish peroxidase transport have identified that only the diagonal band nucleus is a source of the septointerpeduncular projections, but anterograde tracing with labeled amino acids and selective lesions with colchicine have shown that also the posterior septal nuclei project to the interpeduncular nucleus. In the present study, the retrograde labeling in septal nuclei after placing the carbocyanine Dil in the interpeduncular nucleus resulted in the fluorescent labeling of numerous neurons of the diagonal band nucleus. Our results, in addition, showed the labeling of some scattered neurons in the ventral portion of the triangular nucleus of the septal area and in the septofimbrial nucleus, confirming the presence of a previously controversial septointerpeduncular projection.

Topics: Animals; Brain Mapping; Carbocyanines; Fixatives; Fluorescent Dyes; Male; Mesencephalon; Neural Pathways; Neurons, Afferent; Rats; Rats, Wistar; Septum Pellucidum

In tetrapod vertebrates, neural circuitries subserving visceral and somatic reflexes are each represented in distinct columns of cells within the gray area of the spinal cord. To determine the location of visceral elements of the spinal cord of a teleost fish, crystals of the carbocyanine dye 1,1'dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine (DiI), were placed on either the abdominal sympathetic (mesenteric) nerves, the coeliac ganglia, or on the rostral three somatic spinal nerves, in fixed specimens of the channel catfish, Ictalurus punctatus. In fish in which DiI had been placed on the mesenteric nerves, labeled fibers coursed along the lateral margin of the dorsal horn within the first and second spinal segments, and appeared to terminate in a region at the base of the dorsal horn. In contrast, when DiI crystals were placed on the somatic spinal nerves, labeled primary afferents terminated in the dorsalmost two thirds of the dorsal horn, as well as in ventral and ventromedial areas of the medial funicular nuclear complex. Labeled somata (motor neurons) were situated in the ventral horn. When DiI crystals were placed bilaterally on the coeliac ganglia, labeled piriform and fusiform preganglionic neurons occurred in intermediate positions adjacent to the central canal, corresponding to the paracentral nucleus of Herrick, and in the lateral funiculus. These results demonstrate that somatic and visceral afferent and efferent functional columns are distinct in a teleost fish as they are in amniote vertebrates.

Topics: Animals; Autonomic Fibers, Preganglionic; Carbocyanines; Efferent Pathways; Fluorescent Dyes; Ganglia, Spinal; Ganglia, Sympathetic; Ictaluridae; Mesentery; Nerve Endings; Spinal Cord; Splanchnic Nerves; Terminology as Topic; Viscera

The objective of this study was to develop a reliable and sensitive method of diaminobenzidine (DAB) photoconversion through systematic variation of the objectives and the method of DAB enhancement employed. Results showed that reliable photoconversion of DiI-labeled macaque monkey cortical cells and processes can be obtained consistently by using fresh filtered DAB solutions, clean slides, and a bright fluorescent light source. Using a 20x objective and enhancing the DAB reaction product with Giemsa stain provided the most sensitivity and best resolution in the photoconverted area. Objective size was found to correlate directly with sensitivity and indirectly with both the size of the area photoconverted and the time to complete photoconversion. Nitro blue tetrazolium (NBT) did not produce as much contrast in the photoconverted cells as Giemsa stain, but allowed for the best visualization of finer processes because it somewhat enlarged the fiber diameter. Nickel and cobalt used together, but not separately, were found to provide adequate enhancement. These results are likely to apply to photoconversion and enhancement of all fluorescent material, but may require slight modifications in protocol.

Topics: 3,3'-Diaminobenzidine; Animals; Azure Stains; Carbocyanines; Fluorescence; Fluorescent Dyes; Lighting; Macaca nemestrina; Neurosciences; Nitroblue Tetrazolium; Sensitivity and Specificity; Visual Cortex

Allocation of cells in the marsupial epiblast to embryonic and extra-embryonic domains has to date been studied only histologically. An unresolved issue in marsupial embryology has been the existence of a medullary plate. We re-examined the hypotheses that the medullary plate, or neurectoderm, arises before notochord formation and that the integumentary ectoderm is segregated from the ectoderm after the formation of the medullary plate. By marking epiblast cells in 65 Day-8 embryos of the dasyurid marsupial Sminthopsis macroura, with the lipophilic cell-surface marker, DiI, we demonstrated that the so-called medullary plate contains progenitors of the integumentary ectoderm of the neck, chest, limbs and flank of the embryo. Thus, cell-allocation processes in the epiblast must have segregated the entire complement of embryonic ectoderm in one event, not separate events. It is concluded that the embryonic structure called 'medullary plate' in marsupial embryos is misnamed since, in fact, it consists of both integumentary ectoderm and neurectoderm.

Topics: Animals; Blastocyst; Carbocyanines; Central Nervous System; Ectoderm; Embryo, Mammalian; Embryonic and Fetal Development; Fluorescent Dyes; Marsupialia; Mesoderm

Several neuronal tracing substances were applied to the cut ends of leech cephalic nerves and the resulting backfills into the subesophageal ganglion (sbEG) were mapped. A 12 h incubation in 3 kDa dextrans conjugated either to a fluorochrome or to biotin (subsequently tagged with peroxidase) was satisfactory. In separate experiments, possible targets of cephalic nerve afferents (R3 Retzius neurons) were injected with Lucifer Yellow (LY) to visualize their projections. Comparison of the LY-R3 Retzius neuron map with that of the dextran-backfilled D1 nerve revealed extensive overlap in the sbEG. Experiments were performed combining the two protocols, confirming this observation. Moreover, confocal microscopy placed D1 nerve processes in close proximity to R3 Retzius neuron processes, suggesting that they could make synaptic contact with one another in the sbEG. With modifications, this method could be used to identify such contacts using electron microscopy.

Topics: Animals; Biotin; Carbocyanines; Dextrans; Electrophysiology; Fluorescent Dyes; Ganglia, Invertebrate; Isoquinolines; Leeches; Microelectrodes; Microscopy, Electron

The habenula is a conserved structure in the brain of vertebrates. With the aim of further understanding of the evolution of the habenular system in vertebrates, we studied the afferent and efferent connections of the habenula of the rainbow trout. Experiments included application of the carbocyanine dye 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI) into the habenula, telencephalon, pineal organ, posterior tubercle, and interpeduncular nucleus (IPN). The results obtained reveal a consistent pattern of habenular connections. Most afferents originate from three nuclei, one extending from the preoptic region to the rostral thalamus (the entopeduncular nucleus), the second located in the region of the hypothalamus-posterior tubercle and consisting of large bipolar cells (tuberculohabenular nucleus), and the third in the preoptic region (preoptic nucleus). A few large neurons of the locus coeruleus appeared to be labeled in some cases. The trout habenula also receives pineal and parapineal projections. Small labeled glial cells were observed in the thalamus around the fasciculus retroflexus and, sometimes, around the IPN. The most conspicuous efferents coursed in the fasciculus retroflexus to the IPN, the isthmal raphe, and the central gray. The existence of olfactohabenular or habenulotelencephalic projections is discussed.

Topics: Afferent Pathways; Animals; Carbocyanines; Efferent Pathways; Fluorescent Dyes; Habenula; Neurons, Afferent; Oncorhynchus mykiss

The enteric nervous system (ENS) of the grasshopper Schistocerca americana is organized into four ganglia located in the foregut (the dorsal unpaired frontal and hypocerebral ganglia, and the paired ingluvial ganglia), and two plexuses that innervate the foregut and midgut. A dorsomedial recurrent nerve and two lateral esophageal nerves connect the ganglia. The midgut plexus is arranged in four nerves running along the midgut surface. In this study, we have focused on the embryonic development of the grasshopper ENS; we have studied the proliferation pattern, morphogenesis, and some aspects of neuronal differentiation by using a number of specific molecular markers. The grasshopper ENS develops early in embryogenesis (25-30%) from three neurogenic zones (NZs) located on the roof of the stomodeum. These NZs slightly invaginate from an epithelial placode. The expression pattern of specific cell surface proteins and the analysis of the mitotic activity showed that NZs cells delaminate from the epithelium, become neuronal precursors, divide symmetrically, and then actively migrate to their final position in the enteric ganglia or plexuses. The grasshopper enteric ganglia are composed of mixed populations of cells from different NZs. The foregut and midgut plexuses are formed by the dispersal of cells from the developing hypocerebral and ingluvial ganglia. The main ENS nerves are pioneered by axons extending anteriorly from hypocerebral and ingluvial neurons. The insect ENS exhibits an enormous variation in design. Several features of the grasshopper program of neurogenesis and pattern of cell migration are compared to other insects, and some evolutionary implications are discussed.

Topics: Animals; Bromodeoxyuridine; Carbocyanines; Cell Differentiation; Digestive System; Embryo, Nonmammalian; Enteric Nervous System; Fluorescent Dyes; Ganglia, Invertebrate; Grasshoppers; Immunohistochemistry; Iontophoresis; Morphogenesis; Neural Pathways; Neurons

The epigenetic signals and progenitor cell species involved in progressive neural maturation in the mammalian brain are poorly understood. Although these complex developmental issues can be examined in cultures of generative zone progenitor cells, analysis of signaling relationships in complex progenitor cell systems requires the meticulous definition of the cellular complement at each developmental stage. The presence of microglia within the generative zone cultures would further complicate these developmental analyses. Utilizing the microglial markers Griffonia simplicifolia B4 isolectin, carbocyanine dye-acetylated low density lipoprotein, F4/80, and Mac-1 we now report the presence of microglia within cultures of late embryonic murine epidermal growth factor-derived generative zone progenitor cells. Cytokine treatment of serially passaged epidermal growth factor-generated neurospheres altered the phenotype of the microglia in culture. Macrophage colony-stimulating factor treatment promoted the expression of spindle-shaped microglia, whereas granulocytemacrophage colony-stimulating factor treatment promoted the elaboration of flat and amoeboid microglia. Treatment with microglial-conditioned medium or 10% non-heat inactivated fetal calf serum led to an increased complement of both phenotypes. Microglia could be generated from single isolated neurospheres, and there were differences in the number of microglial lineage species obtained from distinct oligopotent progenitor cells, raising the possibility that a complement of this cellular lineage may be derived from a progenitor cell present within the generative zones. These observations indicate that microglia are present within the generative zone progenitor cell system, and this system thus represents an important experimental resource to examine the progenitor cell maturation and the origin of the microglial lineage.

Topics: Animals; Carbocyanines; Cell Lineage; Cells, Cultured; Epidermal Growth Factor; Immunohistochemistry; Lectins; Mice; Mice, Inbred Strains; Microglia; Neurons; Phenotype; Plant Lectins; Stem Cells

The aim of this study was to investigate how different levels of K(+)-induced depolarization affect the survival and growth of isolated, cultured thalamic explants from mice aged embryonic day 13 to postnatal day 2. K+ was added to explants in serum-free culture medium. After culture for three days, explants were sectioned and Nissl-stained or photographed under phase contrast for quantification of neurite outgrowth. Viable and pyknotic cells were counted in sectioned material. The results revealed that, with no added K+, both viability and neurite outgrowth decreased as the age of the thalamic explant increased: most cells survived in embryonic day 13 explants, most died in postnatal day 2 explants. Adding K+ had an age- and dose-dependent effect on viability and neurite outgrowth. The greatest viability-promoting effect of adding K+ was at embryonic day 19: adding 5 mM K+ rescued the majority of these cells, although there was no effect on neurite outgrowth at this age (i.e., enhanced viability did not necessarily produce increased outgrowth). This same dose of K+ had its greatest effect on neurite outgrowth at embryonic day 17. No dose of added K+ had a stimulatory effect on viability and neurite outgrowth after embryonic day 19. The highest dose of K+ used here (50 mM) inhibited thalamic cell survival. We suggest that the survival and growth of the prenatal thalamus can occur without external influences. This intrinsic control may use an autocrine mechanism that becomes increasingly reliant on neural activity for its maintenance as it ages. After birth, when thalamic cells may switch their dependence to cortex-derived growth factors, this intrinsic control may become ineffective.

Topics: Animals; Carbocyanines; Cell Survival; Female; Histocytochemistry; Membrane Potentials; Mice; Mice, Inbred BALB C; Neurites; Neurofilament Proteins; Organ Culture Techniques; Osmolar Concentration; Potassium; Pregnancy; Stimulation, Chemical; Thalamus

The molecular mechanisms that underlie dentate granule cell axon (i.e., mossy fiber) growth during development and following seizure-induced hippocampal injury remain unknown. Part of this process may involve specific factors that support dentate granule cells during differentiation, and molecular cues that allow the appropriate growth of mossy fiber axons toward their targets. To study this process, we developed an in vitro assay system to measure the activity of putative trophic, chemoattractant and chemorepulsive factors. Two-hundred-micrometer-thick transverse hippocampal sections were prepared from neonatal rats and microdissected to isolate the middle one-third of the superior blade of the dentate granule cell layer. These were embedded in a three-dimensional collagen matrix either alone or with microdissected regions of the CA2 pyramidal cell layer. Cultures were maintained in a defined medium and grown for two to three days in a standard culture environment. Results showed that numerous processes grew primarily from the hilar side of explants into the collagen matrix, often in excess of 500 microns in length. These were determined to be axons based on: (i) morphological criteria including size and presence of growth cones, (ii) synaptophysin and growth-associated protein-43 immunoreactivity, (iii) lack of glial fibrillary acidic protein immunoreactivity and (iv) contiguity of biocytin-filled processes with neuronal soma within the explant. Treatment of cultures with brain-derived neurotrophic factor caused a significant increase in axon number and length, and this effect was partially reversed by the addition of a trkB-immunoglobulin fusion protein that blocks the activity of brain-derived neurotrophic factor and neurotrophin-4/5. Basic fibroblast growth factor also caused a marked increase in axon number and length, and caused a migration of neuron-like cells out of the explant into the collagen. These results show that cultured dentate granule cell layer explants are capable of growing mossy fibers into a neutral collagen matrix, and the growth of axons can be modified by the addition of exogenous growth factors. Furthermore, since target tissue and point sources of purified factors can easily be co-cultured with the explants, this new system provides a direct means for testing the molecular cues that influence mossy fiber growth.

Topics: Animals; Axons; Brain-Derived Neurotrophic Factor; Carbocyanines; Collagen; Cytoplasmic Granules; Dentate Gyrus; Fibroblast Growth Factor 2; Immunohistochemistry; Lysine; Neural Pathways; Organ Culture Techniques; Pyramidal Cells; Rats; Receptor, Ciliary Neurotrophic Factor; Receptors, Nerve Growth Factor

Fibers coming from the olfactory epithelium and entering the olfactory bulb expressed peripherin. These were not sensory olfactory fibers because: (1) they did not form glomeruli, (2) they did not express olfactory marker protein (OMP), (3) they entered deeply into the olfactory bulb, reaching its white matter. Cells and fibers in the olfactory bulb expressing luteinizing hormone-releasing hormone (LHRH) differed from those expressing peripherin because LHRH and peripherin did not coexist and the distributions of LHRH-positive and peripherin-positive fibers were found to be different.

Topics: Animals; Carbocyanines; Eye Proteins; Gonadotropin-Releasing Hormone; Immunohistochemistry; Intermediate Filament Proteins; Membrane Glycoproteins; Nerve Fibers; Nerve Tissue Proteins; Olfactory Pathways; Peripherins; Rats; Rats, Wistar

Lipoproteins labeled with 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI) are widely used to visualize LDL-and scavenger-receptor activity in cultured cells. The purpose of this study was to evaluate a new single-step fluorometric assay with high sensitivity for the quantitative determination of the LDL- or scavenger-receptor activity in adherent and non-adherent cells. We used an aqueous solution of 1 g/l SDS dissolved in 0.1 M NaOH to lyse the cells after incubation with DiI-LDL or DiI-acetylated LDL. This allows for the first time the determination of fluorescence intensity and cell protein in the same sample without prior lipid extraction of the fluorochrome. Fluorescence of the cell lysates was determined in microtiter plates with excitation-emission set at 520 and 580 nm, respectively. This rapid method demonstrates high specificity for determining the LDL- and scavenger-receptor activity in cultured cells (e.g., human skin fibroblasts from patients with and without familial hypercholesterolemia; human U-937 monocyte and murine P388 D1 macrophage cell lines). The validity of our fluorescence assay is demonstrated by comparison of cellular uptake and metabolism of lipoproteins labeled with both, DiI and 125iodine. The rapidity and accuracy of this assay allows its routine application for studying receptor-mediated lipoprotein uptake in various cell types.

Topics: Animals; Carbocyanines; Cell Adhesion; Cell Line; Cells, Cultured; Fibroblasts; Fluorescent Dyes; Humans; Hyperlipoproteinemia Type II; Macrophages; Membrane Proteins; Mice; Monocytes; Receptors, Immunologic; Receptors, LDL; Receptors, Lipoprotein; Receptors, Scavenger; Scavenger Receptors, Class B; Skin; Spectrometry, Fluorescence

We show that the lipophilic, cationic fluorescent dyes R18 and Dil translocate from one monolayer of a phospholipid bilayer membrane to the other in a concentration and voltage-dependent manner. When the probes were incorporated into voltage-clamped planar membranes and potentials were applied, displacement currents resulted. The charged probes sensed a large fraction of the applied field. When these probes were added to only one monolayer, displacement currents were symmetrical around 0 mV, indicating that the probes distributed equally between the two monolayers. Charge translocation required that the bilayer be fluid. When membranes were in a condensed gel phase, displacement currents were not observed; raising the temperature to above the gel-liquid crystalline transition restored the currents. Translocation of R18 was also shown by fluorescence measurements. When R18 was in the bilayer at high, self-quenching concentrations, voltage pulses led to voltage-dependent fluorescence changes. The kinetics of the fluorescence changes and charge translocations correlated. Adding the quencher I- to one aqueous phase caused fluorescence to decrease or increase when voltage moved R18 toward or away from the quencher at low, nonquenching concentrations of R18. In contrast to R18, Dil incorporated into bilayers was a carrier fo I-, and hence I- altered Dil currents. Voltage-driven translocations allow R18 and Dil to be used to probe membrane potential changes.

Topics: Carbocyanines; Fluorescent Dyes; Iodides; Lipid Bilayers; Membrane Lipids; Membrane Potentials; Membranes, Artificial; Phosphatidylcholines; Rhodamines; Solubility; Spectrometry, Fluorescence

This study in lampreys (Lampetra fluviatilis L.) is concerned with the phylogenetic age of the suprachiasmatic nucleus of the hypothalamus which in mammals receives input from the retinohypothalamic tract and acts as circadian pacemaker. Tracing experiments with the fluorescent dye DiI demonstrated a retinohypothalamic projection in lampreys which terminates in a distinct area of the hypothalamus corresponding to the infrachiasmatic part of the nucleus of the postoptic commissure. Immunocytochemical investigations showed vasotocinergic neurons and a relatively high concentration of neuropeptide Y-, substance P- and serotonin-immunoreactive nerve fibers in this region. These hodological and immunocytochemical data suggest that the infrachiasmatic part of the nucleus of the postoptic commissure of lampreys might be homologous to the suprachiasmatic nucleus of other craniates.

Topics: Animals; Carbocyanines; Hypothalamus; Immunohistochemistry; Lampreys; Neural Pathways; Neuropeptides; Serotonin; Suprachiasmatic Nucleus

In the present paper, Dil-labeled myelin-forming cells were traced after their transplantation at a distance from a lysolecithin induced lesion in the adult wild-type and shiverer mouse spinal cord. Optical and ultrastructural observations indicate that after their transplantation, Dil-labeled Schwann cells and oligodendrocyte progenitors were found at the level of the graft as well as at the level of the lesion thus confirming that myelin-forming cells were able to migrate in the adult lesioned CNS (Gout et al., Neurosci Lett 87:195-199, 1988). Between the graft and the lesion, labeled Schwann cells and oligodendrocyte progenitors were absent in the gray matter, but were found as previously described, in specific locations (Baron-Van Evercooren et al., J Neurosci Res 35:428-438, 1993; Vignais et al., J Dev Neurosci 11:603-612, 1993). Both cell types were found along blood vessel walls and more precisely in the Virchow-Robin perivascular spaces. They were identified in the meninges among meningeal cells, collagen fibers, or occasionally in direct contact with the basement membrane forming the glia limitans. In addition to these findings, three major observations were made. In the ependymal region, myelin-forming cells were localized between or at the basal pole of ependymocytes. While Dil-labeled oligodendrocyte progenitors were noted to migrate along the outer surface of myelin sheats in CNS wild-type and shiverer white matter, Schwann cells were excluded from this structure in the wild-type mouse spinal cord. Moreover, in the shiverer mouse, migrating Schwann cells did not seem to interact directly with myelin sheats nor with mature oligodendrocytes. Finally, both cell types were seen to invade extensively the spinal peripheral roots. Our ultrastructural observations clearly suggest that multiple cell-cell and cell-substrate interactions rule the migration of myelin-forming cells in the adult CNS infering that multiple mechanisms are involved in this process.

Topics: Animals; Carbocyanines; Cell Movement; Cell Transplantation; Cells, Cultured; Demyelinating Diseases; Lysophosphatidylcholines; Mice; Mice, Inbred Strains; Myelin Sheath; Oligodendroglia; Rats; Schwann Cells; Spinal Cord; Stem Cell Transplantation; Stem Cells

Secondary macrophage cell cultures were generated from the primary culture of epiblasts of 8-d-old pig blastocysts. The epiblast-derived macrophagelike (EDM) cells have a morphology and ameboid behavior that is typical of tissue histocytes. The cells reacted positively with monoclonal antibodies specific for pig granulocyte-macrophage lineage cells, and were not reactive with monoclonal antibodies specific for pig B and T lymphocytes. Marked phagocytic behavior and the formation of phagosomes were demonstrated following incubation with FITC-labeled bacteria. The EDM cells stained positively for nonspecific acid esterase that was not inhibited by sodium fluoride. DiI-acetylated-LDL was rapidly taken up by the cells. Transmission electron microscopy of the EDM cells showed phagolysosomes, numerous cytoplasmic vacuoles, large, lobed nuclei, and numerous pseudopods or filopodia at the cell surface. Strong reactivity of the cells with anti-CD14 monoclonal antibody was observed. Further, cytotoxic activity was produced from the EDM cells after exposure to lipopolysaccharide in a concentration and time-dependent manner. The cultures could be maintained and expanded for several months on STO co-culture. Their derivation from the epiblast of the pig demonstrates the possibility of obtaining hemopoietic cell cultures from the preimplantation blastocysts of all mammals.

Topics: Acetylation; Animals; B-Lymphocytes; Carbocyanines; Cells, Cultured; Cytotoxicity Tests, Immunologic; Histocompatibility Antigens Class I; Histocompatibility Antigens Class II; Lipopolysaccharide Receptors; Lipoproteins, LDL; Macrophages; Microscopy, Electron; Naphthol AS D Esterase; Phagocytosis; Receptors, Immunologic; Swine; T-Lymphocytes; Tumor Necrosis Factor-alpha

Living organotypic cocultures of rat thalamic and cortical explants were used to examine the effects of blocking action potential activity on the morphological development of axons in the mammalian neocortex. Studies in vivo have suggested that blocking sodium channel-dependent activity influences the growth characteristics of thalamocortical axons during development. We have extended these observations by using an in vitro system that affords more direct observational analysis of the early events of axonal growth in an accessible cellular environment DiI-labeled thalamocortical axons grow exuberantly into the target cortex and establish axonal connections that reflect the events of early thalamocortical afferent development. Within these cocultures, the morphological features of DiI-labeled axons can be readily distinguished. Tracings of thalamocortical axons were quantitated with respect to number, length, and termination pattern of axonal branches, as well as number of varicosities. Addition of the voltage-dependent sodium channel blocker, tetrodotoxin, to cocultures did not change the general pattern of thalamocortical axonal ingrowth or the average length of collateral branches of these axons. However, in the presence of tetrodotoxin, axons were more highly branched, with an increased number of varicosities as compared to untreated cocultures. This pattern of axonal growth and branching may reflect the activity-dependent fine-tuning and trimming of collaterals that occur as thalamic afferents begin to refine their cortical territory. Our observations in thalamocortical cocultures are consistent with the view that neuronal activity modulates the pattern of axonal growth and development.

Topics: Animals; Axons; Carbocyanines; Cerebral Cortex; Coculture Techniques; Fluorescent Dyes; Image Processing, Computer-Assisted; Microscopy, Fluorescence; Rats; Rats, Sprague-Dawley; Sodium Channels; Tetrodotoxin; Thalamus

Previous studies have suggested that monocytes or macrophages may mediate internal virus spread. For the present study, the tissue distribution and infectious potential of dye-labelled and/or lactate dehydrogenase-elevating virus (LDV)-infected murine macrophages were determined. Murine peritoneal macrophages were labelled with the fluorescent carbocyanine tracking dye Dil, injected into mice, and the tissue distribution of Dil-labelled cells was determined by fluorescence analysis of frozen sections. Mice receiving intravenous (i.v.) or intraperitoneal injections of Dil-labelled macrophages displayed rapid and broad tissue distribution of the labelled cells. Intravaginal injection of Dil-labelled macrophages resulted in penetration into the placentas, but not the fetuses, of pregnant mice. When macrophages were LDV-infected and Dil-labelled prior to i.v. injection into pregnant mice, they homed to various tissues including the placenta, but were not found in fetuses. Intravaginal injection of LDV-infected macrophages resulted in systemic LDV infection, even though the free-virus dose was less than the minimum infectious dose by this route. Neither polyclonal nor monoclonal IgG anti-LDV antibodies protected mice from vaginal infection with cell-associated virus, and LDV-immune complexes were themselves infectious by the vaginal route. These results show that exogenous macrophages are widely distributed following parenteral injection, penetrate locally to placentas after intravaginal injection, and are capable of acting vaginally as relatively efficient virus infection-delivery vehicles. Thus, 'Trojan Horse' macrophages are potentially infectious vehicles both for internal virus spread and for animal-to-animal transmission.

Topics: Animals; Antibodies, Viral; Arterivirus Infections; Carbocyanines; Drug Administration Routes; Female; Fluorescent Dyes; Injections, Intraperitoneal; Injections, Intravenous; Lactate dehydrogenase-elevating virus; Macrophages, Peritoneal; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Placenta; Pregnancy; Tissue Distribution; Vagina

A double immunocytochemical procedure, with two different chromogens, was used to compare the respective distribution of estrogen receptor-immunoreactive cells and tyrosine hydroxylase-immunoreactive neurons on the same sections of the preoptic region of adult female rainbow trout (Oncorhynchus mykiss). Estrogen receptor-immunoreactive cells were observed in the anterior preoptic region surrounding the preoptic recess and its large lateral extensions. Tyrosine hydroxylase-immunoreactive cells were consistently detected in the ventral and ventrolateral walls of the preoptic recess, in an area that was named nucleus preopticus pars anteroventralis. Dopamine immunohistochemistry and Dil retrograde transport studies indicated that part of these catecholaminergic neurons are dopaminergic and could project to the pituitary. Double staining studies showed consistently that most estrogen receptor-positive cells located ventral to the large extensions of the preoptic recess are also tyrosine hydroxylase-positive, indicating that this region is a major target for estradiol feedback. The results are discussed in relation to the role of the nucleus preopticus pars anteroventralis in mediating the negative feedback actions of estradiol on the secretion of gonadotrophin (GTH2) secretion. A hypothesis is drawn in order to explain the synchronizing role of estradiol at the time of ovulation in rainbow trout.

Topics: Animals; Antibody Specificity; Carbocyanines; Dopamine; Female; Immunohistochemistry; Neurons; Oncorhynchus mykiss; Preoptic Area; Receptors, Estrogen; Tyrosine 3-Monooxygenase

In this study we investigated changes to horizontal cells in human retinae affected by glaucoma. Glaucoma is characterized by raised intraocular pressure and is responsible for retinal ganglion cell and, possibly, photoreceptor degeneration. It was therefore assumed that horizontal cells might also be affected. The carbocyanine dye DiI was placed at discrete points on fixed, whole-mounted retinae obtained from normal and glaucomatous patients. After allowing 6-24 weeks for intramembranous diffusion within the lipid layers of the nerve cells and, therefore, fluorescent labeling, we measured horizontal cell soma and dendritic field sizes. Selected cells were then embedded in Araldite and cut at 4 microns. Horizontal cells in glaucomatous eyes appeared larger and had a granulated outline as compared with cells from normal retinae. Analysis of the mean cell soma size indicated that cells were 26% larger in the glaucomatous retinae and that this increase was significantly different from that seen in normal retinae (P < 0.05). The dendritic field size was unaffected (P > 0.05). As seen in cross section there was a clear loss of photoreceptor outer segments, and shrunken silhouettes of photoreceptor inner segments with pyknotic nuclei were observed. It is proposed that the increase in some size is indicative of horizontal cell responses that are likely to culminate in degeneration as a result of heightened intraocular pressure. In addition, this paper provides further evidence that photoreceptors are affected by advanced glaucoma.

Topics: Carbocyanines; Cell Size; Dendrites; Fluorescent Dyes; Glaucoma; Humans; Neurons; Photoreceptor Cells; Retinal Degeneration

The transport pathways of low density lipoproteins (LDL) across the endothelium at the branched and unbranched regions of the artery were studied in high cholesterol diet-fed rats. Rat tissues were analyzed by perfusing in situ human or rat LDL labeled with colloidal gold or fluorescein 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI). Results indicated that more LDL-DiI accumulated in the branched regions than in the unbranched regions of the artery. LDL-gold conjugates were observed in the plasmalemmal vesicles, multivesicular bodies and in the subendothelial space in both the branched and the unbranched regions of the arteries. Quantitative study revealed that the volume densities of plasmalemmal vesicles which contained the LDL-gold particles in the branched regions of the aortic arch were significantly (P < 0.05) higher than the density value in the unbranched regions of the thoracic aorta, whereas there was no marked difference in the density value of multivesicular bodies between these two regions. The open junctions with gap widths of 30-450 nm between adjacent endothelial cells were only observed in the branched regions of the aortic arch, whereas no open junctions were present in the unbranched regions of the thoracic aorta. Moreover, the LDL-gold conjugates were present within most of these open junctions. In all specimens examined, no gold particles were found in the normal intercellular channels (i.e., 25 nm and less) of both regions. These results indicated that the major visible routes for transport of LDL across the endothelium in the branched regions of the arteries are open junctions as well as plasmalemmal vesicles. The region-associated permeability changes of LDL might account for the incidence of atherosclerosis in the branched areas of arteries.

Topics: Animals; Aorta, Thoracic; Biological Transport; Body Weight; Carbocyanines; Cholesterol, Dietary; Diet, Atherogenic; Endothelium, Vascular; Fluorescent Dyes; Humans; Hypercholesterolemia; Immunohistochemistry; Intercellular Junctions; Lipoproteins, LDL; Male; Microscopy, Fluorescence; Rats; Rats, Sprague-Dawley

Sensory innervation of gingival tissue can cause neurogenic inflammation that depends on the extent of the branching area of the peripheral nerve fibers. We designed the present study to determine whether single trigeminal axons branch to both the buccal and palatal gingiva of maxillary molars of adult rats. Accumulation via retrograde transport of DiI (red) or DiA (green) fluorescent carbocyanine dyes in neurons of trigeminal ganglia was evaluated 7 days after applying one dye to the buccal sulcus and the other to the palatal sulcus. Both dyes were absorbed through the junctional epithelium, and the two sites each labeled similar numbers and sizes of neurons in the lateral zone of the maxillary division (44% from buccal and 46% from palatal gingiva). Double-labeled neurons had the same size (32.5 +/- 6.70 microns, mean circumference +/- S.D.) and location as single-labeled neurons, and they were 9% of the total. This study shows that exogenous dyes can diffuse into mucosa and thereby allow in vivo mapping of sensory nerve branching patterns to several intact tissues per animal. We found that 9% of the labeled cells extended to both the buccal and palatal gingiva. Thus, inflammation that spreads from one gingival region to the other could have a neurogenic mechanism involving trigeminal sensory neurons that extend their peripheral branches to innervate both buccal and palatal gingiva of adult rat molars.

Topics: Absorption; Animals; Axonal Transport; Axons; Carbocyanines; Epithelial Attachment; Fluorescent Dyes; Gingiva; Gingivitis; Male; Molar; Mouth Mucosa; Nerve Fibers; Neurons; Neurons, Afferent; Palate; Pyridinium Compounds; Rats; Rats, Sprague-Dawley; Trigeminal Ganglion; Trigeminal Nerve

There has been controversy about the length and structure of the odontoblast process within dentin since the earliest histologic studies of teeth. Our objective was to use the fluorescent carbocyanine dye Di-I combined with a new gelatin embedment procedure and confocal microscopy to determine the structure and extent of odontoblast processes in developing and mature rat teeth, injured rat molars, reparative dentin, and adult monkey teeth. We found that odontoblast processes do not extend into outer dentin or to the dentin-enamel junction except during early stages of development. Those in innervated regions of crown are long and straight, whereas those in roots are extensively branched and shorter. Cavity injury to crown dentin caused odontoblast fragments to be aspirated into outer dentin. In reparative dentin the odontoblast processes were branched and similar to those in roots. We used photoconversion and electron microscopy to show that Di-I fills the entire odontoblast after gelatin embedment, including the cytoplasm. This is a different type of carbocyanine staining from any previously reported, and it also stains other cells in adjacent hard tissues such as bone and cementum. The Di-I-gelatin method is a new way to use carbocyanine dyes. It has enabled us to solve a long-standing controversy about the histology of teeth, and it should be useful for many other studies of cell structure.

Topics: Animals; Carbocyanines; Dental Caries; Dentin; Dentin, Secondary; Female; Fluorescent Dyes; Gelatin; Incisor; Macaca mulatta; Male; Microscopy, Confocal; Molar; Odontoblasts; Rats; Rats, Sprague-Dawley; Staining and Labeling; Tissue Embedding; Vimentin

The chicken locus coeruleus contains a population of noradrenergic neurons which express the neurotrophin receptor p75 (von Bartheld and Bothwell, 1992). To determine which neurotrophin may regulate the development of noradrenergic neurons in the chicken locus coeruleus, expression of trk receptors, retrograde transport of neurotrophins, and responses to NGF were examined. P75-expressing noradrenergic neurons were found to project to the basal forebrain. They transport radio-iodinated NGF after injections into this target. The retrograde transport of NGF is specific to the noradrenergic neuronal population as evidenced by double labeling with antibodies against dopamine-beta-hydroxylase. The same neuronal population expresses trkA receptor mRNA. The size of noradrenergic neurons in the locus coeruleus proper, but not in the nucleus subcoeruleus, is significantly increased after injections of NGF into the telencephalon, consistent with the hypothesis that target-derived NGF provides trophic support. Noradrenergic coeruleus neurons are rescued from toxic effects of 6-hydroxydopamine injected into the telencephalon when NGF is injected into the midbrain. NGF has no rescue effect when it is coinjected with 6-hydroxy-dopamine into the telencephalon. In explant or dissociated cultures, noradrenergic coeruleus neurons do not respond to elevated levels of NGF with increased neurite outgrowth. Taken together, these results suggest that NGF plays a role in the development and maintenance of noradrenergic coeruleus neurons in the chick brain. The data also support our previous conclusion that major species differences exist between birds (chicken) and mammals with regard to trophic regulation of presumptive homologous neuronal populations.

Topics: Animals; Axonal Transport; Base Sequence; Biological Evolution; Brain Mapping; Carbocyanines; Chick Embryo; Chickens; Dopamine beta-Hydroxylase; Enzyme Induction; Gene Expression Regulation; Locus Coeruleus; Mammals; Molecular Sequence Data; Nerve Growth Factors; Neurites; Neurons; Norepinephrine; Oxidopamine; Peripheral Nerves; Proto-Oncogene Proteins; Receptor Protein-Tyrosine Kinases; Receptor, trkA; Receptors, Nerve Growth Factor; RNA, Messenger; Species Specificity

Previous studies have suggested that the rostrocaudal patterning of branchial arches in the vertebrate embryo derives from a coordinate segmental specification of gene expression in rhombomeres (r) and neural crest. However, expression of the Krox-20 gene is restricted to neural crest cells migrating to the third branchial arch, apparently from r5, whereas this rhombomere contributes cells to both the second and third arches. We examined in the chick embryo how this spatially restricted expression is established. Expression occurs in precursors in both r5 and r6, and we show by cell labelling that both rhombomeres contribute to Krox-20-expressing neural crest, emigration occurring first from r6 and later caudally from r5. Krox-20 transcripts are not detected in some precursors in rostral r5, presaging the lack of expression in cells migrating rostrally from this rhombomere. After transposition of r6 to the position of r4 or r5, many Krox-20-expressing cells migrate rostral to the otic vesicle, whereas when r5 is transplanted to the position of r4, only a small number of migrating cells express Krox-20. These results indicate that, in the chick, Krox-20 expression in branchial neural crest does not correlate with rhombomeric segmentation, and that there may be intrinsic differences in regulation between the r5 and r6 Krox-20-expressing populations.

Topics: Animals; Branchial Region; Carbocyanines; Cell Movement; Chick Embryo; DNA-Binding Proteins; Early Growth Response Protein 2; Fluorescent Dyes; In Situ Hybridization; Models, Biological; Morphogenesis; Neural Crest; Rhombencephalon; Staining and Labeling; Tissue Distribution; Tissue Transplantation; Transcription Factors

The median eminence/pituitary stalk represents the final common pathway for fibers from neurons that project to the pituitary gland. We have used the lipophilic fluorescent tracer 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI) to determine the location of neurons projecting to the median eminence/pituitary stalk in ring doves. The tracer can be precisely applied to fixed tissue, in areas to which it is otherwise difficult to gain access. Following application of DiI to the median eminence/pituitary stalk, labeled neurons were detected in six distinct regions: the ventro-medial hypothalamic nucleus, paraventricular nucleus, supraoptic nucleus, in and ventral to the lateral forebrain bundle, preoptic area, and lateral septum. Labeled fibers branched extensively in the diencephalon, particularly along the third ventricle and in the septal-preoptic area. Sparse fiber labeling occurred caudal to the tuberal hypothalamus, even though these regions were close to the application site of the tracer. Labeled cerebrospinal-fluid-contracting cells were seen in the paraventricular region of the third ventricle. The results indicate that the avian neuronal system that projects to the median eminence and neural lobe occurs in diffuse clusters largely along the midline region of the hypothalamic septal-preoptic area. The paucity of fiber staining caudal to the tuberal hypothalamic region indicates that cells of these regions do not project to the median eminence/pituitary.

Topics: Afferent Pathways; Animals; Axonal Transport; Brain Mapping; Carbocyanines; Columbidae; Female; Fluorescent Antibody Technique; Hypothalamo-Hypophyseal System; Hypothalamus, Anterior; Male; Median Eminence; Neurons; Paraventricular Hypothalamic Nucleus; Pituitary Gland; Preoptic Area; Septal Nuclei; Supraoptic Nucleus; Ventromedial Hypothalamic Nucleus

We examined relationships between the pattern of geniculocortical innervation and the dendritic fields of cells in layer 4 of in cat primary visual cortex. Experiments were performed on normal animals and on cats in which the geniculocortical projection was altered by monocular deprivation or by the induction of divergent squint during the critical period. Thalamic afferents providing the input from the contralateral eye were anterogradely labeled by injecting the fluorescent tracer Dil into lamina A of the lateral geniculate nucleus. Intracellular staining with Lucifer yellow in slice preparations allowed simultaneous visualization of the morphology of individual cells and the thalamic afferents. Our results demonstrate that spiny stellate cells close to the upper and lower margin of the geniculocortical input have highly asymmetric dendritic fields, and thereby confine their dendrites to the termination zone of these afferents. This effect was specific for the cell class; it was not observed in pyramidal neurons. These dendritic asymmetries perpendicular to the laminar borders of spiny stellate cells were not altered by monocular deprivation or strabismus. In contrast, visual deprivation strongly influenced the dendritic arbors of spiny stellate cells near the borders between adjacent ocular dominance columns. In normal animals, the dendrites of cells near columnar borders remained preferentially within one column. These dendritic asymmetries became much more pronounced in strabismic animals. Monocular deprivation weakened the influence of the columnar borders on dendritic fields. Spiny stellate cells within the columns of the open eye exhibited a slight tendency to confine their dendrites to these columns. Cells in the columns of the deprived eye showed the opposite effect; they extended their dendrites preferentially into the adjacent columns of the open eye. These results demonstrate that the segregation of geniculocortical afferents into ocular dominance columns and its perturbation by manipulation of the visual input plays an important role in defining the morphology of cortical target cells. Thus, activity-dependent structural changes not only occur at the level of the presynaptic terminals, but also at the level of the postsynaptic target cells, and thereby contribute to build up the functional architecture of the cortex.

Topics: Animals; Axonal Transport; Carbocyanines; Cats; Dendrites; Female; Fluorescent Dyes; In Vitro Techniques; Isoquinolines; Male; Neurons; Pyramidal Cells; Reference Values; Strabismus; Vision, Monocular; Visual Cortex

The frontal nerve arises from the frontal organ, which represents the extracranial component of the pineal complex in some lower vertebrates, and interconnects the frontal organ with the epiphysis and the brain. The existence of a previously unreported nerve branch of the frontal nerve is described here in the frog Rana esculenta and called the lateral nerve. The course of the lateral nerve and its junction with the frontal nerve have been consistently detected by means of different techniques: toluidine blue staining in semithin sections, the Landau-Ignesti method for myelinated nerve fibers, the methylene blue intravital staining for peripheral nerves, and in vitro tracing with the carbocyanine Dil. A method to preserve intact the delicate lateral nerve during dissection is also described. The lateral nerve was consistently found to be unilateral, and to join the frontal nerve at one end (either on the left or the right side), whereas the other extremity was found to be dispersed in the dermis. Thus, the lateral nerve could represent a new pathway interconnecting the skin and the brain and/or the frontal organ in the frog.

Topics: Animals; Carbocyanines; Fluorescent Dyes; Methylene Blue; Nerve Fibers; Nerve Fibers, Myelinated; Paraffin Embedding; Rana esculenta; Skin; Staining and Labeling; Tolonium Chloride

Migration of dopamine (DA)-containing neurons and its guiding cues were histologically examined in the embryonic mesencephalon of normal mice. Cells immunoreactive (ir) for tyrosine hydroxylase (TH), a DA-synthesizing enzyme, were first detected on embryonic day 10 (E10) in the medio-basal part of the mesencephalon and were distributed throughout the entire length of the ventral mesencephalic wall at E12. By E14, TH-ir cells were located laterally along the ventral pial surface to form the primordia of the substantia nigra. Experiments with a single injection of bromodeoxyuridine, a thymidine analog, demonstrated that cells generated in the ventricular surface of the ventral mesencephalon at E11 migrated ventrally and then moved laterally to form the substantia nigra and the ventral tegmental area. Electron microscopic examination of the ventral mesencephalon of E12 mice disclosed that in the dorsal part ventrally migrating immature neurons made close contacts with the processes of radial glial cells. The expression of tenascin was transiently seen on radial glial processes between E10 and E13 coincident with the period of the ventral migration of mesencephalic DA neurons. By double immunostaining of E13 mesencephalon, ventrally migrating TH-ir cells were seen to be apposed to tenascin-bearing radial glial processes. On the other hand, laterally migrating neurons in the basal part of the mesencephalon were observed by electron microscopy to contact with tangentially arranged nerve fibers which were immunopositive for the 160 kDa neurofilament polypeptide at the light microscopic level from E10. Double immunostaining of E13 mesencephalon demonstrated that laterally migrating TH-ir cells were intermingled among neurofilament-ir fiber bundles. The cells of origin of the tangential nerve fibers were detected in the lateral part of the mesencephalon, when a fluorescent dye, 1,1'-dioctadecyl-3,3,3',3'-tetramethyl-indocarbocyanine perchlorate (DiI) was injected into the basal part of the mesencephalon of fixed E12 mice. The present results suggest that guiding cues of the radial migration of mesencephalic DA neurons represent processes of radial glial cells which express tenascin. On the other hand, tangentially arranged nerve fibers originating from the lateral part of the mesencephalon may provide a scaffolding along which the mesencephalic DA neurons subsequently migrate laterally to form the ventral tegmental area and the substantia nigra.

Topics: Animals; Bromodeoxyuridine; Carbocyanines; Cell Adhesion Molecules, Neuronal; Cell Movement; Dopamine; Extracellular Matrix Proteins; Mesencephalon; Mice; Mice, Inbred ICR; Microscopy, Electron; Nerve Tissue Proteins; Neurofilament Proteins; Neurons; Tenascin; Tyrosine 3-Monooxygenase

Esterase activity of endogenous transiently expressed acetylcholinesterase was locally suppressed in visual cortex of infant rats for 2-5 days by the irreversible inhibitor phospholine iodide, delivered from Elvax implants. Tissue processed for anterograde movement of the carbocyanine dye DiI or anterograde transneuronal transport of wheat germ agglutinin-horseradish peroxidase revealed normal geniculocortical growth into layer IV of visual cortex. These results suggest that the catalytic activity of transiently expressed acetylcholinesterase may play little, if any, role in early development of thalamocortical systems.

Topics: Animals; Animals, Newborn; Carbocyanines; Cholinesterase Inhibitors; Echothiophate Iodide; Female; Fluorescent Dyes; Geniculate Bodies; Horseradish Peroxidase; Male; Neural Pathways; Polyvinyls; Rats; Rats, Sprague-Dawley; Visual Cortex; Wheat Germ Agglutinin-Horseradish Peroxidase Conjugate; Wheat Germ Agglutinins

Oxidized low density lipoprotein (LDL) has a variety of hazardous influences on biological systems. Oxidative modification of LDL by diesel exhaust particles (DEP) was studied in vitro to assess its in vivo health effects. DEP suspensions (1, 10 and 100 micrograms/ml) were incubated for 1 h with LDL (1 mg protein/ml) at 37 degrees C. Conjugated diene formation and negative charges in LDL were increased by DEP-treatment in a dose-dependent manner. When native LDL and DEP-treated LDL (DEP-LDL) were incubated for 18 h with macrophage, J774A.1 cell at 37 degrees C, significantly more DEP-LDL was taken up into cells than native LDL. Accumulation of cholesterol ester in cells incubated with DEP-LDL was 4 to 8 times higher than that with native LDL while there was no significant difference between them in free cholesterol content. Incubation (18 h) of J774A.1 with DEP-LDL caused an increase in leakage of lactate dehydrogenase from cells in a DEP-concentration dependent manner, but the incubation with native LDL or DEP per se did not increase the leakage except at the highest concentration of DEP. These results suggest that DEP oxidatively modified LDL giving it cytotoxic, inflammatory and atherogenic properties characteristic of so-called oxidized LDL; these initial modifications of LDL may be one of the underlying mechanisms for diseases associated with DEP.

Topics: Animals; Carbocyanines; Cell Survival; Cholesterol Esters; Electrophoresis, Polyacrylamide Gel; Female; Fluorescent Dyes; Gasoline; In Vitro Techniques; Lipoproteins, LDL; Macrophages; Mice; Mice, Inbred BALB C; Oxidation-Reduction; Swine; Thiobarbituric Acid Reactive Substances; Vehicle Emissions

We have examined the developmental changes of glial cell organization in the superior colliculus of embryonic and neonatal hamsters in reference to the known sequence of retinal axon ingrowth and arborization in the midbrain. Immunolocalization of vimentin, a marker for neuronal and glial cell precursors, reveals a uniform distribution of radially oriented cells, with perikarya located at the ventricular surface and thin, elongated processes fanning out toward the pia. These vimentin-positive cells, referred to as the lateral radial cells, are present in the tectum from embryonic day (E) 10 (earliest day examined) until approximately postnatal day (P) 5. Vimentin expression in the lateral radial cells decreases markedly during the second week of postnatal life: application of DiI to the ventricular surface reveals that the pial attachment of the lateral radial cells is withdrawn and that the radial processes are gradually pulled back toward the ventricular zone. By P14, virtually no vimentin-positive radial cells are detectable in the superior colliculus. At no time during development are the lateral radial cells immunopositive for the glial fibrillary acidic protein (GFAP); however, shorter, vimentin-positive astrocytic profiles can be seen in the tectum around the time the radial fibers have been withdrawn, suggesting that at least some radial cells are transformed into astrocytes that will colonize the mature colliculus. At approximately E12, a second group of cells, referred to as the midline radial glia, is detected at the tectal midline. These cells are tightly bundled, forming a raphe in the tectum. They are intensely vimentin positive from E13 until at least P14. From the time of birth, the midline radial cells also exhibit intense immunoreactivity for GFAP. The lateral radial cells are present in the superior colliculus prior to and during the period of neurogenesis but remain well past the time when collicular neuronal migration is completed. Pial processes of the lateral radial cells are present within the superficial tectal layers during the time retinal axons are entering this target; they may be involved in directing the growth and initial collateralization of retinotectal axons. Their withdrawal from retinorecipient collicular zones begins at about the time arbors are being elaborated on retinal axons. In contrast, the midline glia become distinct just prior to the time retinal axons enter the superior colliculus and persist during the time

Topics: Animals; Axons; Carbocyanines; Cricetinae; Embryonic and Fetal Development; Fluorescent Dyes; Glial Fibrillary Acidic Protein; Immunohistochemistry; Mesocricetus; Neuroglia; Retina; Staining and Labeling; Superior Colliculi; Time Factors; Vimentin

The effect of homozygotic Wnt-1-/- mutations on the development of ocular motoneurons was examined with the lipophilic dye DiI and compared to control and phenotypic wild-type mouse embryos. A piece of DiI-soaked filter paper was inserted into the orbit, the midbrain, or rhombomere 5 of the hindbrain in six paraformaldehyde-fixed litters (10.5, 12.5, and 14.5 days postcoitum) containing Wnt-1, Wnt+/-, and Wnt-1+/+ individuals and three control litters. We labeled all ocular motoneurons retrogradely and all relevant nerves anterogradely in all control and phenotypic wild-type animals. In all phenotypically identified Wnt-1-/- mutants we could always label the abducens nerve and motoneurons and the optic fibers to the thalamus, but we were unable to label oculomotor or trochlear nerves or motoneurons. In addition to Wnt-1 knockout mutants, we also labeled mice from the WZT9B transgenic line carrying a lacZ reporter gene driven by the Wnt-1 gene enhancer. In these embryos we tested for co-localization of Wnt-1 expression in biotinylated dextran amine-labeled ocular motoneurons using a newly developed technique. In younger embryos we obtained evidence for co-localization of the beta-galactosidase reaction product derived from lacZ gene activity in some retrogradely filled oculomotor motoneurons and adjacent to other oculomotor and the trochlear motoneurons. Acetylcholine esterase, a marker of early differentiating cholinergic neurons, showed a similar topology with respect to the lacZ reaction product. Thus, at least some future oculomotor motoneurons express Wnt-1, whereas others and the trochlear motoneurons caudal to the ventral midbrain expression of Wnt-1 may be exposed to the short range diffusion of the Wnt-1 gene product. Thus, the Wnt-1-/- mutation precludes formation or survival of midbrain and anterior hindbrain neurons, including oculomotor and trochlear motoneurons.

Topics: Animals; Carbocyanines; Cranial Nerves; Eye; Fluorescent Dyes; Homozygote; Mesencephalon; Mice; Mice, Knockout; Motor Neurons; Neural Pathways; Phenotype; Reference Values; Retina; Rhombencephalon; Thalamus

We have previously reported on our investigation of motoneuron cell death (MCD) in the rat nucleus ambiguus (NA). This article focuses on the other major upper respiratory tract motor nucleus: the hypoglossal. The hypoglossal nucleus (XII) contains motoneurons to the tongue and, as such, plays a critical role in defining patterns of respiration, deglutition, and vocalization. Motoneuron counts were made in XII in a developmental series of rats. In addition, the neural tracer fast DiI was used to ensure that all hypoglossal motoneurons had migrated into the nucleus at the time cell death was assessed. Furthermore, an antibody to gamma-aminobutyric acid (GABA) was used to determine the potential effect of inadvertently counting large interneurons on motoneuron counts. Cell death in XII was shown to occur entirely prenatally with a loss of 35% of cells between embryonic day 16 (E16) and birth. Fast DiI tracings of the prenatal hypoglossal nerve indicated that all motoneurons were present in a well-defined nucleus by E15. Immunocytochemical staining for GABA demonstrated considerably fewer interneurons than motoneurons in XII. These findings in XII, in comparison with those previously reported for NA, demonstrate differences in the timing and amount of cell death between upper respiratory tract motor nuclei. These differences establish periods during which one nucleus may be preferentially insulted by environmental or teratogenic factors. Preferential insults may underlie some of the upper respiratory tract incoordination pathologies seen in the newborn such as the sudden infant death syndrome (SIDS).

Topics: Animals; Apoptosis; Carbocyanines; Fluorescent Dyes; gamma-Aminobutyric Acid; Hypoglossal Nerve; Immunohistochemistry; Interneurons; Motor Neurons; Rats; Rats, Sprague-Dawley; Respiratory System

The epsilon 4 allele of apolipoprotein E (apoE) is a major risk factor for Alzheimer disease, suggesting that apoE may directly influence neurons in the aging brain. Recent data suggest that apoE-containing lipoproteins can influence neurite outgrowth in an isoform-specific fashion. The neuronal mediators of apoE effects have not been clarified. We show here that in a central nervous system-derived neuronal cell line, apoE3 but not apoE4 increases neurite extension. The effect of apoE3 was blocked at low nanomolar concentrations by purified 39-kDa protein that regulates ligand binding to the low density lipoprotein receptor-related protein (LRP). Anti-LRP antibody also completely abolished the neurite-promoting effect of apoE3. Understanding isoform-specific cell biological processes mediated by apoE-LRP interactions in central nervous system neurons may provide insight into Alzheimer disease pathogenesis.

Topics: Apolipoprotein E3; Apolipoproteins E; Carbocyanines; Cell Line; Cross-Linking Reagents; Fluorescent Antibody Technique, Indirect; Fluorescent Dyes; Hypothalamus; Low Density Lipoprotein Receptor-Related Protein-1; Microscopy, Confocal; Microscopy, Fluorescence; Neurites; Neurons; Receptors, Immunologic; Receptors, LDL; Recombinant Proteins

1. Repair and recovery following spinal cord injury (complete spinal cord crush) has been studied in vitro in neonatal opossum (Monodelphis domestica), fetal rat and in vivo in neonatal opossum. 2. Crush injury of the cultured spinal cord of isolated entire central nervous system (CNS) of neonatal opossum (P4-10) or fetal rats (E15-E16) was followed by profuse growth of fibres and recovery of conduction of impulses through the crush. Previous studies of injured immature mammalian spinal cord have described fibre growth occurring only around the lesion, unless implanted with fetal CNS. 3. The period during which successful growth occurred in response to a crush is developmentally regulated. No such growth was obtained after P12 in spinal cords crushed in vitro at the level of C7-8. 4. In vivo, in the neonatal (P4-8) marsupial opossum, growth of fibres through, and restoration of, impulse conduction across the crush was apparent 1-2 weeks after injury. With longer periods of time after crushing a considerable degree of normal locomotor function developed. 5. By the time the operated animals reached adulthood, the morphological structure of the spinal cord, both in the region of the crush and on either side of the site of the lesion, appeared grossly normal. 6. The results are discussed in relation to the eventual longterm possibility of devising effective treatments for patients with spinal cord injuries.

Topics: Animals; Animals, Newborn; Behavior, Animal; Carbocyanines; Electrophysiology; Female; Fluorescent Dyes; Immunohistochemistry; Microscopy, Electron; Neural Conduction; Neurons; Opossums; Pregnancy; Spinal Cord; Spinal Cord Injuries

Flow cytometric methods for measurement of low-density lipoprotein (LDL) receptor activity on peripheral blood mononuclear cells (PBMC) may be used to identify patients with familial hypercholesterolemia (FH). However, cellular LDL receptor activities measured in FH heterozygotes may overlap with those of healthy subjects. Analytical variation is probably responsible for some of this overlap. We have examined several technical details that may affect analytical variation. In each analysis, we included one standard and two control cell preparations. These were cells isolated from three donors and stored in aliquots at -135 degrees C. Use of standard cells reduced between-series analytical variation of the controls by approximately 50%. Preincubation-conditions used to induce the maximum number of receptors, the concentration of fluorochrome 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine-perchlorate (DiI)-LDL, labelling time, and conditions during storage of labelled cells before flow cytometry were also examined in order to reduce analytical variation. Having standardized the assay, we found among 20 healthy subjects a median receptor activity of 100% vs. 51% among 26 patients who fulfilled clinical criteria for FH. However, four of the patients showed distinctly normal receptor activities, which may suggest either the presence of some other biochemical defect or that in vivo dysfunctional receptors may be measured as normal in some patients with our assay.

Topics: Adult; Apolipoproteins B; Carbocyanines; Cholesterol; Flow Cytometry; Fluorescent Dyes; Genetic Carrier Screening; Humans; Hyperlipoproteinemia Type II; Leukocytes, Mononuclear; Middle Aged; Receptors, LDL

By combining anterograde and retrograde axonal tracing with AChE histochemistry, we demonstrate the sources of AChE-positive afferents to embryonic neocortex, the pathways they use, their time of arrival into cortex, and their initial invasion of the cortical plate. Acetylcholinesterase (AChE) is expressed by two populations of cortical afferents: AChE is permanently present in basal forebrain fibers and has been reported to be transiently localized in axons of the principal sensory thalamic nuclei over the first few postnatal weeks beginning at the middle of the first week. We first detect AChE-positive afferents histochemically in neocortex on embryonic day seventeen (E17) and determine that they arise from the principal sensory thalamic nuclei. AChE histochemistry labels the entire length of developing thalamocortical axons, including their growth cones and branches. These AChE-positive afferents enter the neocortex by the internal capsule and take an intracortical pathway centered on the subplate layer. As soon as these axons are detected, some have already begun to extend AChE-positive collateral branches superficially toward the cortical plate. By E19, a few collaterals have entered the deep part of the cortical plate and by E21 have densely invaded all but its most superficial undifferentiated part. AChE-positive afferents from basal forebrain structures reach the neocortex by three routes: the external capsule, the internal capsule, and the cingulate bundle. Among basal forebrain components, only the substantia innominata and nucleus basalis of Meynert reach the cortex by the internal capsule. Afferents from these two sources reach neocortex on E18, but are a very minor component of the total population of AChE-positive afferents at this age. Afferents from other basal forebrain components do not reach neocortex until several days later. The spatial and temporal patterns of AChE expression in developing thalamocortical axons indicate that it is useful for delineating their innervation of the primary sensory areas of embryonic neocortex, and suggest that AChE may function in axon extension and cortical differentiation.

Topics: Acetylcholinesterase; Amidines; Animals; Axons; Carbocyanines; Female; Fluorescent Dyes; Histocytochemistry; Nerve Fibers; Neurons, Afferent; Pregnancy; Prosencephalon; Rats; Rats, Sprague-Dawley; Thalamus

The neuroanatomical organization of neurons projecting to the pituitary and the origin of pituitary dopamine and nonapeptides were investigated in the brain of the Atlantic salmon (Salmo salar). Carbocyanine tract tracing in combination with tyrosine hydroxylase, arginine vasotocin and isotocin immunocytochemistry for double labelling revealed a previously unknown organization of hypophysiotrophic cell groups and their extrahypothalamic projections, and provide the first direct identification in a teleost fish of the origin of the dopaminergic and nonapeptidergic innervation of the pituitary. The present data include identification of (1) hypophysiotrophic neurons in the ventral telencephalon and in the periventricular preoptic nucleus, (2) large (magnocellular) vasotocinergic hypophysiotrophic neurons in the most rostral extension of the preoptic area, (3) a distinct neuronal group located in a supraoptic/suprachiasmatic position in the anterior periventricular nucleus, that seems to be the major source of dopaminergic innervation of the pituitary, (4) the nonapeptidergic hypophysiotrophic neurons in the preoptic nucleus, (5) hypophysiotrophic neurons in the ventral and posterior hypothalamus of which some are of liquor-contacting type, (6) projections from hypophysiotrophic and non-hypophysiotrophic neurons in the preoptic nucleus to extrahypothalamic areas such as thalamic and periventricular pretectal nuclei, and (7) subdivisions within the preoptic nucleus that exhibit different combinations of hypophysiotrophic and extrahypothalamic efferent connections. Together with previous studies of retinohypothalamic projections and neurochemical organization of hypothalamic/preoptic areas, the present data suggest that the preoptic nucleus and the anterior periventricular nucleus in teleosts possess functional subdivisions with features that resemble those of the paraventricular, supraoptic and suprachiasmatic nuclei of other vertebrates. In the Atlantic salmon, specific dopaminergic and nonapeptidergic neuronal subdivisions are proposed to play a role for photoperiod control of endocrine activity.

Topics: Animals; Brain Chemistry; Carbocyanines; Dopamine; Fluorescent Dyes; Hypothalamus; Immunoenzyme Techniques; Neurons; Neuropeptides; Neurosecretory Systems; Oligopeptides; Pituitary Gland; Preoptic Area; Salmon; Telencephalon

The ability of immune complexes of LDL or acetylated LDL (acLDL), together with antibodies to LDL, to induce a proatherogenic phenotype in human monocytic cells has been explored. Treatment of THP-1 monocytic cells or peripheral human monocytes with LDL immune complexes containing intact anti-LDL markedly enhanced the ability of these cells to subsequently bind and take up LDL, whereas aggregated LDL or LDL immune complexes prepared with F(ab')2 fragments of anti-LDL had no significant effect. Activation of THP-1 cells with intact LDL immune complexes also stimulated mRNA expression for the scavenger receptor. Additionally, activation of THP-1 cells with insoluble immune complexes of LDL or LDL stimulated generation of reactive oxygen intermediates that, in turn, could oxidize exogenous LDL. These results indicate that the binding of lipoprotein immune complexes to Fc receptors on monocytic cells activates a series of responses that could accelerate the initiation or progression of atherosclerosis.

Topics: Antigen-Antibody Complex; Arteriosclerosis; Base Sequence; Carbocyanines; Fluorescent Dyes; Humans; Leukemia, Myeloid; Lipoproteins, LDL; Membrane Proteins; Molecular Sequence Data; Monocytes; Oxidation-Reduction; Polymerase Chain Reaction; Receptors, IgG; Receptors, Immunologic; Receptors, Lipoprotein; Receptors, Scavenger; RNA, Messenger; Scavenger Receptors, Class B; Tumor Cells, Cultured

Adult rat parasympathetic ciliary ganglion (CG) neurons were retrogradely labelled by intraocular injection of the carbocyanine fluorescent dye 1,1-dioleyl-3,3,3',3'-tetramethylindocarbocyanine methanesulfonate (DiI). Whole-cell and nystatin perforated patch recording techniques were then used to examine the electrophysiological properties of labelled CG neurons growing in primary culture. The resting membrane potential of CG neurons in dissociated cell culture was -50 +/- 8 mV, and isolated neurons fired overshooting action potentials in response to depolarizing current injection. Voltage-clamp recordings of membrane currents revealed a transient tetrodotoxin-sensitive Na+ inward current and both sustained and transient outward K+ currents. Sustained outward K+ current was reduced (55-77%) by 5 mM tetraethylammonium and to a lesser extent (42-46%) by superfusion with nominally Ca2+ free external solution. Transient outward current was blocked by 100 microns 4-aminopyridine and exhibited steady-state inactivation at potentials depolarized to -50 mV. These data demonstrate that identified adult mammalian CG neurons can be successfully maintained in culture. Cultured CG neurons retain electrical excitability, with voltage-sensitive Na+ and K+ currents giving rise to action potentials.

Topics: Action Potentials; Animals; Carbocyanines; Cells, Cultured; Electrophysiology; Fluorescent Dyes; Ganglia, Parasympathetic; Male; Membrane Potentials; Neurons; Patch-Clamp Techniques; Potassium Channels; Rats; Rats, Sprague-Dawley; Sodium Channels; Tetrodotoxin

The tissue movements of epithelial spreading and mesenchymal contraction play key roles in many aspects of embryonic morphogenesis. One way of studying these movements in a controlled manner is to make an excisional skin wound to an embryo and watch the wound heal. In this paper we report our studies of healing of a simple excisional lesion made to the limb bud stage mouse embryo. The wounded, living embryo is cultured in a roller bottle; under such conditions the wound heals with a highly reproducible time course and is completely closed by 24 hr. During the healing period the environment bathing the wound can be simply manipulated by adding drugs or factors to the culture medium. We have used DiI to label mesenchymal cells exposed at the margin of the initial wound and, by following their fate and measuring the area of mesenchyme remaining exposed at various time points during the healing process, we have quantified both the extent of mesenchymal contraction and the extent of reepithelialisation by movement of epidermis over mesenchyme. We show that the two types of tissue movement contribute almost equally (50:50) to the total wound closure rate. We have gone on to investigate the cell machinery underlying these processes. In adult wounds the epidermis migrates by means of lamellipodial crawling, but we show that reepithelialisation in the embryo is achieved instead by purse-string contraction of a cable of filamentous actin which assembles in the basal layer of cells at the free edge of the epidermis. Addition of cytochalasin D to the culture medium blocks formation of this actin cable and leads to failure of reepithelialisation. Contraction of adult wound connective tissue appears to be driven by conversion of dermal fibroblasts into a specialist smooth muscle-like fibroblast, the myofibroblast. However, using an antibody recognising the alpha-isoform of smooth muscle actin and specific for smooth muscle cells and myofibroblasts, we show that a similar conversion into myofibroblasts does not occur at any stage during the embryonic wound healing process. These observations indicate that both of the tissue movements of embryonic wound healing utilise cell machinery fundamentally different from that driving the analogous tissue movements of adult healing.

Topics: Actins; Animals; Carbocyanines; Connective Tissue; Cytochalasin D; Embryo, Mammalian; Epidermis; Epithelium; Fibroblasts; Hindlimb; Mesoderm; Mice; Microscopy, Electron, Scanning; Morphogenesis; Organ Culture Techniques; Prenatal Injuries; Reproducibility of Results; Wound Healing

The mammalian upper respiratory tract (URT) serves as the common modality for aspects of respiration, deglutition, and vocalization. Although these actions are dependent on coordinated and specific neuromuscular control, little is known about the development of URT control centers. As such, this study investigated the occurrence of naturally occurring motoneuron cell death (MCD) in the nucleus ambiguus (NA) of a developmental series of rats. Standard histological techniques were used to count motoneurons in the ventrolateral brainstem where the mature NA is found. In addition, the neural tracer, fast DiI, was used to determine whether motoneurons were still migrating into the region of the NA during the period that cell counts were first taken. Furthermore, to elucidate the potential effect of inadvertently counting large interneurons on the assessment of motoneuron numbers, an antibody to gamma-aminobutyric acid (GABA) was used. The results of this study have, for the first time, demonstrated that MCD occurs in a URT-related motor nucleus. Approximately a 50% cell death was observed during the prenatal development of NA, with no further loss seen postnatally. The fast DiI studies showed that by embryonic day 17, NA was fully formed, suggesting that motoneuron migration from the basal plate was complete. In addition, use of the GABA antibody showed a lack of inhibitory interneurons within the NA. The finding of MCD in the NA helps define a critical period in the formation of URT neuromuscular control. As the course of MCD is modifiable by epigenetic signals, insult to the organism during this prenatal period may compromise future URT control.

Topics: Animals; Brain Stem; Carbocyanines; Cell Death; Fluorescent Dyes; gamma-Aminobutyric Acid; Immunohistochemistry; Interneurons; Laryngeal Nerves; Motor Neurons; Pharynx; Rats; Rats, Sprague-Dawley; Respiratory System

The early development of the uncrossed tectobulbar and the crossed tectospinal tracts was studied. These two projections arise from the same structure, the mesencephalon, and develop during the same time period, but follow divergent courses. We have traced the pathways followed by these projections and identified the positions at which axon guidance decisions are made. The first neurons differentiate either side of the entire rostrocaudal extent of the dorsal midline and initiate axons that extend dorsoventrally across the surface of the tectum. At the ventral edge of the tectum these axons turn abruptly and fasciculate to form a caudal descending projection to the hindbrain. These axons extend to the caudal hindbrain and do not project to the periphery along cranial nerve roots. We therefore consider this tract to be the tectobular, rather than the mesencephalic division of the trigeminal. While the tectobulbar projection is still developing, a second wave of axons is initiated, which arises from only the rostral part of the tectum. These axons grow beyond the tectobulbar turn point and continue toward the ventral midline, where they cross the floor plate, before turning caudally at the lateral edge of the main descending hindbrain tract, the ventrolateral tract. We discuss the development of these tracts with reference to possible guidance cues mediating their course.

Topics: Animals; Axons; Carbocyanines; Chick Embryo; Efferent Pathways; Fluorescent Dyes; Immunohistochemistry; Medulla Oblongata; Spinal Cord; Tectum Mesencephali

We performed experiments using retrograde and anterograde labeling with DiI to examine the development of basal forebrain (BFB) projections to the visual cortex in postnatal rats. DiI placed in occipital cortex led to retrograde labeling of BFB neurons as early as postnatal day 0 (P0); labeled cells were found mainly in the diagonal band complex but also in the medial septum, globus pallidus, and substantia innominata. The retrogradely labeled BFB cells displayed remarkably well-developed dendritic arbors, even in younger animals, and showed increases in soma size, dendritic arbors, and dendritic spines over the first 2 postnatal weeks. DiI placements in the diagonal band led to anterogradely labeled axons in cortex. At early ages (P0-P1), labeled axons were largely confined to white matter. With increasing age, greater numbers of labeled axons were seen in the white matter and in deep cortical layers, and labeled axons extended into superficial layers. The leading edge of labeled fibers reached layer V of visual cortex by P2 and layer IV by P4 and were found throughout the cortical layers by P6. Numbers and densities of labeled axons in visual cortex were greater in older animals, at least through P14. The time of ingrowth of labeled BFB axons into visual cortex indicates that these afferents grow into particular cortical layers after those layers have differentiated from the cortical plate. These data indicate that basal forebrain projections arrive in occipital cortex after cortical lamination is well underway and after the entry of primary thalamocortical projections.

Topics: Animals; Axons; Carbocyanines; Dendrites; Fluorescent Dyes; Neurons; Phenotype; Prosencephalon; Rats; Rats, Sprague-Dawley; Visual Cortex; Visual Pathways

The development of spinal cord circuitry in humans is poorly characterized, primarily because standard anatomical tracers must be actively transported, which requires living tissue. Intensely fluorescent lipid-soluble tracers have largely eliminated this problem, at least for circuits that can be traced over short distances. We have, therefore, used the carbocyanine dye DiI (1,1-dioctadecyl-3,3,3,3,-tetramethyl-indocarbocyanine perchlorate) to study the development of the dorsal root afferent projection to fetal human spinal cord between 8 and 19 weeks of gestation. We show here that the dorsal root afferents enter the gray matter of the spinal cord very early in gestation. By 8 weeks, a few axons have already reached the motor pools. These axons, presumably spindle afferents, traverse the length of the spinal gray matter in fascicles to reach different groups of motor neurons. As development progresses, these axons project to the ventral horn and branch in a restricted area in the intermediate zone as well as in the motor pools. Between 11 and 19 weeks of gestation, axons in the ventral horn elaborate boutons that appear to be in proximity to the motor neuron somata and their proximal dendrites. Other groups of axons penetrate the gray matter of the spinal cord all along the mediolateral extent of the dorsal horn. These axons descend to lamina IV, and then turn upward to terminate in laminae III and IV, arborizing primarily rostrocaudally. The time course of the development of these axons parallels that of the axons projecting to the ventral horn.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Afferent Pathways; Carbocyanines; Embryonic and Fetal Development; Fluorescent Dyes; Gestational Age; Humans; Spinal Cord

A retrograde labeling method is described in which rat embryonic (E18, E21) and postnatal (P1) medial septal neurons were labeled with succinyl wheat germ agglutin-fluorescein, fluorescent green microspheres, or 1,1'-dioctadecyl-3,3,3',3'-tetramethyl-indocarbocyanine perchlorate (Dil) following in vitro hippocampal injections. The brains were removed and immediately immersed in oxygenated Tyrode solution. Dye was pressure injected into the hippocampus bilaterally. After incubating the brain in oxygenated Tyrode, the medial septal region was removed. The neurons were dissociated and cultured at medium density in 35 mm dishes with a hole in the bottom covered by a coverslip with a grid. The neurons were observed with a low light system, and cell counts were made at 5, 24, and 48 h. Labeled and unlabeled neurons showed considerable neurite outgrowth and acetylcholinesterase activity in culture. Highly reproducible labeling was obtained, with Dil giving the best results. Dil labeled the neurons in vitro, was retained during culture for 1 week, and was compatible with cell survival.

Topics: Acetylcholinesterase; Animals; Brain; Carbocyanines; Cells, Cultured; Fluorescein-5-isothiocyanate; Fluorescent Dyes; Hippocampus; Histocytochemistry; Image Processing, Computer-Assisted; Interneurons; Microspheres; Rats; Wheat Germ Agglutinins

These studies were designed to determine the pattern of initial afferent fiber ingrowth into the prenatal spinal gray matter and the establishment of the topographic organization of the presynaptic neuropil in the dorsal horn. A total of 113 lumbar dorsal root ganglia were labeled with carbocyanine fluorescent dye DiI or DiA in 67 rat embryos and neonatal pups aged embryonic day 13 to postnatal day 0 (E13-P0). The initial fiber penetration of the lumbar spinal gray began at E15 and was restricted to the segments of entry. Subsequent growth of fibers into gray matter of adjacent segments began approximately one day later, and this delay was continued, about one day for each successive segment. A second wave of ingrowth of putative small-diameter afferents into the substantia gelatinosa began at E19 and also displayed the same rostrocaudal delay. Fiber ingrowth was specific and occupied the somatotopic area appropriate for the adult, from the earliest stages (E18) in which dorsal horn laminae could be adequately defined. The somatotopic organization of the presynaptic neuropil in laminae III and IV did not change significantly throughout embryonic development as the amount of overlap between adjacent and non-adjacent ganglion projections remained constant throughout embryonic development. In addition, it was found that fibers innervating the proximal and distal hindlimb entered the spinal gray simultaneously at E15 before the innervation of the distal toes was established. The results of these studies indicate that the somatotopic organization of the presynaptic neuropil is established very early in development and requires little refinement to match that seen in the adult. The simultaneous penetration of the fibers originating from the proximal and distal areas of the limb before innervation is complete suggests that this ingrowth may be independent of the establishment of specific peripheral connections.

Topics: Animals; Carbocyanines; Female; Fluorescent Dyes; Ganglia, Spinal; Hindlimb; Nerve Fibers; Neural Pathways; Neurons, Afferent; Pregnancy; Rats; Rats, Sprague-Dawley; Receptors, Presynaptic; Somatosensory Cortex; Spinal Cord

The aim of this study was to investigate (a) whether embryonic retinal transplants can sprout fibers into a lesioned adult host retina and (b) if these fibers established synaptic connections with the host. Embryonic rat (E16-22) or human (9-13 weeks) retinal cells were transplanted to adult rats. Normal Long-Evans rats received rat transplants. The hosts for human transplants were athymic nude rats. After varying survival times (3 to 11 months), animals were perfused with 4% paraformaldehyde (sometimes with added 0.1% glutaraldehyde). Glass microneedles, coated with DiI (a carbocyanine dye) were placed into the transplants which were then stored at room temperature in 2% paraformaldehyde for 3-15 months. This filled the cells that had processes in the area where the needle had been placed. Gelatin-embedded eyecups were cut on a vibratome. DiI-labeled transplant cells exhibited fiber outgrowth into the host retina. After photoconversion of the dye to an electron-dense precipitate, these neuronal processes could be followed with better resolution than with fluorescence. Occasionally, host cells could also be labeled by DiI placed into the graft, indicating fiber ingrowth of host fibers into the transplants. Selected photoconverted sections were embedded for electron microscopy. Synapses could be found along transplant processes that had grown into the host inner plexiform layer. These results indicate that neuronal fibers originating from embryonic retinal transplants form synapses in the host retina.

Topics: Animals; Carbocyanines; Fetal Tissue Transplantation; Fluorescent Dyes; Humans; Nerve Fibers; Rats; Rats, Inbred Strains; Rats, Nude; Retina; Synapses; Visual Pathways

Retinal ganglion cells (RGCs) of adult cats were labeled by injection of diI into the proximal stump of completely transected optic nerves. Approximately 2% to 5% of the RGC population appeared viable 2 months after these axotomies, based on diI retention. The morphological type and dendritic arbor of these surviving RGCs were examined after intracellular injections of Lucifer Yellow into diI-labeled RGCs. Postaxotomy survival rate was much higher for alpha-like cells than for beta-like cells. However, in one of four retinas examined, a large number of RGCs seemed to survive axotomy, and among these, beta cells survived at an unusually high rate. Dendritic arbors of surviving RGCs were also examined after intracellular injection of horseradish peroxidase. Some dendrites of these RGCs lacked branches and were thin in caliber. Other dendrites displayed many spiny processes and bulbous swellings. Essentially, these results confirm the previous suggestion that alpha cells survive axotomy longer than beta cells. The ability of alpha cells to regenerate axons may thus be attributable to their relatively high resistance to axotomy. The atypical dendritic profiles seen after optic nerve transection may reflect either degeneration or regrowth of dendrites.

Topics: Animals; Axons; Carbocyanines; Cats; Cell Count; Cell Survival; Dendrites; Female; Fluorescent Dyes; Horseradish Peroxidase; Isoquinolines; Male; Optic Nerve; Retinal Ganglion Cells

Cell volume is an important physiologic parameter but is not directly accessible by conventional microscopy in vivo or in slice preparations. After staining of rat hippocampal slices with 1,1'-dioctadecyl-3,3,3',3'-tetramethylinso-carbocyanineperchlor ate (DiI), living single CA1 pyramidal cells were visualized by confocal microscopy while others in the illuminated area were recorded intracellularly. Resting membrane potential and action potentials were not affected by staining with DiI (4 microM) and repeated laser illumination (up to 600 times). A time- and dose-dependent increase of cell volume succeeds membrane depolarization upon bath application of kainate (40 microM and 100 microM) with a delay of several minutes indicating active regulation of cell volume. We conclude that cell swelling as visualized by confocal microscopy is a relatively late event of kainate excitotoxicity.

Topics: Animals; Carbocyanines; Cell Size; Fluorescent Dyes; Hippocampus; In Vitro Techniques; Kainic Acid; Male; Membrane Potentials; Membranes; Microscopy, Confocal; Pyramidal Cells; Rats; Rats, Wistar; Receptors, GABA

Axon navigation during vertebrate limb innervation has been shown to be associated with position-dependent changes in size and complexity of the axon growth cones, and sometimes with bifurcation of terminal growth cones and axon branching (Hollyday and Morgan-Carr, companion paper). We have further examined axon branching and asked whether it extends to the projection of collaterals to different nerves. Injections of horseradish peroxidase or Dil were made into individual peripheral nerves in the wings of chick embryos at stages 28-35, and the trajectories of solidly labeled axons were traced proximally from the injection site in tissue sections. During stages when the peripheral nerves were first forming in the shoulder region, collaterals of retrogradely labeled axons were frequently observed to project into uninjected nerves proximal to the injection site. These two-nerve collaterals were formed by a small percentage of axons in a high percentage of the embryos studied and could occur in both motor and sensory axons. Two-nerve collateral projections were observed between nerves separated along both the proximodistal and anteroposterior axes of the limb, but they were limited in spatial extent to nerves supplying adjacent limb regions and were never seen between nerves projecting to widely disparate regions of the limb. Collaterals were not seen at the plexus projecting to both dorsal and ventral pathways. The apparent frequency of two-nerve collaterals was found to decline progressively from stage 28-29 to stage 32; no two-nerve collaterals were seen in the proximal wing at stage 33 and older. The mechanism of their elimination is presently unknown.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Animals; Axons; Carbocyanines; Chick Embryo; Fluorescent Dyes; Horseradish Peroxidase; Peripheral Nerves; Wings, Animal

Monocular enucleations were done in ferret embryos before or during the earliest stages of development of the retinofugal pathway (E23-E26). The effects on the development of the uncrossed pathway from the surviving eye were assessed on embryonic day 30. This stage was chosen for two reasons: (1) we show that in normal development a substantial uncrossed component from the temporal crescent has developed by E30; and (2) the pathway cannot yet have been affected by the cell death that normally occurs in the retina in the perinatal period. Using DiI labelling from either the temporal crescent or the optic nerve head, we have shown that such early enucleations prevent the formation of the uncrossed pathway from the temporal crescent of the surviving eye. Enucleation at E23/24, before or during the period when the first axons reach the chiasm, prevents the formation of the uncrossed projection. The axons that would normally take an uncrossed course stall lateral to the midline of the optic chiasm. At E26, when many axons have reached the optic chiasm, but none yet come from the temporal crescent, enucleation causes a dramatic reduction in the uncrossed projection, and the complete abolition of the normal uncrossed pathway from the temporal crescent. This demonstrates that there is a requirement for an interaction between the axons of the two eyes at the optic chiasm to establish the normal formation of the uncrossed pathway at the optic chiasm.

Topics: Animals; Axons; Carbocyanines; Eye Enucleation; Female; Ferrets; Fluorescent Dyes; Histocytochemistry; Optic Chiasm; Pregnancy; Retina; Vision, Monocular; Visual Pathways

We previously reported that mevalonate starvation elicited by hydroxymethyl glutaryl coenzyme A (HMG-CoA) reductase inhibitors reduced cholesterol accumulation promoted in murine macrophages by acetylated LDL (AcLDL). In the present study we investigated the cellular mechanism of this effect. Our results indicate that the HMG-CoA reductase inhibitors fluvastatin and simvastatin reduce, in a concentration-dependent manner, more than 50% of the 125I-AcLDL degradation by macrophages. This effect was not due to a decrease of lysosomal enzyme activity, and it was paralleled by the retention of AcLDL-associated cholesteryl ester in the incubation medium. The ability of fluvastatin to inhibit AcLDL degradation was completely overcome by mevalonate and its derivative geranylgeraniol. Evaluation at 4 degrees C of 125I-AcLDL binding to plasma membrane suggested that the inhibitory effect of fluvastatin on lipoprotein catabolism was not due to a decreased expression of scavenger receptors. Fluorescent microscope analysis of cellular internalization of AcLDL labeled with the fluorochrome 3,3'-dioctadecyl indocarbocyanine demonstrated that fluvastatin inhibits lipoprotein endocytosis, an effect reversed by mevalonate. Studies performed with native 125I-LDL indicated that fluvastatin did not inhibit but rather increased the degradation of LDL taken up by the normal LDL receptor. These results exclude a generalized depression of the cellular endocytotic activity by the drug. The ability of fluvastatin to reduce AcLDL catabolism and cholesterol esterification was more pronounced in cholesterol-enriched macrophages compared with normal cells. In conclusion, the present results demonstrate that HMG-CoA reductase inhibitors may reduce the in vitro cholesterol accumulation in macrophages by inhibiting AcLDL endocytosis.

Topics: Animals; Carbocyanines; Cholesterol Esters; Diterpenes; Endocytosis; Fatty Acids, Monounsaturated; Fluorescent Dyes; Fluvastatin; Humans; Hydroxymethylglutaryl-CoA Reductase Inhibitors; Indoles; Lipoproteins, LDL; Lovastatin; Macrophages, Peritoneal; Mevalonic Acid; Mice; Mice, Inbred BALB C; Microscopy, Fluorescence; Simvastatin

The membrane properties of visually identified, DiI retrogradely labeled bulbospinal neurons of the C1 adrenergic cell group were studied by whole-cell recordings in brainstem slices from 7- to 10-day-old rats. A post-hoc histochemical analysis allowed us to evaluate the electrophysiological properties of the C1 adrenergic neurons, a group of cells known to project to the sympathetic preganglionic neurons. Two types of cells were labeled: pacemaker and non-pacemaker neurons. In voltage-clamp mode, C1 pacemaker neurons exhibited a TTX-sensitive, persistent inward current that was activated between -55 and -50 mV and reached a peak between -40 and -30 mV. This current was significantly larger in the pacemaker neurons as compared to the non-pacemaker neurons and appeared to be a principal conductance driving the C1 pacemaker activity. Two other conductances modulated the frequency of pacemaker discharge: (1) an anomalous rectifier accelerated pacemaker frequency by three synergistic actions: (a) depolarizing it at rest, (b) increasing the slope of the pacemaker potentials, and (c) limiting hyperpolarizing membrane excursions; and (2) an A-type current which had two opposing actions: (a) slowing it by decreasing the slope of the pacemaker potential, and (b) accelerating it by repolarizing the fast action potential. Persistent sodium current functions as the driver potential responsible for the tonic firing pattern of the C1 bulbospinal neurons providing a cellular mechanism responsible for the descending excitatory drive imposed onto sympathetic preganglionic neurons. Thus, it may explain how C1 neurons may function to maintain vasomotor tone or modulate other autonomic functions. This study is the first attempt to analyze voltage-activated membrane conductances of RVLM neurons of known phenotype and axonal connections.

Topics: Action Potentials; Animals; Biological Clocks; Carbocyanines; Electric Conductivity; Fluorescent Dyes; In Vitro Techniques; Ions; Medulla Oblongata; Membrane Potentials; Muscle Tonus; Neurons; Rats; Rats, Sprague-Dawley; Spinal Cord; Sympathetic Nervous System; Vasomotor System

Neural crest cell migration in the hindbrain is segmental, with prominent streams of migrating cells adjacent to rhombomeres (r) r2, r4 and r6, but not r3 or r5. This migratory pattern cannot be explained by the failure of r3 and r5 to produce neural crest, since focal injections of the lipophilic dye, DiI, into the neural folds clearly demonstrate that all rhombomeres produce neural crest cells. Here, we examine the dynamics of hindbrain neural crest cell emigration and movement by iontophoretically injecting DiI into small numbers of cells. The intensely labeled cells and their progeny were repeatedly imaged using low-light-level epifluorescence microscopy, permitting their movement to be followed in living embryos over time. These intravital images definitively show that neural crest cells move both rostrally and caudally from r3 and r5 to emerge as a part of the streams adjacent to r2, r4, and/or r6. Within the first few hours, cells labeled in r3 move within and/or along the dorsal neural tube surface, either rostrally toward the r2/3 border or caudally toward the r3/4 border. The labeled cells exit the surface of the neural tube near these borders and migrate toward the first or second branchial arches several hours after initial labeling. Focal DiI injections into r5 resulted in neural crest cell contributions to both the second and third branchial arches, again via rostrocaudal movements of the cells before migration into the periphery. These results demonstrate conclusively that all rhombomeres give rise to neural crest cells, and that rostrocaudal rearrangement of the cells contributes to the segmental migration of neural crest cells adjacent to r2, r4, and r6. Furthermore, it appears that there are consistent exit points of neural crest cell emigration; for example, cells arising from r3 emigrate almost exclusively from the rostral or caudal borders of that rhombomere.

Topics: Animals; Carbocyanines; Cell Movement; Chick Embryo; Fluorescent Dyes; Iontophoresis; Microscopy, Fluorescence; Neural Crest; Rhombencephalon

The lipophilic dye, DiI, was placed into the macula lagena of paraformaldehyde-fixed embryonic chicks. Retrogradely labeled cells were found bilaterally in the pontine reticular formation (RF) between the dorsal facial nucleus and the abducens nerve root. This location is similar to that of the dorsomedial group of efferent cells that project to the basilar papilla. No lagenar efferent neurons, however, were found near the superior olivary nucleus where the ventrolateral group of cochlear efferents is located. Whether efferent neurons in the pontine RF send collaterals to both the basilar papilla and to the macula lagena has yet to be determined.

Topics: Acoustic Maculae; Animals; Carbocyanines; Chick Embryo; Cochlea; Fixatives; Fluorescent Dyes; Formaldehyde; Neurons, Efferent; Polymers; Tissue Fixation

The literature on the anatomical organization of primary sensory afferents, though extensive, contains relatively little information about the longitudinal extent of the central collateral projections. Our understanding of intersegmental sensorimotor integration in the spinal cord and of the developmental mechanisms that establish its underlying circuitry could be significantly enhanced by a more complete description of these projections. To address this issue from a developmental perspective, we labeled the central projections of lumbar primary afferents in fixed preparations of the chicken embryo with the lipophilic tracer DiI. At late embryonic stages, the afferent projections had the following characteristics: Primary afferents originating from a single lumbar dorsal root ganglion bifurcated to project longitudinally in the dorsal funiculus or Lissauer's tract. Dorsal funiculus axons extended up to seven segments caudally and to at least ten segments rostrally, whereas axons in Lissauer's tract extended up to seven segments in each direction. Collaterals branched off the longitudinal axons over a range of about seven segments in each direction. Within this range, collaterals to specific terminal fields exhibited more restricted ranges. The development of these longitudinal patterns during earlier embryonic stages was followed from the time the afferents first reached the neural tube on day 4 of embryogenesis. The longitudinal axons lengthened as a single bundle up to day 10, with medial axons consistently longer than lateral axons. After day 10, the longitudinal axons were segregated into the dorsal funiculus and Lissauer's tract. Collaterals sprouted after about 2 days of longitudinal axon growth, by which time the axons had extended several segments in each direction. The segmental range over which collaterals were present reached a maximum of 20 segments at day 10. Collaterals to the different terminal areas differed in their segmental ranges already by this time. After day 10, the total segmental range of collaterals decreased to the stable level of about seven segments in each direction, which is characteristic of late-stage embryos.

Topics: Afferent Pathways; Animals; Carbocyanines; Chick Embryo; Fluorescent Dyes; Lumbosacral Region; Neurons, Afferent; Spinal Cord

To elucidate guidance mechanisms of brain commissural axons, we examined the navigation of cerebellofugal axons. Axons were labeled by implantation of the fluorescent tracer Dil into the cerebellar plate (CP) of fixed, flat whole-mount embryonic rat brain. Axons initially grew straight toward the ventral midline floor plate (FP) in the rostral hindbrain and then, after crossing it, made a right-angled turn to grow either caudally or rostrally along the longitudinal axis. In collagen gel culture, CP axons showed directed growth toward both FP explants and heterologous cells expressing netrin-1, a FP-derived chemoattractant for spinal commissural axons. These results suggest that CP axons are guided to the midline by FP-derived chemoattractant(s) and then reoriented, possibly by another guidance cue, for longitudinal extension. Considering that the basic structures of the neural tube, including the FP, extend up to the caudal diencephalon, these results suggest that common guidance mechanisms operate for ventrally decussating commissural axons in both the brain and spinal cord.

Topics: Animals; Axons; Carbocyanines; Cell Line; Cerebellum; Culture Techniques; Female; Fluorescent Dyes; Gene Expression; Nerve Growth Factors; Netrin-1; Pregnancy; Rats; Rats, Wistar; Transfection; Tumor Suppressor Proteins

Despite numerous descriptive studies, the embryonic origin of vertebrate taste buds has never been experimentally determined. A number of different alternatives have been suggested for taste bud origins, including epibranchial placodes, the neural crest, and the local epithelium of the oropharyngeal cavity. The role of a series of epibranchial placodes and the cephalic neural crest, which together give rise to the cranial nerves innervating taste buds, was examined with regard to the development of oropharyngeal taste buds in an ambystomatid salamander, the axolotl. When pigmented placodal ectoderm or neural folds were grafted isotopically and isochronically into nonpigmented host embryos, known derivatives of each tissue contained pigmented cells, but labeled taste buds were never encountered. Thus, neither epibranchial placodes nor neural crest contribute cells to taste buds during embryogenesis. The majority of the oropharyngeal cavity of ambystomatid salamanders is lined by an endodermal epithelium. In order to demonstrate conclusively that taste buds arise from this local epithelium, the presumptive cephalic endoderm of early axolotl gastrulae was microinjected with the lipophilic dye, DiI. In the oropharyngeal epithelium of all larvae examined, both taste buds and general epithelial cells were labeled with DiI, indicating their common endodermal origin. Our findings are novel in that this is the first experimental demonstration of the endodermal origin of a vertebrate sensory receptor cell class.

Topics: Ambystoma; Animals; Carbocyanines; Ectoderm; Embryonic Induction; Endoderm; Ganglia; Head; Mouth; Nervous System; Neural Crest; Taste Buds; Tissue Transplantation

In most male vertebrates, the müllerian ducts, which are the embryonic oviducts, regress completely during embryogenesis or shortly thereafter. Müllerian duct regression is caused by the testicular hormone müllerian inhibiting substance. While the exact mechanism of müllerian duct regression is not well understood, it is theorized to involve active migration of at least some of the ductal epithelial cells following breakdown of the basement membrane, possibly through an interaction between the epithelial cells and the surrounding extracellular matrix. The purpose of the present study was (1) to confirm that müllerian duct regression is characterized by epithelial cell migration and (2) to determine the destination of these cells. The ductal epithelium of male alligator embryos was labelled with the fluorescent cell marker DiI and then allowed to undergo regression in vitro. DiI-labeled müllerian ducts from female embryos served as controls. An in vivo experiment also was performed in which the müllerian ducts of female hatchlings were injected with DiI prior to each female receiving a testicular implant from a male hatchling. Control females received a kidney implant instead. In both the in vitro and the in vivo experiments, DiI-labeled epithelial cells were observed migrating out from the duct during regression. In the in vitro experiment in which regression proceeded more rapidly, epithelial cells entered the mesonephric kidneys and were incorporated into the nephric tubule epithelium. No regression or cell migration was observed in the control müllerian ducts.

Topics: Alligators and Crocodiles; Animals; Carbocyanines; Cell Movement; Epithelial Cells; Epithelium; Female; Kidney Transplantation; Male; Mesonephros; Microinjections; Morphogenesis; Mullerian Ducts; Organ Transplantation; Oviducts; Sex Characteristics; Testis

Enteric AH neurons, with multipolar Dogiel type II morphology, project around the circumference of the intestine to myenteric ganglia, the submucosa and mucosa. Using retrograde labeling in vitro, intracellular recording, dye filling and immunohistochemistry, the projections of these neurons along the intestine were studied. When the retrograde tracer, Dil, was applied to the myenteric plexus, labeled nerve cell bodies were located up to 111 mm orally but only 13 mm aborally, demonstrating a marked difference in the lengths of projections up and down the small intestine. Of labeled nerve cell bodies located 2-110 mm orally, 43% had Dogiel type II morphology and of these, 70% were immunoreactive for calbindin, a calcium binding protein exclusive to Dogiel type II neurons. Intracellular filling with neurobiotin revealed several long circumferentially directed nerve fibers and short, filamentous dendrites; thus these were "dendritic" Dogiel type II neurons. This class accounts for approximately 3-4% of all myenteric neurons, and about 10% of all Dogiel type II neurons. Intracellular recordings revealed AH cell characteristics, with long afterhyperpolarizations following their action potentials, pronounced slow excitatory synaptic inputs and a lack of fast excitatory synaptic inputs. Antidromic action potentials could be evoked from the Dil application site in some cells, confirming their aboral projection. This is the first account of a major aboral projection of AH/Dogiel type II neurons and suggests an important role in aborally directed reflexes in the intestine.

Topics: Action Potentials; Animals; Biotin; Calbindins; Carbocyanines; Dendrites; Electric Stimulation; Female; Fluorescent Dyes; Ganglia, Autonomic; Guinea Pigs; Immunohistochemistry; Intestinal Mucosa; Intestine, Small; Male; Muscle, Smooth; Myenteric Plexus; Nerve Fibers; Nerve Tissue Proteins; Neurons; Organ Culture Techniques; S100 Calcium Binding Protein G; Synapses; Synaptic Transmission

Several mechanisms have been postulated to explain the relatively high immunogenicity of antigens presented in immune-stimulating complexes (iscom). Their potency can in part be explained by the specific targeting of these structures to cells presenting antigens to the immune system. However, until now no method for the subcellular detection of iscom in situ was available. In the present study, a novel, fast and simple method for the detection of iscoms in situ is demonstrated. By making use of the lipophilic fluorescent carbocyanine dyes, 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI) and 3,3'-dioctadecyloxacarbocyanine perchlorate (DiO), rabies virus antigen and iscom prepared with this antigen were visualized with fluorescence microscopy. The labeled antigen and iscoms were observed in macrophages of spleen and liver of mice within 1-2 h after intravenous administration. When administered intramuscularly or in the footpad, uptake in macrophages of draining lymph nodes could be demonstrated. In the spleen, labeled inactivated virus antigen localized preferentially in the marginal zone macrophages and to a lesser extent in the red pulp macrophages. In contrast, antigen presented in iscom was taken up mainly by the marginal metallophilic macrophages and to a much lesser extend by marginal zone macrophages or follicular-dendritic and -B cells. This method enables the detection of iscom and membrane viruses and allows the analysis of their relation to antigen-presenting cells in situ. Here, we demonstrate that iscom containing rabies virus antigen are taken up by a subset of macrophages in the spleen distinct from those that take up inactivated rabies virus antigen not presented in iscom, thereby possibly explaining the observed difference in immunogenicity of these antigen preparations. Furthermore, we show a lower efficiency on the induction of humoral and cellular responses after intravenous immunization for both types of antigen when compared with subcutaneous immunization.

Topics: Animals; Antibodies, Monoclonal; Antigen Presentation; Antigens, Viral; B-Lymphocytes; Carbocyanines; Dendritic Cells; Female; Fluorescent Dyes; Immunization; Injections, Intramuscular; Injections, Intraperitoneal; Injections, Intravenous; ISCOMs; Liver; Lymphocyte Activation; Macrophages; Metals; Mice; Mice, Inbred BALB C; Microscopy, Fluorescence; Pharmacokinetics; Rabies virus; Spleen

We describe a simple method for evaluating gap junctional communication (GJC) between cells in culture. The procedure involves pre-loading cells with two fluorescent dyes: calcein and DiI. Calcein is able to pass through gap junctions, while DiI is not. These pre-loaded cells are then plated with unlabeled cells. The number of cells receiving calcein from each pre-loaded cell can then be quantified after the cells settle on the plate. Potent and reversible inhibitors of GJC can be used in this system to evaluate dye transfer within a given period of time.

Topics: Carbenoxolone; Carbocyanines; Cell Communication; Cell Line; Cell Line, Transformed; Connexin 43; Fluoresceins; Fluorescent Dyes; Gap Junctions; Glycyrrhetinic Acid; Microscopy, Fluorescence; Oncogene Protein pp60(v-src); Stereoisomerism; Succinates

Currently we can observe an increasing interest for epithelial olfactory neurones. Several laboratories are involved in elucidation of the odorant-molecule recognition process and transduction cascade which brings information to the olfactory bulb. Others use this model, unique in mammals, to accumulate new knowledge on the neurogenesis phenomenon. Here we describe a simple and efficient method to purify mature olfactory neurone populations extracted from the nasal cavity. The approach relies on retrograde axonal tracing followed by flow cytometry sorting. For this purpose we inject a fluorescent dye (Fast Blue or diI C18(3)) in the olfactory bulb of adult rats. Seven days later, we extract the nasal turbinates and separate the mucosa from the subjacent lamina propria. The tissue is enzymatically dissociated and the labelled cells are sorted with a flow cytometer. Purification of the mature olfactory neurones varies from 80 to 99%.

Topics: Amidines; Animals; Carbocyanines; Coloring Agents; Flow Cytometry; Fluorescent Dyes; Immunohistochemistry; Nasal Mucosa; Nerve Tissue Proteins; Neurons, Afferent; Olfactory Bulb; Olfactory Marker Protein; Rats; Smell

Somatosensory thalamus and cortex in rodents contain topological representations of the facial whisker pad. The thalamic representation of a single whisker ("barreloid") is presumed to project exclusively to the cortical representation ("barrel") of the same whisker; however, it was not known when this correspondence is established during early development, nor how precise the thalamocortical projection is at birth, before formation of barrels and barreloids. To answer these questions, we retrogradely labeled thalamocortical projection neurons in fixed brain slices from 0-8 d old (P0-P8) mice, by placing paired deposits of two fluorescent dyes in adjacent barrels or (before barrel formation) in adjacent loci in upper cortical layers. At all ages studied, a negligible fraction of the retrogradely labeled cells was double labeled, implying that branches of single thalamocortical axons never extended within layer IV over an area wider than a single barrel. In P0 preparations, 70% of paired dye deposits placed 75-200 microns apart resulted in statistically significant segregation of labeled cell clusters in the thalamus. Quantitative analysis indicated that on P0 about 70% of thalamocortical axons were within 1.3 presumptive barrel diameters from their topologically precise target. In P4-P8 preparations, the great majority of thalamic cells retrogradely labeled from a single barrel were found in a single barreloid, implying a 1:1 projection of barreloids to barrels. The postnatal increase in topological precision was reproduced by a computer simulation, which assumed that many aberrant axons corrected their initial targeting error by extending terminal arborizations asymmetrically, towards the center of their appropriate barrel.

Topics: Aging; Animals; Animals, Newborn; Brain Mapping; Carbocyanines; Fluorescent Dyes; Mice; Synaptic Transmission; Thalamus; Vibrissae

During embryonic development, neural crest cells give rise to many structures in peripheral tissues. Other neural tube cells are thought to contribute only to structures within the CNS. In contrast to this idea, we report a second wave of migration of cells away from the spinal cord occurring after the emigration of crest cells is complete. Neuroepithelial cells from spinal cords in E5 chicken embryos migrate into the periphery and differentiate into neurons and satellite cells within sensory ganglia and into melanocytes in skin and feathers. These results show that some cell types previously considered to be the descendants exclusively of neural crest cells are also derived from neuroepithelial cells in the spinal cord.

Topics: Animals; Carbocyanines; Cell Differentiation; Cell Movement; Chick Embryo; Chimera; Epithelial Cells; Epithelium; Fluorescent Dyes; Ganglia, Sensory; Ganglia, Spinal; Immunohistochemistry; Melanocytes; Neural Crest; Neurons; Quail; Spinal Cord

In some developing systems, growing axons are attracted to their target site by a diffusible molecule released by the target tissue. In the chick hindlimb, this mechanism could explain how axons, after having reached the plexus region, grow to muscle or to skin. To begin to test this possibility for limb-innervating sensory neurons, we cocultured dorsal root ganglion explants and potential target tissues in three-dimensional collagen gels. In particular, we wanted to know if target tissues, at early stages of development, specifically attract axons of only the appropriate type of sensory neuron. To identify each type of sensory neuron, we used DiI to retrogradely label either cutaneous or muscle sensory neurons in the embryo, prior to culturing. The results showed that dermal and muscle explants could each enhance the outgrowth of both cutaneous and muscle sensory axons. In contrast, the epidermis and the connective tissue associated with developing muscle appeared to inhibit the outgrowth of both cutaneous and muscle sensory axons. These results suggest that, in the embryo, the dermis and muscle cells both release diffusible factors that cause sensory axons to diverge from the plexus, extend toward the sources of these factors, and thereby form discrete peripheral nerves. The inhibitory effects of epidermis and muscle-associated connective tissue may serve to limit the growth of sensory axons to the structures, i.e., dermis and muscle cells, that ultimately receive sensory innervation. However, since for each of the difference types of limb tissue, the responses of cutaneous and muscle sensory neurons were always similar to one another, sensory axons must not be responding to target-derived factors when they decide whether to grow to skin or to muscle.

Topics: Animals; Axons; Carbocyanines; Cell Death; Chick Embryo; Collagen; Culture Techniques; Fluorescent Dyes; Ganglia, Spinal; Gels; Hindlimb; Muscle, Skeletal; Nerve Growth Factors; Neurites; Neurons, Afferent; Skin

In the developing chick hindlimb, axons that will project along a given peripheral nerve sort out together, as they traverse the plexus region at the base of the limb, and become segregated from axons that will project along other peripheral nerves. This sorting out may involve, at least in part, the differential expression of various cell adhesion molecules (CAMs). To begin to explore this possibility, we have compared the relative levels of immunofluorescent labeling on the growth cones of two populations of sensory neurons whose axons become segregated from one another: cutaneous and muscle sensory neurons. We took a tissue culture approach, since this allowed us to readily visualize the immunofluorescent labeling of individual growth cones, and identified the two cell types by previous retrograde labeling with DiI. Two dorsal root ganglion explants, one containing DiI-labeled cutaneous neurons and the other containing DiI-labeled muscle sensory neurons, from opposite sides of the same embryo, were plated together in each culture dish. For all the CAMs were studied (NCAM, polysialylated NCAM, G4/L1, axonin-1, SC1/DM-GRASP/BEN, and N-cadherin), the intensity of immunofluorescent labeling typically was fairly uniform on the growth cone, it filopodia, and the portion of the neurite just proximal to the growth cone. Only one CAM, axonin-1, exhibited labeling that was especially intense at sites of interneuronal contact. Quantification of labeling intensities using image analysis showed that cutaneous and muscle sensory growth cones did not consistently differ from one another in their levels of expression of G4/L1 or of axonin-1. The latter finding stands in contrast to recent reports claiming that axonin-1 is not expressed on muscle sensory neurons. Each of the other CAMs (NCAM, polysialylated NCAM, SC1/DM-GRASP/BEN, and N-cadherin) also showed considerable overlap in the distribution of labeling intensities between the two populations, but overall, expression levels on muscle sensory growth cones were greater than on cutaneous growth cones. How the differential expression of some CAMs could potentially contribute to the way that cutaneous and muscle sensory growth cones become segregated from one another and the implications of these results for sensory neuron specification are discussed.

Topics: Animals; Axons; Carbocyanines; Cell Adhesion Molecules, Neuronal; Chick Embryo; Culture Techniques; Fluorescent Dyes; Ganglia, Spinal; Hindlimb; Microscopy, Fluorescence; Muscle, Skeletal; Neurons, Afferent; Skin; Tissue Distribution

To investigate further the factors involved in the development of cerebral cortical circuitry, we examined the organization of corticocortical connections of heterotopic grafts of fetal cortex placed into neonatal rat cortices. Presumptive perirhinal or sensorimotor areas of the cerebral wall were removed as slabs from embryonic day 12 (E12), E13, or E14 rats and transplanted heterotopically into either rostral perirhinal or sensorimotor cortical areas of neonatal rats. Two weeks later, the afferent cortical connections of the grafts were labeled by placing DilC18(-3) (Dil) into each transplant site. Both the E12 and E13 heterotopic transplants of presumptive perirhinal cortex contain mostly precursor cells. When these grafts are placed into sensorimotor cortex, callosal connections are formed primarily with the contralateral sensorimotor (Sml) area, the normal projection of Sml cortex. In contrast, the E14 heterotopic transplants of the perirhinal cortical wall, containing many more postmitotic neurons, developed contralateral connections with both sensorimotor and rostral perirhinal cortices. Thus, when precursor cells are transplanted heterotopically, by using E12/E13 donor tissue, the grafts receive projections that are similar to those of the host cortical area. When older cortical neurons, together with precursors, are transplanted into a heterotopic cortical area, by using E14 donor tissue, their cortical connections exhibit both host and original donor phenotypes. The data are consistent with our previous analysis of thalamocortical connections of grafts (Barbe and Levitt, 1992b) and suggest the existence of a cell-cell recognition system for thalamocortical and corticocortical circuit formation, whose mechanisms of action may be linked to the timing of neurogenesis.

Topics: Animals; Animals, Newborn; Carbocyanines; Cerebral Cortex; Fetal Tissue Transplantation; Motor Cortex; Neurons, Afferent; Rats; Rats, Sprague-Dawley; Sensation

The fate of the anterior neural ridge was studied by following the relative movements of simultaneous spot applications of DiI and DiO from stage 15 through stage 45. These dye movements were mapped onto the neuroepithelium of the developing brain whose shape was gleaned from whole-mount in situs to neural cell adhesion molecule and dissections of the developing nervous system. The result is a model of the cell movements that drive the morphogenesis of the forebrain. The midanterior ridge moves inside and drops down along the most anterior wall of the neural tube. It then pushes forward a bit, rotates ventrally during forebrain flexing, and gives rise to the chiasmatic ridge and anterior hypothalamus. The midanterior plate drops, forming the floor of the forebrain ventricle, and, keeping its place behind the ridge, it gives rise to the posterior hypothalamus or infundibulum. The midlateral anterior ridge slides into the lateral anterior wall of the neural tube and stretches laterally into the optic stalk and retina, and then rotates into a ventral position. The lateral anterior ridge converges to the most anterior part of the dorsal midline during neural tube closure, then rotates anteriorly, and gives rise to telencephalic structures. Whole-mount bromodeoxyuridine labeling at these stages showed that cell division is widespread and relatively uniform throughout the brain during the late neurula and early tailbud stages, but that during late tailbud stages cell division becomes restricted to specific proliferative zones. We conclude that the early morphogenesis of the brain is carried out largely by choreographed cell movements and that later morphogenesis depends on spatially restricted patterns of cell division.

Topics: Animals; Antimetabolites; Bromodeoxyuridine; Carbocyanines; Cell Division; Embryonic Development; Fluorescent Dyes; Histocytochemistry; In Situ Hybridization; Neural Cell Adhesion Molecules; Prosencephalon; Xenopus laevis

We have explored basic rules guiding the early development of topographically organized projections, employing the rat corticopontine projection as a model system. Using anterograde in vivo tracing with 1,1',dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI), we studied the distribution of labelled fibers in the pontine nuclei in relation to cortical site of origin during the first postnatal week. Labelled corticopontine fibers enter the pontine nuclei in distinct, sharply defined zones. The putative terminal fibers typically occupy lamella-like subspaces. Related to changes in cortical site of origin, we describe mediolateral, internal to external, and caudorostral distribution gradients in the pontine nuclei. Fibers originating in the anterolateral cortex occupy an internal central core, while implantations at increasing distance from the anterolateral cortex produce 1) more externally located lamellae, and 2) a caudal to rostral shift in fiber location. Previous investigations have shown that pontocerebellar neurons migrate into the ventral pons in a temporal sequence (Altman and Bayer [1987] J. Comp. Neurol. 257:529). The earliest arriving neurons occupy the central core and later arriving neurons settle in more externally and rostrally located subspaces. We hypothesize that the earliest arriving corticopontine fibers grow into the then only available zone of pontocerebellar neurons (central core), attracted by a diffusible chemotropic cue. Later arriving fibers grow into correspondingly later and more externally and rostrally located contingents of pontocerebellar neurons. Thus, we propose that the topographical organization in the early postnatal corticopontine projection is determined by simple temporal and spatial gradients operative within source (cerebral cortex) and target region (pontine nuclei).

Topics: Animals; Animals, Newborn; Axons; Brain Mapping; Carbocyanines; Cerebral Cortex; Computer Graphics; Fluorescent Dyes; Image Processing, Computer-Assisted; Nerve Fibers; Neural Pathways; Pons; Rats; Rats, Wistar

Fusion between monocytes and tumor cells has been suggested as a cause for tumor metastasis. The aim of the present study was to establish an in vitro fusion model representing the in vivo situation as close as possible. For this purpose fusion between cells was induced by cytokine containing conditioned medium. In order to prove that hybrid formation between tumor cells and monocytes occurs, a two-color-fusion-assay based on membrane labeling with the fluorochromes PKH 2 (green) and PKH 26 (red) was established. These fusion experiments were analyzed by microscopy and, in addition, by flow cytometry. The attempt to induce fusion between monocytes and several tumor cell lines of hematopoietic origin revealed quite diverse results. The most extensive hybrid formations were seen with TALL, a T-lymphocytic tumor line. The monocytic tumor line HL60 and the B-lymphocytic tumor line BL41 also clearly yielded hybrids with monocytes but in smaller numbers. With some other hematopoietic tumor lines no evidence for hybrid formation was detected. These studies indicate that fusion of normal monocytes with certain tumor cells may be induced under conditions that may occur in comparable manner in vivo.

Topics: Adult; Carbocyanines; Cell Fusion; Cell Line; Cell Membrane; Cytokines; Fluorescein-5-isothiocyanate; Fluorescent Dyes; HL-60 Cells; Humans; Lymphocytes; Membrane Proteins; Microscopy; Monocytes; Organic Chemicals; Rhodamines; Tumor Cells, Cultured

In this study, we have isolated newly formed somites from the caudal regions of 8.5 day mouse embryos and transplanted them orthotopically into correspondingly staged hosts at the level of the prospective limb-forming region. The experimental embryos were then cultured intact for 32-36 hr. The donor somites used were pre-labelled with DiI, a fluorescent lipophilic dye, or were obtained from transgenic embryos that carried a 1 kb 5' regulatory sequence of the desmin gene linked to the gene encoding Escherichia coli beta-galactosidase. The transgene is specifically expressed in skeletal muscles (Li et al. [1993] Development 117:947-959). The aim of these experiments was to show definitively that the musculature of the mammalian limb is derived from the somites. The results demonstrated that DiI-labelled cells from the implanted somites were able to invade the proximal region of the fore-limb bud during the course of development. The use of transgenic somites as grafts confirmed that some of the somitic cells found in the limbs were myogenic cells. To determine whether the displacement of somitic cells is an active or passive process, somatopleure obtained from the prospective limb-forming regions of day 8.5 day embryos was implanted into 8.5 day hosts. We did not detect the presence of DiI-labelled somatopleural cells in the fore-limb after 32-36 hr of culture. This suggests that somitic cells reached the limb bud via active locomotion rather than as a result of being passively dragged there, as the limb elongates during development. In addition, we injected latex beads into the somites, as probes, to determine whether extracellular matrix-driven translocation plays a role in driving the somitic cells to the limb bud. In a majority of the specimens examined, we could not detect the presence of these beads in the limb bud. However, in the trunk of these embryos, the beads were found dispersed throughout the ventral neural crest pathway.

Topics: Animals; Carbocyanines; Cell Movement; Embryo, Mammalian; Embryonic and Fetal Development; Fluorescent Dyes; Lac Operon; Latex; Limb Buds; Mesoderm; Mice; Mice, Inbred C57BL; Mice, Transgenic; Microspheres; Morphogenesis; Muscle Fibers, Skeletal; Recombinant Proteins

Confrontation cultures between glioma spheroids and brain cell aggregates are well established in glioma research, and the model reflects several similarities to the in vivo brain tumour invasive process. The lipid-binding fluorescent carbocyanine dyes DiO (3,3'-dioctadecyloxacarbocyanine perchlorate) and DiI (1,1'-dioctadecyl-3,3,3,'3,'-tetramethylinocarbocyanine perchlorate) are widely used in cell biology as tracers for studying cell movement. Mature brain cell aggregates grown from fetal rat brain cells, and spheroids initiated from two glioma cell lines (GaMg and D-54Mg) were stained with DiO and DiI, respectively. Penetration of DiI and DiO into the tumour spheroids and brain aggregates was studied by confocal laser scanning microscopy (CLSM). After 48 h of dye exposures, the tracers had almost completely penetrated the tumour spheroids and brain aggregates. Light-microscopic sections of the specimens indicated that the dye incorporation had little effect on cellular morphology. Cell migration from DiI stained D-54Mg and GaMg spheroids was similar to that observed from unstained spheroids. Growth was also unaffected after 48 h of DiI exposure. Gioma cell invasion was assessed by CLSM using co-cultures of DiI -stained spheroids and DiO-stained brain cell aggregates. Optical sections revealed a gradual decrease in remaining brain volume, indicating a progressive invasive process. Single tumour cells were identified deep within the brain aggregates. In addition normal brain cells were also identified in the tumour spheroids. It is concluded that vital staining can be used to identify both normal cells and tumour cells during tumour cell invasion in vitro. The method may provide the possibility for studying the kinetics of single normal and tumour cell movement in individual tumour/brain co-cultures.

Topics: Animals; Brain; Brain Neoplasms; Carbocyanines; Cell Aggregation; Cell Division; Cell Movement; Coculture Techniques; Fetus; Fluorescent Dyes; Glioma; Humans; Microscopy, Confocal; Neoplasm Invasiveness; Rats; Rats, Inbred Strains; Tumor Cells, Cultured

1. Responses of 89 single olfactory bulb neurons from 43 channel catfish, Ictalurus punctatus, to amino acid odorants were recorded in vivo simultaneously with the electro-olfactogram (EOG). Recording time for individual neurons ranged from 16 to 344 min. The averaged spontaneous frequency ranged from < 1 to 16 action potentials/s with a mean frequency of 5.2 +/- 3.6 (SD) action potentials/s. 2. Histological examinations of carbocyanine dye 1,1'diocadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI)-labeled olfactory bulbs and electrical stimulation of the olfactory tracts in a subset of experiments suggested that the majority of the recorded olfactory bulb neurons in this study were mitral cells. 3. Olfactory bulb neurons responded to amino acids with either an excitation or suppression of the background neural activity. Of the 337 stimulus applications, 28% of the responses were excitatory, and 33% were suppressive. The approximately 1:1 ratio of excitatory to suppressive responses for all stimulus applications suggests that suppressive responses also play important roles in the coding of odorant information in the channel catfish. 4. Responses of single olfactory bulb neurons were highly reproducible over time (up to 5 h). Responses to any amino acid never changed from excitation to suppression, or vice versa over time. 5. Single olfactory bulb neurons responded with excitation or suppression to more than one amino acid previously indicated to bind to independent receptors. 6. Estimated threshold concentrations for activation of an olfactory bulb neuron ranged from 10(-7) to 10(-3) M and were different from neuron to neuron for a particular stimulus and from stimulus to stimulus for a particular neuron. Responses of single olfactory bulb neurons to a given amino acid did not change from excitation to suppression, or vice versa, across different suprathreshold concentrations.

Topics: Amino Acids; Animals; Carbocyanines; Differential Threshold; Electric Stimulation; Electrophysiology; Ictaluridae; Neurons; Odorants; Olfactory Bulb; Osmolar Concentration; Reproducibility of Results

In an attempt to identify the distribution and structure of vagal fibers and terminals in the gastroduodenal junction, vagal efferents were labeled in vivo by multiple injections of the fluorescent carbocyanine dye DiA into the dorsal motor nucleus (dmnX), and vagal afferents were anterogradely labeled by injections of DiI into the nodose ganglia of the same or separate rats. Thick frontal cryostat sections were analysed either with conventional or laser scanning confocal microscopy, using appropriate filter combinations and/or different wavelength laser excitation to distinguish the fluorescent tracers. Vagal efferent terminal-like structures were present in small ganglia within the circular sphincter muscle, which, in the absence of a well-developed, true myenteric plexus at this level, represent the myenteric ganglia. Furthermore, vagal efferent terminals were also present in submucosal ganglia, but were absent from mucosa, Brunner's glands and circular muscle fibers. Vagal afferent fibers and terminal-like structures were more abundant than efferents. The most prominent afferent terminals were profusely branching, large net-like aggregates of varicose fibers running within the connective tissue matrix predominantly parallel to the circular sphincter muscle bundles. Profusely arborizing, highly varicose endings were also present in large myenteric ganglia of the antrum and duodenum, in the modified intramuscular ganglia, and in submucosal ganglia. Additionally, afferent fibers and terminals were present throughout the mucosal lining of the gastroduodenal junction. The branching patterns of some vagal afferents suggested that individual axons produced multiple collaterals in different compartments. NADPH-diaphorase positive, possibly nitroxergic neurons were present in myenteric ganglia of the immediately adjacent antrum and duodenum, and fine varicose fibers entered the sphincter muscle from both sides, delineating the potential vagal inhibitory postganglionic innervation. These morphological results support the view of a rich and differentiated extrinsic neural control of this important gut region as suggested by functional studies.

Topics: Animals; Axonal Transport; Biomarkers; Calcitonin Gene-Related Peptide; Carbocyanines; Fluorescent Dyes; Lasers; Male; Microscopy; Muscle, Smooth; NADPH Dehydrogenase; Nerve Tissue Proteins; Pylorus; Pyridinium Compounds; Rats; Rats, Wistar; Stilbamidines; Vagus Nerve

The adult cerebral cortex extends axons to a variety of subcortical targets, including the thalamus and superior colliculus. These descending projections are pioneered during development by the axons of a transient population of subplate neurons (McConnell et al., 1989). We show here that the descending axons of cortical plate neurons appear to be delayed significantly in their outgrowth, compared with those of subplate neurons. To assess the possible role of subplate neurons in the formation of these pathways, subplate neurons were ablated during the embryonic period. In all cases, an axon pathway formed from visual cortex through the internal capsule and into the thalamus. In half of all cases, however, cortical axons failed to invade their normal subcortical targets. In the other half, targets were innervated normally. Subplate neurons are therefore likely to provide important cues that aid the process by which cortical axons grow toward, select, and invade their subcortical targets.

Topics: Afferent Pathways; Animals; Animals, Newborn; Axonal Transport; Axons; Carbocyanines; Cats; Cerebral Cortex; Efferent Pathways; Fetus; Fluorescent Dyes; Models, Neurological; Neurons; Temporal Lobe; Thalamus; Visual Cortex

Previous cell lineage studies indicate that the repeated neuromeres of the chick hindbrain, the rhombomeres, are cell lineage restriction compartments. We have extended these results and tested if the restrictions are absolute. Two different cell marking techniques were used to label cells shortly after rhombomeres form (stage 9+ to 13) so that the resultant clones could be followed up to stage 25. Either small groups of cells were labelled with the lipophilic dye DiI or single cells were injected intracellularly with fluorescent dextran. The majority of the descendants labelled by either technique were restricted to within a single rhombomere. However, in a small but reproducible proportion of the cases (greater than 5%), the clones expanded across a rhombomere boundary. Neither the stage of injection, the stage of analysis, the dorsoventral position, nor the rhombomere identity correlated with the boundary crossing. Judging from the morphology of the cells, both neurons and non-neuronal cells were able to expand over a boundary. These results demonstrate that the rhombomere boundaries represent cell lineage restriction barriers which are not impenetrable in normal development.

Topics: Animals; Carbocyanines; Cell Differentiation; Chick Embryo; Dextrans; Fluorescent Dyes; Microscopy, Fluorescence; Rhodamines; Rhombencephalon

In the present study, we demonstrate the existence of numerous peptidergic afferents to the bed nucleus of the stria terminalis (BNST) using the retrograde transport of gold-labeled wheat germ agglutinin-apo-peroxidase (G-WGA-HRP) combined with the indirect immunoperoxidase method after intraparenchymatous injections of colchicine. At first, we show that local injections of colchicine alone into the BNST are able to induce the retrograde accumulation of peptides until the nerve cell bodies of origin, probably because of the blockade of axonal transport in nerve terminal arborizations innervating this nucleus. The actual existence of putative peptidergic afferents to the BNST indicated by the local injections of colchicine was established using: a) the retrograde transport of G-WGA-HRP from the BNST combined with immunocytochemistry after administration of colchicine at the same place, b) the anterograde "transport" of the fluorescent tracer DiI from selected nuclei of the forebrain. We demonstrate that the neurons immunoreactive for enkephalins, neurotensin, or substance P that innervate the BNST are localized mainly in the central amygdaloid nucleus, the paraventricular thalamic nucleus, and the ventromedial hypothalamic nucleus ipsilateral to the injection, as well as bilaterally in the magnocellular paraventricular and perifornical regions of the hypothalamus. From these results it may be concluded that intracerebral injections of colchicine constitute a powerful tool to search for multiple peptidergic afferents to a given brain nucleus using only immunohistochemistry. The existence of these pathways, however, must be verified by other neuroanatomical methods because of the problem of nerve fibers of passage.

Topics: Animals; Axonal Transport; Carbocyanines; Colchicine; Enkephalin, Methionine; Enkephalins; Horseradish Peroxidase; Ibotenic Acid; Immunohistochemistry; Male; Neurons, Afferent; Neuropeptides; Neurotensin; Rats; Rats, Wistar; Substance P; Thalamic Nuclei; Tyrosine 3-Monooxygenase; Wheat Germ Agglutinin-Horseradish Peroxidase Conjugate; Wheat Germ Agglutinins

Substance P, calcitonin gene-related peptide and somatostatin immunoreactivities have been demonstrated in putative afferent renal nerve fibers in the rat. Utilizing retrograde-tracing and immunohistochemistry, we labeled afferent renal nerve soma throughout dorsal root ganglia T9 to L1. Most (85%) of afferent renal nerve perikarya were immunoreactive for calcitonin gene-related peptide, 21% had substance P immunoreactivity and none had somatostatin immunoreactivity. All renal afferents immunoreactive for substance P also contained calcitonin gene-related peptide. These results provide evidence that calcitonin gene-related peptide and substance P are present and co-localized in afferent renal nerves, and therefore, mediate transmission of afferent renal input to the spinal cord in the rat.

Topics: Amidines; Animals; Antibody Specificity; Calcitonin Gene-Related Peptide; Carbocyanines; Fluorescent Dyes; Ganglia, Spinal; Immunohistochemistry; Kidney; Male; Neurons, Afferent; Rats; Rats, Sprague-Dawley; Stilbamidines; Substance P

The amphibian Xenopus laevis is able to adapt the colour of its skin to the light intensity of the background, by releasing alpha-melanophore-stimulating hormone from the pars intermedia of the hypophysis. In this control various inhibitory (dopamine, gamma-aminobutyric acid, neuropeptide Y, noradrenaline) and stimulatory (thyrotropin-releasing hormone and corticotropin-releasing hormone) neural factors are involved. Dopamine, gamma-aminobutyric acid and neuropeptide Y are present in suprachiasmatic neurons and co-exist in synaptic contacts on the melanotrope cells in the pars intermedia, whereas noradrenaline occurs in the locus coeruleus and noradrenaline-containing fibres innervate the pars intermedia. Thyrotropin-releasing hormone and corticotropin-releasing hormone occur in axon terminals in the pars nervosa. In the present study, the neuronal origins of these factors have been identified using axonal tract tracing. Application of the tracers 1,1'dioctadecyl-3,3,3',3' tetramethyl indocarbocyanine and horseradish peroxidase into the pars intermedia resulted in labelled neurons in two brain areas, which were immunocytochemically identified as the suprachiasmatic nucleus and the locus coeruleus, indicating that these areas are involved in neural inhibition of the melanotrope cells. Thyrotropin-releasing hormone and corticotropin-releasing hormone were demonstrated immunocytochemically in the magnocellular nucleus. This area appeared to be labelled upon tracer application into the pars nervosa. This finding is in line with the idea that corticotropin-releasing hormone and thyrotropin-releasing hormone stimulate melanotrope cell activity after diffusion from the neural lobe to the pars intermedia. After anterograde filling of the optic nerve with horseradish peroxidase, labelled axons were traced up to the suprachiasmatic area where they showed to be in contact with suprachiasmatic neurons. These neurons showed a positive reaction with anti-neuropeptide Y and the same held for staining with anti-tyrosine hydroxylase. It is suggested that a retino-suprachiasmatic pathway is involved in the control of the melanotrope cells during the process of background adaptation.

Topics: Adaptation, Physiological; Afferent Pathways; alpha-MSH; Animals; Axonal Transport; Brain Mapping; Carbocyanines; Corticotropin-Releasing Hormone; Horseradish Peroxidase; Hypothalamo-Hypophyseal System; Locus Coeruleus; Melanophores; Neurotransmitter Agents; Optic Nerve; Pituitary Gland, Anterior; Preoptic Area; Retina; Skin Pigmentation; Suprachiasmatic Nucleus; Thyrotropin-Releasing Hormone; Xenopus laevis

The starfish oocyte is relatively clear optically, and its nucleus, termed the germinal vesicle, is large. These characteristics allowed studies by confocal microscopy of germinal vesicle breakdown during maturation in living oocytes. Three fluorescent probes for cytoplasmic components were used: fluorescein 70 kDa dextran, which does not cross the nuclear pore of immature oocytes and probably behaves in the same way as soluble cytosolic proteins, YOYO-1, which was used to localize ribosomes, and DiI which labels the nuclear envelope and endoplasmic reticulum. The first change observable by transmitted light microscopy during maturation is a wrinkling of the germinal vesicle envelope. Several minutes before the wrinkling, the 70 kDa dextran began to enter the germinal vesicle; the ribosomes did not enter during this period. The dextran is likely to be passing through nuclear pores whose size limit has increased but which still exclude ribosomes. At the time of the wrinkling of the germinal vesicle envelope, both 70 kDa dextran and ribosomes entered as a massive wave. The characteristics of this entry indicate that the permeability barrier of the nuclear envelope bilayer has been disrupted. The disruption of the permeability barrier occurred in a local region rather than around the entire periphery. Also, the disruption was observed more often on the animal pole side of the germinal vesicle (26/34 oocytes). The endoplasmic reticulum entered the nuclear region more slowly. Cytochalasin B inhibited this movement and also inhibited characteristic endoplasmic reticulum movements seen at high magnification. The effects of cytochalasin indicate that mixing of endoplasmic reticulum with nuclear space is an active process involving actin filaments.

Topics: Animals; Benzoxazoles; Carbocyanines; Cytoplasm; Cytosol; Dextrans; Endoplasmic Reticulum; Female; Fluoresceins; Fluorescent Dyes; Microscopy, Confocal; Nuclear Envelope; Oocytes; Quinolinium Compounds; Ribosomes; Starfish

Increasing interest in endothelialization of synthetic and tissue cardiovascular prostheses in vitro emphasizes the need for simple and rapid methods to evaluate presence of endothelium on surfaces. Scanning electron microscopy is a commonly used method for this purpose. In this study we investigated alternative and more rapid staining methods. Human saphenous vein endothelial cells in culture and on cardiovascular prosthetic materials (pyrolytic carbon, cusps of bioprosthetic heart valves, pig aorta, and expanded polytetrafluoroethylene) were labeled by exposing them to medium containing 5-chloromethylfluorescein diacetate or 1,1-dioctadecyl-3,3,3',3'-tetramethylindo carbocyanine perchlorate. For comparison, specimens were also fixed and processed for scanning electron microscopy. A bright fluorescence of endothelial cells labeled with 5-chlormethylfluorescein diacetate or 1,1-deoctadecyl-3,3,3',3'-tetramethylindo carbocyanine perchlorate wre clearly visualized in culture, on pyrolytic carbon, and on expanded polytetrafluoroethylene. Unfixed, prelabeled cells could be visualized immediately and unlabeled cells could be investigated for viability within 1 hour. Cells seeded on biologic tissue specimens could be visualized within 15 minutes with a modified hematoxylin-eosin staining. We suggest the use of these methods for rapid visualization of endothelium present on surfaces of cardiovascular prosthetic materials where they can partly replace the use of scanning electron microscopy.

Topics: Bioprosthesis; Blood Vessel Prosthesis; Carbocyanines; Cell Survival; Cells, Cultured; Endothelium, Vascular; Fluoresceins; Fluorescent Dyes; Heart Valve Prosthesis; Humans; Microscopy, Electron, Scanning; Microscopy, Fluorescence; Prosthesis Design; Saphenous Vein; Staining and Labeling; Surface Properties

The labelling efficacies of 7 retrograde tracers were evaluated following cut nerve exposure or intramuscular injection into the serially compartmentalized neck muscle, biventer cervicis. Tested tracers included Fast Blue (FB), Fluorogold (FG), dextran conjugated to fluorescein (FD), dextran conjugated to rhodamine (Fluororuby (FR), 3000 and 10,000 MW), fluorescent latex microspheres, horseradish peroxidase coupled to colloidal gold, and 1,1'-dioctadecyl-3,3,3',3'-tetramethyl indocarbocyanine perchlorate (DiI). In 2 animals, horseradish peroxidase was also employed and spinal cords were processed for peroxidase activity to evaluate its effect on the appearance of cells labelled with fluorescent tracers. Four tracers, FB, FG, FD and FR, could be observed in motoneurones under the conditions of our study. FB and FG labelled comparable numbers of motoneurones following cut nerve exposure, but dissimilar numbers following intramuscular injection. FG diffused extensively following injection and was found in motoneurones not only in the appropriate ipsilateral segment but also adjacent ipsilateral and contralateral segments. Intramuscular injections of FB usually labelled fewer cells than cut nerve exposure, but evidence for spurious labelling following intramuscular injection could also be found. FD or FR labelled motoneurones following cut nerve exposure but not following intramuscular injection. The conjugated dextrans labelled more variable numbers of cells than FB or FG, but the labelled cells had similar patterns of distribution. The remaining tracers were ineffective as retrograde markers in our study, and the possible reasons for these failures are discussed.

Topics: Amidines; Animals; Axonal Transport; Carbocyanines; Cats; Coloring Agents; Dextrans; Evaluation Studies as Topic; Fluoresceins; Fluorescent Dyes; Gold Colloid; Horseradish Peroxidase; Latex; Microspheres; Motor Neurons; Neck Muscles; Rhodamines; Stilbamidines

Corpus callosum (CC) axons in visual cortex were labeled anterogradely by in vivo biotinylated dextran amine (BDA) in neonatal cat at postnatal day (PND) 6, 10 and 15. Labeled CC axons were distributed throughout the visual cortex including medial area 17. The number of CC axons in medial area 17 increased from PND 6 to PND 10, and then decreased from PND 10 to PND 15. At PND 15, few CC axons could be followed into the grey matter in medial area 17. Thus, BDA labels transitory CC axons that extend through all cortical layers in medial area 17, confirming the results revealed by in vitro DiI labeling.

Topics: Animals; Animals, Newborn; Animals, Suckling; Axonal Transport; Axons; Biotin; Carbocyanines; Cats; Corpus Callosum; Dextrans; Visual Cortex

Studies employing axoplasmic transport techniques have suggested that the central arbors of vibrissae-related primary afferents are rapidly and permanently lost from the trigeminal (V) brainstem complex after transection of the intraorbital nerve (ION). The present study reexamined this issue using immunocytochemistry for galanin (GAL) and anterograde labelling with Di-I to evaluate V brainstem organization in rats that sustained damage to the ION or individual vibrissae follicles in infancy or adulthood. After adult nerve damage, GAL-positive fibers are increased in layers I and II of V subnucleus caudalis (SpC). This was apparent by 3 days after the lesion. In rats that sustained nerve damage at birth (P0), GAL immunoreactivity (IR) appeared throughout the V brainstem complex and had a patchy distribution similar to that of vibrissae-related V primary afferents in normal rats. Increased GAL-IR in rostral portions of the V brainstem complex was observed in rats that sustained ION damage as late as P14. Additional experiments in which nerve damage was followed by destruction of the V ganglion demonstrated that this GAL-IR was contained in primary afferents. Damage to single vibrissa follicles or to a row of follicles produced a single patch or row of GAL-IR terminals in the somatotopically appropriate portion of the ipsilateral V brainstem complex. Di-I labelling in neonatally nerve-damaged rats demonstrated that primary afferent axons filled the central territory normally innervated by this nerve and that their terminal distribution was patchy. These results suggest that the V ganglion cells that survive neonatal axotomy may retain somatotopically organized projections to the V brainstem complex for at least a limited postnatal period.

Topics: Afferent Pathways; Aging; Animals; Animals, Newborn; Axonal Transport; Brain Stem; Carbocyanines; Cell Survival; Fluorescent Dyes; Galanin; Immunohistochemistry; Neuropeptides; Oculomotor Nerve; Peptides; Rats; Reference Values; Trigeminal Ganglion; Vibrissae

The projections of Dogiel type II myenteric neurons to the mucosa of the guinea-pig ileum were quantified by combining retrograde transport of DiI, in vitro, with immunohistochemistry. After DiI application to the mucosa over an area of 1.5 x 10 mm2, virtually all (> 97%) calbindin-immunoreactive Dogiel type II neurons in the myenteric plexus underneath the mucosal DiI application site were labelled, indicating that essentially all of these neurons project to the mucosa. From cell counts, on average 5 calbindin-immunoreactive neurons project to each villus, and each calbindin-immunoreactive neuron supplies on average 10 villi. Since Dogiel type II neurons that were not immunoreactive for calbindin (19% of all labelled nerve cells) also projected to the mucosa, it is likely that all Dogiel type II neurons, which are putative sensory neurons of the gut, project to the mucosa.

Topics: Animals; Axonal Transport; Calbindins; Carbocyanines; Cell Count; Guinea Pigs; Intestinal Mucosa; Microvilli; Myenteric Plexus; Nerve Tissue Proteins; Neurons, Afferent; S100 Calcium Binding Protein G

Primary sensory neurons bridge the sensory periphery to the central nervous system (CNS) via their two axonal processes. The morphological patterning of the terminals of each process in its respective target is unique. Whether the differences between peripheral and central axons result from an intrinsic developmental program of the ganglion cell body, or from target-derived signals is not known. To explore this issue, we have used an explant coculture system in which embryonic (E15) trigeminal ganglion explants were placed between a vibrissa pad and a brainstem explant, but the explants were rotated 180 degrees relative to their normal orientation. In other experiments, individual ganglia were placed between two vibrissa pad explants or between two slices taken through the brainstem. The cultures were fixed after several days and ganglion cell processes were labeled with the lipophilic tracer DiI. Results of the ganglion rotation experiments suggest that trigeminal axons which would be directed centrally in vivo can regenerate into peripheral targets, and peripheral axons can grow into CNS tissue. Similarly, in cocultures with two peripheral or two central targets, both processes of trigeminal ganglion cells can simultaneously invade vibrissa pad explants or project into brainstem slices. Moreover, in all cocultures the differentiation of each set of processes is specific to the target innervated by it. These results show that the axons of embryonic sensory neurons are not selective in their choice of targets, and that their morphological patterning is dictated by target-derived signals.

Topics: Animals; Brain Stem; Carbocyanines; Cell Communication; Embryonic and Fetal Development; Fluorescent Dyes; Nerve Regeneration; Neurites; Organ Culture Techniques; Orientation; Rats; Trigeminal Ganglion; Vibrissae

This study examines the connections of the thalamic reticular and perireticular cell groups in developing ferrets. Small crystals of Dil (1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate) were implanted into either the dorsal thalamus or the cerebral cortex of aldehyde-fixed prenatal and postnatal ferret brains. A small implant of Dil into the presumptive lateral geniculate nucleus during early prenatal development [between embryonic day 23 (E23) and E25] reveals many retrogradely labelled cells in the reticular nucleus. At E40, just before birth, the number of cells retrogradely labelled in the reticular nucleus has become reduced compared to earlier prenatal implants, whether from small or large implants of Dil into the lateral geniculate nucleus. By postnatal day 7, an adult-like pattern of retrograde labelling is seen in the reticular nucleus; at this age, a small implant of Dil limited to the lateral geniculate nucleus retrogradely labels a discrete group of cells located in the caudal regions of the reticular nucleus. In the internal capsule, adjacent to the reticular nucleus, there are two distinct groups of neurons. One group, called the large-celled perireticular zone (LPR), enters the internal capsule very early in development (from E25; Mitrofanis, J., Eur. J. Neurosci., 6, 253-263, 1994) and is not labelled from the lateral geniculate nucleus at any developmental stage. Small implants of Dil into presumptive visual and somatosensory cortices shows that the LPR lies in a distinct region of the primordial internal capsule. Corticothalamic and thalamocortical axons turn sharply in the region of the LPR, whilst corticospinal and corticobulbar axons pass straight through the LPR on towards their more caudal targets. Later, after both sets of axons have reached their targets, the LPR is not seen in the internal capsule. The other group of cells in the internal capsule, called the small-celled perireticular zone (SPR), forms a distinct band of cells lying midway between the reticular nucleus and the globus pallidus. These cells enter the internal capsule much later in development, at about E40. Unlike the cells in the LPR, cells in the SPR are retrogradely labelled after an implant of Dil into the lateral geniculate nucleus, and there are many which remain in the adult (Clemence, A. E. and Mitrofanis, J., J. Comp. Neurol., 322, 167-181, 1992).

Topics: Aging; Animals; Animals, Newborn; Carbocyanines; Female; Ferrets; Fluorescent Dyes; Histocytochemistry; Neural Pathways; Pregnancy; Reticular Formation; Thalamus

Recent investigations revealed the localized distribution of the motoneuron subgroups within the mammalian oculomotor nucleus. In this study, we examined the motor neuron pool of the superior rectus muscle (SR) in 12 albino rats by injecting the retrograde fluorescent tracers Fluoro-Gold (FG) into one SR and Dil into the contralateral SR. We also examined the topographic and functional correlation between the neurons controlling the muscles of the upward gaze, i.e., SR and the inferior oblique muscle (IO) in another 5 albino rats by means of the same tracers. Our results demonstrated that: 1) the average total number of the neurons in the motor neuron pool of SR was 322.7 +/- 40.1; 2) topographically, about 94.65% of the motoneurons controlling SR were located in the contralateral side, while the remaining 5.35% lay ipsilaterally; 3) all the IO motoneurons were present on the same side of the muscle they innervate; 4) the motoneurons of SR and IO did not form separate subnuclei, but were intermingling with each other; 5) functionally, about 94.59%, 4.26% and 1.15% of the total motoneurons in the SR subnucleus of either oculomotor nucleus were projecting contralaterally, ipsilaterally and bilaterally, respectively; 6) although the distribution of the perikarya was not homogeneous, the bilaterally projecting (i.e., to both SR) cells were disposed centrally, and their double labeling indicated that their axons innervate the bilateral SR via axonal bifurcation and/or collateral branching; and 7) the motoneurons of SR and IO were functionally segregated from each other as no bilaterally projecting neurons (i.e., to SR and IO) were found.

Topics: Animals; Carbocyanines; Female; Fluorescent Dyes; Male; Motor Neurons; Oculomotor Muscles; Rats; Rats, Wistar; Stilbamidines

The general architecture of the visual system is similar for all species of mammal. To determine if the development of connections in the visual system might be under the influence of conserved molecules, we co-cultured explants of the murine lateral geniculate nucleus with slices from either murine or feline occipital cortex. Neurite outgrowth from embryonic murine geniculate explants was significantly enhanced by slices of newborn mouse occipital cortex or kitten visual cortex or by medium previously conditioned by these slices. Slices of similar volume but from sites other than occipital cortex had less or no effect on the murine geniculate explants. Fibers from murine geniculate explants grew freely on cortical slices from the kitten. They terminated mainly in layer 4 and also in layer 6, in both murine and feline visual and frontal cortical slices, irrespective of whether they entered through the white matter or pial side. Only the deep layers of the kitten's cortex sent projections to co-cultured murine geniculate explants. We suggest that the diffusible factors released by the cortex that stimulate the growth of axons from the lateral geniculate nucleus and the molecules that mark specific cortical laminae as targets for ingrowing afferents, are conserved in divergent species. We also found that murine geniculate axons grew freely on feline cerebellar slices. It is known from previous co-culture experiments that rodent geniculate axons are inhibited on rodent cerebellum and we suggest that the inhibitory factors involved are not conserved.

Topics: Animals; Axons; Carbocyanines; Cats; Culture Techniques; Embryonic Induction; Female; Fluorescent Dyes; Geniculate Bodies; Mice; Nerve Growth Factors; Occipital Lobe; Pregnancy; Presynaptic Terminals; Signal Transduction; Species Specificity; Visual Cortex; Visual Pathways

Fluorescently labeled low density lipoproteins (LDL) and influenza virus particles were bound to the surface of human fibroblasts and imaged with a cooled slow-scan CCD camera attached to a fluorescence microscope. Particles were also imaged after attachment to polylysine-coated microscope slides. The digital images were analyzed by fitting data points in the region of fluorescent spots by a two-dimensional Gaussian function, thus obtaining a measure of spot intensity with correction for local background. The intensity distributions for particles bound to polylysine slides were mainly accounted for by particle size distributions as determined by electron microscopy. In the case of LDL, the intensity distributions for particles bound to fibroblasts were considerably broadened, indicative of clustering. The on-cell intensity distributions were deconvolved into 1-particle, 2-particle, 3-particle, etc. components using the data obtained with LDL bound to polylysine-coated slides as an empirical measure of the single particle intensity distribution. This procedure yielded a reasonably accurate measure of the proportion of single particles, but large errors were encountered in the proportions of larger cluster sizes. The possibility of studying the dynamics of clustering was investigated by binding LDL to cells at 4 degrees C and observing changes in the intensity distribution with time after warming to 20 degrees C.

Topics: Biophysical Phenomena; Biophysics; Carbocyanines; Cell Line; Cell Membrane; Fibroblasts; Fluorescent Dyes; Humans; Lipoproteins, LDL; Microscopy, Electron; Microscopy, Fluorescence; Orthomyxoviridae; Particle Size; Receptor Aggregation; Rhodamines; Skin; Temperature; Time Factors

The hindbrain of the chick embryo contains three classes of motor neurons: somatic, visceral, and branchial motor. During development, somata of neurons in the last two classes undergo a laterally directed migration within the neuroepithelium; somata translocate towards the nerve exit points, through which motor axons are beginning to extend into the periphery. All classes of motor neuron are immunopositive for the SC1/DM-GRASP cell surface glycoprotein. We have examined the relationship between patterns of motor neuron migration, axon outgrowth, and expression of the SC1/DM-GRASP mRNA and protein, using anterograde or retrograde axonal tracing, immunohistochemistry, and in situ hybridization. We find that as motor neurons migrate laterally, SC1/DM-GRASP is down-regulated, both on neuronal somata and axonal surfaces. Within individual motor nuclei, these lateral, more mature neurons are found to possess longer axons than the young, medial cells of the population. Labelling of sensory or motor axons growing into the second branchial arch also shows that motor axons reach the muscle plate first, and that SC1/DM-GRASP is expressed on the muscle at the time growth cones arrive.

Topics: Activated-Leukocyte Cell Adhesion Molecule; Animals; Axons; Brain Stem; Carbocyanines; Cell Adhesion Molecules, Neuronal; Chick Embryo; Down-Regulation; Extracellular Matrix Proteins; Fluorescent Dyes; Horseradish Peroxidase; Immunohistochemistry; In Situ Hybridization; Motor Neurons; Nerve Tissue Proteins

Fluorescent carbocyanine dye (diI), applied to the glossopharyngeal (IX) nerve of the axolotl, transneuronally labeled solitary cells in the non-taste lingual epithelium. With diaminobenzidine (DAB), the diI was photoconverted to a dark, electron-dense product. The labeled cell had a large nucleus with invaginations, dense-cored vesicles in the cytoplasm, and finger-like processes. These are reminiscent of morphological features of cutaneous Merkel cells, suggesting that solitary cells innervated by the IX nerve are associated with mechanosensory function of the IX nerve system.

Topics: Ambystoma mexicanum; Animals; Carbocyanines; Epithelium; Fluorescent Dyes; Mechanoreceptors; Microscopy, Electron; Microscopy, Fluorescence; Nerve Fibers; Tongue

Low density lipoprotein receptors (LDL-R) on Daudi Burkitt's lymphoma (BL) cells were assessed using fluorescent DiI (3,3'-dioctadecylindocarbocyanine iodide)-LDL and flow cytometric analyses. Receptor-specific binding of DiI-LDL is followed by internalization of the bound complex and lysosomal hydrolysis of the ligand. Increase in the fluorescence intensity per cell is hence used as a measure of DiI-LDL uptake and, implicitly, as an indication of LDL-R presence. Our results show that uptake was observed in > 98% of the Daudi cells, and the level of uptake was significant and clearly distinguishable from autofluorescence, suggesting that: (a) this assay is comparable to the iodinated LDL uptake assay, although the ED50 values for the ligands are different; (b) this assay is comparable to the flow-cytometric detection of LDL-R using a commercial antibody directed against the receptor itself, and superior to a similar assay based on an antibody directed against membrane-bound LDL; (c) LDL uptake could be monitored along with transferrin uptake, suggesting that multiple endocytic receptor activities can be concurrently studied; (d) DiI-LDL uptake can be examined along with fluorescein-conjugated anti-CD10, -CD19, and -CD71, with little cross-interference, offering the added advantage that endocytic uptake and phenotyping can be simultaneously monitored; (e) the expression of LDL-R is intrinsically elevated in diverse cell lines such as Daudi, Raji, Ramos, Jurkat, and WIL2-NS, but not in normal lymphocytes. Our results therefore indicate that flow cytometric analysis of DiI-LDL uptake has potentially useful applications in the detection and study of endocytic receptor LDL-R in B and T lymphocytic cell lines.

Topics: B-Lymphocytes; Biological Transport; Carbocyanines; Cell Line; Flow Cytometry; Fluorescent Dyes; Humans; Lipoproteins, LDL; Receptors, LDL; T-Lymphocytes; Transferrin

1. An in vitro brainstem slice preparation of the superior olivary complex has been developed permitting patch recording from a presynaptic terminal (calyx of Held) and from its postsynaptic target--the principal neurone of the medial nucleus of the trapezoid body (MNTB). 2. The fluorescent stain DiI (1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate) was used in fixed tissue and Lucifer Yellow in living slices, to identify calices enclosing single MNTB neuronal somata. 3. Whole-cell recording from the MNTB neurone shows evoked EPSCs preceded by a prespike, corresponding to the presynaptic action potential (AP). In some cases one patch pipette recorded from both pre- and postsynaptic elements, but confirmation of exclusively presynaptic recording was obtained using pipettes containing Lucifer Yellow in a further eleven cases. 4. Under current clamp, the pre- and postsynaptic sites could be distinguished by their response to step depolarizations; presynaptic terminals generated a train of APs at frequencies up to 200 Hz, while MNTB neurones gave a single AP. Each presynaptic AP had an after-hyperpolarization lasting less than 2 ms. 5. Under voltage clamp, step depolarizations of presynaptic terminals generated a tetrodotoxin-sensitive inward current followed by rapidly activating outward potassium currents at potentials more positive than -60 mV. The outward current exhibited little inactivation over the 150 ms steps and 4-aminopyridine (200 microM) blocked 63.0 +/- 14.5% (mean +/- S.D., n = 3) of the sustained current at 0 mV. Like the squid giant synapse, mammalian terminals express rapidly activating 'delayed rectifier'-type potassium currents.

Topics: Action Potentials; Animals; Brain; Carbocyanines; Electrophysiology; Fluorescent Dyes; Glutamic Acid; Isoquinolines; Microscopy, Confocal; Neurons; Patch-Clamp Techniques; Pons; Presynaptic Terminals; Rats; Receptors, AMPA; Receptors, N-Methyl-D-Aspartate; Tetrodotoxin

Early commissural axons in the zebrafish spinal cord extend along a pathway consisting of a ventrally directed ipsilateral, a contralateral diagonal, and a contralateral longitudinal segment. The midline floor plate cell is one important cue at the transition from the ipsilateral to the contralateral pathway segments. In order to identify additional guidance cues, the interactions between commissural growth cones and their substrates were examined at the electron microscopic level in the different pathway segments. The growth cones extended near the superficial margin of the spinal cord, within filopodial reach of three bilateral longitudinal axon pathways that were ignored irrespective of whether other axons were already present. Ultimately the commissural growth cones pioneered an additional independent longitudinal pathway in the dorsolateral spinal cord. Neuroepithelial cells were extensively contacted in the lateral marginal zone of the dorsal spinal cord and are thus in a position to contribute to the establishment of the longitudinal commissural pathway segment. The extent of contact with neuroepithelial cells in the ventral spinal cord was dependent on whether commissural growth cones had already crossed the ventral midline: ipsilateral, but not contralateral, growth cones showed extensive contacts with neuroepithelial processes and minor contacts with the basal lamina. In marked contrast, commissural growth cones that had already crossed the ventral midline and entered the diagonal pathway segment showed major appositions to the basal lamina. Extensive contact with the basal lamina was first established in the ventral midline region, where crossing growth cones always inserted between the basal lamina and the base of the midline floor plate cells. This indicates that a change occurs in the response characteristics of commissural growth cones as they cross the ventral midline of the spinal cord. Such a change could help to explain why the growth cones extend first toward but then away from the ventral midline.

Topics: Animals; Axons; Carbocyanines; Embryonic and Fetal Development; Fluorescent Dyes; Microscopy, Electron; Neural Pathways; Spinal Cord; Zebrafish

Midbrains from 43 fresh human embryos and fetuses at 8-22 weeks of gestation were processed for routine histology, Golgi staining, tyrosine hydroxylase (TH) immunolabelling and retrograde tracing with the fluorescent dye DiI. Cells were immature and densely packed between 8 and 10 weeks. By 13 weeks cells could be identified as neurons and glia. Neurons matured gradually and achieved adult characteristics by 20-22 weeks. Neurons in the paramedian regions of the tegmentum, raphae region and substantia nigra were positive for TH from 13 weeks onwards, the earliest age group used for this technique. The presence of TH-positive neurons in the paramedian part of the tegmentum until 18 weeks and radial glial fibers extending from the aqueductal lining to the ventral part until 20 weeks were suggestive of migration of neurons to the ventral mesencephalon region. DiI labelling of the neurons and fibers of ipsilateral nigra from the caudate as early as 10 weeks demonstrated early nigrostriatal connections. The mature nature of the neurons appeared only by 13 weeks by this method. The present study shows that the nigral neurons in the human migrate and mature until mid-gestation. The nigrostriatal connection at 10 weeks suggests a trophic influence of nigra on the proliferating and maturing neurons of the striatum.

Topics: Carbocyanines; Embryo, Mammalian; Embryonic and Fetal Development; Female; Fetus; Fluorescent Dyes; Gestational Age; Golgi Apparatus; Humans; Immunohistochemistry; Mesencephalon; Neurons; Pregnancy; Substantia Nigra

Development of the node and formation of the notochordal plate in gestational day 7-9 mice (Theiler stages 10-14) has been documented principally with scanning electron microscopy (SEM) and cell fate analyses utilizing DiI and/or CFSE as a cell label. With SEM, cells composing these two populations are initially identifiable at stage 10 at the ventral midline of the rostral half of the embryo. They can be recognized by their relatively small ventral surface area, as compared to that of the peripherally adjacent prospective gut endodermal cells, and by the presence on the ventral side of each cell of a prominent single, central cilium, which is lacking on endodermal cells. At stage 10, the node is located at the apex of the cup-shaped embryo. It represents the rostral end of the primitive streak (although its structure differs from that of the rest of the streak), and it consists of a localized two-layered area (i.e., epiblast and the most caudal aspect of the notochordal plate). By stage 11, the notochordal plate constitutes a relatively broad, circular area (at the level of the node) that tapers rostrally into a narrower midline strip (beneath the future floor plate of the neural tube). The tip of the notochordal plate terminates rostrally at the much broader prechordal plate, which underlies the future forebrain level of the neuraxis. The prechordal plate cells, like the ventral node and notochordal plate cells, each have a relatively small ventral surface area and displays a single central cilium on their ventral surface. The most caudal aspect of the notochordal plate remains morphologically distinct on the dorsal, midline surface of the open gut through stage 13; the more rostral levels progressively fold off from the roof of the gut to form the definitive notochord. Videomicroscopy reveals that the cilia extending from the ventral surfaces of the cells of node and of the prechordal and notochordal plates are motile. The potential significance of this motile behavior remains unknown. Labeling studies, which marked cells in both the dorsal and ventral layers of the node, reveal that the stage-10 node contributes cells to the notochordal plate and overlying midline ectodermal cells of the neural plate, the future floor plate of the neural tube.(ABSTRACT TRUNCATED AT 400 WORDS)

Topics: Animals; Carbocyanines; Chick Embryo; Female; Fluoresceins; Fluorescent Dyes; Gastrula; Gestational Age; Mice; Mice, Inbred C57BL; Microscopy, Electron, Scanning; Notochord; Pregnancy; Species Specificity; Succinimides

Fluorescent dyes were used in the present study to map three rostrocaudal levels of the primitive streak of mouse embryos at 7.5 days of gestation. Injections at the most rostral level, the 1/4 streak level, principally labeled cells of the paraxial mesoderm, including mesenchyme in the head, and somites and segmental plate mesoderm in the trunk. Injections at the intermediate level, the 1/2 streak level, principally labeled cells of the lateral plate mesoderm of the trunk. Injections at the most caudal level, the 3/4 streak level, principally labeled cells of the extraembryonic mesoderm at the periphery (i.e., caudolateral portion) of the embryo adjacent to the developing amnion, yolk sac, and allantois. However, injections placed at all three primitive-streak levels provided some labeled cells to the areas of the ingressed mesoderm derived from immediately adjacent levels, suggesting that prospective mesodermal areas are not fully spatially segregated from one another at this stage. These data combined with those from a companion study in which the structure of the node was defined and its derivatives were mapped (Sulik et al. [1994] Dev. Dyn. 201:260-278) allowed us to construct a prospective fate map of the mouse primitive streak at 7.5 days of gestation. This map revealed that mediolateral subdivisions of the ingressed mesoderm were arrayed in rostrocaudal sequence within the primitive streak, such that more rostral levels of the streak contained prospective medial cells (e.g., notochord and paraxial mesoderm), intermediate levels contained prospective intermediate cells (e.g., intermediate mesoderm or nephrotome and lateral plate mesoderm), and more caudal levels contained prospective lateromost cells (e.g., extraembryonic mesoderm). Prospective mesodermal cells, with the exception of those of the notochord, ingressed bilaterally from the primitive streak, contributing to the mesoderm on both the right and left sides of the axis. Our prospective fate map of the mouse primitive streak at 7.5 days of gestation, when compared to a map of the avian primitive streak at a comparable stage of development (Schoenwolf et al. [1992] Dev. Dyn. 193:235-248), demonstrates that considerable congruity exists between the locations of mesoderm precursor cells in birds and mammals. Thus, similar morphogenetic movements underlie mesoderm formation during avian and mammalian gastrulation. Moreover, prospective mesodermal locations within the mouse primitive streak corr

Topics: Animals; Carbocyanines; Cell Movement; Chick Embryo; Embryonic and Fetal Development; Female; Fluoresceins; Fluorescent Dyes; Gastrula; Gestational Age; Mice; Mice, Inbred C57BL; Pregnancy; Species Specificity; Succinimides

Retinal arbors in the tectum of living adult goldfish were imaged to determine whether the structural remodelling and refinement that occurs during development continues in adulthood. Individual optic fibers were labelled by making small injections of the lipophilic fluorescent dye DiI into ventral retina and viewing the exposed tectum through a fluorescence microscope equipped with a cooled CCD camera. Arbors were imaged in the living fish every 30-60 minutes for up to 7 hours. Normal adult goldfish showed no evidence of arbor remodelling during this period, though dynamic movements of varicosities present along axon segments were observed. For comparison, regenerating optic fibers were similarly imaged in fish that had undergone optic nerve crush 2-6 weeks previously. In these fish, dynamic structural changes were seen, including branch remodelling, extension and retraction of growth cones, and movement of varicosities.

Topics: Animals; Carbocyanines; Goldfish; Image Processing, Computer-Assisted; Microscopy, Fluorescence; Nerve Crush; Nerve Fibers; Nerve Regeneration; Optic Nerve; Superior Colliculi

We tested a variety of antioxidants as possible therapeutic agents in an acute CCl4 mouse model of hepatotoxicity. Liver damage, gauged by the amount of serum aminotransferase released into the blood, morphological changes, lethal dose response, and presence of thiobarbituric acid-reactive substances (TBARS), were significantly inhibited in a dose-dependent manner by liposomes containing vitamin E (LVE) or by Rocavit E, a water-soluble emulsion of alpha-tocopherol. Serum aminotransferase levels in LVE- or Rocavit E-treated animals were always > 10-fold lower than levels in corresponding CCl4 controls. Other liposome-associated antioxidants, butylated hydroxytoluene, vitamin E succinate, catalase, desferoxamine, superoxide dismutase, and ascorbic acid 6-palmitate, were also able to elicit a decrease in damage; however, they were substantially less effective. Intravenous therapy with LVE decreased mortality by nearly 90% when a lethal dose of CCl4 was given. When the biodistribution of the liposomes was examined, it was determined that the vast majority were localized in the Kupffer cell population. This approach of delivering nontoxic therapeutic agents selectively to the liver offers a variety of clinical applications in humans.

Topics: Animals; Antioxidants; Carbocyanines; Carbon Tetrachloride; Dose-Response Relationship, Drug; Female; Fluorescent Dyes; Injections, Intraperitoneal; Lipid Peroxides; Liposomes; Liver; Mice; Mice, Inbred Strains; Necrosis; Survival Analysis; Thiobarbituric Acid Reactive Substances; Tissue Distribution; Vitamin E

Several lines of evidence implicate a crucial role for thalamic afferents from the ventroposterior nucleus (VP) in the development of barrels and their characteristic pattern in the primary somatosensory cortex (S1) of rodents. We sought to determine the stage in development when VP thalamocortical afferents are first distributed in a periphery-related pattern and the sequence of events that culminate in a mature pattern. Using acetylcholinesterase (AChE) histochemistry, an early marker for VP thalamocortical afferents, and the anterograde axon tracer DiI, we show that VP thalamocortical afferents become distributed into a periphery-related pattern earlier than was previously reported, including their parcellation into a barrel-related pattern that mirrors the distribution of sensory hairs on the face. The earliest periphery-related patterning observed is transiently present in the deep cortical layers prior to the emergence of layer 4, the layer in which barrels later develop. AChE histochemistry reveals a clear sequence of maturation of the barrel pattern in the distribution of VP afferents: An initially patternless distribution of AChE-reactive afferents is followed by their distribution in a nascent trigeminal representation, from which rows subsequently emerge; barrel-related clusters of afferents then emerge from the rows. This process begins before birth, and the transition from row-related to barrel-related distributions of VP afferents is evident during the first postnatal day (P0). This demonstration of a periphery-related pattern in developing rat S1 precedes by about 2 days that revealed by any other marker reported to delineate barrels. These findings confirm that VP thalamocortical afferents are the first barrel component to have a periphery-related pattern and support the hypothesis that thalamocortical afferents provide to immature S1 the patterning information that initiates the formation of barrels and their characteristic array. Furthermore because these findings show an earlier onset for barrel formation than was previously realized, they necessitate a reevaluation of conclusions drawn from experiments examining developmental plasticity in barrel patterning.

Topics: Acetylcholinesterase; Afferent Pathways; Animals; Brain Mapping; Carbocyanines; Embryonic and Fetal Development; Histocytochemistry; Neuronal Plasticity; Rats; Rats, Sprague-Dawley; Somatosensory Cortex; Thalamus

There is little detailed information about retinal ganglion cells which project to specific central targets in the brain. The present study examined retinal ganglion cells projecting to the nucleus of the basal optic root, a major accessory retinal target in the turtle. These ganglion cells were first selectively labeled using retrograde transport of rhodamine injected stereotaxically into the nucleus of the basal optic root. The number and distribution of the retrogradely labeled cells in the retina was then determined. Some of these retrogradely labeled cells were then injected intracellularly with Lucifer Yellow, photoconverted using diaminobenzidine, and drawn in detail using a camera lucida attachment. There were approximately 1500 ganglion cells in each retina which projected to the nucleus of the basal optic root, of which 20% had cell bodies displaced to the inner nuclear layer. More than 50% of the total population was concentrated in the visual streak region. All ganglion cells projecting to the nucleus of the basal optic root, both normal and displaced, had monostratified dendritic arborizations in stratum 1 of the inner plexiform layer. About 41% of these ganglion cells had elongated dendritic arborizations with distinct orientations, which may suggest a correlation of morphology and function. There were similarities in the overall appearance, and in the type and stratification of the dendritic arborizations of all ganglion cells projecting to the nucleus of the basal optic root. These anatomical similarities are consistent with the previously demonstrated similarities in physiology and may reflect a common role for these ganglion cells in visual processing.

Topics: Animals; Carbocyanines; Dendrites; Isoquinolines; Mesencephalon; Retinal Ganglion Cells; Rhodamines; Tissue Fixation; Turtles; Visual Pathways

It is unknown how neuronal connections are specified in the olfactory system. To define rules of connectivity in this system, we investigated whether the projection of sensory neurons from the olfactory epithelium to the olfactory bulb is topographically ordered. By backtracking with 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI), we find that neurons projecting into a single identified glomerulus are widely dispersed over the olfactory epithelium. Their positions in the sensory surface do not predict their glomerulus specificity and are probably random. A statistical analysis reveals that neurons connected to the same glomerulus are spaced at distances of several cell diameters from each other. The convergence of projections to one point in the target area from neurons that are widely and evenly distributed in the sensory surface constitutes an unusual type of connectional topography that contrasts with the precise topological (neighborhood-preserving) maps found in other sensory systems. It may maximize the probability to detect odorants that activate a single glomerular unit.

Topics: Animals; Carbocyanines; Fluorescent Dyes; Image Processing, Computer-Assisted; Olfactory Bulb; Olfactory Pathways; Olfactory Receptor Neurons; Zebrafish

Junctional epithelium (JE) is the special attachment tissue between gingiva and teeth, and it is well innervated by sensory nerve fibers. We have found that the fluorescent carbocyanine dye, Di-I, can penetrate quickly into intact JE and spread into connective tissue. Di-I containing neurons in trigeminal ganglion were found at 3-7 days and were mostly gone by 3 weeks. We conclude that substances such as Di-I can penetrate through permeable epithelia such as intact JE where they are picked up by sensory nerve fibers and carried to nerve cell bodies.

Topics: Animals; Carbocyanines; Diffusion; Epithelial Attachment; Fluorescent Dyes; Male; Microscopy, Fluorescence; Neurons, Afferent; Rats; Rats, Sprague-Dawley; Time Factors; Trigeminal Ganglion

Neurotrophin 3 (NT-3) is one of four related polypeptide growth factors that share structural and functional homology to nerve growth factor (NGF). NT-3 and its receptor, called neurotrophic tyrosine kinase receptor type 3 (Ntrk3; also called TrkC), are expressed early and throughout embryogenesis. We have inactivated the NT-3 gene in embryonic stem (ES) cells by homologous recombination. The mutated allele has been transmitted through the mouse germ line, and heterozygote intercrosses have yielded homozygous mutant newborn pups. The NT-3-deficient mutants fail to thrive and exhibit severe neurological dysfunction. Analysis of mutant embryos uncovers loss of Ntrk3/TrkC-expressing sensory neurons and abnormalities at early stages of sensory neuronal development. NT-3-deficient mice will permit further study of the role of this neurotrophin in neural development.

Topics: Animals; Carbocyanines; Cells, Cultured; Embryo, Mammalian; Failure to Thrive; Fluorescent Dyes; Ganglia, Spinal; Homozygote; Mice; Mice, Inbred C57BL; Mice, Neurologic Mutants; Muscles; Mutagenesis; Nerve Growth Factors; Neurons, Afferent; Neurotrophin 3; Reference Values; Restriction Mapping; Spinal Cord; Stem Cells

Topics: Animals; Carbocyanines; Cell Transplantation; Cells, Cultured; Fluorescent Dyes; Glucuronosyltransferase; Liver; Microscopy, Fluorescence; Rats; Rats, Gunn; Rats, Wistar

Five different populations of interlayer neurones (ILNs) can be described after DiI/Di-ASP tracing in rat superior colliculus (SC). All of these labelled neurones preferentially lay in the rostro-medial part of the SC. Most of them are located in the stratum opticum and in the stratum griseum superficiale. Our results indicate that ILNs represent a minority of neurones in the superficial layers but may constitute a substantial population of neurones in the stratum opticum connecting the visual and the multimodal collicular layers.

Topics: Animals; Carbocyanines; Fluorescent Dyes; In Vitro Techniques; Neurons; Pyridinium Compounds; Rats; Rats, Wistar; Superior Colliculi

In both laboratory and clinical studies, injection of doxorubicin directly into the eyelid results in permanent muscle loss of the majority of fibers within treated eyelids. A first clinical trial of this technique in blepharospasm and hemifacial spasm patients has been performed. All patients who completed a full course of doxorubicin treatment showed a permanent decrease in eyelid strength, with over 50% of these patients requiring no further treatment. Doxorubicin is known to be carried by retrograde axonal transport to the brain and is a known neurotoxin. This raises the question of the effect of these treatments on the facial neurons which innervate the orbicularis oculi muscle in the eyelids. The effect on the number of facial neurons present after injection of doxorubicin into the eyelid of rabbits was determined using both HRP and diI retrograde labeling techniques. Despite the extensive and permanent muscle loss caused by the doxorubicin treatments, there was no measurable loss of facial neurons on the doxorubicin treated sides. DiI was shown to be myotoxic at high concentrations and amplified the myotoxic effect of doxorubicin. Lack of neuronal loss may offer assurance of clinical safety to the facial motor neurons of muscle spasm patients who receive doxorubicin injections into their eyelids.

Topics: Affinity Labels; Animals; Carbocyanines; Doxorubicin; Eyelids; Facial Nerve; Horseradish Peroxidase; Injections; Motor Neurons; Muscle Contraction; Rabbits

The projection pattern of the ventral thalamic reticular nucleus onto the dorsal thalamus was studied in the lizard Gallotia galloti using in vitro horseradish peroxidase and fluorescent carbocyanine labelling techniques. Localized label deposits at three dorsoventrally spaced sites in the dorsal thalamus elicited retrograde transport into separate, though partly overlapping, medial, dorsolateral and ventrolateral sectors within an extended cytoarchitectonic complex which may be globally identifiable as the reticular nucleus. Neurons found in the dorsolateral and ventrolateral sectors mainly corresponded to the cell group named nucleus ventromedialis (or nucleus of the dorsal supraoptic decussation) in the literature, whereas neurons labelled in the medial sector corresponded to the so-called dorsal hypothalamic nucleus. Sparser cells appear labelled in the superficially placed nucleus suprapeduncularis. Thalamotelencephalic fibers arising from the injected dorsal thalamic nuclei also project to the corresponding retrogradely labeled sectors within the reticular nucleus. These findings reveal a rough topographic organization in the connections of the extended reticular nucleus complex with the whole dorsal thalamus. This supports the hypothesis of hodological homology between this ventral thalamic formation in Gallotia and the mammalian thalamic reticular nucleus.

Topics: Animals; Brain Mapping; Carbocyanines; Fluorescent Dyes; Horseradish Peroxidase; Lizards; Neural Pathways; Thalamic Nuclei; Thalamus

Afferent projections to the pituitary pars intermedia were studied using the DiI tract-tracing technique in two amphibian species, the urodelan Triturus carnifex, and the anuran Rana esculenta. After DiI crystal application into the pituitary intermediate lobe, in both species cells were retrogradely labeled in the preoptic nucleus, in the supra- and retro-chiasmatic hypothalamus and in the brainstem (especially in the area indicated as locus coeruleus). The findings are discussed in relation to data on the neurochemical nature of the innervation of the pars intermedia in amphibians.

Topics: Animals; Brain Stem; Carbocyanines; Female; Fluorescent Dyes; Immunohistochemistry; Male; Neural Pathways; Neurons, Afferent; Pituitary Gland; Rana esculenta; Species Specificity; Suprachiasmatic Nucleus; Triturus

Retinotopic map development in nonmammalian vertebrates appears to be controlled by molecules that guide or restrict retinal axons to correct locations in their targets. However, the retinotopic map in the superior colliculus (SC) of the rat is developed instead by a topographic bias in collateral branching and arborization. Temporal retinal axons extending across alternating membranes from the topographically correct rostral SC or the incorrect caudal SC of embryonic rats preferentially branch on rostral membranes. Branching preference is due to an inhibitory phosphatidylinositol-linked molecule in the caudal SC. Thus, position-encoding membrane-bound molecules may establish retinotopic maps in mammals by regulating axon branching, not by directing axon growth.

Topics: Animals; Axons; Carbocyanines; Cells, Cultured; Embryonic and Fetal Development; Fluorescent Dyes; Phosphatidylinositol Diacylglycerol-Lyase; Phosphoric Diester Hydrolases; Rats; Rats, Sprague-Dawley; Retinal Ganglion Cells; Superior Colliculi

To examine the possible migration of luteinizing hormone-releasing hormone (LHRH) neurons from the olfactory placode to the brain, the epithelial cells of the olfactory placode of the chick embryos were labeled with a fluorescent carbocyanine dye DiI at embryonic days 3.5-4.0. The embryos treated with DiI were incubated in ovo for 1 to 7 days. The DiI-labeled cells were first detected in the olfactory epithelium and olfactory nerve one day after the application of DiI. Two to four days after the application of the dye, the labeled cells sequentially appeared in the rostral and medial forebrain, and in the septo-preoptic area. The distribution pattern of DiI-labeled cells closely resembled that of LHRH neurons. Double staining for DiI and LHRH demonstrated that DiI-labeled cells co-expressed LHRH. However, there were a number of DiI-labeled cells which did not co-express LHRH. These results provide evidence for the actual migration of LHRH neurons from the olfactory placode to the septo-preoptic area during the development, and also suggest that some non-LHRH neurons in the forebrain are of olfactory placodal origin.

Topics: Animals; Carbocyanines; Cell Movement; Chick Embryo; Epithelial Cells; Epithelium; Fluorescent Dyes; Gonadotropin-Releasing Hormone; Microscopy, Fluorescence; Nerve Fibers; Neurons; Olfactory Pathways; Prosencephalon; Telencephalon

We have developed a technique to detect, recognize, and track each individual low density lipoprotein receptor (LDL-R) molecule and small receptor clusters on the surface of human skin fibroblasts. Molecular recognition and high precision (30 nm) simultaneous automatic tracking of all of the individual receptors in the cell surface population utilize quantitative time-lapse low light level digital video fluorescence microscopy analyzed by purpose-designed algorithms executed on an image processing work station. The LDL-Rs are labeled with the biologically active, fluorescent LDL derivative dil-LDL. Individual LDL-Rs and unresolved small clusters are identified by measuring the fluorescence power radiated by the sub-resolution fluorescent spots in the image; identification of single particles is ascertained by four independent techniques. An automated tracking routine was developed to track simultaneously, and without user intervention, a multitude of fluorescent particles through a sequence of hundreds of time-lapse image frames. The limitations on tracking precision were found to depend on the signal-to-noise ratio of the tracked particle image and mechanical drift of the microscope system. We describe the methods involved in (i) time-lapse acquisition of the low-light level images, (ii) simultaneous automated tracking of the fluorescent diffraction limited punctate images, (iii) localizing particles with high precision and limitations, and (iv) detecting and identifying single and clustered LDL-Rs. These methods are generally applicable and provide a powerful tool to visualize and measure dynamics and interactions of individual integral membrane proteins on living cell surfaces.

Topics: Algorithms; Biophysical Phenomena; Biophysics; Carbocyanines; Cell Line; Cell Membrane; Computer Simulation; Diffusion; Fibroblasts; Fluorescent Dyes; Humans; Image Processing, Computer-Assisted; Lipoproteins, LDL; Microscopy, Fluorescence; Models, Biological; Receptor Aggregation; Receptors, LDL; Skin

The medial preoptic nucleus of the Japanese quail is a testosterone-sensitive structure that is involved in the control of male copulatory behavior. The full understanding of the role played by this nucleus in the control of reproduction requires the identification of its afferent and efferent connections. In order to identify neural circuits involved in the control of the medial preoptic nucleus, we used the lipophilic fluorescent tracer DiI implanted in aldheyde-fixed tissue. Different strategies of brain dissection and different implantation sites were used to establish and confirm afferent and efferent connections of the nucleus. Anterograde projections reached the tuberal hypothalamus, the area ventralis of Tsai, and the substantia grisea centralis. Dense networks of fluorescent fibers were also seen in several hypothalamic nuclei, such as the anterior medialis hypothalami, the paraventricularis magnocellularis, and the ventromedialis hypothalami. A major projection in the dorsal direction was also observed from the medial preoptic nucleus toward the nucleus septalis lateralis and medialis. Afferents to the nucleus were seen from all these regions. Implantation of DiI into the substantia grisea centralis also revealed massive bidirectional connections with a large number of more caudal mesencephalic and pontine structures. The substantia grisea centralis therefore appears to be an important center connecting anterior levels of the brain to brain-stem nuclei that may be involved in the control of male copulatory behavior.

Topics: Afferent Pathways; Animals; Biological Transport, Active; Carbocyanines; Coturnix; Efferent Pathways; Female; Fluorescent Dyes; In Vitro Techniques; Male; Preoptic Area; Sex Characteristics; Sexual Behavior, Animal

We have analyzed the early development of the main hippocampal afferents in the mouse. Following injections of the lipophilic tracer 1-1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI) in the entorhinal cortex, entorhinal axons were observed for the first time in the hippocampus at E15, in the white matter. At E17, entorhinal fibers arborized within the stratum lacunosum-moleculare. At subsequent stages entorhinal axons formed dense networks that were restricted to their appropriate termination zone in the lacunosum-moleculare. The first axons invading the fascia dentata were noticed at E19, their density increasing at later stages. These axons were mainly present in the outer molecular layer. This onset of entorhinohippocampal projections was corroborated by retrograde labeling data after injections in the hippocampus. Commissural fibers first entered the contralateral hippocampus at E18, their number increasing at the following stages. Commissural axons arborized within the stratum oriens and radiatum in the hippocampus proper. In the fascia dentata, the earliest commissural fibers were seen at P2, terminating in the inner zone of the molecular layer and in the hilus. We conclude that developing entorhinal and commissural axons show a high degree of laminar specificity from the earliest stages of formation, which is compatible with the notion that distinct subsets of early maturing neurons populating the hippocampal plexiform layers may attract particular fiber systems. Hippocamposeptal fibers develop at E15, before the first septal fibers can be detected in the hippocampus. These early hippocamposeptal fibers originated from nonpyramidal neurons and terminated in the medial septal area, which is the main source of septal afferents to the hippocampus. In contrast, septohippocampal fibers were not seen in the hippocampus until E17. At perinatal stages, the hippocamposeptal connection reshapes, sending axons to the dorsolateral septal area as the innervation of the medial septum becomes less conspicuous. This sequence suggests that hippocampal neurons pioneer the formation of septohippocampal connections.

Topics: Animals; Axons; Carbocyanines; Female; Fluorescent Dyes; Hippocampus; Histocytochemistry; Mice; Neural Pathways; Neurons, Afferent; Synapses

In order to define the extent to which retinotopic order in the optic pathways may contribute to fiber segregation at the chiasm or to the formation of central maps, the arrangement of fibers in the optic nerve and tract of rat embryos, on embryonic days 16.5 and 18.5, has been studied by placing a small granule of DiI in one of the four quadrants of the retina and tracing the filled fibers through transverse sections of the retinofugal pathway with confocal microscopy. There is a distinct quadrant-specific order in the optic stalk immediately behind the eye, with fibers from the ventral nasal, dorsal nasal, dorsal temporal, and ventral temporal retina arranged sequentially across the rostrocaudal axis of the cross section of the stalk. However, this distinct order is not maintained very far. There is a gradual increase in the degree of overlap between fibers from the different quadrants as the fibers pass towards the chiasm. The dorsal groups of fibers intermingle extensively along almost the entire length of the stalk, but the fibers from ventral sectors remain separate until they reach the prechiasmatic region, where the ventral temporal and the ventral nasal fibers spread throughout the rostrocaudal extent of the stalk and the chiasm. The initial quadrant-specific order is completely lost at the chiasm. However, beyond the optic chiasm, the fibers are reorganized into another distinct order. In the optic tract, there is a segregation of dorsal from ventral fibers, but the nasal and temporal groups remain intermingled. The results of this study indicate that the earliest fibers in the developing optic tract are arranged according to topographical rules that differ from those obtaining behind the eye. Since all topographical order is lost between these two levels, there must be an active sorting mechanism in the region where the chiasm joins the tract. Possibly this mechanism is related to the development of the dorsoventral axis of the topographic maps in the central visual targets.

Topics: Animals; Axons; Carbocyanines; Female; Fluorescent Dyes; Nerve Fibers; Optic Chiasm; Optic Nerve; Pregnancy; Rats; Rats, Sprague-Dawley; Retina; Tissue Fixation; Visual Pathways

Recent studies in adult rodents have shown that mossy fibers, the axons of hippocampal granule cells, sprout into the inner molecular layer of adult rats when hilar cell death occurs following kainate-induced seizure activity. This pattern of hilar cell death and mossy fiber sprouting is not observed in young rats at 15 postnatal days of age. Since granule cells are generated postnatally, one may assume that a lack of a mature mossy fiber input to hilar neurons at 15 days of age is a possible cause for this observed difference. Neo-Timm preparations were made from rats at 5, 10, 12, 15, 20, 21, 25, 30 and 32 postnatal days of age to study the postnatal development of mossy fibers. The adult pattern of Timm-labeled mossy fiber innervation in the granule cell layer was observed by 25 days. The Timm reaction product forms large dense granules in CA3 of 15 day old rats but the hilus at this age lacks this type of large granule. Instead, the hilus displays only small labeled boutons, suggesting that mossy terminals have not yet reached a mature size. Electron microscopic preparations of the deep hilus and the subgranular zone of the hilus at 7, 12, 15, 21 and 30 days were analyzed to study the development of synapses formed by axons of granule cells. At 7 days the deep hilus showed only a few asymmetric synapses formed by the developing mossy fibers.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Aging; Animals; Axons; Carbocyanines; Cell Death; Hippocampus; Kainic Acid; Microscopy, Electron; Nerve Fibers; Neurons; Rats; Rats, Sprague-Dawley; Synapses

Fetal as well as mature neural cells were homografted into the right cerebral hemisphere of adult BD-IX rats. The animals were sacrificed 7 d after implantation, and the localization of implanted cells was visualized by fluorescence and light microscopy. The cell implants were prestained with the fluorescent vital dye 1,1'-Dioctadecyl-3,3,3'3'-tetramethylindocarbocyanine perchlorate (DiI) to discriminate between implanted cells and host brain tissue. At the implantation site, the fetal brain cells as well as the cells from immature brain cell aggregates showed diffuse infiltration into the surrounding host brain tissue of up to 0.5 mm. Extensive cell migration along the corpus callosum for up to 5 mm in the coronal and to a lesser extent in the sagittal plane was also observed. In addition, fetal cells were distributed in the subarachnoid space of both cerebral hemispheres and showed a distinct association with larger blood vessels. Cells from mature brain aggregates did not migrate as far as fetal cells and showed only a local infiltration into the host neuropil. Fluorescent microspheres as well as fixed fetal brain cells were implanted, either alone or in combination with vital cells to distinguish between active cell migration and passive cell displacement. The microspheres and the fixed cells were found either localized to the implantation pathway or distributed in the corpus callosum for up to 2 mm in the coronal plane without any dispersion in the sagittal plane. The microspheres also showed an extensive displacement in the subarachnoid space. In vitro co-culture experiments between two immature aggregates showed a complete fusion of the two aggregates during a 96 h culture period. In co-cultures between two mature aggregates complete fusion was not prominent, although the confrontation zone appeared diffuse. Confrontations between a mature and an immature aggregate showed the same pattern of interaction as seen for the two mature aggregates. It is concluded that carbocyanine dyes may be used as a tracer for transplanted cells. Cells from fetal rat brain cell aggregates, opposed to those from mature aggregates, showed extensive migration along well defined anatomical structures in the mature along well defined anatomical structures in the mature brain. Some of the spread of cells following implantation is probably due to passive movement since inert microspheres will spread into certain areas of the CNS.

Topics: Animals; Brain; Brain Tissue Transplantation; Carbocyanines; Cell Division; Cells, Cultured; Female; Fetal Tissue Transplantation; Fluorescent Dyes; Male; Microscopy, Fluorescence; Microspheres; Phagocytosis; Rats; Rats, Inbred Strains

The hypothalamic arcuate nucleus is known to be reciprocally connected to various limbic regions, such as the bed nucleus of the stria terminalis (BST). The route of this reciprocal connection, in particular with the BST, remains unknown. In order to visualize this pathway, we used the fluorescent tracer carbocyanine dye (DiI), that was inserted in the arcuate nucleus in fixed and dissected brains. This allowed us to label an arcuate-BST pathway DiI-labeled coursing through the stria terminalis. Immunohistochemistry for the arcuate-derived peptide adrenocortico-tropin (ACTH) revealed the presence of ACTH-immunoreactive axons in the stria terminalis. Together, these results provide arguments in favour of the existence of an arcuatofugal projection to the BST via the stria terminalis.

Topics: Adrenocorticotropic Hormone; Animals; Arcuate Nucleus of Hypothalamus; Axons; Carbocyanines; Efferent Pathways; Immunohistochemistry; Male; Pro-Opiomelanocortin; Rats; Rats, Wistar; Thalamic Nuclei

Previous studies from this laboratory have used antisera to aldehyde-conjugated ovalbumin to localize ovalbumin-like immunoreactivity within a subpopulation of sensory neurons. We have now combined retrograde tracing and immunohistochemical procedures to identify the tissues innervated by sensory neurons which are either immunoreactive or non-immunoreactive for ovalbumin. The fluorescent tracer Di-I was administered to feather follicles, flexor ulnar muscle, subdermis, expansor secundariorum, heart and liver and identified seven days later within corresponding dorsal root ganglia. Most neurons innervating the follicles had large cell somata, and fewer than 3% were immunoreactive for ovalbumin. In contrast, most sensory neurons projecting to subdermis, muscle and expansor secundariorum muscle were of a medium diameter. Approximately 25% of those neurons projecting to the expansor secundariorum, and 60% projecting to the subdermis and muscle, were immunoreactive for ovalbumin. Sensory neurons innervating heart and liver were the smallest, and only 8% were immunoreactive for ovalbumin. The study indicates that sensory neurons innervating different organs have somata with significantly different sizes, suggesting a functional specificity. Moreover, neurons demonstrating either the ovalbumin-IR positive or negative phenotypes show distinct peripheral projections, suggesting that this phenotype may be at least partially controlled by retrograde signals derived from the cells they innervate.

Topics: Afferent Pathways; Animals; Carbocyanines; Cell Size; Chickens; Feathers; Fluorescent Antibody Technique; Fluorescent Dyes; Ganglia, Spinal; Heart; Liver; Muscles; Nerve Tissue Proteins; Neurons, Afferent; Organ Specificity; Ovalbumin; Phenotype; Wings, Animal

Previous investigators have documented the postnatal development of alpha and beta type ganglion cells in cat retinae (Ramoa et al. [1987] Science 237:522-525; Ramoa et al. [1988] J. Neurosci. 8:4239-4261; Dann et al. [1987] Neurosci. Lett. 80:21-26; Dann et al. [1988] J. Neurosci. 8(5):1485-1499). The development of the remaining cells (about 50%), which constitute a heterogeneous group and are referred to here collectively as gamma cells (Boycott and Wässle, '74), has not been studied in detail. The purpose of this study was to compare the postnatal development of alpha, beta, and gamma cells in kitten and adult retinae using horseradish peroxidase histochemistry and the fluorescent dye DiI. In the kitten, alpha, beta, and gamma cells are recognizable. We find, as have others, that kitten alpha and beta cell bodies and dendritic fields are significantly smaller than in the adult. However, kitten gamma cells are nearly adult sized. In fact, at birth the cell bodies of beta cells throughout the retina are significantly smaller than those of gamma cells. During the first 12 weeks of life, alpha and beta cell bodies increase in size from 90% to 680% depending upon eccentricity. Gamma cells hardly increase in size at all. Also, the normal adult center-to-peripheral cell size gradient for alpha and beta cells is not seen in the neonate. Gamma cells show no such gradient in the neonate or adult. Our results suggest that the morphological development of alpha and beta cells occurs later than that of gamma cells and may explain some of the differences in the effects of visual deprivation and surgical manipulation upon the parallel Y-, X-, and W-cell pathways.

Topics: Animals; Animals, Newborn; Carbocyanines; Cats; Dendrites; Geniculate Bodies; Histocytochemistry; Horseradish Peroxidase; Retina; Retinal Ganglion Cells; Superior Colliculi

Cultured vascular smooth muscle cells (SMCs) containing retrovirally introduced genes are a potential vehicle for gene replacement therapy. Because the cultured SMCs are selected for their ability to proliferate in vitro, it is possible that the SMCs might be permanently altered and lose their capacity to respond to growth-suppressing conditions after being seeded back into blood vessels. To investigate this possibility we measured SMC proliferation and intimal thickening in balloon-injured Fischer 344 rat carotid arteries seeded with SMCs stained with the fluorescent marker 1,1'-dioctadecyl-3,3,3',3'-tetramethylindo-carbocyanine perchlorate (DiI) and infected with replication-defective retrovirus expressing human adenosine deaminase or human placental alkaline phosphatase. The majority of the seeded SMCs remained in the intima while a few of the cells appeared to migrate into the first layer of the media. Intimal SMC proliferation returned to background levels (< 0.1% thymidine labeling index) by 28 d. At late times (1 and 12 mo) the morphological appearance of the intima was the same for balloon-injured arteries with or without seeded SMC, except that the seeded arteries continued to express human adenosine deaminase or alkaline phosphatase. These results support the conclusion that cultured SMC infected with a replication-defective virus containing human adenosine deaminase or alkaline phosphatase are not phenotypically altered and do not become transformed. After seeding onto the surface of an injured artery, they stop replicating but continue to express the introduced human genes even over the long term.

Topics: Adenosine Deaminase; Alkaline Phosphatase; Animals; Carbocyanines; Carotid Arteries; Carotid Artery Injuries; Catheterization; Cells, Cultured; Fluorescent Dyes; Genetic Therapy; Genetic Vectors; Humans; Male; Microscopy, Electron, Scanning; Muscle, Smooth, Vascular; Rats; Rats, Inbred F344; Retroviridae; Transfection

Neurogenesis, migration and maturation of ganglion cells in the posterior pole of chick retina have been studied using embryonic incorporation of [3H]thymidine, immunocytochemistry and retrograde labeling. Unlike previous studies, we have examined the neurogenesis of independently identified ganglion cells that have survived the period of naturally occurring cell death (embryonic days 11-16). Embryos were labeled with [3H]thymidine at different embryonic ages (embryonic days 3, 5 and 7). After the chicks hatched, ganglion cells were retrogradely labeled with rhodamine microspheres and the retinas were processed for autoradiography and fluorescent microscopy. The results indicate that 40% of the ganglion cells in the posterior pole undergo a final mitosis by embryonic day 3 and that more than 25% of the ganglion cells are born on or after embryonic day 7. These results also suggest that naturally occurring cell death does not preferentially affect ganglion cells born on specific embryonic days. Using immunocytochemistry with an antibody against neuron-specific beta-tubulin and retrograde labeling with the carbocyanine dye DiI we show that ganglion cells begin to differentiate before the completion of their migration to the presumptive ganglion cell layer. These results suggest the following developmental sequence. (1) Ganglion cells of the posterior pole undergo their final mitosis near the ventricular margin between embryonic days 2 and 8. (2) They maintain contacts with both retinal surfaces and their nuclei move toward the ganglion cell layer. At this time they start to differentiate, expressing a form of neuron-specific tubulin and growing axons that can reach the optic chiasm. (3) Once migration is completed dendritic development commences.

Topics: Animals; Autoradiography; Axons; Carbocyanines; Cell Death; Cell Differentiation; Cell Movement; Chick Embryo; Immunohistochemistry; Optic Nerve; Retina; Retinal Ganglion Cells; Thymidine; Tubulin

During development, axons of the mammalian corpus callosum must navigate across the midline to establish connections with corresponding targets in the contralateral cerebral cortex. To gain insight into how growth cones of callosal axons respond to putative guidance cues along this CNS pathway, we have used time-lapse video microscopy to observe dynamic behaviors of individual callosal growth cones extending in living brain slices from neonatal hamster sensorimotor cortex. Crystals of the lipophilic dye 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (Dil) were inserted into the cortex in vivo to label small populations of callosal axons and their growth cones. Subsequently, 400 microns brain slices that included the injection site, the corpus callosum, and the target cortex were placed in culture and viewed under low-light-level conditions with a silicon-intensified target (SIT) camera. Time-lapse video observations revealed striking differences in growth cone behaviors in different regions of the callosal pathway. In the tract, which is defined as the region of the callosal pathway from the injection site to the corresponding target cortex, growth cones advanced rapidly, displaying continual lamellipodial shape changes and filopodial exploration. Forward advance was sometimes interrupted by brief pauses or retraction. Growth cones in the target cortex had almost uniform compact shapes that were consistently smaller than those in the tract. In cortex, axons adhered to straight radial trajectories and their growth cones extended at only half the speed of those in the tract. Growth cones in subtarget regions of the callosum beneath cortical targets displayed complex behaviors characterized by long pauses, extension of transitory branches, and repeated cycles of collapse, withdrawal, and resurgence. Video observations suggested that extension of axons into cortical targets could occur by interstitial branching from callosal axons rather than by turning behaviors of the primary growth cones. These results suggest the existence of guidance cues distinct for each of these callosal regions that elicit characteristic growth cone behaviors.

Topics: Animals; Animals, Newborn; Axons; Carbocyanines; Cerebral Cortex; Corpus Callosum; Cricetinae; Fluorescent Dyes; In Vitro Techniques; Mesocricetus; Time Factors; Video Recording

Discharge of nematocysts from cnidocytes occurs in response to appropriate chemical and mechanical stimulation. In sea anemone tentacles, activating chemoreceptors for N-acetylated sugars shifts maximal discharge into vibrating targets to low frequencies corresponding to prey movements and induces hair bundles to elongate by approximately 1-2 microns. Until now, only indirect, correlative evidence linked these two events. Using cytochalasin D, we provide evidence that bundle elongation is necessary for the frequency shift. Moreover, we find that only bundles associated with sensory cell/supporting cell complexes elongate with chemosensitization, and not bundles associated with cnidocyte/supporting cell complexes as was previously thought. Cytochemical labeling of sensory cells, purported to be bipolar neurons connected to the nerve net, suggests that sensory cells may interconnect with each other and with cnidocytes. Taken together, these findings are incompatible with the classical view that cnidocytes are independent effectors of nematocyst discharge and, furthermore, implicate the involvement of morphodynamic neurons in fine-tuning vibration-dependent discharge of nematocysts into swimming prey.

Topics: Animals; Carbocyanines; Chemoreceptor Cells; N-Acetylneuraminic Acid; Neural Pathways; Neurons, Afferent; Sea Anemones; Seawater; Sialic Acids; Silver Staining

Considerable progress has been made in recent years in identifying molecules with restricted expression in mammalian spinal cord at early developmental stages. However, the significance of the different expression patterns for most of these molecules is unclear because so little is known about the development of various classes of spinal interneurons. Recently, we have characterized the development of rat spinal cord interneurons with an axon that crosses in the ventral commissure (Silos-Santiago and Snider, J. Comp. Neurol., 325:514, 1992). In the current study, we describe the morphological development of ipsilaterally projecting spinal interneurons in laminae V-VIII of the thoracic spinal cord. These neurons were labelled by retrograde lateral diffusion of DiI after crystals were placed in various locations in the embryonic thoracic cord. By E14, approximately 48 hours after the first interneurons are generated, eight different groups of ipsilateral interneurons are present in the spinal cord. By E15, these groups of ipsilateral interneurons have reached distinct locations within the gray matter. Even at this early stage, different groups of cells have elaborated characteristic dendritic arborizations. By E19, at least 17 different types of ipsilateral interneurons can be identified on the basis of location and dendritic morphology. In general, ipsilateral interneurons are located more dorsally and laterally than commissural interneurons at all stages of embryonic development. Furthermore, in comparison with commissural neurons, fewer ipsilateral interneurons have dendritic arbors with a mediolateral orientation in the transverse plane. This work demonstrates that rat embryonic spinal cord contains a large number of morphologically distinct classes of interneurons that extend axons into the ipsilateral lateral funiculus. These neurons can be distinguished from commissural neurons on the basis of location and morphology. These results, taken together with those from our previous study, provide a framework for the localization of gene expression to different classes of spinal interneurons at early developmental stages.

Topics: Animals; Carbocyanines; Dendrites; Female; Fluorescent Dyes; Histocytochemistry; Interneurons; Neural Pathways; Pregnancy; Pyramidal Cells; Rats; Rats, Sprague-Dawley; Spinal Cord

Topographically distinct populations of radial glial cells in the diencephalon and mesencephalon of neonatal rats and hamsters were transcellularly labeled with wheat germ agglutinin conjugated to horseradish peroxidase (WGA-HRP) and with the lipophilic tracer DiI. A comparison of the histological distribution of the two tracers is suggestive of two different mechanisms of transcellular labeling. Intraocular injections of WGA-HRP resulted in the uptake of exogenously applied WGA-HRP by retinal ganglion cells, followed by anterograde axonal transport and exocytosis within the optic target nuclei. In addition to the transneuronal labeling, which is typical of such injections, we observed the transcellular labeling of the processes and somata of radial glial cells that were topographically associated with the terminal fields of the labeled axons. Similar transcellular labeling of radial glial cells associated with the axon terminal fields of the colliculogeniculate projection to the medial geniculate nucleus was observed following injections of WGA-HRP in the inferior colliculus. The transcellular labeling within the radial glial cells was discontinuous and somatopetally concentrated, indicating the existence of a retrograde active transport mechanism within the radial glial processes subsequent to its uptake following release of tracer from axons. This type of labeling can be referred to as transcellular retrograde glioplasmic transport. In contrast, DiI was used as a tracer through its capacity to diffuse within the plasmalemma. Topographically distinct populations of radial glial cells were transcellularly labeled following placements of DiI in the retina, inferior colliculus, or dorsal thalamus of fixed brains. The radial processes of labeled radial glial cells consistently extended into regions that also contained labeled axons. It is likely that the transcellular radial glial labeling with DiI occurred via transmembranous diffusion. These data indicate that a close structural and functional relation exists between axons and glial cells in the developing brain.

Topics: Aging; Animals; Animals, Newborn; Axonal Transport; Axons; Carbocyanines; Cricetinae; Fluorescent Dyes; Functional Laterality; Geniculate Bodies; Horseradish Peroxidase; Inferior Colliculi; Neuroglia; Rats; Rats, Sprague-Dawley; Retinal Ganglion Cells; Superior Colliculi; Visual Pathways; Wheat Germ Agglutinin-Horseradish Peroxidase Conjugate; Wheat Germ Agglutinins

The carbocyanine dye Dil (1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate) not only serves as an excellent neuronal tracer, but also produces staining of neurons similar to the results of the Golgi technique. In one aspect staining with Dil seems superior to the Golgi technique: the axons are well stained and show morphological details of their structure. The dendrites and the spines can also be studied easily so that this technique seems to be a promising alternative of the Golgi technique. The fading of the fluorescence could probably be overcome by photoconversion of the stained neurons.

Topics: Brain; Carbocyanines; Fluorescent Dyes; Golgi Apparatus; Neurons; Silver; Staining and Labeling

The differentiation of facial motoneurons and inner ear (octaval) efferents was examined in chicken embryos by applying DiI or dextran amines to the cut VII/VIII nerve (peripheral label) or to the basal/floor plate of rhombomeres 4/5 (central label). Central labeling found axons of these efferent neurons to leave the brain as early as 2.5 days of incubation. Peripheral labeling identified cell bodies ipsilaterally in rhombomeres 4 and 5 at 2.5 days. Central labeling at 3.5 days showed these fibers to have fully segregated into separate pathways to the facial nerve and the inner ear and that the octaval efferent axons had reached the otocyst wall. By 3.5 days many peripherally labeled octaval efferent somata were found in the floor plate and by 5 days they were found bilaterally. At 6 days, selective peripheral labeling of either the VIIth or VIIIth nerve showed that the contralateral population consisted of octaval efferents and central label applied to the floor plate of rhombomeres 4/5 identified fibers that entered the octaval nerve via the facial root and entered the vestibular sensory epithelia. Together these data suggest an initial mingling of two different motoneuron populations (facial and octaval) in rhombomeres 4/5 and a subsequent segregation by differential migration. Our data also find a much earlier arrival of octaval efferent axons at the otic vesicle than previously described and suggest a contralateral migration of many octaval efferents beginning shortly after their axons reach the facial nerve root.

Topics: Animals; Biotin; Carbocyanines; Cell Differentiation; Central Nervous System; Chick Embryo; Dextrans; Ear, Inner; Efferent Pathways; Facial Nerve; Histocytochemistry; Motor Neurons; Nerve Fibers; Peripheral Nervous System

The colonization of limb buds by neural crest cells was studied in quail-chick chimeras and in chick embryos using HNK-1 and DiI staining and the LD-DOPA reaction. Two populations of neural crest cells were found to colonize the limb bud. They migrate successively and use different routes of migration. The first population migrates within the limb bud subectodermally at stages before the limb is innervated. In the wing bud the migration route is localized postaxially and in the leg bud preaxially. Two cell types were identified differentiating from this first population: melanoblasts and Merkel cells. The second population of crest cells invades the limb bud at a later stage. These cells follow the routes of ingrowing nerves and migrate along a dorsal and a ventral path which correspond to the position of nerves for extensor and flexor muscles. Crest cells were found here also in the absence of nerves. Schwann cells and terminal glial cells develop from this second population of neural crest cells.

Topics: Animals; Antigens, CD; Antigens, Differentiation, T-Lymphocyte; Carbocyanines; CD57 Antigens; Cell Movement; Chick Embryo; Chimera; Coturnix; Dihydroxyphenylalanine; Extremities; Fluorescent Dyes; Immunohistochemistry; Neural Crest

The proposed pathways of chick cranial neural crest migration and their relationship to the rhombomeres of the hindbrain have been somewhat controversial, with differing results emerging from grafting and DiI-labelling analyses. To resolve this discrepancy, we have examined cranial neural crest migratory pathways using the combination of neurofilament immunocytochemistry, which recognizes early hindbrain neural crest cells, and labelling with the vital dye, DiI. Neurofilament-positive cells with the appearance of premigratory and early-migrating neural crest cells were noted at all axial levels of the hindbrain. At slightly later stages, neural crest cell migration in this region appeared segmented, with no neural crest cells obvious in the mesenchyme lateral to rhombomere 3 (r3) and between the neural tube and the otic vesicle lateral to r5. Focal injections of DiI at the levels of r3 and r5 demonstrated that both of these rhombomeres generated neural crest cells. The segmental distribution of neural crest cells resulted from the DiI-labelled cells that originated in r3 and r5 deviating rostrally or caudally and failing to enter the adjacent preotic mesoderm or otic vesicle region. The observation that neural crest cells originating from r3 and r5 avoided specific neighboring domains raises the intriguing possibility that, as in the trunk, extrinsic factors play a major role in the axial patterning of the cranial neural crest and the neural crest-derived peripheral nervous system.

Topics: Actin Cytoskeleton; Animals; Antigens, CD; Antigens, Differentiation, T-Lymphocyte; Carbocyanines; CD57 Antigens; Cell Movement; Chick Embryo; Coturnix; Embryonic and Fetal Development; Nerve Tissue Proteins; Neural Crest; Neurofilament Proteins; Rhombencephalon

Activation of the endogenous opioid system can suppress pain without affecting other sensations, but the cellular mechanism of this selectivity is unclear. The analgesia might be due to inhibitory synapses arranged only on neurons whose activity leads to pain sensations. Alternatively, opioids might be released broadly, with neurons involved in pain sensation being especially sensitive. Therefore, we asked whether different subsets of rat dorsal root ganglion (DRG) sensory neurons vary in their sensitivity to opioids. Dissociated neurons were subdivided according to the spinal laminae to which they likely had projected, and whether they had innervated muscle. Using the patch-clamp method, we measured the inhibition of Ca2+ current by DAGO (Tyr-D-Ala-Gly-MePhe-Gly-ol), a peptide that selectively activates the mu (morphine) receptor. We also investigated the presence of different types of Ca2+ channels. In DRG neurons chosen at random, Ca2+ currents were inhibited by DAGO to widely varying degrees, with an average inhibition of 38%. Ca2+ currents in neurons in a subset that projects to laminae I and II had a lower average inhibition, and unlike the randomly selected cells, the responses were predictable and tightly distributed about the mean. This indicates that the variability of opioid sensitivity among DRG neurons reflects the presence of different subsets of cells. Since neurons projecting to laminae I and II, the projection site of nociceptive neurons, did not show high opioid sensitivity, there is no evidence that nociceptive neurons have stronger responses to opioids. But a firm conclusion is impossible because projection site does not strictly define sensory modality.

Topics: Amino Acid Sequence; Animals; Antibodies, Monoclonal; Axonal Transport; Calcium Channels; Carbocyanines; Cells, Cultured; Electric Conductivity; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalins; Fluorescent Dyes; Ganglia, Spinal; Molecular Sequence Data; Muscles; Neurons, Afferent; Nociceptors; Rats; Rats, Sprague-Dawley; Receptors, Opioid, mu

Fetal allogeneic dorsal root ganglia (DRG) between 13 and 15 days (E13-E15) were transplanted into the enucleated fourth and fifth lumbar (L4, L5) ganglionic capsules of adult rat hosts. Some of the grafts were prelabeled with the vital carbocyanine dye DiI. Three to 9 months later, neuroanatomic tracers were applied singly or in combination to the sciatic nerve at a transection site 2-3 cm distal to the ganglion and to the dorsal quadrant of the spinal cord. Tissues in selected cases were stained with antibodies to calcitonin gene-related peptide (CGRP) or to neurofilament protein (antibody RT-97) as evidence of neuronal differentiation and axonal growth. In two grafted animals serial sections were made across the root-cord junction which was examined by light and electron microscopy. This material was compared to similarly prepared sections from two nongrafted animals subjected to dorsal root crush. Some grafted ganglion neurons survived for the 3-9 months of the study. Many of these cells became labeled after tracers were applied to the peripheral nerve, to the lumbar spinal cord, or to both. Additional signs of differentiation included expression of CGRP and neurofilament protein immunoreactivity in neuronal cell bodies and processes. Electron microscopic examination showed many small diameter fibers, both myelinated and unmyelinated, in the grafted root on both sides of the PNS/CNS junction. The results with this orthotopic transplantation model show that fetal DRG neurons can differentiate in an adult host and grow axonal branches into peripheral nerves as well as centrally through the dorsal root toward the spinal cord. In addition, our findings suggest that some of the centrally growing fibers cross the PNS/CNS border into the mature spinal cord.

Topics: Animals; Axons; Carbocyanines; Cell Differentiation; Female; Fetal Tissue Transplantation; Fluorescent Dyes; Ganglia, Spinal; Horseradish Peroxidase; Immunohistochemistry; Microscopy, Electron; Neurons; Rats; Rats, Sprague-Dawley; Sciatic Nerve; Staining and Labeling; Transplantation, Homologous; Wheat Germ Agglutinin-Horseradish Peroxidase Conjugate; Wheat Germ Agglutinins

The origin of the long descending propriospinal (LDP) projections have been studied in adult and developing opossums, Monodelphis domestica. This species has been chosen because of the considerable immaturity of the hindlimbs at birth, the postnatal appearance of their motility and the late development of coordination between them and the forelimbs. Neuroanatomical tracing has indicated that some LDP projections form postnatally. The ones present at birth arise from the regions of the cord where they are the most numerous in the adult opossum, presumptive laminae VII and VIII of the brachial enlargement. Subsequently, LDP projections arise from neurons located in adjacent laminae (IV to VI and IX and X) and at more rostral cervical levels. The origin of LDP projections in the adult opossums generally matches that reported for other mammals. These long propriospinal projections are in place well before the behavioral appearance of coordination between the hindlimbs and the forelimbs, but the timing of their synaptogenesis is not yet known.

Topics: Aging; Animals; Animals, Newborn; Carbocyanines; Fluorescent Dyes; Horseradish Peroxidase; Neurons; Opossums; Proprioception; Spinal Cord; Synaptic Transmission; Wheat Germ Agglutinin-Horseradish Peroxidase Conjugate; Wheat Germ Agglutinins

The factors determining the development of specific fiber tracts in the central nervous system as well as the interactions of growth cones with the surrounding micromilieu are largely unknown. Here we investigated the ontogenetic development of the septohippocampal projection in the rat with the lipophilic carbocyanine dye DiI which is transported anterogradely and retrogradely in neurons and can be applied to fixed embryonic tissue. Photoconversion of anterogradely labeled fibers allowed us to study individual growth cones by electron microscopy. The first axons originating from the septal complex were found in the hippocampus as early as on embryonic day (ED) 19, reaching the fimbrial pole of the hippocampus on ED 18. However, on ED 17 we consistently found retrogradely labeled cells in the hippocampus, indicating that the development of the hippocamposeptal projection precedes that of the septohippocampal projection. On ED 19, the majority of the axons directed toward the hippocampal formation passed the hippocampus and grew further into the subicular complex and entorhinal cortex. These axons gave off collaterals that invaded the hippocampus proper. A fairly adult pattern of the septohippocampal projection was reached on postnatal day 10, although may growth cones were still found. A comparative analysis of individual growth cones found in the fimbria and the hippocampus proper revealed no striking differences in their morphology. Electron microscopic analysis showed that growth cones in the fimbria were mainly contacted by other axons, whereas growth cones in the hippocampus had contact with all available elements. This may indicate that growing septohippocampal fibers are guided by axons of the earlier formed hippocamposeptal projection. In the hippocampus proper, other cues, probably derived from the target itself, may guide the septohippocampal axons to their appropriate target cells.

Topics: Afferent Pathways; Animals; Animals, Newborn; Axonal Transport; Axons; Carbocyanines; Gestational Age; Hippocampus; Microscopy, Electron; Morphogenesis; Rats; Rats, Sprague-Dawley; Septum Pellucidum

Brain nuclei that control song are larger in male canaries, which sing, than in females, which sing rarely or not at all. Treatment of adult female canaries with testosterone (T) induces song production and causes song-control nuclei to grow, approaching the volumes observed in males. For example, the higher vocal center (HVC) of adult females approximately doubles in size by 1 month following the onset of T treatment. Male HVC projects to a second telencephalic nucleus, RA (the robust nucleus of the archistriatum), which projects in turn to the vocal motor neurons. Whether HVC makes a similar connection in female canaries is not known, although HVC and RA are not functionally connected in female zebra finches, a species in which testosterone does not induce neural or behavioral changes in the adult song system. This experiment investigated whether HVC makes an efferent projection to RA in normal adult female canaries, or if T is necessary to induce the growth of this connection. In addition, we examined whether T-induced changes in adult female canary brain are reversible. Adult female canaries received systemic T implants that were removed after 4 weeks; these birds were killed 4 weeks after T removal (Testosterone-Removal, T-R). Separate groups of control birds received either (a) T implants for 4 weeks which were not removed (Testosterone-Control, T-C) or (b) empty implants (Untreated-Control, O-C). Crystals of the fluorescent tracer DiI were placed in the song-control nucleus HVC in order to anterogradely label both efferent targets of HVC, RA and Area X. Projections from HVC to RA and Area X were present in all treatment groups including untreated controls, and did not appear to differ either qualitatively or quantitatively. Thus, formation of efferent connections from HVC may be prerequisite to hormone-induced expression of song behavior in adult songbirds. The volumes of RA and Area X were measured using the distribution of anterograde label as well as their appearance in Nissl-stained tissue. RA was larger in T-treated control birds than in untreated controls. Experimental birds in which T was given and then removed (T-R) had RA volumes closer in size to untreated controls (O-C). Because the volume of RA in T-treated controls (T-C) was larger than that of birds that did not receive T (O-C), we conclude that the volume of RA increased in both T-C and T-R birds but regressed upon removal of T in T-R birds.(ABSTRACT TRUNCATED AT 400 WORDS)

Topics: Animals; Birds; Brain; Carbocyanines; Female; Histocytochemistry; Neural Pathways; Neuronal Plasticity; Optic Chiasm; Sex Differentiation; Staining and Labeling; Telencephalon; Testosterone; Vocalization, Animal

The differentiation of vagal motor neurons and their emerging central relationship with vagal sensory afferents was examined in fetal rats. To identify peripherally projecting sensory and motor neurons, 1,1'-dioctadecyl-3,3,3'3'-tetramethylindocarbocyanine perchlorate (DiI) was inserted into the proximal gut or cervical vagus nerve in fixed preparations. At embryonic day (E) 12, labeled vagal sensory neurons are present in the nodose ganglia and a few sensory axons project into the dorsolateral medulla. Central sensory processes become increasingly prevalent between E13 and E14 but remain restricted to the solitary tract. Vagal motor neurons are first labeled at E13, clustered within a region corresponding to the nucleus ambiguus (NA). Additional motor neurons appear to be migrating toward the NA from the germinal zone of the fourth ventricle. Motor neurons in the dorsal motor nucleus of the vagus (DMV) first project to the gut at E14 and have processes that remain in physical contact with the ventricular zone through E16. Sensory axons emerge from the solitary tract at E15 and project medially through the region of the nucleus of the solitary tract (NST) to end in the ventricular zone. A possible substrate for direct vagovagal, sensorimotor interaction appears at E16, when vagal sensory fibers arborize within the DMV and DMV dendrites extend into the NST. By E18, the vagal nuclei appear remarkably mature. These data suggest specific and discrete targeting of vagal sensory afferents and motor neuron dendrites in fetal rats and define an orderly sequence of developmental events that precedes the establishment of vagal sensorimotor circuits.

Topics: Animals; Carbocyanines; Dendrites; Female; Histocytochemistry; Horseradish Peroxidase; Medulla Oblongata; Motor Neurons; Neurons, Afferent; Nodose Ganglion; Pregnancy; Rats; Rats, Sprague-Dawley; Staining and Labeling; Vagus Nerve

Anterograde movement of DiI and transneuronal transport of wheat germ agglutinin-horseradish peroxidase (WGA-HRP) were used to study the temporal and laminar patterns of ingrowth of the geniculocortical projection to visual cortex in fetal and postnatal rats. The development of this projection was compared to patterns of migration and settling of [3H]-thymidine-labeled neurons destined for cortical layer IV, and to geniculocortical synapse formation. DiI-labeled geniculocortical axons were found in the intermediate zone beneath the lateral cerebral mantle at embryonic day (E)17 and in the subplate layer underlying visual cortex by E18. On E19 they appeared to accumulate and grow radially into an expanding subplate layer and into the deep part of developing cortical layer VI. By postnatal day (P)0, DiI or WGA-HRP-labeled geniculocortical axons were found in developing cortical layers VI and V. By P1, they invaded the deep portion of the cell-dense cortical plate, where they were in position to make initial contact with neurons that would later form layer IV. A few axons traversed the cortical plate to reach the marginal zone. Layer IV became an identifiable layer on P2, and a clear projection to layer IV was evident by P3. These results suggest that geniculocortical afferents grow continuously from the intermediate zone, initially into an expanding subplate layer and then sequentially into each of the developing cortical layers without evidence of "waiting." Electron microscopic data suggest that geniculocortical axons begin to form immature synapses with dendrites and neuronal perikarya as they first encounter cortical neurons, first in the subplate layer and then in developing layers VI, V and marginal zone, in addition to the primary target layer IV. The precise targeting and overall temporal and laminar patterns of ingrowth and synaptogenesis suggest that geniculocortical axons are directed to the visual cortex by guidance cues within the internal capsule and subplate. Further, they reach the occipital pole early enough to influence the specification and histogenesis of cortical area 17, perhaps by exerting an influence on the deep-to-superficial "wave" of neuronal differentiation in sequentially developing subplate and cortical layers VI, V and IV.

Topics: Afferent Pathways; Animals; Animals, Newborn; Axons; Carbocyanines; Fluorescent Dyes; Geniculate Bodies; Horseradish Peroxidase; Neural Pathways; Rats; Rats, Sprague-Dawley; Synapses; Visual Cortex; Wheat Germ Agglutinin-Horseradish Peroxidase Conjugate; Wheat Germ Agglutinins

In normal E6 (sixth day of incubation) quail mesencephalons, circumferential axons originate in the tectum (the dorsal part of a mesencephalon), course in a ventral direction and split into three trajectories in the ventral region of the mesencephalon; two of them turned on the ipsilateral side of the mesencephalon, one rostrally and the other caudally, while the third crossed the ventral midline and turned caudally on the contralateral side. In this study, we examined whether the ventral part of the mesencephalon has plasticity in its rostrocaudal polarity formation. We transplanted quail mesencephalons at the 9-10 somite stage (E2) with reversed rostrocaudal orientation prior to axon outgrowth. In reversely transplanted mesencephalons, circumferential axons at E6 took the same trajectory pattern as that of normal embryos; one turned rostrally and the second caudally on the ipsilateral side, and the third one turned caudally on the contralateral side. These results indicate that the rostrocaudal polarity of the mesencephalon is not fixed at E2, and that it may develop under the influence of tissues surrounding the mesencephalon.

Topics: Animals; Axonal Transport; Axons; Carbocyanines; Chick Embryo; Coturnix; Embryo, Nonmammalian; Fetal Tissue Transplantation; Fluorescent Dyes; Mesencephalon; Neuronal Plasticity; Superior Colliculi; Transplantation, Heterologous

Although the Xenopus embryo has served as an important model system for both molecular and cellular studies of vertebrate development, comparatively little is known about its neural crest. Here, we take advantage of the ease of manipulation and relative transparency of Xenopus laevis embryos to follow neural crest cell migration and differentiation in living embryos. We use two techniques to study the lineage and migratory patterns of frog neural crest cells: (1) injections of DiI or lysinated rhodamine dextran (LRD) into small populations of neural crest cells to follow movement and (2) injections of LRD into single cells to follow cell lineage. By using non-invasive approaches that allow observations in living embryos and control of the time and position of labelling, we have been able to expand upon the results of previous grafting experiments. Migration and differentiation of the labelled cells were observed over time in individual living embryos, and later in sections to determine precise position and morphology. Derivatives populated by the neural crest are the fins, pigment stripes, spinal ganglia, adrenal medulla, pronephric duct, enteric nuclei and the posterior portion of the dorsal aorta. In the rostral to mid-trunk levels, most neural crest cells migrate along two paths: a dorsal pathway into the fin, followed by presumptive fin cells, and a ventral pathway along the neural tube and notochord, followed by presumptive pigment, sensory ganglion, sympathetic ganglion and adrenal medullary cells. In the caudal trunk, two additional paths were noted. One group of cells moves circumferentially within the fin, in an arc from dorsal to ventral; another progresses ventrally to the anus and subsequently populates the ventral fin. By labelling individual precursor cells, we find that neural tube and neural crest cells often share a common precursor. The majority of clones contain labelled progeny cells in the dorsal fin. The remainder have progeny in multiple derivatives including spinal ganglion cells, pigment cells, enteric cells, fin cells and/or neural tube cells in all combinations, suggesting that many premigratory Xenopus neural crest precursors are multipotent.

Topics: Affinity Labels; Animals; Carbocyanines; Cell Differentiation; Cell Movement; Dextrans; Microinjections; Morphogenesis; Neural Crest; Phenotype; Rhodamines; Xenopus laevis

The anatomical connections of the rat paraventricular thalamic nucleus (PaVT) have been studied using the fluorescent dye DiI. The fact that this compound is able to dissolve and to diffuse in plasma membranes in formalin-fixed tissues (Godement et al., 1987) allowed us to depose precisely tiny quantities of DiI on the PaVT, in order to study its neuronal connections. Ventralward, the forebrain projections of the PaVT were directed to the reuniens nucleus of the thalamus, the dorso-medial hypothalamic region, the zona incerta and the lateral hypothalamic area. In the rostral direction, the PaVT appeared to innervate mainly the medial preoptic area, the bed nucleus of the stria terminalis (particularly the ventral and medial regions) and the medial septum. The forebrain afferent connections of the PaVT were found to originate mainly in the lateral and periventricular hypothalamic area, the medial preoptic area, the bed nucleus of the stria terminalis and the medial septum where the fluorescent nerve cell bodies appeared particularly numerous. From these results, it is concluded that DiI is a useful tool for demonstrating the neuroanatomical connections of small brain nuclei, as well as for post-mortem neuropathological studies.

Topics: Animals; Carbocyanines; Fluorescent Dyes; Histocytochemistry; Hypothalamus; Nerve Fibers; Neural Pathways; Neurons, Efferent; Prosencephalon; Rats; Rats, Wistar; Thalamic Nuclei

The nervous system consists of two classes of cells, neurons and glia, which differ in morphology and function. They derive from precursors located in the neurogenic region of the ectoderm. In this study, we present the complete embryonic lineage of a neuroectodermal precursor in Drosophila that gives rise to neurons as well as glia in the abdominal CNS. This lineage is conserved among different Drosophila species. We show that neuronal and glial cell types in this clone derive from one segregating precursor, previously described as NB1-1. Thus, in addition to neuroblasts and glioblasts, there exists a third class of CNS precursors in Drosophila, which we call neuroglioblasts. We further show that the NB 1-1 lineage exhibits characteristic segment-specific differences on the cellular level.

Topics: Abdomen; Animals; Carbocyanines; Cell Differentiation; Cell Movement; Cell Transplantation; Central Nervous System; Drosophila; Drosophila melanogaster; Ectoderm; Gastrula; Horseradish Peroxidase; Morphogenesis; Neuroglia; Neurons; Species Specificity; Stem Cells; Thorax

In order to obtain a detailed understanding of the chemical identity of callosal neurons and of their synaptic targets during development of the rat, a technique was developed combining anterograde and retrograde transport of the carbocyanine dye, DiI, previously applied in living or fixed tissue with conventional immunocytochemistry for peptides. It is reported here that photoconversion of the fluorescent DiI label to a stable diaminobenzidine reaction product is fully compatible with the application of the most widely used immunocytochemical techniques peroxidase-antiperoxidase (PAP) or avidin-biotin (ABC) on the same tissue section, for correlated light and electron microscopic studies. Advantages of this double-labeling procedure over previously described techniques which permit concurrent visualization of projection systems and chemically defined neuronal elements are discussed.

Topics: Animals; Carbocyanines; Corpus Callosum; Fluorescent Dyes; Immunohistochemistry; Methods; Microscopy, Electron; Neurons; p-Dimethylaminoazobenzene; Rats; Rats, Wistar; Somatostatin; Vasoactive Intestinal Peptide

We have investigated the neuroprotective effects of recombinant human ciliary neurotrophic factor (CNTF) for injured dopaminergic neurons of the adult rat substantia nigra compacta. Fourteen days after a unilateral transection of the nigrostriatal pathway two-thirds of the neurons (identified by retrograde labeling) had degenerated. In sharp contrast, 73% (a few cases, > 90%) of this cell loss was prevented by continuous infusion of CNTF close to the injured neurons. However, CNTF did not prevent the disappearance of the transmitter-synthesizing enzyme tyrosine hydroxylase. Thus, CNTF has potent neurotrophic effects for injured adult rat dopaminergic substantia nigra neurons, whose degeneration plays a major causative role in Parkinson disease.

Topics: Animals; Carbocyanines; Ciliary Neurotrophic Factor; Dopamine; Female; Nerve Growth Factors; Nerve Tissue Proteins; Neurons; Rats; Rats, Sprague-Dawley; Substantia Nigra; Tyrosine 3-Monooxygenase

Embryonic motoneurons were fluorescently-labelled with carbocyanine (diI) by means of retrograde transport and then grafted into the adult mouse spinal cord (L2) and brain (striatum) for 2-10 weeks. The motoneurons were grafted either following purification on the fluorescence-activated cell sorter or in the presence of embryonic glial cells and interneurons from the spinal cord. In both conditions of grafting, motoneurons were found to survive and develop in both grey and white matter and were found to migrate long distances in both regions of the central nervous system. Migration of neurons after grafting remains a controversial issue, therefore we have discussed the work of other groups that have described the same phenomenon.

Topics: Animals; Brain; Brain Tissue Transplantation; Carbocyanines; Cell Movement; Cell Survival; Cell Transplantation; Fetal Tissue Transplantation; Histocytochemistry; Mice; Mice, Inbred C57BL; Motor Neurons; Spinal Cord

This study examines the connections of the thalamic reticular and perireticular nuclei during development. In addition, because these nuclei lie directly in the path of corticofugal and corticopetal axons during development, we have examined the relationship of these growing axons to the reticular and perireticular cell groups. Neurones were labelled by applying DiI, wheat germ agglutinin conjugated to horseradish peroxidase (WGA-HRP), or HRP to the dorsal thalamus and/or cerebral cortex of rats at different stages of development. The axons of neurons in the reticular nucleus reach the dorsal thalamus as early as embryonic day (E) 14. At this age, and during later prenatal development, a small DiI implant limited to the presumptive lateral geniculate nucleus labels reticulothalamic and thalamocortical axons which travel in a clearly defined bundle through the thalamus. During late gestation, thalamocortical (approximately E15) and corticothalamic (approximately E17) axons pass directly through the reticular nucleus toward their targets. It is not until birth that collaterals are seen extending into the nucleus from the parent axons. Neurones in the perireticular nucleus, in contrast to those in the reticular nucleus, are not labelled from the lateral geniculate nucleus until after birth. The perireticular nucleus is very large at a stage when the first thalamocortical axons leave and when the first corticothalamic axons approach the thalamus. These axons are seen to change course sharply in the region of the internal capsule, where there are many perireticular cells. Corticothalamic axons turn toward the reticular nucleus, and thalamocortical axons turn toward the cortical subplate. Corticospinal and corticobulbar axons, on the other hand, pass directly through the perireticular region toward their more caudal targets. After these axons have reached their targets, the perireticular nucleus reduces dramatically in size.

Topics: Afferent Pathways; Animals; Axons; Brain Mapping; Carbocyanines; Efferent Pathways; Horseradish Peroxidase; Rats; Thalamic Nuclei; Wheat Germ Agglutinins

The distribution of the central neural connections of the pineal organ of the larval sea lamprey was investigated by means of anterograde and retrograde tracing with the fluorescent lipophilic dye, DiI (1,1'-dioctadecyl 3,3,3',3'-tetramethylindocarbocyanine perchlorate). Pinealofugal projections are well developed in larvae, extending from the posterior commissure into the diencephalon and mesencephalon. Small numbers of neurons were retrogradely labelled in the transition zone between the diencephalon and the mesencephalic tegmentum. These cells may constitute the first pinealopetal system described in anamniotes.

Topics: Animals; Carbocyanines; Diencephalon; Lampreys; Larva; Neural Pathways; Photoreceptor Cells, Invertebrate; Pineal Gland; Tegmentum Mesencephali

Layer V pyramidal neurons in the occipital part of the rat cerebral cortex project to both the cervical spinal cord and the tectum early in postnatal development. The occipito-spinal projection is transient and is subsequently withdrawn, while a permanent connection is maintained with the tectum. The withdrawal of the transient occipital corticospinal axons may be due to their inability to respond to target-derived influences. In the current study we co-cultured explants of the occipital cortex and cervical spinal gray matter or tectum in 3-D collagen gels. Directional growth of the cortical axons towards either the cervical spinal gray or tectal explant was observed. This indicates that the failure of neurons located in the occipital cortex to maintain collaterals within the spinal cord in vivo is not due to their inability to respond to a target-derived factor, but must be regulated by other extrinsic factors.

Topics: Animals; Axons; Carbocyanines; Histocytochemistry; In Vitro Techniques; Neural Pathways; Occipital Lobe; Pyramidal Cells; Rats; Spinal Cord; Superior Colliculi

After crushing one optic nerve in a bony fish, retinal fibers regenerate to both tecta. Anterograde labelling indicates that the ipsilaterally regenerating fibers have a rather straight growth, apparently along the undamaged fibers of the contralateral retina. In contrast, the contralaterally regenerating fibers deviate widely from a straight course. Retrograde labelling shows a mirror-symmetric distribution of regenerated ipsilateral and resident contralateral ganglion cells in a comparable annulus. In contrast, ganglion cells in the regenerated contralateral retina show no topological order after comparable small Dil applications to the ventrolateral tectum. These data suggest that regenerating fibers can orient on the undisturbed, contralateral fibers.

Topics: Animals; Axons; Brain; Carbocyanines; Fishes; Histocytochemistry; Nerve Crush; Nerve Fibers; Nerve Regeneration; Retina; Retinal Ganglion Cells; Superior Colliculi

Co-cultures of rat basal forebrain and cerebral cortex were maintained from 1 to 5 weeks in vitro with serum-free defined medium. The formation of synaptic connections between basal forebrain afferent fibres and cortical neurons was studied by specific labelling with three staining techniques, including (i) neuronal tract tracing with the fluorescent dye 1,1'-dioctodecyl-3,3,3'3'- tetramethylindocarbocyanine perchlorate, (ii) acetylcholinesterase histochemistry, and (iii) choline acetyltransferase immunocytochemistry. Both basal forebrain and cerebral cortex tissue displayed organotypic characteristics in culture. Cerebral cortex revealed a dense innervation by axonal projections from the basal forebrain. All three labelling techniques produced similar results at the light microscopic level, with densest innervation located in the marginal zone. At the fine structural level, the 1,1'-dioctodecyl-3,3,3'3'-tetramethylindocarbocyanine perchlorate-, acetylcholinesterase- and choline acetyltransferase-stained basal forebrain afferents all revealed a number of synaptic contacts with cortical neurons. The contacts displayed consistent synaptic features, including presynaptic accumulation of small round vesicles, cleft widening, and postsynaptic densities forming symmetric synapses. These morphological characteristics of connections formed in vitro are similar to basal forebrain cholinergic projections to cerebral cortex in normal brain. Based on these results, this tissue culture model appears to be an useful tool for investigations of the development of cholinergic innervation of cerebral cortex.

Topics: Acetylcholinesterase; Afferent Pathways; Animals; Carbocyanines; Cerebral Cortex; Choline O-Acetyltransferase; Fluorescent Dyes; Histocytochemistry; Immunohistochemistry; Microscopy, Electron; Neurons; Organ Culture Techniques; Prosencephalon; Rats; Synapses

In regenerating skeletal muscle, sarcoplasmic extensions containing variable numbers of nuclei, widely referred to as 'buds' or 'stumps', are formed at the ends of damaged myofibres. In this paper we investigated whether the nuclei seen in the buds results from fusion of myogenic cells or from migration of myonuclei to the sealed ends of damaged myofibres in murine muscle regenerating after crush injury. The fusion of mononuclear and multinucleate myogenic cells to the buds was demonstrated by transmission electron microscopy. In order to elucidate the frequency and kinetics of cytoplasmic continuity between myotubes and sealed myofibres, we labelled the damaged myofibres with carbocyanine dye DiI (which inserts into the lipid bilayer and travels down continuous membranes) and the samples were then examined by confocal scanning microscopy. This technique showed that there was little fusion between myotubes and myofibres during the first 6 days after crush injury, but significant fusion had occurred by the tenth day especially at the newly sealed region of the damaged myofibre. A scheme for the repair of damaged skeletal muscle is presented.

Topics: Animals; Carbocyanines; Cell Membrane; Histocytochemistry; Lipid Bilayers; Mice; Microscopy, Electron; Muscles; Regeneration; Sarcolemma

Early development of retinal ganglion cell morphology has been studied in human fetuses of gestational age ranging from 7-8 weeks to 19-20 weeks by retrograde labelling with a lipophilic fluorescent dye, DiI. The retinal ganglion cells with simple and relatively uniform appearance having few dendritic processes between 7 to 11 weeks of gestation show progressive growth and elaboration of dendrites at 17-18 weeks revealing some morphological variation in shapes. It is by 19-20 weeks of gestation that the three major ganglion cell types resembling the alpha, beta and gamma classes of cat ganglion cells are identifiable in the developing human retina. The dendrites of some ganglion cells exhibit varicosities, filiform processes and spines even as early as 10-11 weeks indicating a possibility of involvement in synaptic connectivity.

Topics: Animals; Carbocyanines; Cats; Dendrites; Fluorescent Dyes; Gestational Age; Humans; Morphogenesis; Retina; Retinal Ganglion Cells; Species Specificity

Cells in the cerebral cortex project to many distant regions in the brain. Each cortical target receives input from a specific population of cells which have a characteristic morphology and which are located in a distinct cortical layer. In an attempt to learn about the mechanisms by which this stereotypic output pattern is generated during development, we have studied the formation of cortical projections in an in vitro system. Slices from developing rat visual cortex were cocultured with slices from the superior colliculus, the major target of cells in layer 5, and the lateral geniculate nucleus, the major target of cells in layer 6. Cortical neurons which established connections with tectal and thalamic explants were retrogradely labelled with fluorescent dyes. It was found that, in vitro, different populations of neurons project to these two targets, and that the laminar position and cellular morphology of the projecting cells were similar to their in vivo counterparts. These specific connections were established when the target explants were placed either next to the white matter or next to the pial side of cortical slice cultures. The axons of cells projecting to ectopic positioned explants reoriented their trajectories and grew through the cortical grey matter directly towards their targets. Thus subcortical targets exert an orienting effect specifically on their innervating cells and attract growing axons of the appropriate cells at a distance. These results suggest that different targets release different molecules that act selectively on specific populations of neurons. Therefore, chemotropic guidance is likely to play a significant role in the development of specific connections between cortical neurons and their target areas.

Topics: Animals; Animals, Newborn; Carbocyanines; Chemotactic Factors; Culture Techniques; Efferent Pathways; Fluorescent Dyes; Geniculate Bodies; Rats; Superior Colliculi; Synaptic Transmission; Thalamus; Visual Cortex

In newborn kittens, cells in the striate cortex (visual area 17) that project to area 18 (part of extrastriate cortex) are distributed with uniform density in the superficial and in the deep layers. During postnatal weeks 2-3, some of these corticocortical connections are removed to generate an adult-like projection in which association cells are clustered mainly in the superficial layers of area 17. Axonal elimination, without cell death, is the major factor sculpting patches of corticocortical cells in superficial layers. In adult cats, few cells in area 17 (approximately 5%) have axons that bifurcate to multiple extrastriate areas. We have studied the possibility that the early exuberant innervation of area 18 by neurons in area 17 is largely from the transient collaterals of axons that also project to other visual areas. Kittens aged 2-21 days were each injected with a pair of retrogradely transported tracers, either diamidino yellow and fast blue, or diamidino yellow and a carbocyanine dye, at retinotopically corresponding points in area 18 and either area 19 or the posteromedial lateral suprasylvian cortex (PMLS). As for injections in area 18, those in area 19 and PMLS in kittens aged < or = 5 days labelled cells in continuous bands in area 17; in older kittens neurons projecting from area 17 to extrastriate regions were in patches, mainly in superficial layers. In each animal, the labelling from the two injections overlapped by 51-92%.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Amidines; Animals; Animals, Newborn; Carbocyanines; Cats; Cerebral Cortex; Fluorescent Dyes; Neural Pathways; Visual Cortex

The development of the uncrossed retinofugal pathways in normally pigmented and albino rats, aged from embryonic day (E) 14.5 to E18.5, was investigated. DiI was placed into one optic tract and the retinal origin of the uncrossed component, as well as its course in the optic stalk, was studied. The results show that, as in the mouse, the uncrossed retinal projection has two components. The first component is seen at E15.5 in normally pigmented animals. It develops exclusively in the central parts of the retina and is normal in albino littermates. The second component, which arises from the peripheral parts of the ventrotemporal retina, is seen two days later at E17.5 in all animals but is significantly smaller in albinos than in their pigmented littermates. Studies of axons in the optic stalk labelled retrogradely with DiI placed in the optic tract indicate that the uncrossed axons have no preference for any position in the stalk except when they approach the chiasm, where they tend to accumulate at the caudal region of the stalk. The uncrossed axons intermingle with the crossed axons along the entire length of the stalk. In albino embryos, no obvious difference in the prechiasmatic course of uncrossed axons was seen at any age examined. It is concluded that the albino mutation in rats affects the late ventrotemporal component of the uncrossed pathway selectively. It does not act on the early central component. Further, the intermingling of crossed and uncrossed axons in the stalk and the apparently unaffected prechiasmatic course of uncrossed axons in albinos indicate that the albino gene has its primary action in the retina.

Topics: Albinism; Animals; Axons; Carbocyanines; Embryonic and Fetal Development; Fluorescent Dyes; Gestational Age; Rats; Rats, Mutant Strains; Rats, Sprague-Dawley; Retina; Retinal Ganglion Cells; Species Specificity; Visual Pathways

The cytoarchitecture, fiber connections, and ultrastructure of the nucleus pretectalis superficialis pars magnocellularis (PSm) were studied in cypriniform teleosts (Cyprinus carpio). The PSm is an oval nucleus in the pretectum. Medium-sized cells and synaptic glomeruli are the main components of the nucleus. A lesser number of small cells are also present. Most of the medium-sized cells form one or two cell layers on the periphery of the nucleus, and some cells are scattered among synaptic glomeruli in the nucleus. Cell bodies in the peripheral cell layer are pyriform and sprout a thick dendrite directed inward. The dendrite gives off fine dendritic branches, which are postsynaptic elements in synaptic glomeruli. The PSm projects to the ipsilateral corpus mamillare (CM) and sends collaterals to the ipsilateral nucleus lateralis valvulae (NLV). Axons of the PSm neurons have terminals with many varicosities in the CM, and collaterals in the NLV have cup-shaped terminals around the cell bodies of the NLV neurons. Following horseradish peroxidase (HRP) injections into the PSm, HRP-labeled cells are found ipsilaterally in the optic tectum, the nucleus tractus rotundus of Schnitzlein, and the nucleus ruber of Goldstein. The tecto-PSm projections are topographically organized. The rostral optic tectum projects mainly to the rostral portion of the PSm, and the caudal tectum projects to the caudal portion of the PSm. The ventral tectum sends fibers mainly to the ventral part of the PSm. The dorsomedial tectum projects to the medial part of the PSm, and the dorsolateral tectum projects to the lateral part of the PSm. Tectal projection neurons to the PSm are of only one type. The tectal cell body is pyriform and is situated in the superficial part of the ipsilateral stratum periventriculare (SPV). The tectal neurons have a long perpendicular dendrite, which branches out in the stratum opticum (SO). An axon emerges from the branching site in the SO. Judging from the dendritic branching pattern of the tectal projection neurons, we concluded that the PSm receives visual information from the optic tectum.

Topics: Animals; Carbocyanines; Carps; Fluorescent Dyes; Histocytochemistry; Horseradish Peroxidase; Nerve Fibers; Superior Colliculi

Fluorescent carbocyanine dye was applied to the glossopharyngeal nerve of axolotls fixed with paraformaldehyde. Three cell types in the lingual epithelium were transneuronally labeled: chemosensory taste receptor cells and presumably mechanosensory basal cells in the taste buds, and epithelial cells with unknown function in the non-taste lingual epithelium.

Topics: Ambystoma mexicanum; Animals; Carbocyanines; Epithelial Cells; Fluorescent Dyes; Glossopharyngeal Nerve; Histocytochemistry; Lasers; Microscopy, Fluorescence; Neurons, Afferent; Taste Buds; Tongue

Acutely isolated slices of developing rat hippocampus have been used to study axon growth and synapse formation. Mossy fibers, which are the axons of dentate granule cells, were labeled in living brain slices by injection of a fluorescent membrane dye (DiI or DiO) into the dentate gyrus. Time-lapse observations were made in area CA3 at a time when mossy fibers are normally growing in and forming en passant synapses with pyramidal neurons. Single scan images were collected at 1-2 min intervals over a period of several hours using a scanning laser confocal microscope. At the tips of growing mossy fibers were highly motile growth cones with several filopodia and small lamellae. Labeled fibers typically extended at rates up to 15 microns/h, but occasionally individual axons abruptly stopped elongating and the leading growth cone became quiescent. In addition, dynamic filopodia-like structures were found to be associated with axonal varicosities proximal to the leading growth cone. We are currently pursuing methods to determine whether these motile activities correlate with synapse formation.

Topics: Animals; Animals, Newborn; Carbocyanines; Fluorescent Dyes; Hippocampus; In Vitro Techniques; Microscopy, Fluorescence; Nerve Fibers; Pyramidal Cells; Rats; Rats, Sprague-Dawley; Synapses

The extent of the secondary olfactory projections shows great variation among different groups of craniates. Gnathostomes typically display restricted secondary olfactory projections, whereas lampreys have more extensive projections. Any attempt to determine the phylogenetic polarity of these characters, that is, to decide which is primitive and which is derived, requires an investigation of the secondary olfactory system in the sister group of lampreys and gnathostomes, the hagfishes. Therefore the secondary olfactory projections of the Pacific hagfish, Eptatretus stouti, were traced with the use of horseradish peroxidase and the lipophilic fluorescent tracing compound DiI. The projections are bilateral and massive to all pallial areas and the septum, moderate to the striatum, and relatively weak to the preoptic and infundibular regions of the hypothalamus, reaching caudally to the diencephalic-mesencephalic boundary. Afferents to the olfactory bulb arise from the pallium, the preoptic area, and the ventral thalamus. We compare the secondary olfactory projections in hagfishes with those in lampreys and in gnathostomes, and we conclude that the presence of extensive secondary olfactory projections is a primitive character of craniate brains.

Topics: Animals; Brain Mapping; Carbocyanines; Hagfishes; Histocytochemistry; Horseradish Peroxidase; Nerve Fibers; Olfactory Pathways; Phylogeny; Prosencephalon

DiI, a fluorescent lipophilic dye, was micro-injected into the brachial somites of 10.5 day rat embryos to determine whether these somites can contribute cells to the development of the fore-limb bud. The injected embryos were cultured and harvested at the 20-25-somite stage. The dye did not interfere with somitogenesis because, at the injection site, the DiI-labelled somites were able to differentiate into dermomyotome and sclerotome. We have analyzed cryo-sections of 20-21-somite stage embryos and were unable detect the presence of DiI-labelled cells in the fore-limb buds. However, at the 22-somite stage, a few DiI-positive cells were found in the proximal region of the limb bud. These labelled cells had migrated into the limb from the lateral border of the dermomyotome. From the 23-somite stage onwards, there were even more DiI-positive cells inside the limb. We have performed an additional set of experiments to confirm that the somitic cells do have the ability to invade and colonize the limb bud. This was achieved by first labelling newly formed somites isolated from the caudal region of 10.5 day embryos with DiI and then grafting them into corresponding regions in 8-11-somite stage hosts. The donor somites were not orientated when they were implanted into the host. However, this did not disrupt their ability to undergo normal somitogenesis. We have detected the presence of DiI-positive cells in the limb buds of approximately 71% of the 19-30-somite stage embryos that have been examined. This is similar to what we obtained for the injected embryos. Nevertheless, there is one slight difference and that is the stage the somitic cells begin their invasion of the limb. For the injected embryos, migration began at the 22-somite stage but in the transplanted embryos, it commenced as early as the 18-somite stage. We have also investigated the myogenic potential of the fore-limb bud at various stages of development to ascertain whether there is a correlation between the stage the somitic cells first appear in the limb bud and the stage the bud acquires the capacity to form skeletal muscles. This was realized by culturing fore-limb buds excised from 18-30-somite stage embryos conventionally and in the kidney capsules of adult rats. In both methods, bone and cartilage were present in all of the cultures whereas skeletal muscles were only present in cultured explants older than the 21-22-somite stage.(ABSTRACT TRUNCATED AT 400 WORDS)

Topics: Animals; Carbocyanines; Cell Differentiation; Cell Membrane; Cells, Cultured; Chimera; Embryo, Mammalian; Embryonic and Fetal Development; Extremities; Female; Immunohistochemistry; Kidney; Mesoderm; Muscles; Myosins; Pregnancy; Rats; Rats, Sprague-Dawley; Time Factors

Crystals of the lipophilic tracer DiI were applied to discrete regions of the olfactory epithelium of goldfish to trace the primary sensory projection to the olfactory bulb. Receptors from the anterior half of the sensory sheet project primarily to glomeruli in the medial half of the bulb and receptors in the posterior half terminate mainly within the lateral half of the bulb. This pattern disappeared following ablation of selected, discrete epithelial regions. In order to investigate reorganization of secondary olfactory projections, unoperated control and unilaterally bulbectomized animals received injections of [3H]proline into the right olfactory bulb. Densities of silver grains per unit area were determined within six different forebrain nuclei in both the right and left hemispheres of each animal. Of the six areas examined, three demonstrated a significantly greater density of afferent innervation from the ipsilateral versus contralateral bulb; a difference which disappeared in two of these three regions after bulbectomy. Thus, for at least two forebrain nuclei, bulb removal caused a change in the afferent input from the spared olfactory bulb to those regions. We conclude that both primary and secondary olfactory projections in goldfish are capable of some degree of reorganization following insult.

Topics: Animals; Brain Mapping; Carbocyanines; Fluorescent Dyes; Goldfish; Mucous Membrane; Neuronal Plasticity; Olfactory Bulb; Olfactory Pathways; Prosencephalon; Reference Values

Innervation of the axolotl lingual epithelium by the glossopharyngeal nerve was examined to reveal its sensory target cells. The carbocyanine dye diI was applied to the nerve stump in the tongue fixed with paraformaldehyde. After a diffusion period of several months, the tongues were examined with a conventional epifluorescence microscope and a confocal laser scanning microscope (LSM) in wholemounts or preparations sectioned with a vibratome. Beneath the epithelium the labeled nerve fibers spread horizontally to form a meshwork of fibers, from which fascicles of fibers extended upward perpendicularly to the epithelium to innervate taste buds. Numerous taste buds were labeled by possible transcellular diffusion of diI. At the base of the taste bud, the nerve fibers branched and formed a basal plexus of fine fibers, on which numerous varicosities were seen. One or at most several taste cells were labeled in a taste bud. In the basal part of taste buds, the cell without an apical process, the basal cell, was also labeled. In the epithelium, between the taste buds, a few solitary cells were labeled. In some cases, a single fascicle of fibers innervating these cells was clearly shown by the LSM. In addition, fine fibers apparently formed free nerve endings in the epithelial cell layer. The results showed that the IX nerve innervated not only taste cells, but also presumed mechanosensory basal cells in the taste bud and the solitary cells of unknown function in the non-taste lingual epithelium. Afferent nerve responses to mechanical stimulation of the tongue may be explained by these non-taste cellular elements in the epithelium.

Topics: Ambystoma mexicanum; Animals; Carbocyanines; Epithelial Cells; Epithelium; Glossopharyngeal Nerve; Histocytochemistry; Nerve Fibers; Neurons, Afferent; Taste Buds; Tissue Fixation; Tongue

The present study in the male Djungarian hamster determined the neuroanatomical distribution and morphology of gonadotropin-releasing hormone (GnRH) neurons which innervate the medial basal hypothalamus during sexual maturation. Prepubertal, peripubertal, and postpubertal males were perfused, brains were removed, and crystals of the fluorescent tract tracer, DiI, were implanted directly into the median eminence of the brain. Eight weeks later, brains were sectioned and processed for GnRH immunofluorescence. At all ages, GnRH cell bodies were bipolar or unipolar; both subtypes were labeled with DiI in proportion to their respective numbers in each brain region. GnRH perikarya were distributed in a diffuse ventromedial continuum from the septum through the anterior hypothalamus. In prepubertal males, DiI was present in the majority of GnRH neurons (54% of total) that were located in brain regions rostral to and including the medial preoptic area. In lateral and caudal brain areas, fewer GnRH perikarya contained DiI (28% of total or less). With sexual maturation, fewer GnRH somata were labeled with DiI in areas rostral to the hypothalamus. The data suggest that bipolar and unipolar GnRH neurons in the forebrain, rostral to the preoptic area, are major contributors to the GnRH innervation of the median eminence in the male Djungarian hamster. With the onset of puberty, the finding that decreasing numbers of GnRH perikarya directly project to the medial basal hypothalamus suggests that fewer GnRH neurons constitute the final common pathway that controls gonadotropin secretion.

Topics: Aging; Animals; Carbocyanines; Cricetinae; Fluorescent Dyes; Gonadotropin-Releasing Hormone; Hypothalamus; Male; Neural Pathways; Neurons; Phodopus; Sexual Maturation

The development of the olfactory organ of the zebrafish, from the forming of early placode to the adult organ, was investigated by electron microscopy and DiI labeling. The olfactory placode is formed by a subepidermal layer of cells. These cells differ from those of the epidermis as well as from brain cells, and they do not mingle either with epidermal or with brain cells. No migration of cells from the brain or the epidermis towards the subepidermal cell layer has been observed. The cells of the subepidermal layer seem to form all cell types of the olfactory mucosa, i.e., basal cells, ciliated and microvillous receptor cells, supporting cells, and ciliated nonsensory cells. Axons grow into the forebrain at a very early stage when the epidermis still covers the placode completely. Dendrites grow out when the epidermis separates, building the olfactory pit. This process implicates neither cell lysis nor cell degeneration. The olfactory pit forms a rosette with a midline raphe and olfactory lamellae. The incurrent nostril is separated from the excurrent nostril by a funnel-shaped structure. Differentiation of the olfactory placode in the embryo is accomplished very quickly, whereas the development into the adult organ during larval stages is a slow process.

Topics: Animals; Carbocyanines; Embryonic and Fetal Development; Fluorescent Dyes; Microscopy, Electron; Microscopy, Electron, Scanning; Olfactory Pathways; Zebrafish

Functional studies of the development of the corpus callosum in the cat have shown that an intact callosum during postnatal month 1 is necessary for normal visual development. In vivo tracing techniques have not provided enough information on corpus callosum connectivity to fully evaluate the evidence for a morphological mechanism for the functional effects of neonatal callosum section. However, lipophilic in vitro membrane tracers permit a more detailed search for such evidence because the entire limit of many cells can be labeled simultaneously. To investigate the morphological basis for the observed functional results in cats, the corpus callosum was labeled in vitro with the carbocyanine dye, DiI. Crystals of DiI were placed in the midsagittal callosum in tissue from 2 to 277-day-old cats. Tissue was coronally sectioned 3-22 months later. Sections were photographed and reconstructed to show the overall distribution of corpus callosum projections, as well as the locations of individual corpus callosum axons and their presumed terminals. The distribution of corpus callosum projections, examined in cortical areas 17-19, 7, and posterior medial lateral suprasylvian cortex, changes significantly during development. During postnatal week 1, callosal axons extend throughout these cortical areas to layer I. Numerous varicosities on callosal axons are located en passant and at axon terminals in layer I. During postnatal week 2, the density of callosal projections is reduced in all cortical areas, although many axons still extend to layer I. By postnatal month 2, the callosal axons extending to layer I are predominantly near the border with adjacent cortical areas; in the nonborder regions of these areas, many axons extend to layer VI while a much smaller number of axons extend to layers II-V. By postnatal month 3, the callosal projections to supragranular layers are almost exclusively restricted to cytoarchitectonic border regions; in the remaining regions, including medial area 17, there are occasional axons extending to the supragranular layers and only a moderate number of axons extending to infragranular layers. Thus, a substantial number of elaborately formed transitory corpus callosum axons, distributed throughout visual cortex, exist for several weeks during postnatal development; in area 17, these axons are found in central through peripheral visual field representations. The transitory callosal axons appear to have axon terminals in layer I as well as

Topics: Aging; Animals; Animals, Newborn; Axons; Carbocyanines; Cats; Corpus Callosum; Fluorescent Dyes; Nerve Fibers; Neural Pathways; Time Factors; Visual Cortex

The neural cell adhesion molecule NCAM exists as several related peptides formed by alternative splicing of the single NCAM gene. Here the ability of NCAM containing and lacking the alternatively spliced VASE exon to act as a permissive growth substrate was tested by examining retinal axon outgrowth on normal L cell fibroblasts and L cells expressing stably transfected 140 kD NCAM +/- VASE. L cells expressing either NCAM form were a more permissive substrate than control L cells. At higher substrate cell densities, greater axon outgrowth occurred on substrate cells expressing NCAM - VASE than on those expressing NCAM + VASE. Similar experiments tested retinal axon growth on neuronal substrates by utilizing clonal B35 cells, C3 cells that are NCAM lacking variants of B35, and C3 cells into which 140 kD NCAM +/- VASE has been restored by transfection. Axon growth on C3 cells transfected with NCAM - VASE was greater than that on all other substrates including cells transfected with NCAM + VASE. In these experiments C3 cells and transfected C3 expressing NCAM + VASE cell promoted similar outgrowth. The influence on neurite growth of the NCAM isoform of the neurite itself was tested by examining neurite formation using combinations of C3 cells and C3 NCAM transfectants both in the growth monolayer and as responding cells. C3 cells were able to extend neurites, indicating NCAM is not required for neurite growth. However, C3 derivatives transfected with NCAM +/- VASE had greater neurite outgrowth. The most extensive neurite growth was found when NCAM - VASE was expressed by both substrate cells and the responding neurite growing cells. Thus NCAM enhances axon or neurite outgrowth when present either in the growth substrate or on the growing axon. NCAM - VASE has a significantly greater growth promoting capability than NCAM + VASE. The expression of NCAM + VASE by more mature neural cells could thus be a significant factor in the reduced axonation capabilities of mature neurons.

Topics: Animals; Axons; Carbocyanines; Cell Adhesion Molecules, Neuronal; Cell Line; Cells, Cultured; Chick Embryo; Exons; Gene Expression; Immunohistochemistry; L Cells; Mice; Microscopy, Fluorescence; Neurites; Retinal Ganglion Cells; Transfection

The fluorescent stain Di-I combined with confocal microscopy identified an abnormal neuritic pattern in the cortices of frontal and anterior temporal lobes in postmortem tissues from patients with Pick's disease (PD). Focal, dense neuritic aggregates 50 to 200 microns in diameter were scattered throughout all cortical layers. The three-dimensional analysis provided by confocal microscopy revealed the neuritic clusters to consist of enlarged, randomly arrayed, dystrophic neurites that were not associated with amyloid deposits, astrocytic processes or capillaries. In the intervening neuropil, there were fewer neurites compared to controls. The occipital cortex, which is unaffected in PD, showed a neuritic architecture comparable to normal controls as did affected brain tissues from patients with Alzheimer's disease or remote, ischemic infarction. This neuritic pattern is, thus far, unique to PD and may reflect the loss of specific subpopulations of cortical neurons and proliferation of neurites of the remaining neurons.

Topics: Aged; Aged, 80 and over; Brain; Carbocyanines; Dementia; Female; Fluorescent Dyes; Humans; Immunohistochemistry; Male; Microscopy, Electron; Microscopy, Fluorescence; Middle Aged; Neurites; Reference Values

Red blood cells labeled with the carbocyanine dyes, 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI) and 3,3'-dioctadecyloxacarbocyanine perchlorate (DiO), were evaluated for use in making microvascular measurements in rat small intestine and spinotrapezius muscle. We determined the minimum concentration of each dye which produced near maximal fluorescent intensity and labeled cell fraction. These dyes, which have excitation and emission spectra similar to fluorescein and rhodamine derivatives, have a number of advantages over the isothiocyanates: (1) the labeling procedure is quicker, easier, and less expensive; (2) the labeled cell fraction and the fluorescent intensity of DiI and DiO cells are stable for long periods of time in the rat circulation; and (3) DiI-labeled cells are brighter and transmit light through overlying erythrocytes better than rhodamine X isothiocyanate. However, in vitro and in vivo evaluations illustrate the potential limiting effects of vessel diameter and cell velocity on the accuracy of microvascular measurements made using this technique. In the small intestine and spinotrapezius muscle preparations, measurements of labeled cell flux were readily reproducible and could be partly automated with image analysis only in capillaries and small venules. Counting labeled cells in larger vessels by human observation or with automation was not reproducible, presumably due to absorption and dispersion of the fluorescent signal by overlying erythrocytes and smearing of the cell image at high cell velocities.

Topics: Animals; Carbocyanines; Erythrocytes; Fluorescent Dyes; Image Processing, Computer-Assisted; Intestine, Small; Microcirculation; Muscles; Rats

In this report we studied the translocation of fluorescent particulate antigens to the draining lymph node, and the migration of fluorescent labeled alveolar macrophages (AM) and peritoneal macrophages (PM) in mice. The results show that intratracheally (IT) instilled particulate antigens translocate to the paracortical T cell area of the draining lymph node. When labeled AM were injected IT, they were found to migrate from the alveolar space into the paracortical T cell area of the draining lymph node. An identical localisation was found after IT injection of labeled PM. When either labeled AM, or PM were injected into the peritoneal cavity, a different migration pattern was observed. Via this route the labeled macrophages migrated to the subcapsular sinus and medulla of the draining lymph nodes. It is shown that the migrated cells are not dendritic cells (DC) present in the cell preparations. A possible role for the micro-environment of the injection site, and the significance of the specific migration pattern of AM is discussed.

Topics: Animals; Antigens; Carbocyanines; Cell Movement; Drug Administration Routes; Liposomes; Lymph Nodes; Macrophages; Macrophages, Alveolar; Mice; Mice, Inbred BALB C; Peritoneal Cavity; T-Lymphocytes; Tissue Distribution; Trachea

DiI-LDL (3,3'-dioctadecylindocarbocyanine-low density lipoprotein) has been extensively used in morphological and microscopic studies of receptor-mediated metabolism of LDL in many cell lines. To date the use of this fluorescent probe in a quantitative assay of LDL receptor activity has not been widely used in studies with multiple samples due to the lack of a practical method for quantitatively recovering cell-associated DiI. Therefore, detection by 125I-labeled LDL has remained the method of choice for assaying LDL receptor activity rapidly and reliably. In this paper, we describe a rapid, simple, and nonradioactive assay of LDL receptor activity using DiI-LDL. The increased sensitivity of this method was achieved by modifications to the labeling procedure of LDL and to the extraction of DiI from cells for subsequent fluorescence determination. These modifications did not affect the affinity of DiI-LDL toward HepG2 cells, and the assay was easily adapted to a rapid screen for LDL receptor modulators in this cell model.

Topics: Binding, Competitive; Carbocyanines; Carcinoma, Hepatocellular; Fluorescent Dyes; Humans; Hydroxycholesterols; Iodine Radioisotopes; Lipoproteins, LDL; Liver Neoplasms; Lovastatin; Receptors, LDL; Tumor Cells, Cultured

The bilateral efferent supply to the inner ear receptor fields is located in the hindbrain. In ovo injections of Dil into the common facial/vestibulo-acoustic nerve root at 3 days of chick development (stage 16) followed by analysis at 7 days has revealed the origin of the contralateral efferent neurons of the inner ear and their relation to the transient hindbrain rhombomeres. These neurons have a rhombomere 4-specific origin and form their commissure not by axonal outgrowth but, unusually, by transmedian cell migration into the contralateral rhombomere 4 and rhombomere 5. Neurons first project their axons from the ipsilateral basal plate through the VII/VIIIth nerve exit point and then migrate in the opposite direction, crossing the floor plate at stage 19-21. This rhombomere-specific cell behavior provides evidence at the cellular level that segmentation is intimately involved in establishing the pattern of this region of the CNS.

Topics: Animals; Auditory Pathways; Carbocyanines; Cell Line; Cell Movement; Chick Embryo; Efferent Pathways; Embryonic and Fetal Development; Facial Nerve; Fluorescent Dyes; Neurons, Efferent; Synaptic Transmission; Vestibule, Labyrinth; Vestibulocochlear Nerve

The startle response is a bilateral response even when elicited by unilateral acoustic or tactile stimuli. Similarly, unilateral electrical stimulation of the reticular formation also elicits a bilateral startle-like response. To examine whether crossed reticular formation connections can distribute the effects of unilateral stimulation across the midline, we delivered one pulse to the caudal pontine (RPC) or medullary reticular formation (MRF) and a second pulse to the opposite side of the brain, at various interpulse intervals. The symmetric collision effects suggest that axons which produce at least 37% (range 23-53%) of the startle response efficacy cross from RPC to RPC with a mean conduction velocity of 13 m/s. Similar collision effects were observed between RPC and MRF sites but at shorter conduction times. To examine which axons might cause these collision effect, the axonally transported label DiI was injected post mortem into 37 RPC sites. Many coarse axons were observed to cross in fascicles between bilateral RPC sites and then separate in the contralateral RPC. The fiber diameters and trajectories of these DiI-labelled axons are consistent with the conduction velocities and trajectories of the substrates mediating the startle-like response determined in collision tests.

Topics: Animals; Axons; Carbocyanines; Electric Stimulation; Fluorescent Dyes; Rats; Reaction Time; Reflex, Startle; Reticular Formation

During metamorphosis of the moth, Manduca sexta, an identified leg motor neuron, the femoral extensor motor neuron (FeExt MN) undergoes dramatic reorganization. Larval dendrites occupy two distinct regions of neuropil, one in the lateral leg neuropil and a second in dorsomedial neuropil. Adult dendrites occupy a greater volume of lateral leg neuropil but do not extend to the dorsomedial region of the ganglion. The adult dendritic morphology is acquired by extreme dendritic regression followed by extensive dendritic growth. Towards the end of larval life, MN dendrites begin to regress, but the most dramatic loss of dendrites occurs in the 3 days following pupation, such that only a few sparse dendrites are retained in the lateral region of leg neuropil. Extensive dendritic growth occurs over the subsequent days such that the MN acquires an adult-like morphology between 12 and 14 days after pupation. This basic process of dendritic remodeling is not dependent upon the presence of the adult leg, suggesting that neither contact with the new target muscle nor inputs from new leg sensory neurons are necessary for triggering dendritic changes. The final distribution of MN dendrites in the adult, however, is altered when the adult leg is absent, suggesting that cues from the adult leg are involved in directing or shaping the growth of MN dendrites to specific regions of neuropil.

Topics: Animals; Carbocyanines; Dendrites; Femur; Fluorescent Dyes; Larva; Metamorphosis, Biological; Moths; Motor Neurons

The development and regeneration of the ipsilateral retinopetal projection of the nucleus olfactoretinalis (NOR) in the cichlid fish Haplochromis burtoni was studied with 1,1'-dioctadecyl-3,3,3',3'-tetramethyl indocarbocyanine perchlorate (DiI) in fixed tissue. Throughout development most NOR cells projected to the contralateral retina. Only an insignificant, transient elevation of a projection to the ipsilateral retina was found in a few animals; however, after severing the contralateral processes of NOR cells by either enucleation or nerve crush, many animals had significantly more NOR cells with a regenerated process to the ipsilateral retina. Nevertheless, within a few weeks of surgery, the number of animals with ipsilaterally projecting cells were reduced to control values. The transiently enhanced ipsilateral projections to the retina imply changes in the guiding mechanism after these operations and the existence of control mechanisms against unusual connections to the retina in this bony fish.

Topics: Animals; Carbocyanines; Eye Enucleation; Fluorescent Dyes; Nerve Crush; Nerve Regeneration; Neural Pathways; Olfactory Pathways; Perches; Retina

The development of the adult visual system of Drosophila requires the establishment of precise retinotopic connections between retinal photoreceptor cell axons and their synaptic partners in the optic lobe of the brain. To assess the role of axon-axon interactions in retinal axon guidance, we used genetic methods to disrupt the normal spatiotemporal order of retinal axon ingrowth. We examined retinal axon projections to the developing first optic ganglion, the lamina, in two mutants in which reduced numbers of ommatidia develop in the eye imaginal disk. We find that in the developing lamina of these mutants, sine oculis and Ellipse, retinal axons project to proper dorsoventral positions despite the absence of the usual array of neighboring retinal axons. In a second approach, we examined animals that were somatic mosaics for the mutation, glass. In glass- animals, retinal axons project aberrantly and the larval optic nerve is absent. We find that in the developing lamina of glass mosaic animals, wild-type retinal axons project to proper dorsoventral positions despite the misrouted projections of neighboring glass- retinal axons. In addition, wild-type retinal axons project normally in the absence of the larval optic nerve, indicating that the latter is not an essential pioneer for retinal axon navigation. Our observations support the proposal that axon fascicles can make at least some pathfinding decisions independently of other retinal axon fascicles. We suggest that positional guidance cues that might label axon pathways and target destinations contribute to retinotopic pattern formation in the Drosophila visual system.

Topics: Animals; Carbocyanines; Cell Differentiation; Coloring Agents; Drosophila; Immunohistochemistry; Larva; Mutation; Neural Pathways; Optic Lobe, Nonmammalian; Optic Nerve; Photoreceptor Cells; Retina

A theory describing the shapes of polarized fluorescence photobleaching recovery (PFPR) curves for a population of fluorophores undergoing restricted rotational diffusion in two-dimensional systems such as planar membranes has been developed. In this model, restricted rotational diffusion of the fluorophores is described by using reflective boundary conditions, in which the fluorophores are assumed to diffuse freely but only within an angular space of width 2 omega. The magnitude and apparent rate of the PFPR postbleach fluorescence curves are a function of both omega and the angle between the bleaching and observation beam polarizations psi. It is shown that estimates of the degree of rotational restriction omega may be obtained from changes in the psi-dependent postbleach fluorescence intensities. Using angle-dependent PFPR, slow rotational reorientations of the fluorescent lipid analogue 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine in distearoylphosphatidylcholine Langmuir-Blodgett monolayers deposited on octadecyltrichlorosilane-treated fused quartz were measured. As theoretically predicted for a rotationally restricted fluorophore population, both the initial F psi (0) and final F psi (infinity) postbleach fluorescence intensities varied as a function of psi, and no measurable change in the postbleach fluorescence intensities was observed for psi = 45 degrees. Using the theory for restricted rotational motion, the psi-dependent variations of the final fluorescence intensities F psi (infinity) obtained at two bleaching intensities gave an average apparent omega approximately 52 degrees. However, to adequately fit the F psi (0) data, inclusion of the theoretical effects of rapid (faster than the duration of the photobleaching pulse) fluorophore dynamics was also required. Best fits of the F psi (0) and F psi (infinity) data were obtained when the fluorophores were assumed to rapidly wobble within a cone of semiangle delta approximately 30 degrees-50 degrees while slowly rotating within an angular space defined by semiangle omega approximately 35 degrees-60 degrees. Subsequent analysis of the time- and psi-dependent changes in the post-bleach fluorescence curves F psi (t) gave apparent diffusion coefficients ranging from D approximately 10(-3) s-1 to 4 x 10(-2) s-1.

Topics: Carbocyanines; Diffusion; Fluorescent Dyes; Liposomes; Mathematics; Models, Theoretical; Molecular Conformation; Phosphatidylcholines; Spectrometry, Fluorescence

The precursor crest cells of the spinal dorsal root ganglia (DRG) in the tilapia, Oreochromis mossambicus, were analysed by HNK-1 antibody staining, scanning electron microscopy, and DiI labeling techniques. The ontogeny of the DRG was followed in the embryos and young fry of the fish. Neural crest cells which contribute to the formation of the DRG were observed to commence their migration in the trunk region after 40 hours postfertilization. They do not penetrate the somites but travel through the space between the neural tube and the somite. Crest cells destined to become the DRG accumulate at the midsomitic region where the ventral root exits. At 50 to 80 hours postfertilization, they differentiate and become bipolar sensory cells. The DRG continues to grow and develop right through hatching at 115 hours. During the early larval stages, crest cells accumulate around the ventral root and the DRG eventually fuses with the motor root, giving rise to a situation in which the DRG contains not only the sensory cells but also motor fibres. The mixed nature of the DRG was confirmed by HRP retrograde labeling. We believe that this is the first report in describing the formation of the DRG in a teleost.

Topics: Animals; Carbocyanines; Cell Movement; Embryo, Nonmammalian; Fluorescent Antibody Technique; Ganglia, Spinal; Horseradish Peroxidase; Microscopy, Electron, Scanning; Morphogenesis; Motor Neurons; Neural Crest; Neurons, Afferent; Tilapia

We measured the lateral mobility of two fluorescent lipid probes dioctadecylindocarbocyanine (diI) and tetramethyl rhodamine phosphatidylethanolamine (R-PE) in the plasma membranes of Saccharomyces cerevisiae ino1 and opi3 spheroplasts. These are well-characterized strains with mutations in the inositol and phosphatidylcholine biosynthetic pathways. Membrane phospholipid composition was altered by growing these mutants in the presence or absence of inositol and choline. Lateral mobility was measured by fluorescence recovery after photobleaching (FRAP). Microscopic fluorescence polarization employing CCD digital imaging produced an ordered orientation distribution of the lipid probe diI, confirming that at least one of the probes was largely incorporated into the bilayer membrane. Our results demonstrated anomalously slow mobility of both lipid probes for both mutants, regardless of whether the lipid composition was near normal or dramatically altered in relative composition of phosphatidylinositol and phosphatidylcholine. Trypsinization of the spheroplasts to remove surface proteins resulted in markedly increased lateral mobility. However, even in trypsinized spheroplasts, mobility was still somewhat lower than the mobility observed in the membrane of mammalian cells, such as rat smooth muscle culture cells tested here for comparison.

Topics: Carbocyanines; Cell Membrane; Fluorescence Polarization; Fluorescent Dyes; Membrane Lipids; Phosphatidylethanolamines; Rhodamines; Saccharomyces cerevisiae

In order to shed light on the controversial issue of vagal innervation of the solar plexus ganglia, vagal efferent preganglionic fibers were anterogradely labeled by injecting the fluorescent carbocyanine dye Dil into the dorsal motor nucleus (dmnX). Additionally, Fluorogold was used to label the ganglia in toto, providing a counterstain and the possibility of UV light-guided dissection of the various ganglia. Using optical sectioning of whole mounted intact ganglia by means of laser scanning confocal microscopy, a considerable number of Dil-labeled vagal terminal-like structures were found in the major ganglia (celiac, superior mesenteric and suprarenal). Additionally, vagal efferent terminals were regularly found in microganglia associated with the periarterial plexuses of the celiac and superior mesenteric arteries, and in a few cases in small ganglia of the intermesenteric and renal plexuses. By using animals with prior selective vagal branch vagotomies, leaving only one (or a pair) of the three major abdominal divisions intact, it was concluded that the two celiac branches contribute the bulk of this vagal innervation, with the two gastric and the unpaired hepatic branch providing a small contribution mostly limited to the celiac ganglia. From control experiments, which involved Dil injections (1) into the dmnX in animals whose visceral afferents had been previously destroyed by capsaicin; (2) into the nodose ganglia, in order to anterogradely label vagal afferents; and (3) into the cervical vagus nerve as a control for uptake by fibers of passage, it was concluded that the identified terminal-like structures were vagal efferents and not inadvertently labeled afferents. We suggest that these vagal terminals have to be regarded either as ectopic parasympathetic junctions, or as part of a vagal mechanism for gating of sympathetic ganglionic transmission. Functionally, the parasympathetic innervation of the solar plexus may provide not only the classic vagal influence on gastrointestinal targets, but also vagal control of the adrenal glands and possibly other abdominal organs that have not been traditionally regarded as vagal targets.

Topics: Afferent Pathways; Animals; Carbocyanines; Efferent Pathways; Fluorescent Dyes; Ganglia; Ganglia, Sympathetic; Kidney; Male; Rats; Rats, Sprague-Dawley; Stilbamidines; Vagus Nerve

Cholinergic neurons in the medial septum/diagonal band complex project to the hippocampus and fascia dentata and establish characteristic types of synapses on a variety of target neurons. At present we do not know the principles that underlie the development of this projection and the formation of the cholinergic synapses. Here we have used co-cultured slices of septum and hippocampus of one- to six-day-old rat pups to study the development of the septohippocampal pathway and the formation of cholinergic synapses on hippocampal target neurons in vitro. Slices of septum and hippocampus were incubated together for 10-46 days applying the roller-tube technique. The fluorescent dye dioctadecyltetramethylindocarbocyanine perchlorate and histochemical staining for acetylcholinesterase labeled many fibers connecting both explants. Combined light- and electron-microscopic immunocytochemistry for choline acetyltransferase, the acetylcholine-synthesizing enzyme, revealed multipolar immunopositive neurons with long aspiny dendrites in the septal culture. Numerous varicose immunoreactive, supposedly cholinergic fibers could be followed from the septal to the hippocampal culture where they ramified and formed a three-dimensional network. As in situ, cholinergic terminals formed characteristic symmetric synapses on cell bodies, spines and, most often, on dendritic shafts of the hippocampal target neurons. No immunoreactive fibers and synapses were observed in single cultures of hippocampus. These results demonstrate that the cholinergic septohippocampal projection develops in vitro and that similar types of cholinergic synapses are established on co-cultured hippocampal target neurons as observed in situ.

Topics: Acetylcholinesterase; Animals; Animals, Newborn; Brain; Carbocyanines; Cell Communication; Cell Survival; Choline O-Acetyltransferase; Dendrites; Fluorescent Dyes; Hippocampus; Microscopy, Immunoelectron; Nerve Fibers; Neurons; Organ Culture Techniques; Rats; Rats, Sprague-Dawley; Synapses

Human low-density lipoproteins (LDL) bind specifically and saturably to the surface of the trematode parasite, Schistosoma mansoni, in vitro. Here we have tested whether human monocytes process the bound LDL. Monocytes obtained by leukapheresis generate H2O2, kill schistosomula, and were seen here endocytosing fluorescently labeled human LDL that was bound to the surface of the parasites. Compounds known to inhibit uptake of LDL via the scavenger receptor, namely, acetylated LDL, polyinosinic acid, dextran sulfate, fucoidan, and polyvinyl sulfate, inhibited both endocytosis of LDL and cell-mediated killing. Non-functional analogs of these inhibitors, namely, polycytidylic acid and dextran, did not inhibit either endocytosis or killing. Monocytes obtained from whole blood after venipuncture neither killed the parasite nor endocytosed LDL from the worm surface. Thus, human monocyte killing of schistosomula may involve removal of LDL from the parasite surface via scavenger receptors.

Topics: Animals; Carbocyanines; Endocytosis; Fluorescent Dyes; Humans; Lipoproteins, LDL; Membrane Proteins; Monocytes; Receptors, Immunologic; Receptors, Lipoprotein; Receptors, Scavenger; Scavenger Receptors, Class B; Schistosoma mansoni

We have reinvestigated the time of arrival of efferent fibers at the developing otocyst of mice employing diffusion of the lipophilic dye DiI in fixed tissue. In contrast to almost all previous reports, our data indicate a prenatal arrival of efferent fibers. A few efferent fibers were found to enter the eighth nerve root at embryonic day (ED) 10 1/2. Retrogradely labelled efferent cell bodies were at this stage coextensive with those of the facial motor nucleus, but started to segregate by ED 12. In contrast to retrogradely labelled facial motor neurons, labelled efferent neurons were bilaterally distributed in the hindbrain with a few projecting to both otocysts as early as ED 12. Anterograde labelling from the brain showed efferent fibers in the vestibular ganglion by ED 11. Invasion of the future vestibular sensory epithelia started by ED 12. Growth cones of efferent fibers had also reached the future cochlear sensory epithelium but invasion was only achieved by a few filopodia at this stage. The early arrival of efferents at the future sensory epithelia demonstrated here may allow an as yet unexplored interaction of efferent fibers with the proliferating and/or differentiating hair cells.

Topics: Animals; Auditory Pathways; Brain; Carbocyanines; Cell Differentiation; Cochlea; Ear, Inner; Facial Nerve; Hair Cells, Auditory; Mice; Motor Neurons; Neurons, Afferent; Neurons, Efferent; Otoacoustic Emissions, Spontaneous; Vestibular Nerve

One of the early events in the establishment of regional diversity in brain is the subdivision of the forebrain into the cerebral cortex and underlying basal ganglia. This subdivision is of special interest, owing to the striking difference in cellular patterning in these two regions. Whereas the dorsal aspect of the telencephalon gives rise to the laminar, cortical regions of brain, the basal aspect gives rise to nuclear, subcortical regions. To examine early events in the regionalization of the forebrain, we visualized cell movement within the ventricular zones of the dorsal and basal regions of the E15 murine telencephalon. Over an 8-24-hour observation period, labelled cells moved extensively in the plane of the cortical ventricular zone. Cell dispersion was restricted, however, at the border between the cortical ventricular zone and the lateral ganglionic eminence, the basal telencephalic ventricular zone. We suggest that this restriction of cell movements establishes a regional pattern of neurogenesis in the developing brain.

Topics: Animals; Carbocyanines; Cell Movement; Culture Techniques; Fluorescent Dyes; Image Processing, Computer-Assisted; Mice; Neurons; Stem Cells; Telencephalon

Recent studies on the distribution of optic axons in the mature visual pathways, as well as on the genesis of their ganglion cells of origin, suggest that the time of axonal arrival at the optic chiasm determines the side of the brain to which a temporal retinal axon will project. The present study has examined this issue directly in fetal ferrets, by determining the projection of the temporal retina at different developmental stages. Fetuses of known gestational age were fixed with paraformaldehyde and subsequently implanted with crystals of the carbocyanine dye, DiI, into either the temporal retina, or into one optic tract. The lipophilic diffusion of the dye within the plasma membrane of the axons revealed the course of temporal retinal fibers through the fetal chiasm, as well as the distribution of ganglion cells across the two retinae projecting to one optic tract. During early fetal stages, the temporal retina extends axons preferentially into the ipsilateral optic tract: the early retinal projection shows a classical partial decussation pattern. During later fetal stages, temporal retinal axons can be traced into both optic tracts, and the distribution of cells with crossed and uncrossed optic axons in the temporal retina is overlapping. These results indicate that the mature decussation patterns of retinal ganglion cell classes are not primarily the consequence of regressive phenomena such as cell death; rather, they are formed as axons navigate the chiasmatic region during development. The differences in decussation pattern between cell classes arise from the fact that the mechanisms producing the segregation of nasal and temporal retinal axons at the chiasm must change as development proceeds.

Topics: Animals; Axons; Carbocyanines; Embryo, Mammalian; Embryonic and Fetal Development; Ferrets; Fluorescent Dyes; Optic Chiasm; Retina; Visual Pathways

Afferent and efferent connections of the thalamic eminence of the axolotl were determined using the fluorescent compound DiI as a tracer. The thalamic eminence is connected reciprocally with a number of telencephalic and diencephalic areas, particularly with the medial pallium, the amygdala and the preoptic region. Efferent connections are widespread throughout the ipsilateral diencephalon. These findings are discussed in relation to the homology of this nucleus, especially its homologue in agnathan brains.

Topics: Ambystoma mexicanum; Animals; Carbocyanines; Diencephalon; Horseradish Peroxidase; Neural Pathways; Neurons, Afferent; Neurons, Efferent; Salamandridae; Telencephalon; Thalamus

The ability of neurites to grow through a lesion and form synaptic connections has been analyzed in a developing mammalian spinal cord in vitro. After isolation of the entire central nervous system (CNS) of the newly born South American opossum (Monodelphis domestica) the spinal cord was crushed. Outgrowth through and beyond the lesion was observed in living preparations for 2-5 days by staining axons with carbocyanine dyes. The structure of the acute crush and the growing neurites was examined by light and electron microscopy in tissue fixed immediately after the crush had been made. All axons had been severed and the site was filled with debris and amorphous vesicular structures. By 3 days after injury, numerous labelled neurites had grown into the lesion; by 4 days, many had extended several millimetres beyond it. At this time normal axonal profiles were apparent in electron micrographs of the crush site. Although fewer axons grew across the lesion than had been severed by the crush, the amplitudes of compound action potential volleys conducted across the crush in injured preparations were comparable with those recorded from uninjured spinal cords. Physiological experiments made with raised concentrations of extracellular magnesium in the culture fluid indicated that growing axons had formed synaptic connections. Thus, delayed major peaks of the response were abolished while the small component corresponding to through conduction remained unaffected by magnesium. These experiments demonstrate the development of synaptic interactions by the growing neurites and confirm the far greater powers of repair in neonatal mammals compared to adults. They set the stage for comparing molecular mechanisms involved in development and regeneration of the mammalian CNS.

Topics: Action Potentials; Animals; Animals, Newborn; Axons; Carbocyanines; Culture Techniques; Electrophysiology; Fluorescent Dyes; Magnesium; Microscopy, Electron; Nerve Crush; Opossums; Spinal Cord; Spinal Cord Injuries; Synapses

How we see the world largely depends on the organization of neuronal circuits in visual cortex. Physiological recordings in mammals indicate that circuits develop over a period that extends well into early postnatal ages (LeVay et al., 1980; Albus and Wolf, 1984). Our understanding of how these circuits are assembled during development is still fragmentary (Katz and Callaway, 1992). Here we describe the development of local connections within visual cortex, using the fluorescent dye 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate to trace axonal projections in post-mortem human brains. Vertical (intracolumnar) connections between layers 2/3 and 5, which link neurons representing the same point in the visual field, develop prenatally at 26-29 weeks gestation. In contrast, horizontal (intercolumnar) connections between different points in the visual field develop later. They first emerge prenatally at approximately 37 weeks gestation within layers 4B and 5. After birth (> 40 weeks gestation) the fiber density increases rapidly, showing a uniform plexus of connections at 7 weeks postnatal. The more adult-like patchiness of the projection, however, emerges after 8 weeks postnatal. Long-range horizontal connections within layer 2/3 develop after the connections within layers 4B, 5, and 6. These connections emerge after 16 weeks postnatal, long after cytochrome oxidase blobs have developed, and reach mature from sometime before 15 months of age. Unlike the patchy horizontal projections within layers 4B and 5, which seem to develop through a process of collateral elimination, long-range projections within layer 2/3 are patchy from the outset and seem to develop with greater topographical precision. The finding that intracolumnar connections develop before intercolumnar projections suggests that circuits that process local features of a visual scene develop before circuits necessary to integrate these features into a continuous and coherent neural representation of an image. In addition, the sequential development of horizontal connections within layer 4B before those within layer 2/3 suggests that circuits that may be related to the processing channel for visual motion develop in advance of those that may be more intimately related to the processing of form, color, and precise stereoscopic depth.

Topics: Aging; Carbocyanines; Child Development; Child, Preschool; Embryonic and Fetal Development; Fluorescent Dyes; Humans; Infant; Infant, Newborn; Visual Cortex; Visual Pathways

Hypoglossal motoneurons of 1-2 day-old newborn rats were retrogradely labelled following an injection of fluorescent latex microspheres and carbocyanines. Motoneurons were identified among the cell population of the hypoglossal nuclei during the dissociation and culture procedures. DiI labelled motoneurons could be maintained in culture on poly-L-lysine coating and Dulbecco Minimum Essential Medium with Ham F12 complement, supplemented with additives and 3% fetal calf serum. Neuronal survival as well as extension of neurites, identified by their content in DiI or by the presence of choline acetyltransferase immunoreactivity, was increased in the presence of myoblastic satellite cells originating from tongue muscular explants. Co-cultures of dissociated hypoglossal cells with tongue myoblasts revealed the presence, after 10-15 days in culture, of structures morphologically similar to neuromuscular junctions. Such re-innervated muscular fibres exhibited muscular contractions which were blocked by curare and augmented by glutamate applications, demonstrating the functionality of the observed re-innervations.

Topics: Animals; Animals, Newborn; Carbocyanines; Cell Survival; Cells, Cultured; Choline O-Acetyltransferase; Female; Histocytochemistry; Hypoglossal Nerve; Motor Neurons; Muscles; Neuromuscular Junction; Rats; Rats, Wistar; Tongue

Male canaries revise their vocal repertoire every year. Early work indicated that the volume and neuron number of the song-control nucleus HVC (Higher Vocal Center) declined in late-summer/fall as birds added and deleted syllables from their repertoire, and increased in spring as the set of song syllables stabilized to a fixed number. Seasonal variation in serum testosterone levels suggested that these changes in brain and behavior were regulated by testosterone (T). However, although initial studies describing growth and regression of HVC used Nissl-staining to define its borders, recent experiments that have measured the distribution of identified populations of HVC cells (projection neurons, hormone target cells) suggest that there are no seasonal changes in HVC volume or neuron number. In order to clarify the role of T in the regulation of HVC morphology, we castrated male canaries, maintained them on short (fall-like) days, and treated them with either T, antisteroid drugs, or nothing. After 1 month of treatment, we used a double-labeling technique to characterize HVC projection neurons and androgen target cells. The results showed that hormonal manipulation influenced HVC volume, the density and size of HVC cells, and the absolute number and percentage of androgen target cells in HVC. Hormonal manipulation did not influence the absolute number of cells in HVC. Moreover, the distribution of projection neurons, androgen target cells, and the Nissl-defined borders of HVC were closely aligned in all experimental groups, indicating that exposure to T and/or its metabolites (estradiol and dihydrotestosterone) regulates the overall size of HVC by affecting the distributions of both projection neurons and androgen target cells. Analysis of double-labeling results suggests that T specifically influences both cell size and the ability to accumulate androgen among HVC neurons that project to the robust nucleus of the archistriatum (RA). The results of this study show that steroid hormones exert potent effects on HVC morphology in male canaries, but differences between our results and studies of seasonal males suggest there may be additional factors that can regulate HVC morphology.

Topics: Animals; Birds; Brain; Carbocyanines; Fluorescent Dyes; Histocytochemistry; Hormones; Male; Microscopy, Fluorescence; Neurons; Orchiectomy; Seasons; Testosterone; Vocalization, Animal

The lateral diffusion of the fluorescent lipid analog 3,3'-dioctadecylindocarbocyanine iodide (DiI) was measured in the membranes of murine B lymphocytes treated with the B cell mitogen lipopolysaccharide (LPS). The mobility of DiI, as measured by fluorescence photobleaching recovery (FPR) techniques, was temperature-dependent with a value of 6.1.10(-9) cm2 s-1 at 37 degrees C. Untreated cells exhibited this diffusion coefficient over 72 h in culture. In contrast, DiI mobility decreased to 2.0.10(-9) cm2 s-1 at 37 degrees C in membranes of LPS-stimulated lymphocytes 24 h following LPS exposure. Interestingly, this decreased lipid lateral diffusion was not accompanied by any change in surface immunoglobulin lateral diffusion which remained essentially unchanged at 3.6-4.3.10(-11) cm2 s-1 over 72 h. To determine whether LPS effects on lipid lateral diffusion were due to insertion of LPS into the cell plasma membrane, we examined TRITC-LPS diffusion in B lymphocytes from LPS-responsive Balb/c and C3Heb/FeJ mice and from hypo-responsive C3H/HeJ mice. DiI and TRITC-LPS mobility decreased more than 50% in LPS-stimulated Balb/c and C3Heb/FeJ cells by 72 h. On C3H/HeJ lymphocytes, there was no change in DiI or TRITC-LPS lateral diffusion throughout the incubation period. These data indicate that B lymphocyte membrane composition is altered in LPS-activated lymphoblasts and that the decreased lateral diffusion of lipid probes does not result from membrane perturbation by LPS insertion into the lipid bilayer. Further, similarities between TRITC-LPS and DiI lateral diffusion suggest that most LPS molecules interact non-specifically with B cell membranes, presumably by acyl chain insertion of the lipid A moiety.

Topics: Animals; B-Lymphocytes; Carbocyanines; Cell Membrane; Female; Lipopolysaccharides; Lymphocyte Activation; Membrane Fluidity; Mice; Mice, Inbred BALB C; Mice, Inbred C3H; Rhodamines; Temperature

It is well established that neurons in the dorsal medulla, including the area postrema and the medial nucleus tractus solitarius (mNTS), are involved in the central actions of circulating peptides such as angiotensin II (ANG II) and arginine vasopressin (AVP). This report describes a preparation that permits the identification and maintenance of area postrema/mNTS neurons in culture in which the cellular and potentially subcellular responses to neurotransmitters and neuropeptides on area postrema/mNTS cells can be investigated. Following 15-21 days in culture, the effects of ANG II and AVP on changes in intracellular Ca2+ concentration ([Ca2+]i) were examined. Both ANG II and AVP resulted in a rapid and transient increase in [Ca2+]i reaching maximum in 15 s and returning towards baseline values within 180 s. The ANG II-mediated increase in [Ca2+]i was almost completely abolished by the selective angiotensin AT1 receptor subtype antagonist, losartan (DuP 753). These results suggest that ANG II and AVP modulate area postrema/mNTS neuronal activity by increasing intracellular Ca2+.

Topics: Angiotensin II; Animals; Arginine Vasopressin; Biphenyl Compounds; Calcium; Carbocyanines; Cells, Cultured; Cytosol; Fura-2; Image Processing, Computer-Assisted; Imidazoles; Immunohistochemistry; Losartan; Medulla Oblongata; Nerve Endings; Neuropeptides; Rats; Rats, Sprague-Dawley; Tetrazoles

Intramuscular injections of the retrograde tracers 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI) and horseradish peroxidase (HRP) were used to map the motor pools innervating axial muscles in the cervical and thoracic regions of the chicken. We found that motor pool position is well correlated with the muscle's embryonic origin, and not necessarily with its position. Muscles of myotomal (exclusively somitic) origin were innervated by medially positioned motoneurons in the median motor column, and the motor pools supplying these muscles were somatotopically organized. Muscles having a dual embryonic origin, from the somites and lateral plate, were innervated by motoneurons positioned further laterally within the median motor column. The relationship between motor pool position and embryonic origin of the muscle may be a general principle of ventral horn organization.

Topics: Aging; Animals; Carbocyanines; Chick Embryo; Chickens; Histocytochemistry; Horseradish Peroxidase; Motor Neurons; Muscles; Neck Muscles; Perfusion; Spinal Cord; Thorax

Research in cellular transplantation is frequently compromised by an inability to identify transplanted cells engrafting into orthotopic sites if they exhibit normal morphology and no unique antigenic markers. A method is described for using the fluorescent dye DiI as a marker for cell transplantation studies. This dye is not metabolized or exchanged between cells in vitro or in vivo and enables identification of engrafted cells by fluorescence microscopy, flow cytometry or fluorescence-activated cell sorting. Applications are described in autologous hepatocellular and thyroid follicular cell transplantation.

Topics: Animals; Carbocyanines; Cell Separation; Cell Transplantation; Flow Cytometry; Fluorescent Dyes; Liver; Sheep; Staining and Labeling; Thyroid Gland

Lipid microspheres have been used as carriers of drugs such as prostaglandin E1 (lipo-PGE1) and corticosteroid (liposteroid). Lipo-PGE1 is used for the treatment of chronic arterial occlusive diseases because its activity is far greater than that of free PGE1 in vivo. To verify the fact that the drug carriers, lipid microspheres, are preferentially taken up by endothelial cells, we labeled lipid microspheres with a fluorescent probe, DiI (DiI-LM), and observed them in some in vitro models. Stoichiometric fluorescence was obtainable, and the fluorescence was stable between pH 3.3 and pH 8.9. Human umbilical vein endothelial cells and cells of a human endothelial cell line, ECV304, showed increased uptake of DiI-LM, 81% and 61%, respectively. In contrast, uptakes were less than 7% in human skin fibroblasts, 3T3 cells, and human neutrophils. Prominent perinuclear fluorescence was also observed in endothelial cells by fluorescence microscopy. DiI-LM and flow cytometric analysis will be useful for studies to elucidate the precise mechanism of the selective accumulation of lipid microspheres by cells in blood vessel walls.

Topics: Carbocyanines; Cell Adhesion; Cell Line; Emulsions; Endothelium, Vascular; Fibroblasts; Flow Cytometry; Fluorescent Dyes; Humans; Lipid Metabolism; Microspheres; Neutrophils; Skin; Triglycerides

Unlike the afferent input into the spinal cord, it is generally believed that the motor efferent system in the mammalian spinal cord is more segmental in arrangement. Isolated comments in recent reports suggest that motor neurons in the mammalian spinal cord may be strictly segmental, contrary to many earlier reports using less sophisticated methods. We have attempted to address the question of the segmental arrangement of motor neurons in a mammalian spinal cord using single and double labelling with the fluorescent lipophilic dyes, Di I and Di A. These dyes have the ability to diffuse along and stain the plasma membrane of the neural elements in aldehyde-fixed specimens. Sprague-Dawley rat fetuses and early postnatal pups were used. The study was conducted on the thoracic spinal cord. The results confirm that thoracic spinal motor neurons are restricted to the segments from which their axons exit via the ventral roots in the intrauterine stages of development. The segmental arrangement of motor neurons in the thoracic spinal cord corresponded well with that of the sympathetic preganglionic neurons which were stained simultaneously.

Topics: Animals; Axons; Carbocyanines; Female; Fluorescent Dyes; Immunohistochemistry; Lipids; Motor Neurons; Pregnancy; Rats; Rats, Sprague-Dawley; Spinal Cord

We have used the anterograde axon tracer 1,1'-dioctodecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (Dil) to characterize the development of topographic order in the rat retinocollicular projection. Retinal axons were labeled by Dil injections covering 0.15-2% of peripheral temporal, nasal, superior, or inferior retina, or more central retina, in rats ranging in age from embryonic day 20 to postnatal day (P) 19. At P11-P12 and later, such injections label retinal axons that form overlapping arbors restricted to a topographically correct terminal zone covering about 1% of the superior colliculus (SC) area. At perinatal ages, though, axons labeled from each retinal site are distributed in the SC over much of its medial-lateral axis and extend caudally well beyond the rostral-caudal location of their correct terminal zone; some continue caudally into the inferior colliculus. Axons typically form side branches and often arborize at topographically incorrect positions throughout the SC; however, they appear to branch preferentially in a region that includes, but is much larger than, their correct terminal zone. The mature, retinotopically ordered projection emerges during an early postnatal remodeling period through the rapid remodeling of the early, diffuse projection. This process involves the large-scale removal of axons, axon segments, branches, and arbors from topographically inappropriate positions concurrently with a dramatic increase in branching and arborization at topographically correct locations. Quantitative measurements show that elimination of aberrant branches without loss of the primary axons contributes substantially to the development of order. By P6, fewer mistargeted axons persist, but those that do persist tend to branch or arborize more extensively in topographically inappropriate regions. By P8, the labeling patterns begin to approximate those seen at maturity. Further refinement leads to an adultlike topographic ordering of axonal arborizations by P11-P12. At maturity, some axons take very indirect routes to reach their correct terminal zone. However, such trajectory changes typically correct only small positional inaccuracies, indicating that axons and axon segments that make larger targeting errors do not survive the remodeling phase. Previous retrograde labeling studies indicate that some retinal axons make topographic targeting errors (O'Leary et al., 1986; Yhip and Kirby, 1990), but none have suggested the degree of diffus

Topics: Animals; Axons; Brain Mapping; Carbocyanines; Fluorescent Dyes; Inferior Colliculi; Nasal Cavity; Neuronal Plasticity; Rats; Rats, Inbred Strains; Retina; Retinal Ganglion Cells; Superior Colliculi; Synaptic Transmission

We measured the lateral diffusion of the fluorescent lipid analogue dioctadecylindocarbocyanine iodide (DiI) and of membrane glycoproteins labeled with tetramethylrhodamine (TRITC) succinyl concanavalin A (SConA) via fluorescence photobleaching recovery (FPR) at selected times during a temperature downshift experiment on transformation-defective temperature-sensitive (td-ts) Rous sarcoma virus (RSV) NY68-transformed chicken embryo fibroblasts (CEF) and on identically treated CEF and RSV-transformed CEF. There were no significant differences in the lateral diffusion in DiI at any of the times measured. The lateral diffusion of TRITC-SConA on the RSV-transformed CEF, (1.32 +/- 0.12).10(-10) cm2 s-1, was approximately two times faster than that observed in normal CEF, (0.61 +/- 0.06).10(-10) cm2 s-1. In the cells undergoing RSV NY68-mediated transformation, TRITC-SConA diffusion increased over a 24-h period from a value comparable to that observed in normal CEF, (0.72 +/- 0.13).10(-10) cm2 s-1 to a value comparable to the RSV-CEF transformed cells, (1.74 +/- 0.20).10(-10) cm2 s-1. All diffusion measurements reported were made at the permissive temperature for RSV-NY68 (35 degrees C) unless stated otherwise. The changes in the lateral diffusion of TRITC-SConA occurred between the fifth and twelfth hour of the downshift course and could be associated with cytoskeletal disruption and/or fibronectin degradation, both known to occur at this time in RSV-transformed cells. To assess the contribution of extracellular matrix (ECM) degradation, SConA mobility was measured in normal and RSV-transformed cells treated with trypsin. This treatment increased SConA mobility approximately 4-fold in the normal cells relative to untreated controls and only 2-fold in the RSV-CEF transformed cells. No significant difference in SConA mobility between trypsinized spherical normal and transformed cells was apparent.

Topics: Animals; Avian Sarcoma Viruses; Carbocyanines; Cell Transformation, Viral; Cells, Cultured; Chick Embryo; Concanavalin A; Diffusion; Fibroblasts; Kinetics; Membrane Glycoproteins; Membrane Lipids; Rhodamines; Temperature

The existence of a neural crest cell migration pathway from occipital levels of the hindbrain into the heart was suspected in mammalian embryos because it had previously been identified in avian embryos and because the Di George anomaly, an association between craniofacial and cardiac malformations, is most easily explained on the basis of abnormal neural crest cell migration to all of the affected structures. In order to demonstrate the existence of this pathway, neural crest cells were labelled in situ in rat embryos with the fluorescent dye DiI, and the embryos cultured for up to 48 h. Cells labelled between occipital somites 1 and 2 or 3 and 4 migrated within and dorsal to the third and fourth pharyngeal arches and into the outflow tract of the heart (conus cordis and truncus arteriosus). The cardiac labelling was in individually visible cells, in contrast to the mass of fluorescence seen in the pharyngeal and dorsal mesenchyme. Within the outflow tract wall, the labelled cells were enmeshed by strands of alcian blue-stained extracellular matrix. There was no labelling of cardiac cells following injections just rostral to, or just caudal to, somites one and four. This study establishes the existence and precise levels of origin of the 'cardiac' neural crest in a mammalian embryo.

Topics: Animals; Biomarkers; Carbocyanines; Carrier Proteins; Cell Movement; Chick Embryo; DiGeorge Syndrome; Fetal Heart; Fluorescent Dyes; Morphogenesis; Neural Crest; Organ Culture Techniques; Pharynx; Rats; Rats, Inbred Strains; Receptors, Retinoic Acid; Rhombencephalon; Species Specificity

Low density lipoproteins (LDL) bound to the surface of Schistosoma mansoni may protect the parasite from assault by the immune system and provide essential lipids for the parasite in human schistosomiasis. Here we have characterized the LDL binding sites on the surface of schistosomula by comparing the binding of fluorescently labeled LDL to the parasite with LDL binding proteins as seen by ligand blotting before and after enzymatic treatment of viable parasites. Ligand blotting revealed two LDL binding bands, 17.8 +/- 0.8 and 15.7 +/- 0.6 kDa, in intact schistosomula. Trypsinization eliminated all of the specific and approximately two-thirds of the total LDL binding capacity of schistosomula in a time and concentration-dependent manner. LDL did not bind to any bands on blots of trypsinized, viable worms. Specific LDL binding was also eliminated by phosphatidylinositol-specific phospholipase C (PIPLC). PIPLC treatment removed both LDL binding bands from the worms and caused the appearance of an LDL binding band, 16.6 +/- 0.3 kDa, in the culture medium. LDL binding to the parasite recovered within 24 to 48 h after trypsinization but the recovery was inhibited by either monensin or puromycin. Both LDL binding bands reappeared in ligand blots of cultured worms within 24 h; the reappearance was blocked by puromycin but not by monensin. These studies suggest that the specific binding of human LDL to schistosomula is mediated by GPI-linked low molecular weight proteins that are continually synthesized and transported to the parasite surface.

Topics: Affinity Labels; Animals; Carbocyanines; Carrier Proteins; Fluorescent Dyes; Glycolipids; Glycosylphosphatidylinositols; Helminth Proteins; Lipoproteins, LDL; Membrane Proteins; Peptide Hydrolases; Phosphatidylinositol Diacylglycerol-Lyase; Phosphatidylinositols; Phosphoinositide Phospholipase C; Phosphoric Diester Hydrolases; Schistosoma mansoni; Trypsin

We excised the dorsal part of the optic vesicle of chick embryos at stages 13-14 (embryonic day 2). The remainder of the optic vesicle reoganized and developed a small eye. This study was undertaken to determine how locus specificity is kept within the reorganized retina. The projection pattern from the reorganized eyes was examined with HRP and DiI, a fluorescence axon tracer. Whole retinal fibers labeled with HRP were found on the medial half of the contralateral tectum, thus showing the lateral half of the tectum as being devoid of retinal fibers. DiI labeling revealed a detailed projection map, with the dorsoposterior to ventroanterior axis of the reorganized eye being projected as the rostrocaudal axis on the medial half of the tectum. These results suggest that the positional specificity along the anteroposterior axis of the optic vesicle was already determined at the time of excision, and that the ventral part of the optic vesicle reorganized the "whole" retina, keeping its original anteroposterior polarity.

Topics: Animals; Carbocyanines; Chick Embryo; Eye; Histocytochemistry; Horseradish Peroxidase; Nerve Fibers; Ocular Physiological Phenomena; Retina; Superior Colliculi; Visual Pathways

We mapped sympathetic renal preganglionic neurons in the rabbit spinal cord using Herpes simplex virus retrograde transneuronal tracing after application of the virus to the renal nerve. Virus-positive neurons were found in the spinal cord from T7 to L2, principally in the ipsilateral intermediolateral cell column. Renal and adrenal preganglionic neurons are largely separate populations. Extensive nonspecific spread of virus from infected cells to neighboring neurons does not appear to occur.

Topics: Adrenal Glands; Animals; Carbocyanines; Ganglia, Sympathetic; Kidney; Male; Neuroglia; Neurons; Rabbits; Simplexvirus; Spinal Cord; Staining and Labeling

A myelin basic protein (MBP)-reactive TH cell line capable of inducing experimental allergic encephalomyelitis (EAE), and a MBP-reactive TH cell clone that does not cause EAE were labeled with a fluorescent vital dye, and transferred into naive syngeneic SJL/J mice. Animals were killed before the appearance of symptoms (3 and 4 days post-injection). Sections obtained from the spleen, spinal cord and brain of both groups of animals were examined by fluorescence microscopy to localize labeled TH cells. At all time points examined, the spleens of both groups contained innumerable labeled cells. The spinal cords and brains of animals that had received EAE-causing cells had a basal level of 20 labeled cells/cm2 at 3 days; this number increased rapidly to 150 cells/cm2 in the spinal cord at 4 days. Perivascular infiltrates and small foci of astrogliosis were already apparent in this group 3 days after injection. The spinal cords and brains of animals that had received the non-EAE-causing TH cells contained 50 labeled cells/cm2 at 3 days. The density of these transferred cells, as compared to that of the EAE-causing cells, suggested that they have an unaltered CNS-homing capability. However, by 4 days, the number of non-EAE-causing labeled cells had returned to near basal level. Our findings suggest that discrimination between disease and non-disease causing MBP-responsive TH cells occurs within the first 3 days following transfer, requires the presence in the CNS of a limited number of TH cells, and depends on yet unidentified TH cell factor(s).

Topics: Animals; Autoimmune Diseases; Carbocyanines; Cell Line; Central Nervous System; Encephalomyelitis, Autoimmune, Experimental; Fluorescent Dyes; Mice; Microscopy, Fluorescence; Myelin Basic Protein; Receptors, Lymphocyte Homing; Spleen; T-Lymphocytes

The bladder and other pelvic viscera are innervated in the rat by the major pelvic ganglion (MPG), a mixed sympathetic/parasympathetic population of neurons that participates in lower urinary pathophysiology. Neurons from the MPG of adult females were removed, dissociated and cultured in order to test retention of the neuronal phenotype and whether they responded to Nerve Growth Factor (NGF). The bladder-specific subset of MPG neurons were distinguished by retrograde labeling prior to culture. The adult ganglionic neurons adapted to culture with greater than 80% survival in the best cases. The cultured neurons retained excitability, as determined by measuring voltage-activated ionic currents. They were positive for neuron-specific beta-tubulin and many retained immunoreactivity for characteristic peptides and transmitter synthetic enzyme. The proportion of neurons in the different categories tested varied somewhat from that in vivo, but there was no evidence of selective death of a particular population. The cultured MPG neurons were responsive to NGF and anti-NGF antibody. NGF supported neuronal survival and expression of tyrosine hydroxylase. Added NGF also affected the expression of neuropeptide Y. Hypertrophied neurons from animals with experimental bladder outlet obstruction demonstrated increased responsiveness to NGF. The data suggest that NGF participates in adult neural plasticity due to continued responsiveness to the factor. Furthermore, questions concerning regulation of MPG neurons may be addressed in vitro.

Topics: Animals; Carbocyanines; Cell Survival; Cells, Cultured; Electrophysiology; Female; Ganglia, Sympathetic; Immunohistochemistry; Nerve Growth Factors; Neuronal Plasticity; Neurons; Neuropeptide Y; Phenotype; Rats; Rats, Wistar; Staining and Labeling; Urinary Bladder

Callosally projecting neurons in areas 17 and 18 of the adult cat can be classified into two types on the basis of their dendritic morphology: pyramidal and stellate cells. The latter are nearly exclusively of the spinous type and are predominantly located in upper layer IV. Retrograde transport of the carbocyanine dye DiI, applied to the corpus callosum, showed that, up to P6, all callosally projecting neurons resemble pyramids in the possession of an apical dendrite reaching layer I. At P10, however, callosally projecting neurons with stellate morphology were found. A study was designed to distinguish whether these neurons are late in extending their axons to the corpus callosum or, alternatively, have transient apical dendrites. To this end, callosally projecting neurons were retrogradely labeled by fluorescent beads injected in areas 17 and 18 at P1-P3 and then either relabeled with DiI applied to the corpus callosum at P10 or intracellularly injected with Lucifer Yellow at P57. Double-labeled stellate and pyramidal cells were found in similar proportions to those found for the total, single-labeled population of callosally projecting neurons. It is therefore concluded that callosally projecting spiny stellate cells initially possess an apical dendrite and a pyramidal morphology. At P6, i.e. close to the time when stellate cells appear, layer IV neurons with an atrophic apical dendrite were found, suggestive of an apical dendrite in the process of being eliminated.

Topics: Animals; Animals, Newborn; Carbocyanines; Cats; Corpus Callosum; Histocytochemistry; Horseradish Peroxidase; Microspheres; Neural Pathways; Neurons; Pyramidal Tracts; Visual Cortex; Wheat Germ Agglutinin-Horseradish Peroxidase Conjugate; Wheat Germ Agglutinins

An important goal in neuroanatomical research is to identify the neurotransmitters in specific neural pathways. One step towards this goal is to combine experimental neuronal tracing with immunocytochemistry. Unfortunately, optimal procedures for nerve tracing and immunocytochemistry are not always compatible. Carbocyanine compounds have recently been shown to be efficient tracers both in vivo and in paraformaldehyde-prefixed neural tissue. The possibility to apply them to prefixed tissue make them suitable for tracing of neural pathways that are not easily accessible in vivo. We have optimized the procedures for neural tracing with one carbocyanine compound, DiI (1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate), in the CNS of a teleost fish, and evaluated its compatibility with different immunocytochemical protocols. We have compared several immunocytochemical protocols, taking into account cryostat and vibratome sectioning, glutaraldehyde post-fixation to stabilize DiI, antibodies with different capacity for tissue penetration and the use of detergents, and antibodies with different sensitivity to prolonged paraformaldehyde fixation. We have also evaluated the choice of marker for immunoreactivity and compared indirect immunofluorescence techniques using different fluorophores, and the peroxidase-antiperoxidase (PAP) technique with or without nickel enhancement of the diaminobenzidine reaction product. It appears that DiI tracing of neural connections in the teleost CNS yields very consistent results and that the combination with immunocytochemistry is very reliable. We present four different basic protocols for combined DiI tracing and immunocytochemistry, with notes on their specific applicability. Owing to their reliability, the protocols may prove useful in comparative neuroanatomical studies of other vertebrates, particularly fish and amphibians, as well as in studies of developmental changes and neural plasticity in fish and amphibians.

Topics: Animals; Carbocyanines; Female; Fluorescent Antibody Technique; Immunoenzyme Techniques; Immunohistochemistry; Male; Nervous System; Neurotransmitter Agents; Pineal Gland; Retina; Rhodamines; Salmon; Staining and Labeling; Superior Colliculi; Tyrosine 3-Monooxygenase

Muscle and cutaneous nerves were individually labeled with DiI in chick embryos to examine the development of sensory afferent arborizations in the spinal cord. Initially, cutaneous and muscle arbors were similar; both types first entered the spinal gray matter at stage 28-29 (embryonic day (E) 6). Differences in projections were first observed by late stage 34 (E8.5): muscle afferent collaterals extended almost unbranched to the level of motoneuronal dendrites while cutaneous afferents branched frequently and remained within the dorsal horn. Projections of putative small caliber axons into laminae 1 and 2, located laterally in the chick, did not develop until E13-14.

Topics: Afferent Pathways; Animals; Axonal Transport; Carbocyanines; Chick Embryo; Fluorescent Dyes; Ganglia, Spinal; Muscles; Neurons; Proprioception; Skin; Spinal Cord

The cardiac neural crest is located in a transitional area on the neuraxis between trunk and cephalic regions and gives rise to both the dorsolateral and ventrolateral crest cell populations. Around stage 18 of chick development, a mass of E/C8+ cells surrounds the postotic pharyngeal arches and forms a crescent-shaped arch, termed the circumpharyngeal ridge. Using immunohistochemistry and quail-chick chimeras, it was determined that the E/C8+ cell mass located in the circumpharyngeal ridge derives from the dorsolateral component of the cardiac neural crest. The ventrolateral cell population of the cardiac crest is located more medially and shows long-persistent HNK-1 immunoreactivity dorsolateral to the foregut. The crest cells that populate the gut arise from the caudal portion of the circumpharyngeal crest and are always located caudal to the caudal-most pharyngeal ectomesenchyme. Circumpharyngeal crest cells continuously populate the pharyngeal arch ectomesenchyme and enteric nervous system on the lateral side of the foregut wall, as well as the hypoglossal pathway which develops within the ventral portion of the circumpharyngeal ridge. E/C8 and HNK-1 immunoreactivity are associated with the cells migrating via the dorsolateral (circumpharyngeal) and ventrolateral pathways, respectively, with one exception: there is a population of putative crest cells along the proximal course of the vagal intestinal branch that shows both immunoreactivities around stage 20. DiI labeling of the cells in the circumpharyngeal ridge suggests that the cells are contributed from the circumpharyngeal ridge to this population. Thus, the distribution of the circumpharyngeal crest cells and their derivatives coincides with the peripheral branch distribution of the cranial nerves IX, X, and XII, whose development is selectively affected in the absence of the cardiac neural crest, the source of the circumpharyngeal crest.

Topics: Animals; Carbocyanines; Cell Line; Cell Movement; Chick Embryo; Chimera; Coturnix; Embryonic and Fetal Development; Fluorescent Dyes; Head; Heart Conduction System; Immunohistochemistry; Neural Crest; Pharynx; Staining and Labeling

Topographic order in the rat retinocollicular projection emerges from an initially diffuse projection during an early postnatal remodeling period that is coincident with the period of naturally occurring ganglion cell death. Here, we examine the relationship between a retinal axon's position along the medial-lateral axis as it enters the superior colliculus (SC) and its ability to form an appropriately positioned arbor and survive the remodeling period. At E18-E19, prior to map remodeling, axons labeled with focal DiI injections in the periphery of temporal, nasal, superior or inferior retina are widespread along the medial-lateral SC axis. At P12, after remodeling, the distributions of axons remain widespread over the medial-lateral SC axis relative to the positioning of their terminal arborizations, and resemble the distributions labeled at E18-E19, with the exception that the small proportion of axons most widely mispositioned along the medial-lateral SC axis are less frequent. These data indicate that the most widely mispositioned retinal axons are preferentially eliminated, but that a high proportion of retinal axons mispositioned along the medial-lateral axis as they enter the SC can correct their position, form topographically appropriate arbors, and survive the remodeling period.

Topics: Aging; Animals; Axonal Transport; Axons; Carbocyanines; Fluorescent Dyes; Gestational Age; Rats; Rats, Sprague-Dawley; Retina; Superior Colliculi

Polyacrylamide is widely used for the separation of proteins by electrophoresis. Here the usage of polyacrylamide as an infiltrating and embedding medium for vibratome sectioning of human fetal cerebellum containing DiI-filled axons is reported. The procedure is simple to implement and has several advantages: it does not require prior dehydration and clearing of tissue; it can be carried out at a low temperature of 4 degrees C; and the polyacrylamide neither dissolves the DiI nor does it interfere with the visualizing of DiI-filled profiles under fluorescence microscope.

Topics: Acrylamides; Carbocyanines; Cerebellum; Female; Fetus; Histocytochemistry; Horseradish Peroxidase; Humans; Microtomy; Pregnancy; Tissue Embedding

We show that rat retinal ganglion cell axons exhibit no topographic specificity in growth along the rostral-caudal axis of the embryonic superior colliculus (SC). Position-related, morphological differences are not found between temporal and nasal axon growth cones. However, embryonic retinal axons respond in vitro to a position-dependent molecular property of SC membranes. In vivo, regional specificity in side branching is the earliest indication that axons make topographic distinctions along the rostral-caudal SC axis. Our contrasting in vivo and in vitro results indicate that molecules encoding rostral-caudal position in the SC neither guide nor restrict retinal axon growth, but may promote the development of topographic connections by controlling specificity in the extension or stabilization of branches.

Topics: Animals; Axons; Carbocyanines; Cell Membrane; Fluorescent Dyes; Gestational Age; Neurites; Rats; Rats, Sprague-Dawley; Retina; Retinal Ganglion Cells; Superior Colliculi

The trajectory, developmental time course, and origin of callosal fibres that recross through the anterior commissure were studied in developing hamsters, using carbocyanines in fixed brains on different ages. The bicommissural fibres were found in hamsters from E15 through P7, but disappeared after P7. By double labelling it was found that the neurones of origin of these bicommissural fibres were located in the lateral cortex within the region where the callosal zone of origin overlaps that of the anterior commissure. From these experiments, it was concluded that the axons of a group of cells in the lateral cortex of developing rodents are branched and grow transiently through both the callosum and the anterior commissure.

Topics: Animals; Carbocyanines; Cerebral Cortex; Corpus Callosum; Cricetinae; Female; Nerve Fibers; Neural Pathways; Neurons; Pregnancy

Clustered intrinsic connections in the kitten striate cortex originate from an unclustered, diffusely organized pattern prevailing during the first postnatal week. We have studied the progress of this reorganization and its dependence on visual input by determining the topographies of the intrinsic tangential connections at various postnatal ages by means of axonal tracing methods. Neurons were labeled either by diffusion of the carbocyanin dye DiI in animals ranging in age between 1 day and 30 days, or by retrograde transport of fluorescent microspheres in animals ranging in age between 7 days and 11 months. Quantitative evaluation of retrogradely labeled neurons revealed that during the first postnatal week, intrinsic tangential connections are organized in an unclustered fashion. During the second postnatal week a rapid rearrangement of connections occurs and is complete around postnatal day 11. The main events taking place during the course of this rearrangement are a decrease in the density of tangential connections and an arrangement of them in a clustered fashion. Once the clusters have been formed, the periodicity of the clustered pattern of connections and the size and distinctness of the clusters do not change. This means that the system of clustered tangential connections is adult-like at the end of the second postnatal week. Dark rearing affects neither the rapid rearrangement of horizontal connections into an adult-like system of clusters, nor the integrity of this clustered topography until the end of the first postnatal month. The overall distribution and the lateral extent of the tangential connections remain about the same during the postnatal period and are not affected by dark rearing until the end of the first postnatal month. We conclude that the clustered system of tangential connections in the cat's striate cortex is determined innately.

Topics: Aging; Animals; Behavior, Animal; Carbocyanines; Cats; Darkness; Histocytochemistry; Microspheres; Neural Pathways; Visual Cortex; Visual Pathways

Judgment of abnormalities in fetal cortical axon development is more sensitive when a good standard of normal ontogeny is established. The recent availability of postmortem tract tracing methods has greatly improved the observation of axon extension and growth cone morphology in mouse fetuses, which allows much stronger statements about the timing of crucial steps in the formation of the corpus callosum in particular. The first outgrowth and crossing of midplane by axons of the corpus callosum (CC) were examined in 153 normal mouse embryos and fetuses of the hybrid cross B6D2F2/J with carbocyanine dyes applied to brains fixed by perfusion. In most brains a crystal of DiI was inserted into either frontal, parietal, temporal, or occipital cortex in one hemisphere, and a crystal of DiA was placed into a different site in the opposite hemisphere. Although dye diffusion obscured the emergence of axons, linear regression analysis revealed that the first callosal axons emerged from their cortical cells of origin at about 0.4 g body weight or 15.5 days after conception for all four sites. Subsequent axon growth rate was substantially faster for those from frontal cortex (3.2 mm/day) than occipital cortex (1.8 mm/day). Axons from frontal cortex crossed the cerebral midplane first (0.69 g, E16.3), followed by those from parietal (0.74 g), temporal (0.77 g) and occipital cortex (0.92 g, E16.9). Prior to crossing midplane, the pioneering CC axons were usually 200 microns or less in advance of the main bundle, but when they crossed midplane and encountered CC axons growing from homotopic sites in the opposite hemisphere, the pioneering axons were often 0.5 to 2.5 mm ahead of the main bundle. Growth cones were usually large and complex until they had crossed midplane and were thereafter smaller with simple and flat morphologies. The topography of axons in the CC at midplane was organized according to cortical region of origin from the very beginning, when the CC was only a small cap over the hippocampal commissure and dorsal septum. The quantitative results provide a convenient standard for normal callosal development in mice and should facilitate comparative studies.

Topics: Animals; Axons; Body Weight; Carbocyanines; Coloring Agents; Corpus Callosum; Female; Gestational Age; Histocytochemistry; Male; Mice; Mice, Inbred C57BL; Mice, Inbred DBA; Mice, Inbred Strains; Pregnancy; Regression Analysis

In the present study the early phases of the development of the inferior olive were examined by using immunocytochemical techniques. We observed that, from embryonic day 16 onward, antibodies against the calcium binding proteins parvalbumin and calbindin and the calcitonin gene related peptide stain partially overlapping territories of the inferior olive. This staining delimits a biochemical zonation of the inferior olive which is combinatory and transient. We have previously observed a biochemical parcellation of the cerebellar Purkinje cells which, like that of the inferior olive, is first observed at E16, involves the combined expression of marker proteins and is also transient. In order to know whether the biochemical compartmentations of the cerebellum and inferior olive arise independently, the time course of the development of the olivocerebellar projection was studied by anterograde and retrograde in vitro axonal tracing by using the fluorescent carbocyanine dye DiI. The olivocerebellar axons were found to reach the limit of the cerebellar plate at E16 and to enter it at E17. Even at this age the great majority of the climbing fibers are tightly fasciculated, which minimizes their interactions with the PC clusters. These observations indicate that the topographical heterogeneity of Purkinje cells and inferior olive neurons arise independently. The transient biochemical individualization of subgroups of neurons during development could contribute to recognition mechanisms.

Topics: Animals; Biomarkers; Calbindins; Calcitonin Gene-Related Peptide; Carbocyanines; Cerebellum; Female; Immunohistochemistry; Neural Pathways; Olivary Nucleus; Parvalbumins; Pregnancy; Purkinje Cells; Rats; Rats, Wistar; S100 Calcium Binding Protein G

A fluorescence method is described for the evaluation of cell death induced by cellular cytolytic activity. A green fluorescent membrane dye, D275, was used to label various target cell lines and propidium iodide (PI) uptake was used to assay cell death. Natural killer (NK), lymphokine activated killer (LAK) as well as targeted T cell mediated cellular cytotoxicity were quantified using the fluorescence method and compared to results obtained with the 51chromium (51Cr) release assay. A good correlation was found after an assay period of 4-8 h indicating that the fluorescence method is a reliable alternative to the 51Cr release assay.

Topics: Carbocyanines; Cell Death; Chromium Radioisotopes; Cytotoxicity, Immunologic; Fluoroimmunoassay; Humans; Immunity, Cellular; In Vitro Techniques; Killer Cells, Lymphokine-Activated; Killer Cells, Natural

Retinal projections were experimentally manipulated in a bony fish to reveal conditions under which considerably enlarged ipsilateral projections developed and persisted. Three experimental groups were studied: animals after unilateral enucleation, after unilateral nerve crush, and after enucleation and crush of the remaining optic nerve. At 29 days after unilateral enucleation alone, no enhanced ipsilateral projection had developed. After nerve crush, however, large numbers of retinal fibers regenerated into the ipsilateral tectum. Retrogradely filled, ipsilaterally projecting ganglion cells were distributed throughout the entire retina. After 15 days regenerating retinal fibers covered the entire ipsilateral tectum. At later stages the ipsilateral projection showed progressive reduction in coverage of the tectum. Combining enucleation with nerve crush led to an ipsilateral projection that covered the tectum at 28 days and later. In this experimental situation the development of an ipsilateral projection appears to be a two-step process: (1) Fibers are rerouted to the ipsilateral side at the diencephalon, and (2) ipsilateral fibers persist in the tectum only in the absence of a contralateral projection while they appear to be eliminated in the other cases.

Topics: Animals; Carbocyanines; Eye Enucleation; Histocytochemistry; Horseradish Peroxidase; Nerve Crush; Nerve Fibers; Nerve Regeneration; Optic Nerve; Perches; Retina; Retinal Ganglion Cells; Superior Colliculi; Visual Pathways

The normal development of the retinal projection was studied in a bony fish with Dil. Between 5.5 and 10 days postfertilization the contralateral retinal projection grows from the rostral pole of the tectum across its center. A maximum of 15 retinal fibers reaches the ipsilateral tectum. In 33-day-old juvenile animals, less than 15 ipsilateral fibers terminate in the entire tectum. Ipsilaterally projecting ganglion cells (maximal number = 20 cells) are scattered throughout the entire retina, and the location of ganglion cells in the retina and axonal terminations in the tectum display a large interindividual variability. This suggests that the small adult contingent of ipsilateral fibers in this bony fish develops without an initial exuberant ipsilateral retinal projection that is later pruned back.

Topics: Animals; Axons; Carbocyanines; Histocytochemistry; Horseradish Peroxidase; Larva; Optic Nerve; Perches; Retina; Retinal Ganglion Cells; Superior Colliculi; Visual Pathways

Development of the facial nerve was studied in normal chicken embryos and after surgical disruption of ingrowing sensory facial nerve fibers at 38-72 h of incubation. Disruption of facial nerve fibers by otocyst removal often induced a rostral deviation of the facial nerve and ganglion to the level of the trigeminal ganglion. Cell bodies of the geniculate ganglion trailed their deviating neurites and occupied an abnormal rostral position adjacent to the trigeminal ganglion. Deviating facial nerve fibers were labeled with the carbocyanine fluorescent tracer DiI in fixed tissue. Labeled fibers penetrated the cranium adjacent to the trigeminal ganglion, but they did not follow the trigeminal nerve fibers into the brain stem. Rather, after entering the cranium, they projected caudally to their usual site of entrance and proceeded towards their normal targets. This rostral deviation of the facial nerve was observed only after surgery at 48-72 h of incubation, but not in cases with early otocyst removal (38-48 h). A rostral deviation of the facial nerve was seen in cases with partial otocyst removal when the vestibular nerve was absent. The facial nerve followed its normal course when the vestibular nerve persisted. We conclude that disruption of the developing facial pathway altered the routes of navigating axons, but did not prevent pathfinding and innervation of the normal targets. Pathfinding abilities may not be restricted to pioneering axons of the facial nerve; later-developing facial nerve fibers also appeared to have positional information. Our findings are consistent with the hypothesis that navigating axons may respond to multiple guidance cues during development. These cues appear to differ as a function of position of the navigating axon.

Topics: Acetylcholinesterase; Animals; Carbocyanines; Chick Embryo; Facial Nerve; Geniculate Ganglion; Histocytochemistry; Motor Neurons; Nerve Fibers; Neural Crest; Neural Pathways; Trigeminal Ganglion; Vestibular Nerve

In vivo, kinase C phosphorylation of the growth-associated protein GAP-43 is spatially and temporally associated with the proximity of growing axons to their targets. Here we have used dissociated dorsal root ganglia (DRG)s and an antibody specific for the phosphorylated form of GAP-43 to demonstrate that neurite regeneration in culture also begins in the absence of detectable levels of phosphorylated GAP-43. Since the beta isoform of kinase C was found to be enriched in growth cones before stably phosphorylated GAP-43 was detected, it may normally be inactive during initial neurite outgrowth; however, premature phosphorylation of GAP-43 could be stimulated in newly dissociated DRGs by plating them on cultures in which phosphorylation had already been initiated media conditioned by such cultures caused no response suggesting an effect of either cell-cell or cell-substrate contact. Increased GAP-43 phosphorylation correlated with a reduced extent of neurite outgrowth but not with the rate at which individual growth cones translocated so that motile growth cones contained very low levels of phosphorylated GAP-43, whereas stationary growth cones showed much more immunoreactivity. Downregulation of kinase C by phorbol ester prevented increased GAP-43 phosphorylation and led to growth cone collapse. Finally, phosphorylated GAP-43 was found to be differently distributed within growth cones. Increased immunoreactivity was frequently observed in the neck of the growth cone and was heterogeneously distributed in lamellae and filopodia. These results, which demonstrate the dynamic regulation of GAP-43 phosphorylation in individual growth cones, are discussed with reference to the association between changes in growth cone shape and the ability to translocate and change direction.

Topics: Animals; Carbocyanines; Cell Division; Cells, Cultured; Down-Regulation; Female; Fluorescent Antibody Technique; Ganglia, Spinal; GAP-43 Protein; Histocytochemistry; Isoenzymes; Membrane Glycoproteins; Nerve Tissue Proteins; Phorbol 12,13-Dibutyrate; Phosphorylation; Pregnancy; Protein Kinase C; Rats

Nucleus rostrolateralis, a newly identified nucleus, has been found to date in only three species of ray-finned fishes, two of which are osteoglossomorphs. It is relatively large and well developed in only one of the osteoglossomorphs, Pantodon buchholzi, in which it receives a relatively sparse, primarily contralateral, input from the retina. The present report describes a relatively intense, bilateral projection from the optic tectum to nucleus rostrolateralis.

Topics: Animals; Carbocyanines; Diencephalon; Fishes; Histocytochemistry; Horseradish Peroxidase; Neural Pathways; Superior Colliculi

Little is known about the development of the various populations of interneurons in the mammalian spinal cord. We have utilized the lipid-soluble tracer DiI in fixed tissue to study the migration and dendritic arborization of spinal neurons with axons in the ventral commissure in embryonic rats. Crystals of DiI were placed in various locations in the thoracic spinal cord in order to label commissural neurons within the dorsal horn, intermediate zone, and ventral horn at E13.5, E15, E17, and E19. Seven different groups of commissural interneurons are present in the spinal cord by E13.5. Migration is relatively simple with groups occupying a position along the dorsoventral axis roughly corresponding to their position of origin along the neuroepithelium. By E15, commissural cells are near their final locations and exhibit characteristic morphology. One striking feature is the tendency of cells with similar morphology to cluster in distinct groups. By E19, at least 18 different types of commissural interneurons can be identified on morphological grounds. Although the situation is complex, some generalities about dendritic morphology are apparent. Commissural neurons located in the dorsal horn are small and have highly branched dendrites oriented along the dorsoventral axis. In more ventral regions, commissural neurons are larger and possess dendritic arbors oriented obliquely or parallel to the mediolateral axis with long dendrites extending toward the lateral and ventral funiculi. The number of primary dendrites of most groups is set by E15 and dendritic growth occurs in the transverse plane by lengthening and branching of these primary processes. This study demonstrates that a large number of classes of commissural interneurons can be recognized on the basis of characteristic morphologies and locations within the dorsal horn, intermediate zone and ventral horn of the embryonic rat spinal cord. This finding is consistent with the fact that commissural neurons project to many different targets and mediate a variety of different functions. The demonstration that dendritic arbors of spinal interneurons with characteristic morphologies can be conveniently labelled with DiI should prove useful in future studies on the development of specific circuits in the mammalian spinal cord.

Topics: Animals; Axons; Carbocyanines; Dendrites; Female; Histocytochemistry; Interneurons; Neurons; Pregnancy; Rats; Rats, Sprague-Dawley; Spinal Cord

A serial forebrain pathway in the songbird brain plays a critical role in vocal learning; Area X of the parolfactory lobe (X) projects to the medial portion of the dorsolateral nucleus of the anterior thalamus (DLM), which in turn projects to the lateral magnocellular nucleus of the anterior neostriatum (IMAN). Lesions of this pathway in juvenile birds disrupt vocal development, whereas identical lesions in adult birds do not influence the production of already learned song. During the course of vocal learning, IMAN undergoes a phase of massive neuronal loss, whereas the neuronal population of X more than doubles. In the present study, the development of neuron number in DLM was analyzed and found not to change during the course of vocal learning. Anterograde DiI labeling of DLM efferent fibers was then used to analyze the morphological development of this projection in relation to both the loss of neurons from lMAN and the loss of the ability of X-DLM-lMAN lesions to influence vocal production. We found that DLM axons arrive within lMAN by 15 days of age, prior to both the loss of neurons from lMAN and the onset of vocal production. The volume of anterograde DiI label over lMAN did not change between 15 and 20 days of age, but this volume more than doubled between 20 and 35 days of age. During this phase of exuberant growth, anterograde label matched the dorsal border of lMAN but extended beyond all other borders of lMAN into a surrounding "shell" of parvicellular neurons.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Afferent Pathways; Aging; Animals; Birds; Carbocyanines; Cell Count; Efferent Pathways; Fluorescent Dyes; Male; Neurons; Thalamic Nuclei; Vocalization, Animal

The purpose of our study was twofold: (1) to trace the development of the olfactory axons from early embryonic stages until the mature pattern of connectivity and (2) to determine whether a transient penetration of them exists beyond the olfactory glomeruli. Two techniques were employed: DiI applied in the olfactory epithelium after aldehyde fixation, and olfactory marker protein (OMP) immunostaining. At E13 and E14 olfactory axons were observed spreading over the telencephalic vesicle and entering deeply into the prospective olfactory bulb, extending near the ventricular zone. Growth cones were seen at the end of these axons. At E15, the bundles of olfactory axons form a network, in which axons, growth cones and cells were seen. Some of these axons entered the olfactory bulb. Using OMP immunostaining olfactory axons were observed along the external plexiform layer, the mitral cell layer and in the granular layer from E19 to P6. At P9 some OMP immunoreactive axons were observed in the external plexiform layer. No OMP immunostained axons could be observed outside the glomeruli at P10. Our conclusions are that a transient immature pattern of early invasion over the telencephalic vesicle and of the olfactory bulb by olfactory axons occurs in the olfactory system. By the second postnatal week the glomerular layer reaches its mature configuration, and no olfactory fibers are seen outside the glomerular layer.

Topics: Animals; Animals, Newborn; Axons; Carbocyanines; Nerve Tissue Proteins; Olfactory Bulb; Olfactory Marker Protein; Olfactory Pathways; Rats; Rats, Wistar

To investigate the development of corpus callosal connectivity in the hamster sensorimotor cortex, we have used the sensitive axonal tracer 1,1 dioctadecyl-3,3,3',3', tetramethylindocarbocyanine perchlorate (DiI), which was injected either in vivo or in fixed brains of animals 3-6 days postnatal. First, to study changes in the overall distribution of developing callosal afferents we made large injections of DiI into the corpus callosal tract. We found that the anterogradely labeled callosal axons formed a patchy distribution in the contralateral sensorimotor cortex, which was similar to the pattern of adult connectivity described in earlier studies of the rodent corpus callosum. This result stands in contrast to previous retrograde studies of developing callosal connectivity which showed that the distribution of callosal neurons early in development is homogeneous and that the mature, patchy distribution arises later, primarily as a result of the retraction of exuberant axons. The initial patchy distribution of callosal axon growth into the sensorimotor cortex described in the present study suggests that exuberant axons destined to be eliminated do not enter the cortex. In addition, small injections of DiI into developing cortex resulted in homotopic patterns of callosal topography in which reciprocal regions of sensorimotor cortex are connected, as has been shown in the adult. Second, to study the radial growth of callosal afferents we followed the extension of individual callosal axons into the developing cortex. We found that callosal axons began to invade the contralateral cortex on about postnatal day 3, with little or no waiting period in the callosal tract. Callosal afferents then advanced steadily through the cortex, never actually invading the cortical plate but extending into layers on the first day that they could be distinguished from the cortical plate. The majority of callosal axons grew radially through the cortex and did not exhibit substantial branching until postnatal day 8, the age when the cortical plate disappears and callosal afferents reach the outer layer of cortex. This mode of radial growth through cortex prior to axon branching could serve to align callosal afferents with their radial or columnar targets before arborizing laterally.

Topics: Anesthesia; Animals; Animals, Newborn; Axons; Carbocyanines; Corpus Callosum; Cricetinae; Mesocricetus; Motor Cortex; Neural Pathways; Neurons, Afferent; Somatosensory Cortex

In the mammalian optic chiasm retinal axons from each eye divide into two populations, those that decussate and those that remain uncrossed. In eutherian (placental) mammals, the separation of these pathways is not reflected in the structure of the chiasm. The two populations from each eye are mixed through each hemichiasm, segregating only at the midline, where the uncrossed projection turns back. In this study the optic chiasm of a marsupial, the wallaby, Setonix brachyurus (quokka) has been investigated with staining and neuronal tracing techniques. The chiasm of this mammal is quite different from that of eutherian mammals. In coronal section it can be morphologically subdivided into three regions, a central body in which fasciculated groups of axons from each eye interdigitate across the midline, and two distinct lateral regions, one on each side, which contain the uncrossed retinal projections. In the rostral chiasm the lateral regions are separated from the main body of the chiasm by vertically oriented fibre-free regions. Caudally, the lateral regions increase in size and become less distinct as increasing numbers of contralaterally projecting axons that have crossed the midline project into them. However, the two populations remain predominantly segregated in this region. As the lateral regions develop, the central body of the chiasm becomes thinner and finally detaches at the midline to form the two optic tracts. The routes taken by retinal axons through the eutherian and marsupial chiasm appear to be fundamentally different. Therefore, the developmental factors that determine the laterality of retinal projections are likely to show significant differences in the two mammalian groups.

Topics: Animals; Carbocyanines; Fluorescent Dyes; Horseradish Peroxidase; Injections; Macropodidae; Nerve Fibers; Neural Pathways; Optic Chiasm; Proline; Retina

Much research on the activity and half-life of endothelium-derived substances has entailed the removal of endothelium from arteries by mechanical or enzymatic processes. It has been observed that the technique used for the removal of arterial endothelium may profoundly affect smooth muscle function and release of prostanoids by the vessel wall. The function and patterns of regeneration of arterial endothelium have been extensively described, but there is a relative paucity of information about the venous endothelium, due in part to the difficulty of its removal. We developed a technique for removal of the endothelium of rabbit femoral veins by passing a stream of air through the lumen of the vessel to dry and remove the endothelium. The effectiveness of endothelium removal was verified by the lack of in vitro reactivity to endothelium-dependent relaxing substances, examination of frozen sections of vessels, labeled with fluorescent-tagged acetylated low-density lipoprotein, with fluorescent light microscopy and scanning electron microscopy of vessel segments. Air drying effectively removed the endothelium and abolished mechanical responses to endothelium-dependent vasodilators but did not affect the function of the smooth muscle. We propose the use of air to remove endothelium from veins to be used to study endothelium-derived factors since this method achieves complete removal of endothelium without causing detectable damage (morphological or functional) to the remainder of the vessel wall.

Topics: Air; Animals; Carbocyanines; Endothelium, Vascular; Femoral Vein; Fluorescent Dyes; In Vitro Techniques; Microscopy, Electron, Scanning; Microscopy, Fluorescence; Rabbits; Vasoconstriction

We have used the carbocyanine fluorochrome, DiI, to trace living glial cells (astrocytes, oligodendrocytes and immortalized Schwann cells) after their transplantation into the newborn shiverer and normal mouse brain. DiI fluorescence first detected on vibratome sections, was photoconverted into a stable, non-diffusible and electron-dense diaminobenzidine product. Both fluorescence and precipitate were found in the same cells and were detectable until 60 days after transplantation. At the ultrastructural level, DiI precipitate was contained within cytoplasmic vesicles scattered in the transplanted cell bodies and processes. Photoconversion did not interfere with the cell fine structure or predicted post-transplantation behavior. DiI is thus a suitable marker to trace, at the ultrastructural level, living cells after their transplantation.

Topics: Animals; Animals, Newborn; Brain Tissue Transplantation; Carbocyanines; Cytoplasm; Mice; Mice, Inbred C3H; Mice, Neurologic Mutants; Neuroglia; Plastic Embedding

We have studied the role of electrical activity in the elimination of axonal targeting errors, which is a normal process in brain development. The experiments were focused on the isthmo-optic nucleus (ION), which, in adults, projects in topographical order on the contralateral retina. During embryogenesis, however, a few isthmo-optic neurons project to the ipsilateral retina, and many project to topographically inappropriate parts of the contralateral one; both kinds of targeting error are known to be eliminated by the deaths of the parent neurons. We injected tetrodotoxin (TTX) intraocularly at embryonic days 13 and 15 and, on the latter, applied a retrograde label to the retina of the same eye. Embryos were fixed at embryonic day 17. In some embryos, the label was a peripherally placed fleck of the carbocyanine dye "diI"; the resulting retrogradely labeled neurons in the contralateral ION were much more widely scattered in the TTX-injected embryos than in controls (errors in topography). In other embryos, the label was a solution of rhodamine-B-isothiocyanate (RITC) injected into the vitreous body; this yielded several ipsilaterally labeled isthmo-optic neurons in the TTX-injected embryos, but virtually none in the controls. The numbers of both kinds of aberrantly projecting neuron approached those previously reported near the beginning of the ION's period of neuronal death. We conclude that electrical activity plays an important role in the elimination of axonal targeting errors in the chick embryo's isthmo-optic system.

Topics: Animals; Axons; Carbocyanines; Chick Embryo; Eye; Fluorescent Dyes; Injections; Microscopy, Fluorescence; Neural Pathways; Optic Nerve; Tetrodotoxin

In the primate ovary, luteal steroidogenesis is largely dependent upon cholesterol derived from receptor-mediated uptake of circulating low-density lipoprotein (LDL). However, granulosa cells (GC) of preovulatory follicles possess few LDL binding sites compared to those present in developing and mature corpora lutea. We recently reported (Endocrinology 1991; 129:3247-3253) that uptake of LDL tagged with the fluorescent probe 1,1'-dioctadecyl-3,3,3',3'-tetramethyl-indocarbocyanine perchlorate (DiI) can be monitored in macaque luteal cells by fluorescence-activated flow cytometry. This study was designed to determine whether an ovulatory stimulus induced uptake of DiI-LDL in GC aspirated from preovulatory follicles of macaques undergoing ovarian stimulation. Development of multiple large follicles was stimulated in adult rhesus macaques with human gonadotropin treatment for 9 days. On Day 10, monkeys received either no ovulatory stimulus or 1000 IU hCG to initiate ovulatory events. GC were aspirated on Day 10 in monkeys receiving no ovulatory stimulus (nonluteinized GC) or 27 h or 34 h after hCG injection (luteinizing GC). GC were resuspended in Ham's F-10 medium + 0.1% BSA and incubated with several concentrations (0-25 micrograms/ml) of DiI-LDL (Biomedical Technologies, Stoughton, MA) for various time intervals (2-60 min). DiI-LDL uptake by GC was time- and concentration-dependent. Coincubation of cells with DiI-LDL and unlabeled LDL dose-dependently suppressed the percentage of fluorescent cells. In contrast, coincubation with up to a 250-fold excess of acetylated LDL or high-density lipoprotein did not alter the percentage of fluorescent GC.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Animals; Carbocyanines; Chorionic Gonadotropin; Estradiol; Female; Flow Cytometry; Fluorescent Dyes; Granulosa Cells; Lipoproteins, LDL; Luteinizing Hormone; Macaca mulatta; Ovulation; Progesterone

The carbocyanine membrane probe DiI was applied to cut fibers of the superior cerebellar peduncle in the fixed brains of adult mice. Labeled mossy fibers in the cerebellar cortex formed rosettes only in the most superficial aspect of the granular layer, just beneath the Purkinje cell layer. These results provide evidence for a functional division of the cerebellar granular layer into horizontal laminae, in addition to the commonly recognized lobular and sagittal divisions of the cerebellar cortex.

Topics: Animals; Carbocyanines; Cerebellar Cortex; Cerebellum; Female; Fluorescent Dyes; Mice; Mice, Inbred CBA; Microscopy, Fluorescence; Nerve Fibers

Ganglion cells in the chicken retina fail to regenerate their axons upon mechanical injury. In order to determine whether this failure to regenerate axons is intrinsic to the neurons or is mediated by the environment, we asked whether ganglion cells possess an ability to regrow their injured axons in the absence of their natural environment, namely in vitro. Since the retina contains morphologically different types of ganglion cells, it became desirable to investigate whether all types of ganglion cells contribute to regeneration of axons. Ganglion cells were labelled post-mortem with the fluorescent dye DiI and described morphologically. Morphometric parameters like the sizes of their perikarya, their dendrites, and the patterns of dendritic ramification and stratification were considered for grouping cells. Although a strong classification of the cells could not be achieved because of the high diversity among this population of neurons, the chick retinal ganglion cells could be separated into seven regular groups which have their somata located within the ganglion cell layer and into one group with the somata located within the inner nuclear layer (displaced ganglion cells). The experimental procedure for regeneration combines crush injury of the chick optic nerve in situ with explanation of retinal pieces 1 week later for organ cultures in a serum-free medium. Under these conditions, the ganglion cells extended axons 1 day after explanation on polylysine/laminin. The densities of ganglion cells contributing to the axonal regrowth reached up to 1447 cells mm-2 (mean 1028 cells mm-2; S.D. 237). This density corresponds to 13% of the ganglion cell density in the normal retina, averaged across the total retina area. Although the dendrites of some cells whose axons had regenerated were altered in comparison with the normal ganglion cells, all morphological types of ganglion cells including those with displaced cell bodies contributed proportionally to the regrowth of axons from the explants.

Topics: Affinity Labels; Animals; Axons; Carbocyanines; Cell Count; Chickens; Dendrites; Disease Models, Animal; Fluorescent Dyes; Microscopy, Fluorescence; Nerve Regeneration; Optic Nerve; Organ Culture Techniques; Retinal Ganglion Cells

A human cell line THP-1 was differentiated into macrophages expressing the scavenger receptor for uptake of modified lipoproteins. The cells were exposed to native low-density lipoprotein (n-LDL), acetylated-low-density lipoprotein (Ac-LDL), oxidised-LDL, or 25-OH cholesterol, leading to the accumulation of cholesteryl esters within the cells. Harvested macrophages were studied using three separate probes: 1) 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (diI)-labelled LDL to study lipoprotein uptake; 2) the lipophilic fluorescent dye Nile Red to study cholesteryl ester accumulation within the cells; and 3) the polyene antibiotic Filipin III to study free cholesterol homeostasis. Cells were analysed using fluorescence flow cytometry and the three signals analysed separately. THP-1 macrophages incubated with diI-labelled modified lipoproteins produced a concentration dependent increase in the fluorescence emissions, consistent with accumulation of the labelled particles. Macrophages exposed to unlabelled modified LDLs were demonstrated, by staining with Nile Red, to accumulate cholesteryl esters within their cytoplasm and to alter their cholesterol content as judged by staining with Filipin. The foam-cell forming macrophage and its response to modified lipoproteins is considered a key step in the development of atherosclerosis. The use of these three probes during the formation of foam-cells in vitro offers a way of studying their behaviour at the single cell level.

Topics: Arteriosclerosis; Carbocyanines; Cell Differentiation; Cell Line; Cholesterol Esters; Filipin; Flow Cytometry; Fluorescent Dyes; Foam Cells; Humans; Lipoproteins, LDL; Macrophages; Oxazines; Receptors, LDL

In amphibians and teleosts, retina and tectum grow incongruently. In order to maintain the retinotopy of the retinotectal projection, Gaze, Keating, and Chung (1974) postulated a shifting of terminals throughout growth. In order to test the possibility that ingrowing retinal fibers are the driving force for this shifting, we induced a permanent retinal projection into the ipsilateral tectum in juveniles of the cichlid fish Haplochromis burtoni. The surface of the tectum had increased (11-18 months later) 2.5-5.8 times, and the surface of the retina 8.6-14 times. Filling of ganglion cells with horseradish peroxidase (HRP) retrogradely from the tectum showed ipsilaterally regenerating ganglion cells only in the center of the retina. The position of ganglion cells indicated that the ipsilateral projection derived only from axotomized and regenerating retinal ganglion cells but not from those newly born. Ipsilaterally projecting retinal fibers showed terminals only in the rostral half of the tectum. Comparison of area of terminations of ipsilaterally projecting ganglion cells at various times after the crush provided no evidence for expansion or a shift into caudal tectal areas throughout the period of growth. These findings are compatible with the idea that newly ingrowing fibers induce older terminals to move caudally.

Topics: Animals; Axons; Carbocyanines; Eye Enucleation; Fluorescent Dyes; Ganglia; Horseradish Peroxidase; Nerve Endings; Neural Pathways; Perches; Postoperative Care; Retina; Superior Colliculi

The expression of two cadherins, N- and R-cadherin, was mapped in the CNS of chicken embryos of 6-11 d incubation, focusing on the sensory and motor fiber systems. In the spinal cord, the laterally located fibers of the dorsal funiculus express N-cadherin while the medially located fibers do not. These two fiber systems have a different course within the CNS but associate to form the spinal dorsal roots. In the hindbrain, N-cadherin is expressed by the descending trigeminal (general somatic sensory) tract, which is contiguous with the N-cadherin-positive zone of the dorsal funiculus of the spinal cord. R-cadherin is not expressed by sensory fibers, but is expressed by the visceral motor system of the vagus and glossopharyngeal nerves, which are N-cadherin negative. The motor neurites expressing R-cadherin have a different course within the brain than the sensory neurites expressing N-cadherin, although they form the common sensory/motor roots of the vagus nerve at the surface of the brain. The possibility that N-cadherin provides a guidance cue for sensory axon migration within the CNS by a homophilic adhesion mechanism was investigated in vitro. Explants from sensory spinal ganglia expressing N-cadherin were placed on N-cadherin-transfected neuroblastoma cells, and axon outgrowth was visualized. Results showed that the sensory axons defasciculate and closely follow the cell-cell boundaries between transfected cells where high levels of N-cadherin are expressed. These results show that the two cadherins, like members of the immunoglobulin superfamily of molecules, are expressed in a topographically restricted fashion during chick brain development. They furthermore suggest that N-cadherin expression by neurites may play a role in guiding these neurites along CNS paths that express the same molecule.

Topics: Animals; Axons; Cadherins; Carbocyanines; Central Nervous System; Chick Embryo; Fluorescent Dyes; Neurites; Neurons, Afferent; Rhombencephalon; Spinal Cord

A fast and simple method for the in vivo/in situ detection of liposomes is described. Utilizing lipophilic carbocyanine dyes, DiI and DiO, yellow (or red) and green fluorescent liposomes can be visualised with routinely available filters. The main advantages of the method are (i) the vesicles can be labelled after they are formed and (ii) the label does not interfere with proteins on the surface of the liposomes. Labelled liposomes were found in macrophages of spleen and liver (of mice) within 30 min of intravenous administration. In the spleen, labelled liposomes localised preferentially in the marginal zone macrophages, as confirmed by double staining with FITC-Ficoll. These data correlate well with the fact that empty or haptenated liposomes are thymus-independent antigens, and that other thymus-independent antigens are also specifically taken up by marginal zone macrophages. The immunological role of these macrophages in the processing and presentation of antigen-bearing liposomes can now be studied in more detail. Administration of high doses (1-3 mg lipid) of labelled liposomes showed that uptake occurred preferentially, but not exclusively, by marginal zone macrophages. After the marginal zone macrophages had been 'saturated', the red pulp macrophages took up the liposomes. DiI and DiO have also been successfully used for labelling lymphocytes and bacteria for in vivo homing studies. The fact that liposomes can be labelled after they have been formed is an advantage for retrospective (i.e. liposomes already in use/storage) studies in e.g. targeting of drugs by liposomes.

Topics: Affinity Labels; Animals; Carbocyanines; Female; Fluorescent Antibody Technique; Fluorescent Dyes; Kinetics; Liposomes; Liver; Macrophages; Mice; Mice, Inbred BALB C; Microscopy, Fluorescence; Spleen

Visually responsive neurons in the superficial layers of the hamster's superior colliculus (SC) can be divided into distinct morphological and functional classes. In the preceding paper (Mooney et al., '91), we showed that neonatal enucleation has only slight and insignificant effects upon the structural characteristics of cells within a given class, but results in a significant reduction of neurons (narrow and widefield vertical cells) with dorsally directed dendritic arbors. In an effort to determine whether this change reflected differential transneuronal degeneration of these cell types or alterations in the dendritic arbors of surviving cells, this study re-examined this issue by restricting the analysis to a specific and relatively homogeneous subpopulation of superficial layer neurons, those that project to the lateral posterior nucleus (LP). Physiological recordings demonstrated that most (64.7%) tecto-LP cells in neonatally enucleated hamsters develop somatosensory receptive fields. The combination of retrograde tracing and injection of cells with Lucifer yellow in a fixed slice preparation demonstrated that nearly 75% of tecto-LP cells in normal adult hamsters are widefield vertical cells while less than 25% of the neurons filled in neonatally enucleated adults are in this class. Most of the tecto-LP cells in the neonatally enucleated adult hamsters were either horizontal cells (19.5%), giant stellate cells (24.6%), or had dendrites that were directed only toward the deep SC laminae (10.3%). Differential enucleation-induced cell death could not account for all of these changes. Tecto-LP neurons were retrogradely labelled with the carbocyanine dye, Di-I, in hamsters on postnatal day (P-) 0 (the day of birth) through P-10. As early as P-0, most retrogradely labelled neurons could be identified as either widefield (44.6%) or narrowfield (18.9%) vertical cells. These results, when considered together with those from the normal adult and neonatally enucleated adult hamsters, support the conclusion that neonatal eye removal results in a reorganizaton of the dendritic arbors of some collicular neurons that have already undergone considerable development at the time of the lesion.

Topics: Afferent Pathways; Animals; Animals, Newborn; Carbocyanines; Cricetinae; Electric Stimulation; Eye Enucleation; Fluoresceins; Geniculate Bodies; In Vitro Techniques; Isoquinolines; Microspheres; Neurons, Afferent; Particle Size; Perfusion; Physical Stimulation; Superior Colliculi

The olivocochlear pathway in the developing rat was visualized in fixed material. The fluorescent carbocyanine dye 1,1'-Dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI) was applied to the cut central axons of the olivocochlear neurones at the floor of the fourth ventricle, and the termination pattern within the cochlea was examined after dye diffusion. From the day of birth (P0) to postnatal day 2 (P2), efferent innervation of the cochlea was exclusively in the region of the inner hair cells. Between P2 and P11, progressive outgrowth of neuronal processes to the outer hair cell region occurred; possible connections with the outer hair cells were occasionally seen at P4 and approached the mature pattern by P6. The efferent innervation of the organ of Corti appeared to mature progressively from the cochlear base to the apex, with outgrowth to the outer hair cells occurring earlier in the basal turn of the cochlea than in the second and third cochlear turns. Numerous blind axonal endings were observed in the spiral lamina especially at early postnatal ages. These findings may be consistent with a sequential pattern of arrival of efferent axons at the organ of Corti and ongoing death of efferent neurones in the brainstem during this period of development.

Topics: Animals; Carbocyanines; Cochlea; Efferent Pathways; Rats

The organization of afferents to the pituitary was investigated by applying DiI crystals to the pituitary or pituitary stalk of the gymnotiform electric fish, Apteronotus leptorhynchus. Most hypophysiotrophic cells were found in the hypothalamus and were distributed throughout its rostrocaudal extent: nucleus preopticus periventricularis, pars anterior and posterior; suprachiasmatic nucleus; anterior, dorsal, ventral, lateral, and caudal hypothalamic nuclei; and nucleus tuberis lateralis, pars anterior and posterior. In addition a small number of retrogradely labeled cells were found in the ventral telencephalon (area ventralis, pars ventralis) and, most surprisingly, in a thalamic nucleus (nucleus centralis posterioris). The nucleus preopticus periventricularis pars posterior and the anterior hypothalamic nucleus appear to correspond to the parvicellular and magnocellular divisions of the nucleus preopticus of other teleosts. Integration of these results with immunohistochemical localization of monoamines and neuropeptides in the apteronotid brain suggests many homologies between the hypophysiotrophic nuclei of teleosts and other vertebrates, including mammals. Apteronotus communicates electrically during agonistic and sexual interactions. There are numerous anatomical links between the hypophysiotrophic systems and the brain areas related to electrocommunication.

Topics: Animal Communication; Animals; Carbocyanines; Electric Fish; Electric Organ; Histocytochemistry; Hypothalamus; Neurons; Neurons, Afferent; Pituitary Gland, Posterior; Suprachiasmatic Nucleus; Thalamus

In many exocytic systems, micromolar concentrations of intracellular Ca2+ trigger fusion. We find that aggregates of secretory granules isolated from sea urchin eggs fuse together when perfused with greater than or equal to 10 microM free Ca2+. Mixing of membrane components was demonstrated by transfer of fluorescent lipophilic dye, and melding of granule contents was seen with differential interference microscopy. A technique based upon light scattering was developed to conveniently detect fusion. Two protein modifiers, trypsin and N-ethylmaleimide, inhibit granule-granule fusion at concentrations similar to those that inhibit granule-plasma membrane fusion. We suggest that molecular machinery sufficient for Ca(2+)-triggered fusion resides on secretory granules as purified and that at least some of these essential components are proteinaceous.

Topics: Animals; Calcium; Carbocyanines; Cytoplasmic Granules; Ethylmaleimide; Exocytosis; Female; Fluorescent Dyes; Intracellular Membranes; Kinetics; Light; Membrane Fusion; Microscopy, Fluorescence; Ovum; Scattering, Radiation; Sea Urchins; Trypsin; Video Recording

The mutant hypogonadal (hpg) mouse lacks a functioning gene for the neurohormone gonadotropin releasing hormone (GnRH). Previous studies from our laboratory had indicated that the initiation and maintenance of reproductive function in these mice could be brought about by the implantation of normal fetal grafts into adult hosts. Testicular or ovarian growth and other indicators of normal neurosecretory output were always accompanied by survival of GnRH neurons and growth of GnRH axons into the host median eminence where such axons terminate on the hypophysial portal capillaries. To determine if other connections exist between graft and the host hypothalamus, small crystals of the carbocyanine dye, 1,1'-dioctadecyl-3,3,3',3'-tetramethyl-indocarbocyanine perchlorate (DiI) were applied to either graft or host after fixation of the brain. Tissue sections were analyzed for retrograde and and anterograde movement of the dye. When crystals were placed on the graft, labeled axons were found in the host median eminence or in the host hypothalamus taking an arching trajectory toward the median eminence. Retrogradely labeled neurons in the host were few in number and largely confined to the host arcuate nucleus. With DiI crystals applied to the basal hypothalamus, labeled axons were distributed widely in the host but much sparser in the graft. Axons appeared to enter primarily at sites where the graft and host interface lacked an ependymal lining. Small numbers of retrogradely labeled neurons were also seen in the graft. Most were cells of very simple morphology and were distributed randomly in the graft. When double label experiments were carried out most DiI positive cells in the graft contained GnRH. These results indicate the connectivity between host hypothalamus and the third ventricular preoptic area grafts exists but is limited in nature.

Topics: Afferent Pathways; Animals; Axons; Brain Tissue Transplantation; Carbocyanines; Cerebral Ventricles; Efferent Pathways; Female; Fetal Tissue Transplantation; Gonadotropin-Releasing Hormone; Hypogonadism; Hypothalamus; Male; Mice; Mice, Mutant Strains; Neurons; Organ Size; Ovary; Preoptic Area; Septum Pellucidum; Testis; Transplantation, Heterotopic

The nature and physiological importance of high-density lipoprotein (HDL) binding sites on unstimulated (resting) and mitogen-activated (blast) human peripheral blood lymphocytes were investigated. Specific HDL binding on resting and blast T-lymphocytes was saturable at 50 micrograms of 125I-HDL/ml and of high affinity, with Kd values of 8.1 x 10(-8) M and 6.5 x 10(-8) M, respectively, and Bmax. values of 79 ng and 180 ng/mg of cell protein respectively at 4 degrees C. Binding of HDL double-labelled with fluorescent dioctadecylindocarbocyanine (Dil) and isotope (125I) as well as of single fluorescence- or isotope-labelled HDL was inhibited competitively by HDL apoproteins. Studies of the cholesterol flux between the cells and HDL showed that HDL, low-density lipoprotein (LDL) or BSA at a concentration of 100 micrograms/ml in the tissue culture medium did not result in a significant difference in exogenous [3H]cholesterol efflux from the cell membrane at 37 degrees C. Proliferating T-blasts incorporated more cholesterol from HDL or LDL than did resting lymphocytes. When the cells were pulsed with 125I-HDL and chased in fresh lipid-free medium, up to 80% of the radioactivity released was not precipitable with trichloroacetic acid. This percentage decreased in a competitive manner when unlabelled HDL was present in the chase incubation medium. Finally, cultivation of lymphocytes with conditioned medium from macrophages increased Dil-HDL binding/uptake, while it was decreased by mevinolin-induced inhibition of hydroxymethylglutaryl-coA reductase. In conclusion, human lymphocytes possess a HDL binding site (receptor) responsible for lipid binding/uptake and concomitant internalization and degradation of apoproteins from HDL, but not for reverse cell membrane cholesterol transport. The activity of the binding site is up-regulated during cell proliferation and down-regulated during cell growth suppression.

Topics: Adult; Binding Sites; Biological Transport; Carbocyanines; Cell Division; Cholesterol; Female; Fluorescent Dyes; Humans; Iodine Radioisotopes; Lipid Metabolism; Lipoproteins, HDL; Lipoproteins, LDL; Lymphocytes; Male; Temperature

The ability to modify human hepatocytes genetically is an essential first step in the development of liver-directed ex vivo gene therapy for inherited metabolic disease. The purpose of these studies was to prove that the genome of human hepatocytes can be altered successfully to express foreign genetic material.. Human hepatocytes were plated at 2 or 4 x 10(6) cells/10 cm Primaria (Falcon, Oxnard, Calif.) plates. Fresh virus from the amphotropic viral producer cell line BAG, containing the Escherichia coli beta-galactosidase gene lacZ, was placed directly onto hepatocyte cultures and quantitative analysis of cells staining positive for the lacZ gene was undertaken. In a different human liver, a variety of viruses from producer cell lines containing clones of the human low-density lipoprotein (LDL) receptor were plated directly on cultures of human hepatocytes, and gene transfer was demonstrated by increased uptake of fluorescent-labeled LDL.. Beta-galactosidase production in hepatocytes was assayed histochemically with the chromogenic substrate X-gal. The highest percentage of cells staining positive for expression of enzyme was seen at 4 x 10(6) cells/plate (43.66% +/- 1.02% vs 27.99% +/- 2.31%). Gene transfer was also documented by the uptake of fluorescent-labeled LDL with a variety of different vectors containing the human LDL receptor.. (1) Human hepatocytes can be cultured in vitro and are susceptible to retroviral infection, (2) functional gene transfer is demonstrated by intracellular function of foreign genes, and (3) the level of expression appears dependent on plating density. We conclude that human hepatocytes are suitable targets for genetic manipulation and may play an important role in human gene therapy trials.

Topics: beta-Galactosidase; Carbocyanines; Cell Separation; Cells, Cultured; Fluorescent Dyes; Histocytochemistry; Humans; Lipoproteins, LDL; Liver; Middle Aged; Retroviridae; Transfection

Fetal allogeneic dorsal root ganglion (DRG) transplants from 13-15 day rat embryo's (E13-E15) survived and differentiated when grafted orthopically (within the capsules of the excised 4th and 5th lumbar (L4-L5) ganglia) in adult rats. Survival of grafted neurones was established by prelabeling the grafts with a fluorescent vital dye (DiI) and visualizing the retained fluorescent marker 3 to 9 months later. Simultaneous retrograde tracing using fluorescent tracers applied in the spinal cord and peripheral nerve, respectively, yielded double-labeled dorsal root ganglion neurons, some of which were prelabeled. These findings demonstrate that prelabeled E13-E15 ganglia survive orthopic grafting, organotypically differentiate into mature DRG neurones, and can be double-labeled with fluorescent dyes applied to their peripherally and centrally directed processes. The presence of DiI containing cells which were retrogradely labeled from the spinal cord suggests that fetal (E13-E15) ganglia may have the capability of growing into a mature spinal cord.

Topics: Aging; Animals; Carbocyanines; Female; Fetal Tissue Transplantation; Fluorescent Dyes; Ganglia, Spinal; Gestational Age; Pregnancy; Rats; Rats, Inbred Strains; Spinal Cord; Transplantation, Homologous

During development of the mammalian cerebral cortex, thalamic axons must grow into the telencephalon and select appropriate cortical targets. In order to begin to understand the cellular interactions that are important in cortical target selection by thalamic axons, we have examined the morphology of axons from the lateral geniculate nucleus (LGN) as they navigate their way to the primary visual cortex. The morphology of geniculocortical axons was revealed by placing the lipophilic tracer Dil into the LGN of paraformaldehyde-fixed brains from fetal and neonatal cats between embryonic day 26 (E26; gestation is 65 d) and postnatal day 7 (P7). This morphological approach has led to three major observations. (1) As LGN axons grow within the intermediate zone of the telencephalon toward future visual cortex (E30-40), many give off distinct interstitial axon collaterals that penetrate the subplate of nonvisual cortical areas. These collaterals are transient and are not seen postnatally. (2) There is a prolonged period during which LGN axons are restricted to the visual subplate prior to their ingrowth into the cortical plate; the first LGN axons arrive within visual subplate by E36 but are not detected in layer 6 of visual cortex until about E50. (3) Within the visual subplate, LGN axons extend widespread terminal branches. This represents a marked change in their morphology from the simple growth cones present earlier as LGN axons navigate en route to visual cortex. The presence of interstitial collaterals suggests that there may be ongoing interactions between LGN axons and subplate neurons along the entire intracortical route traversed by the axons. From the extensive branching of LGN axons within the visual subplate during the waiting period, it appears that they are not simply "waiting." Rather, LGN axons may participate in dynamic cellular interactions within the subplate long before they contact their ultimate target neurons in layer 4. These observations confirm the existence of a prolonged waiting period in the development of thalamocortical connections and provide important morphological evidence in support of the previous suggestion that interactions between thalamic axons and subplate neurons are necessary for cortical target selection.

Topics: Aging; Animals; Animals, Newborn; Axons; Carbocyanines; Cats; Fluorescent Dyes; Geniculate Bodies; Gestational Age; Neural Pathways; Telencephalon; Thalamus; Visual Cortex

Ganglion cells in the cat retina were retrogradely labeled by injecting a fluorescent dye (DiI) into either the lateral geniculate nucleus (LGN) or the superior colliculus (SC). Cells were then dissociated enzymatically from the retinal tissue. LGN-projecting ganglion cells consisted of 2 different populations, one with small and the other with large somata, which were identified as W and X cells, respectively. SC-projecting cells consisted of a single group of cells with small somata, identified as W cells. The voltage-gated sodium current (INa) was recorded from isolated ganglion cells under the voltage-clamp condition using a patch pipette in the whole cell configuration. INa was identified by reversible tetrodotoxin block. INa was activated by depolarization of the cell from the holding potential (Vh) of -95 mV to membrane voltages (Vm) more positive than -45 mV. The maximum INa was recorded at around -15 mV. INa flowed outward at Vm more positive than +65 mV. The reversal potential of INa became more negative voltages with low extracellular Na concentration ([Na+]o) with a relation of 58 mV for a 10-fold change in [Na+]o. INa was inactivated with a few milliseconds. Once inactivated, INa recovered by holding the cell membrane hyperpolarized. While the voltage dependence of INa activation and steady-state inactivation were constant from cell to cell, the time course of recovery was not. Cells with a large soma showed a rapid recovery, while cells with a small soma showed slow recovery. Thus, the rate of recovery is faster for X cells than for W cells. Perhaps this helps to explain the 'sluggish' firing of the latter cell type.

Topics: Action Potentials; Animals; Carbocyanines; Cats; Geniculate Bodies; Kinetics; Membrane Potentials; Retinal Ganglion Cells; Sodium Channels; Superior Colliculi; Tetrodotoxin

The rodent primary somatosensory cortex is characterized by aggregates of cellular and axonal elements that replicate the distribution of mystacial vibrissae on the face. The periphery-related cortical pattern ("barrels") is influenced by an amalgam of elements extrinsic (i.e., afferents) and intrinsic (i.e., neurons, glia, and their substrate) to the developing neocortex. To assign the role of some of these elements in cortical pattern formation, we have examined the temporal correlation between periphery-related patterns formed by thalamocortical axons and by extracellular matrix (ECM) molecules associated with neurons and glia in the cortex. Thalamocortical axons were labeled with the lipophilic tracer 1,1'-dioctydecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI) in aldehyde-fixed neonatal rat brains, and the same brains were also prepared for immunohistochemical localization of ECM molecules cytotactin and cytotactin-binding proteoglycan. We present evidence that thalamocortical axons form a periphery-related pattern well before such an organization is detectable in the distribution of ECM molecules. Furthermore, a patterned distribution of ECM molecules results from the down-regulation of these molecules from barrel centers, where thalamic axons have established vibrissa-specific patches. We conclude that thalamic axons convey the blueprint of the sensory periphery onto the neocortex and that ECM molecules do not participate in the initial formation of this pattern.

Topics: Afferent Pathways; Affinity Labels; Animals; Animals, Newborn; Axons; Carbocyanines; Cell Adhesion Molecules, Neuronal; Cerebral Cortex; Extracellular Matrix; Extracellular Matrix Proteins; Immunohistochemistry; Morphogenesis; Rats; Rats, Inbred Strains; Tenascin; Thalamus

Carbocyanine DiI is described as a suitable fluorescent tracer to investigate the connectivity of cortical transplants. DiI was applied in 2 ways: in tissue previously fixed with aldehydes, and in vivo for labelling the donor tissue prior to transplantation. When applied in fixed tissue DiI travelled anterogradely, allowing the study of efferent connections from the transplant to the host. When DiI was applied in vivo, it travelled retrogradely showing the afferents to the transplant from the host.

Topics: Animals; Animals, Newborn; Brain Tissue Transplantation; Carbocyanines; Cerebral Cortex; Female; Fetal Tissue Transplantation; Formaldehyde; Pregnancy; Rats; Rats, Inbred Strains; Staining and Labeling

The distribution of afferents from the dorsal lateral geniculate nucleus (LGNd) and the lateral posterior nucleus (LP) and of cell bodies projecting to these nuclei has been studied in the visual cortex of the wallaby (Macropus eugenii) throughout development to determine how the characteristic laminar distribution of afferents and efferents of the mature cortex is achieved. Young are born after 26-28 days of gestation and do not open their eyes until around 140 days after birth. Horseradish peroxidase conjugated to wheatgerm agglutinin was injected in the visual thalamus in adults and in pouch young aged from 22 days after birth, just after thalamic axons first reach the visual cortex, to 118 days, when cortical lamination resembles the adult. From 22 to 65 days, the developing visual cortex consists of a marginal zone (MZ), cortical plate (CP), and intermediate zone (IZ) including the superficial subplate (SP), subventricular zone, and ventricular zone. There is a thin compact cell zone (CCZ) at the top of the CP and below it a less densely packed region that increases in thickness with age. Retrogradely labelled cells in two bands were first seen at 40 days, one in the CCZ and the other at the base of the CP. Two bands of cells were seen at all subsequent times if the injection covered both LGNd and LP, and by 76 days, these cells were located within cytoarchitectonically recognizable layers V and VI. Anterograde label prior to 45 days was distributed densely and evenly throughout the IZ and the CP up to the CCZ. Label in MZ was first seen at 25 days and was substantial by 54 days. Anterograde label than became gradually reduced in the IZ, whereas in the CP it remained evenly and densely distributed until 82 days. At this age, coincident with the emergence of layer IV, label within the CP first showed variations in density and by 99 days was concentrated over layer IV and, to a lesser extent, over layer VI. By 118 days label resembled the adult after injections covering both LGNd and LP, with label concentrated in layer I, IV, and VI with a less dense projection to lower layer III and upper layer V. There is a relatively earlier initial ingrowth of axons into the visual cortex in the wallaby and throughout development thalamocortical axons appear to be more widely distributed in the depth of the visual cortex than has been demonstrated for placental mammals.(ABSTRACT TRUNCATED AT 400 WORDS)

Topics: Aging; Animals; Axons; Carbocyanines; Female; Geniculate Bodies; Horseradish Peroxidase; Macropodidae; Male; Neural Pathways; Neurons, Afferent; Stereotaxic Techniques; Thalamus; Visual Cortex; Wheat Germ Agglutinin-Horseradish Peroxidase Conjugate; Wheat Germ Agglutinins

Using various histological methods, we investigated the cellular and morphological organization of rat hippocampal slice cultures. Many of the typical features of the hippocampus were retained in vitro over a long period of time. The principal cell types of the hippocampus and dentate gyrus, the pyramidal cells and granule cells, were well preserved and matured in vitro. Nonpyramidal cells and gamma-aminobutyric-acid (GABA) cells were also present in slice cultures and exhibited a strikingly similar dendritic appearance at the light microscopic level. Moreover, GABA-immunoreactive cell bodies and presynaptic terminals could be identified at the electron microscopic level; they expressed typical symmetric synaptic contacts with cell bodies and dendrites. The course of the intrinsic hippocampal fiber pathways--the mossy fibers, Schaffer collaterals, and alveus--was generally retained in vitro. Additional aberrant fiber projections could be identified. Finally, three types of nonneuronal cells could be distinguished on the basis of immunocytochemical methods.

Topics: Animals; Carbocyanines; Fluorescent Antibody Technique; gamma-Aminobutyric Acid; Hippocampus; Immunohistochemistry; Neural Pathways; Neurons; Organ Culture Techniques; Pyramidal Tracts; Rats; Silver; Staining and Labeling

During embryonic development, spinal motor neurons require muscle-derived trophic factors for their survival and growth. We have recently isolated a protein from muscle that is not laminin but that still stimulates neurite outgrowth from embryonic neurons in culture. In the present study, we investigated whether this protein, which we refer to as muscle-derived neurite-promoting factor (NPF), could also promote the survival and growth of motor neurons in culture. Spinal motor neurons were isolated from 6-day-old chicken embryos by a metrizamide step-gradient centrifugation protocol. Most large cells (putative motor neurons) were found in the upper metrizamide fraction (0%-6.8% interface; fraction I). Motor neurons were identified by increased specific activity of choline acetyltransferase (CAT) and by their propensity to transport retrogradely either wheat germ agglutinin-horseradish peroxidase or the fluorescent dye, 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine per chlorate (diI), when those substances were injected into the target field. Labeled motor neurons were 2.6-fold enriched in fraction I and the specific CAT activity was 4.4-fold increased in fraction I as compared to unfractionated cells. When motor neurons were grown on muscle-derived NPF, the protein supported the survival of at least 21% of the neurons for as long as 6 days in culture. The protein showed no significant effect on either CAT specific activity or on high-affinity choline uptake by neurons. There was a substantial increase from 21% to 38% of the survival of motor neurons when a combination of muscle-derived NPF and laminin was used as the substrate. Muscle-derived NPF also promoted the survival of sensory neurons and sympathetic neurons in culture. Our results demonstrate that a neurite-promoting protein derived from muscle promotes both the survival and the outgrowth of neurites from cultured spinal motor neurons as well as from sensory and sympathetic neurons.

Topics: Acetylcholinesterase; Animals; Carbocyanines; Cell Survival; Cells, Cultured; Chick Embryo; Choline; Choline O-Acetyltransferase; Ganglia, Spinal; Ganglia, Sympathetic; Horseradish Peroxidase; Immunohistochemistry; Metrizamide; Motor Neurons; Muscles; Nerve Growth Factors; Neurons; Peptides; Spinal Cord; Wheat Germ Agglutinin-Horseradish Peroxidase Conjugate; Wheat Germ Agglutinins

Vagal efferent innervation of the pancreas was labeled by anterograde transport of Dil injected into the dorsal motor nucleus (dmnX). While over the entire organ only 19 +/- 3 (or 8 +/- 1%) of the 231 +/- 17 interlobular ganglia received Dil-labeled vagal fibers and terminals, the proximal duodenal lobe (or head) was significantly more densely innervated. Laser scanning confocal microscopy revealed further morphological details of the vagal terminals and their target ganglion cells. No vagal fibers or terminals were found in islets and acinar tissue.

Topics: Animals; Axonal Transport; Carbocyanines; Efferent Pathways; Fluorescent Dyes; Ganglia, Sympathetic; Islets of Langerhans; Male; Medulla Oblongata; Pancreas; Rats; Rats, Inbred Strains; Stilbamidines; Vagus Nerve

The suprachiasmatic nuclei (SCN) regulate circadian rhythmicity in many biological and behavioral responses. Hamsters are made permanently arrhythmic by bilateral destruction of the SCN. Circadian locomotor rhythmicity is restored by fetal tissue transplants placed in the 3rd ventricle (3V). If intact animals are implanted with fetal SCN grafts, they maintain locomotor activity rhythms when the host SCN are subsequently destroyed. The mechanism(s) whereby the SCN (either grafted or in situ) regulate locomotor rhythmicity is not known. Evidence from other graft models point to the possibility of efferents to appropriate targets in the host. In the present study, efferent connections of transplanted fetal SCN were examined using the carbocyanine dye, Dil. Intact or SCN-lesioned animals were sacrificed 7 or 40 days after receiving fetal SCN grafts into 3V. Dil crystals were placed on the grafts in fixed brains which were then incubated for 3-6 weeks before sectioning. Sections bearing Dil-labelled efferents from the graft were photographed and then stained for immunoreactive VIP and NP cells to locate donor SCN. Although labelled efferents were observed in a majority of the grafts, most were confined to the limits of the graft. The few labelled efferents that entered the host tissue when the graft seemed to merge with the host did not extend very far regardless of whether the graft contained immunohistochemical evidence for donor SCN or not. The observation of limited graft-host connectivity suggests either that a limited number of efferents is sufficient to support circadian locomotor rhythmicity, or that the mechanism mediating restoration of function entails a diffusible substance.

Topics: Animals; Axonal Transport; Brain Tissue Transplantation; Carbocyanines; Circadian Rhythm; Cricetinae; Efferent Pathways; Fetal Tissue Transplantation; Fluorescent Dyes; Male; Mesocricetus; Motor Activity; Nerve Fibers; Suprachiasmatic Nucleus

Myenteric neurons which innervate the mucosa of the guinea-pig ileum were characterized by combining retrograde transport of DiI in vitro with immunohistochemistry. Of DiI-labelled myenteric neurons, 43% were immunoreactive for calbindin and substance P, 25% were immunoreactive for calbindin alone, and 18% were immunoreactive for substance P alone. These 3 classes of neurons had Dogiel Type II morphology and are probably sensory neurons. Two classes of probable secretomotor neurons were characterized by immunoreactivity for neuropeptide Y (4%) and vasoactive intestinal peptide (2%). These 5 classes of myenteric neurons represent over 90% of the retrogradely labelled myenteric neurons that project to the mucosa.

Topics: Animals; Calbindins; Carbocyanines; Guinea Pigs; Ileum; Immunohistochemistry; Intestinal Mucosa; Microscopy, Fluorescence; Myenteric Plexus; Neural Pathways; Neurons; Neuropeptide Y; S100 Calcium Binding Protein G; Substance P; Vasoactive Intestinal Peptide

The present report describes a method for the simultaneous visualization, in the same structure, of two different sets of afferent pathways and the neurons of origin of some efferent projections. This method has been applied in the cat for studying, in the thalamic anterior intralaminar nuclei, the topographical relationships of afferent arising from the spinal cord and deep cerebellar nuclei with neurons projecting to different cortical areas. Spino- and cerebello-thalamic terminals were anterogradely labeled by injections of the fluorescent dyes fast blue (FB) and 1,1'-dioctadecyl-3,3,3',3'-tetramethyl-indocarbocyanine perchlorate (DiI) in the spinal cord and cerebellum. Thalamo-cortical neurons were retrogradely labeled by injections of fluorescent tracers in the precruciate and anterior suprasylvian cortices. The findings show that spinal and cerebellar afferent fibers and the cells of origin of intralaminocortical projections are organized in a clear modular manner and indicate that the method used here is suitable for analyzing simultaneously, in light microscopy, multiple input-output interrelationships of a single structure.

Topics: Afferent Pathways; Amidines; Animals; Axonal Transport; Carbocyanines; Cats; Cerebellum; Efferent Pathways; Fluorescent Dyes; Spinal Cord; Thalamic Nuclei

We have developed a labeling procedure which accurately and consistently labels the original sensory pools projecting to their respective nerve branches as a model to quantify the accuracy of nerve regeneration at the single neuron level. Adult and juvenile rats had the saphenous branch of the femoral nerve transected just distal to the bifurcation of the nerve into a sensory branch (saphenous nerve) and a motor branch (nerve to the quadriceps muscle) and exposed to a 3% solution of 1,1'-di-octadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI) for 1 h, and then reanastomosed. Two weeks later the sensory branch was redivided proximal to the previous site and labeled with a second tracer (5% fluorogold) using similar procedures. Five days later the animals were killed and cryostat sections were prepared and analyzed with a fluorescence microscope to score single- and double-labeled primary sensory neurons. The results show that the primary sensory neurons which project into the saphenous nerve can be reliably prelabeled by exposure of the saphenous nerve to DiI, and two weeks later approximately 99% of the same population of neurons can be labeled by exposure of the nerve to a second dye, fluorogold. This model system will be very powerful for future studies concerning target reinnervation following nerve regeneration.

Topics: Age Factors; Animals; Axonal Transport; Carbocyanines; Fluorescent Dyes; Ganglia, Spinal; Leg; Male; Microscopy, Fluorescence; Nerve Regeneration; Neurons, Afferent; Rats; Rats, Inbred Strains; Stilbamidines

The prenatal development of axonal connections in the rat olfactory system was studied using DiI. On day 16 (E16), the olfactory and vomeronasal nerves extended from the olfactory epithelia to the olfactory bulb (OB), the terminal nerve to the telencephalic septum, while axons of mitral and tufted cells reached the anterior olfactory nucleus (AO). Axons from the AO were also seen in the anterior commissure. On day E16(8) (at 16 days, 8 h), axons were anterogradely followed from the dorsal OB through the lateral olfactory tract (lo) to the bed nucleus of the accessory olfactory tract. At E18(0), crystals implanted in the olfactory epithelium labeled the mitral cell layer and the lo.

Topics: Aging; Animals; Axons; Brain; Carbocyanines; Diffusion; Female; Olfactory Bulb; Olfactory Mucosa; Olfactory Nerve; Olfactory Pathways; Pregnancy; Rats; Smell; Telencephalon

The carbocyanine dye DiIC18(3) ("DiI") is commonly used for both anterograde and retrograde labeling of neurons, including live neurons in situ and in vitro. In the present experiments, DiIC18(3) was used to label motoneurons in the spinal cords and sensory neurons in the dorsal root ganglia of embryonic rats. When the neurons from these regions were placed in culture, the neurons labeled by the dye were found to die rapidly, suggesting that DiIC18(3) can be toxic to neurons of these types. A related dye, DiIC12(3), was found to be equally suitable for labeling these neurons, and was found not to have detectable toxic effects in vitro.

Topics: Affinity Labels; Animals; Carbocyanines; Cell Survival; Cells, Cultured; Embryo, Mammalian; Flow Cytometry; Fluorescent Dyes; Kinetics; Motor Neurons; Neurons, Afferent; Rats; Rats, Inbred Strains

Anterograde tracing with the carbocyanine dye, Di-I, was used to describe the normal postnatal development of the projection from the trigeminal (V) brainstem complex to the superior colliculus (SC) in hamster. In adult hamsters, this projection is completely restricted to the deep laminae, primarily the stratum album intermedium. Trigeminal fibers were present in the SC by the day of birth, and at this time, they terminated mainly in the deep layers. However, labelled fibers also extended into the superficial laminae (the stratum griseum superficiale and stratum opticum) reaching as far as the SC surface. Trigeminal projections to the superficial SC laminae were visible as late as postnatal day (P-) 6, but were absent by P-8. During the period when V axons were present in the superficial SC laminae, they could not be detected in the dorsal lateral geniculate nucleus.

Topics: Animals; Animals, Newborn; Brain Stem; Carbocyanines; Cricetinae; Geniculate Bodies; Neural Pathways; Ocular Physiological Phenomena; Superior Colliculi; Trigeminal Nuclei

Anterograde and retrograde labelling with the carbocyanine dye, Di-I, was used to assess the development of the visual cortical projection to the superior colliculus (SC) in pre- and postnatal hamsters. Posterior cortical axons arrive in the SC on postnatal (P-) day one (the first 24 hours after birth = P-0) and begin to arborize in the superficial laminae (the stratum griseum superficiale [SGS] and stratum opticum [SO]) within one day after they enter the tectum. Over succeeding days, the density of the projection increases and numerous labelled fibers are visible throughout the depth of the SGS and SO. Beginning on P-6, there is a decrease in the density of labelled fibers in the upper SGS and by P-10, the laminal distribution of the occipital corticotectal pathway appears adult-like. Anterograde tracing with Di-I also revealed the presence of a few corticotectal fibers that crossed the midline in both the SC and posterior commissures to terminate mainly in the superficial tectal laminae contralateral to the injection site. Crossed corticotectal fibers were visible in hamsters aged between P-3 and P-12. Retrograde tracing with Di-I in hamsters killed between P-3 and P-12 demonstrated that both the ipsilateral and crossed corticotectal projections arose exclusively from pyramidal cells in developing lamina V.

Topics: Animals; Axons; Carbocyanines; Cerebral Cortex; Cricetinae; Female; Fetus; Mesencephalon; Neural Pathways; Occipital Lobe; Phytohemagglutinins; Pregnancy; Superior Colliculi

The carbocyanine dye, DiI, was used to demonstrate human retinohypothalamic tract (RHT) projections in 6 normal human postmortem brains. In 5 of 6 brains, labeling was seen extending from the site of implantation in the distal optic nerve to both the ipsilateral and contralateral suprachiasmatic nuclei. This study confirms the presence of RHT projections in humans, and demonstrates the usefulness of DiI for neuronal tracing in human postmortem tissue.

Topics: Cadaver; Carbocyanines; Fluorescent Dyes; Humans; Hypothalamus; Optic Nerve; Retina; Suprachiasmatic Nucleus; Visual Pathways

Fluorescent carbocyanine dye (diI) was used to label the glossopharyngeal (IX) nerve in the fixed preparation of the Mexican salamander, axolotl. When the cell bodies were viewed with a confocal laser scanning microscope and Nomarski optics, the cytoplasm was brightly fluorescent, but not the cell nucleus. The cell bodies which send peripheral axons in the two branches of the IX nerve were mainly distributed in the rostral part of the combined glossopharyngeal-vagus ganglion, but a few cells were also distributed in the middle and caudal parts. This may indicate a relatively undifferentiated organization of the IX nerve in the ganglion.

Topics: Ambystoma; Animals; Carbocyanines; Fluorescent Dyes; Glossopharyngeal Nerve; Microscopy, Fluorescence

The vestibulo-ocular pathways have been examined in embryonic chicks using horseradish peroxidase or diI as retrograde and anterograde tracers. The vestibular neurons project to the rostral, external eye motor nuclei over one or the other of three separate pathways; the ipsilateral and contralateral medial longitudinal fascicle and the contralateral brachium conjunctivum. The brachium conjunctivum component originates dorsally in the superior vestibular region and projects to the contralateral inferior oblique and superior rectus motor nuclei. An ipsilateral component of the medial longitudinal fascicle is labeled from more ventral sites in the vestibulo-cerebellar process and terminates in the ipsilateral superior oblique and inferior rectus nuclei. The contralateral medial longitudinal fascicle component originates still more ventrally and terminates in the contralateral superior oblique and inferior rectus motor nuclei. Accordingly, the vestibulo-ocular pathways in chickens operate predominantly on synergistic pairs of external eye muscles. These selective terminal fields are established within a day or two after the first terminals invade the eye motor nuclei during embryogenesis.

Topics: Affinity Labels; Animals; Carbocyanines; Chick Embryo; Chickens; Horseradish Peroxidase; Microscopy, Fluorescence; Motor Neurons; Reflex, Vestibulo-Ocular; Visual Pathways

Fate maps of chick Hensen's node were generated using DiI and the lineage of individual cells studied by intracellular injection of lysine-rhodamine-dextran (LRD). The cell types contained within the node are organized both spatially and temporally. At the definitive primitive streak stage (Hamburger and Hamilton stage 4), Hensen's node contains presumptive notochord cells mainly in its anterior midline and presumptive somite cells in more lateral regions. Early in development it also contains presumptive endoderm cells. At all stages studied (stages 3-9), some individual cells contribute progeny to more than one of these tissues. The somitic precursors in Hensen's node only contribute to the medial halves of the somites. The lateral halves of the somites are derived from a separate region in the primitive streak, caudal to Hensen's node.

Topics: Animals; Carbocyanines; Cell Differentiation; Chick Embryo; Fluorescent Dyes; Gastrula; Mesoderm; Microscopy, Electron; Notochord

The purpose of this work was to study the development of specific projections from the postero-lateral cortex during the third trimester of gestation in the mouse. To do this, we labeled undifferentiated lateral cortex with the fluorescent carbocyanine dye, Dil, in the embryonic day (E) 16 mouse embryo using exo utero surgical techniques (Muneoka, Wanek, and Bryant, 1986). Embryos were allowed to develop to term (postnatal day 0, P0) at which time the fiber patterns emanating from the marked regions were studied. Dye placement in the undifferentiated postero-ventral cortex produced labeled fibers in the hippocampal formation. A robust projection of the angular bundle into the CA1 region of the hippocampus was heavily labeled. In addition, in some animals, cortical tracts, such as the anterior commissure, corpus callosum, and a corticotectal tract, were labeled. These tracts have been described previously as scaffolding pathways in the fetal cat (McConnell, Ghosh, and Shatz, 1989), and other vertebrates (Wilson, Ross, Parrett, and Easter, 1990). Dye placement in adjacent, more anterior or dorsal areas showed strong labeling in cortical structures but no labeling in the hippocampal formation. These data indicate that, by birth, the temporal cortex is subdivided along the rostro-caudal axis as entorhinal cortex and perirhinal cortex, and along the dorso-ventral axis, as entorhinal cortex and neocortex. Also, these earliest connections are similar to adult connections in their specificity of target area selection. Therefore, these early, yet specific, connections may play a role int he formation of future connections during postnatal development.

Topics: Animals; Brain Mapping; Carbocyanines; Cerebral Cortex; Female; Fetus; Hippocampus; Mice; Neural Pathways; Pregnancy; Silver Staining

The fate of the medial edge epithelial (MEE) cells during palatal fusion has been proposed to be either programmed cell death or epithelial-mesenchymal transformation. Vital cell labeling techniques were used to mark the MEE and observe their fate during palatal fusion in vitro. Fetal mouse palatal shelves were labeled with Dil and allowed to proceed through fusion while maintained in an organ culture system. The tissues were examined at several stages of palatal fusion for the distribution of Dil, presence of specific antigens and ultrastructural appearance of the cells. The MEE labeled with Dil occupied a midline position at all stages of palatal fusion. Initially the cells had keratin intermediate filaments and were separated from the underlying mesenchyme by an intact basement membrane. During the process of fusion the basement membrane was degraded and the Dil-labeled MEE were in contact with the mesenchymal-derived extracellular matrix. In the late stages of fusion the Dil-labeled MEE altered their cellular morphology, had vimentin intermediate filaments, and were not associated with an identifiable basement membrane. Dil-labeled cells, without an epithelial phenotype, remained present in the midline of the completely fused palate. The data indicate that the MEE did not die but underwent a phenotypic transformation to viable mesenchymal cell types, which were retained in the palatal mesenchyme.

Topics: Animals; Carbocyanines; Cell Death; Epithelium; Fluorescent Dyes; Mice; Mice, Inbred Strains; Morphogenesis; Organ Culture Techniques; Palate

We examined the incorporation of fluoresceinated low-density lipoprotein (LDL) and acetylated or acetoacetylated low-density lipoprotein (A-LDL or AA-LDL) by a number of ocular cells in culture. All the cells investigated, including bovine, monkey, human trabecular meshwork cells, human corneal endothelial cells, human corneal stromal cells and human scleral cells, took up fluorescently labeled LDL. The bovine, monkey and human trabecular meshwork cells showed the strongest fluorescence reactions. In addition, we found that the trabecular meshwork cells became fluorescent after incubations with labeled A-LDL or AA-LDL. They were the only cell type examined that possessed this capacity. The fluorescence intensity was markedly diminished by adding to the incubation solution either fucoidin, a competitive inhibitor of modified LDL uptake, unlabeled A-LDL or AA-LDL. The trabecular meshwork cells in situ also became brightly labeled after exposure to fluoresceinated native LDL, A-LDL or AA-LDL. The uptake of modified LDL separated the trabecular meshwork cells from other types of ocular cells, which may be used to aid identification of trabecular meshwork cells in culture as well as in situ. This property also suggested that trabecular meshwork cells may have some functional similarities to macrophages.

Topics: Acetylation; Adult; Affinity Labels; Aged; Animals; Carbocyanines; Cattle; Cells, Cultured; Child; Child, Preschool; Corneal Stroma; Endothelium, Corneal; Endothelium, Vascular; Fluorescent Dyes; Humans; Lipoproteins, LDL; Macaca fascicularis; Middle Aged; Rabbits; Receptors, LDL; Trabecular Meshwork

We investigated whether regenerating mature axons recapitulate embryonic features essential to successful reconnectivity within the injured nervous system. Strips from embryonic and adult chick retinae were cultured, and outgrowing axons were examined morphometrically and immunohistochemically. In addition, the target-recognition properties of adult neurites were analyzed. Regenerating adult axons elongate on a poly-L-lysine/laminin substratum with a speed about one order of magnitude slower than that of embryonic axons. Morphologically, adult axonal tips differ dramatically from embryonic growth cones in that they possess only filopodial extensions whereas embryonic growth cones possess both lamellipodial and filopodial processes. Both embryonic and adult neurites express the growth-associated protein GAP-43. When cultured on alternating stripes of anterior and posterior embryonic tectal membranes, both adult and embryonic retinal axons distinguish between the two membrane preparations. Our results demonstrate that during axonal regeneration the mature neurons express embryonic properties that are involved in the recognition of tectal positional cues.

Topics: Animals; Axons; Carbocyanines; Cells, Cultured; Chick Embryo; Chickens; GAP-43 Protein; Membrane Glycoproteins; Microscopy, Fluorescence; Nerve Tissue Proteins; Regeneration; Retinal Ganglion Cells

The ongoing activity of superior cervical ganglion neurons projecting to the rat submandibular gland was investigated with intracellular techniques. The relevant cells were identified by retrograde labeling with the fluorescent dye DiI placed in the submandibular gland 7-10 days earlier. The majority of labeled cells (89%) were 'silent' during a standard 5-min period of recording. In contrast, only 15% of cells projecting to targets other than the submandibular gland were silent during the standard period of observation. Evidently, ganglion cells innervating different targets have different patterns of activity.

Topics: Animals; Carbocyanines; Electrophysiology; Ganglia, Sympathetic; Neurons; Rats; Rats, Inbred Strains; Submandibular Gland; Synaptic Transmission

A method to culture rat cerebral microvascular endothelial cells (RCMECs) was developed and adapted to concurrently obtain cultures of rat aortic endothelial cells (RAECs) without subculturing, cloning, or "weeding." The attachment and growth requirements of endothelial cell clusters from isolated brain microvessels were first evaluated. RCMECs required fetal bovine serum to attach efficiently. Attachment and growth also depended on the matrix provided (fibronectin approximately laminin much greater than gelatin greater than poly-D-lysine approximately Matrigel greater than hyaluronic acid approximately plastic) and the presence of endothelial cell growth supplement and heparin in the growth medium. Non-endothelial cells are removed by allowing these cells to attach to a matrix that RCMECs attach to poorly (e.g., poly-D-lysine) and then transferring isolated endothelial cell clusters to fibronectin-coated dishes. These cell cultures, labeled with 1,1'-dioctadecyl-3,3,3',3'-tetramethyl-indocarboxyamine perchlorate (DiI-Ac-LDL) and analyzed using flow cytometry, were 97.7 +/- 2.6% (n = 6) pure. By excluding those portions designed to isolate brain microvessels, the method was adapted to obtain RAEC cultures. RAECs do not isolate as clusters and have different morphology in culture, but respond similarly to matrices and growth medium supplements. RCMECs and RAECs have Factor VIII antigen, accumulate DiI-Ac-LDL, contain Weibel-Palade bodies, and have complex junctional structures. The activities of gamma-glutamyl transferase and alkaline phosphatase were measured as a function of time in culture. RCMECs had higher enzymatic activity than RAECs. In both RCMECs and RAECs enzyme activity decreased with time in culture. The function of endothelial cells is specialized depending on its location. This culture method allows comparison of two endothelial cell cultures obtained using very similar culture conditions, and describes their initial characterization. These cultures may provide a model system to study specialized endothelial cell functions and endothelial cell differentiation.

Topics: Animals; Aorta; Brain; Carbocyanines; Cattle; Cell Adhesion; Cell Division; Cell Separation; Cells, Cultured; Culture Media; Endothelium, Vascular; Extracellular Matrix Proteins; Fetal Blood; Flow Cytometry; Fluorescent Dyes; Humans; Microcirculation; Microscopy, Electron; Microscopy, Electron, Scanning; Rats

We examined in rats the relationship between the ordering of retinal axons in the optic pathway and the formation of a retinotopically organized projection to their primary target, the contralateral superior colliculus (SC). We have previously found that axons labeled by focal injections of 1,1'-dioctadecyl 3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI) made in temporal or nasal retina of perinatal rats commonly mistarget along the medial-lateral and rostral-caudal axes of the SC. By postnatal day (P) 11-12, the retinocollicular projection attains an adult-like topography. Incorrectly targeted axons or axon segments are removed; axons that persist terminate in the topographically appropriate part of the SC (Simon and O'Leary: Dev Biol 137:125, 1990). In the present study, we made similar DiI injections, covering less than 2% of the retinal area, in peripheral temporal, nasal, superior, or inferior retina, in rats of two age groups, embryonic day (E) 21 to P (postnatal day) 2 and P11-P17. Whole mounts of retina, optic nerve and tract, and SC, and cross sections of the optic nerve, were examined. In E21-P2 rats, retinal axons labeled from each retinal site are diffusely distributed in the SC, and poorly ordered in the optic pathway. In retina, labeled axons travel in fascicles directly from the injection site to the optic disc, but neighbor relationships begin to degrade as fascicles split and mix. Retinotopic order is virtually lost in the optic nerve; axons labeled from each injection site disperse throughout its cross-sectional area, but the labeled axons tend to be concentrated toward a specific half of the nerve depending upon their retinal origin. This slight tendency toward retinotopic order increases in the optic tract, but axons are still poorly ordered as they leave the tract and enter the SC. Targeting errors along the medial-lateral axis of the SC, but apparently not along its rostral-caudal axis, are related to the positioning of axons across the width of the optic tract. In P11-P17 rats, axons labeled from each injection site arborize only in a small, topographically correct part of the SC. However, the distributions of labeled retinal axons observed in whole mounts of the retina and optic pathway have a degree of disorder similar to those in E21-P2 rats. Further, the scatter of labeled axons in optic nerve cross sections is comparable in both age groups. Therefore, the emergence of topographic order in the retinocollicular projection

Topics: Animals; Axons; Brain Mapping; Carbocyanines; Embryo, Mammalian; Female; Gestational Age; Neural Pathways; Optic Nerve; Pregnancy; Rats; Rats, Inbred Strains; Retina; Rhodamines; Superior Colliculi; Vision, Ocular

Mitral valve interstitial cells (MVICs) are important in the structure and function of the valve. In a cell culture system that used explants to harvest MVICs, we found that after several passages, some of the cultures at confluence formed a monolayer of cobblestone-shaped cells (CB type). The rest of the cultures were, however, different, consisting of elongated cells (EL type) that formed overlapping orthogonal patterns. When compared with the MVICs-EL, cells from cultures of MVICs-CB were less adherent to a plastic substratum initially than were MVICs-EL and were more sensitive to trypsinization following culture for 3 to 7 days. Ultrastructurally, the cultures of MVICs-CB showed prominent Golgi's complexes and endoplasmic reticulum and fewer microfilaments when compared with those of MVICs-EL. Most strikingly, the CB cells contained much less alpha-smooth-muscle cell actin compared with that of EL cells. The MVICs-CB were not endothelial cells as they did not show the characteristic dense peripheral band that was present in endothelial cells in a confluent monolayer and showed only trace amounts of fluorescence when incubated with 1,1-dioctadecyl 1-3,3,3,3 tetramethyl-indocarbocyanine-percholate (Dil-)--acetylated low-density lipoprotein compared with large amounts of fluorescence with endothelial cells. Cells that were morphologically similar to MVICs-CB were present in the in vivo valve as well and also formed small islands of cells even in the primary cultures that grew out of the explant. The findings supported the hypothesis that MVICs-CB represent a distinct phenotype of the MVICs that is different from that of the MVICs-EL and is not that of either endothelial or medial smooth-muscle cells.

Topics: Actin Cytoskeleton; Actins; Animals; Carbocyanines; Cell Adhesion; Cells, Cultured; Connective Tissue; Endothelium, Vascular; Fluorescent Dyes; Interferometry; Lipoproteins, LDL; Microscopy, Electron; Mitral Valve

The ER of eggs of the sea urchin Lytechinus pictus was stained by microinjecting a saturated solution of the fluorescent dicarbocyanine DiIC18(3) (DiI) in soybean oil; the dye spread from the oil drop into ER membranes throughout the egg but not into other organelles. Confocal microscopy revealed large cisternae extending throughout the interior of the egg and a tubular membrane network at the cortex. Since diffusion of DiI is confined to continuous bilayers, the spread of the dye supports the concept that the ER is a cell-wide, interconnected compartment. In time lapse observations, the internal cisternae were seen to be in continuous motion, while the cortical ER was stationary. After fertilization, the internal ER appeared to become more finely divided, beginning as a wave apparently coincident with the calcium wave and becoming most marked by 2-3 min. By 5-8 min the ER returned to an organization similar to that of the unfertilized egg. The cortical network also changed at fertilization; it became disrupted and eventually recovered. DiI labeling allowed continuous observations of the ER during pronuclear migration and mitosis. DiI-stained membranes accumulated in the region of the microtubule array surrounding the sperm nucleus and centriole (the sperm aster) as it migrated to the center of the egg; this accumulation persisted near the centrosomes and zygote nucleus throughout pronuclear fusion and the first two mitotic cycles. We have used a new method to observe the spatial and temporal organization of the ER in a living cell, and we have demonstrated a striking reorganization of the ER at fertilization.

Topics: Animals; Carbocyanines; Endoplasmic Reticulum; Fertilization; Fluorescent Dyes; Intracellular Membranes; Ovum; Sea Urchins

Experiments were done to test the hypothesis that the avian gut is colonized by cells derived from both vagal and sacral regions of the neural crest. A fluorescent dye, diI (1,1-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate), and a replication-deficient retrovirus (LZ10; Galileo et al. 1990) were employed as tracers. Since LZ10 was constructed with lacZ of E. coli as a reporter gene, infected cells were identified by demonstrating beta-galactosidase immunoreactivity. DiI and LZ10 were injected between the neural tube and surface ectoderm (before the migration of crest cells away from the injection sites) at vagal, truncal (diI only), or sacral axial levels. The bowel was examined 4 days later in order to allow crest-derived cells sufficient time to migrate to the gut. Following injections of either tracer into the vagal crest, labelled cells were found in the gizzard and duodenum. When diI or LZ10 was injected into the sacral crest, labelled cells were seen in the post-umbilical bowel and ganglion of Remak. In the hindgut, marked cells were concentrated in the mesenchyme, just internal to the serosa, and were never observed rostral to the umbilicus. No fluorescent cells were ever found in the bowel following truncal injections of diI, although such cells were observed in sympathetic ganglia. Labelled cells were always found in dorsal root ganglia, no matter which tracer or level of the crest was injected. In embryos injected with LZ10, infected cells in the gut and dorsal root ganglia displayed a neural crest marker (NC-1 immunoreactivity). These observations confirm that the gut is colonized by cells from the sacral as well as the vagal region of the neural crest and that the emigrés from the sacral crest are confined to the post-umbilical bowel.

Topics: Animals; Carbocyanines; Cell Movement; Chick Embryo; Fluorescent Dyes; Immunohistochemistry; Intercalating Agents; Intestines; Microscopy, Fluorescence; Neural Crest; Retroviridae; Virus Replication

The perinatal development of anterior commissure projections was studied in hamsters by use of carbocyanine crystals implanted either into the commissure or into the ventrolateral prosencephalon. The earliest fascicles of growing commissural fibers had reached the midline on day 14 of gestation (E14). On E15, these fibers had entered the opposite hemisphere and reached the borders of their target regions. No waiting period was observed, since on E16 axons were already collateralizing into most targets. On P1, labelled cells were seen in all regions projecting through the anterior commissure in adults, namely, the anterior olfactory nucleus, olfactory tubercle, piriform cortex, nucleus of the lateral olfactory tract, bed nucleus of the stria terminalis, insular, perirhinal, entorhinal, and temporal cortices, as well as the amygdaloid complex. No evidence of topographical exuberance was detected. Counts of labelled neurons showed that the number of commissural cells increased gradually after birth. It is concluded that the development of paleocortical connections through the anterior commissure employs progressive strategies, lacking the regressive phenomena that are characteristic of the neocortical projections through the corpus callosum.

Topics: Aging; Animals; Animals, Newborn; Carbocyanines; Cerebral Cortex; Corpus Callosum; Cricetinae; Female; Fluorescence; Gestational Age; Histocytochemistry; Mesocricetus; Neural Pathways; Pregnancy

Glial cells of the human retina participate in various pathologic processes characterized by cell migration, proliferation, and extracellular matrix production. To study these events in vitro, a procedure was developed to obtain primary cultures of human retinal glial cells. The cultures resulting from the processing of 130 globes contained cells with variable morphology including bipolar and multipolar or stellate cells. Most cells in the primary culture were labeled with antisera to the glial fibrillary acidic protein. The cultures were also examined with antibodies directed against factor VIII-related antigen and muscle-specific actin to determine the presence of endothelial cells and pericytes. A variable contamination of cells staining for the latter was found in these cultures (usually less than 10%). Together, these data indicated that the primary cultures arose principally from glial cells of the human retina but did not precisely identify the cell of origin.

Topics: Actins; Carbocyanines; Cells, Cultured; Factor VIII; Fluorescent Antibody Technique; Glial Fibrillary Acidic Protein; Humans; Lipoproteins, LDL; Microscopy, Fluorescence; Neuroglia; Retina

The soma location and peripheral connectivity of motoneurons in abdominal segments of the embryo and larva of the fruitfly, Drosophila melanogaster are described as an initial step in determining the mechanisms by which motoneurons make connections with their target muscles in a genetically accessible organism. Embryonic motoneuron somata were retrogradely labelled by application of the fluorescent dye, DiI, to the whole peripheral nerve or to its separate anterior or posterior fascicles in segments A5-A7 of late stage 15/early stage 16 embryos. This technique reveals a stereotyped, segmentally repeated population of 34 motoneurons per hemisegment, several of which can be individually identified from their soma position. The same set of motoneurons was revealed in third instar larvae of D. melanogaster by cobalt backfilling of abdominal peripheral nerves, although the positions of some of these neurons change during larval development. The peripheral connectivity and axon morphology of several of the abdominal motoneurons was determined by intracellular injection with Lucifer Yellow in stage 16 embryos. For the motoneurons with axons in the anterior fascicle there is no clear relationship between somata groupings and the muscle targets innervated: contrary to earlier claims, these motoneurons arborize over both ventral and dorsal muscles. Individual motoneurons possess a stereotyped pattern of terminal arborization.

Topics: Abdomen; Animals; Axons; Carbocyanines; Cobalt; Drosophila melanogaster; Embryo, Nonmammalian; Isoquinolines; Larva; Motor Neurons; Muscles; Neural Pathways

Tetrodotoxin was injected into one eye of chick embryos so as to block activity in the target territory of the isthmo-optic nucleus (ION) during its period of neuronal death. This markedly reduced the neuronal death and thereby prolonged the survival of some 'aberrantly' projecting neurons which would normally all die. In addition, the cytoarchitecture of the ION developed abnormally. Since these two effects differ markedly in their dose-dependence and in other ways, they cannot both be explained by changes in the strength of a single retrograde signal.

Topics: Animals; Axons; Brain; Carbocyanines; Cell Death; Chick Embryo; Electric Stimulation; Electrophysiology; Neurons; Retina; Tetrodotoxin

Fluorescence microscopy performed on living cells is a valuable technique for elucidating patterns of cell growth in vitro over artificial biomaterials such as vascular grafts, and for in vivo studies such as identification and treatment of atherosclerotic plaques. Two fluorescent dyes of particular value for vital fluorescence studies are Rhodamine-123 and 3,3'-dioctadecylindocarbocyanine-labeled low density lipoprotein (DiI-LDL). We examined the toxicity of these two dyes and of dimethylsulfoxide (DMSO), a solvent used in Rhodamine-123 studies, on the growth of MRC5 human fetal fibroblasts in monolayer culture. Two parameters of cell growth were quantitated: Cell number (a measure of proliferation), and cell area (a measure of cell spreading), based on microscopic images obtained at the start and end of a 48-h growth period after brief exposure (0.5 h) to test solutions. We found that the recommended solvent for solubilization of Rhodamine-123, DMSO, caused cessation of cell proliferation and actual reduction in the area covered by adherent fibroblasts at concentrations of as low as 0.1% (vol:vol). Rhodamine-123 made up from an aqueous stock solution modestly retarded proliferation and spreading, and there was no significant effect of DiI-LDL on these parameters over prolonged periods of exposure (up to 24 h) in culture. These results demonstrate that the most toxic substance for growing fibroblasts was the solvent DMSO. We conclude that both the solvent vehicle and fluorescent dye should be carefully examined for potential toxicity before such dyes are used for vital fluorescence studies of living cells.

Topics: Carbocyanines; Cell Division; Cell Survival; Cells, Cultured; Dimethyl Sulfoxide; Fetus; Fibroblasts; Fluorescent Dyes; Humans; Kinetics; Lipoproteins, LDL; Rhodamine 123; Rhodamines

Three enriched populations of cells [C alpha, non-steroidogenic cells less than or equal to 15 microns in diameter; R1, small (less than or equal to 15 microns) steroidogenic cells; and R3, large (greater than 20 microns) steroidogenic cells] have been isolated from the macque corpus luteum using flow cytometry based on light scatter properties. To determine whether the cell populations differ in their ability to bind and internalize low-density lipoprotein (LDL), collagenase-dispersed cells were prepared from the corpus luteum of rhesus monkeys at midluteal phase of the menstrual cycle. Cells were incubated in Hams F-10 medium containing fluorescent-tagged LDL (DiI-LDL). Optimal labeling occurred at 10 micrograms DiI-LDL/10(6) cells.ml incubated for 20 min at 37 degrees C. Labeled cells were analyzed and sorted by flow cytometry based on light scatter and fluorescence. Only 8.2 +/- 1.0% (n = 10) of C alpha cells exhibited fluorescence intensity greater than the autofluorescence of unlabeled cells. In contrast, 52.3 +/- 3.4% of R1 cells and 83.9 +/- 2.3% of R3 cells were fluorescent. Uptake of DiI-LDL was competitively inhibited when cells were conincubated with either unlabeled monkey or human LDL, but human high-density lipoprotein and very low-density lipoprotein were less effective. Progesterone (P) production by fluorescent [DiI-LDL(+)] R1 luteal cells was increased (P less than 0.001) in the presence of pregnenolone, but not human (h) CG, consistent with earlier results for the R1 population. Surprisingly, basal P production by the nonfluorescent [DiI-LDL(-)] R1 cells was similar to that by fluorescent cells and was stimulated by both hCG (P less than 0.01) and pregnenolone (P less than 0.001). Basal P production by DiI-LDL(+) R3 cells was nearly 10-fold greater than that by DiI-LDL(-) R3 cells. P secretion by both DiI-LDL(+) and (-) R3 cells was stimulated by hCG (P less than 0.01) and pregnenolone (P less than 0.001). DiI-LDL(-) C alpha cells produced barely detectable levels of P, but DiI-LDL(+) C alpha cells secreted P at levels similar to R1 cells. We conclude that: 1) multiparameter (light scatter and fluorescence) cell sorting is a useful method for separating enriched populations of cells from the corpus luteum; and 2) small and large luteal cell populations from the macaque corpus luteum consist of subtypes of steroidogenic cells that differ in lipoprotein uptake and/or gonadotropin sensitivity.

Topics: Animals; Binding, Competitive; Carbocyanines; Cell Separation; Chorionic Gonadotropin; Female; Flow Cytometry; Fluorescent Dyes; Kinetics; Lipoproteins, LDL; Luteal Cells; Macaca mulatta; Microscopy, Electron; Microscopy, Fluorescence; Pregnenolone; Progesterone

Postmortem morphometric investigation of autopsy material is helpful for understanding the alterations cells undergo during life. The present work was undertaken to determine the morphologic features of human retinal ganglion cells during the first 2 days after death. Ganglion cells of 16 retinas were obtained from eyes, the corneas of which had been retrieved for keratoplasties. The ganglion cells were stained with the fluorescent carbocyanine dye Dil and investigated 12 to 36 hours postmortem. All labeled cells identified as ganglion cells had well preserved, type-specific dendritic profiles that made it possible to catalog them according to the established classes of ganglion cells. The various types of cells have in common some typical morphologic changes that occur during the postmortem period of observation: swelling and varicosity in the cell bodies and along the dendritic branches and the axons. These swellings were consistent throughout the postmortem period and did not hinder the identification of particular types of ganglion cells. The two major types (parasol and midget) and various subtypes of ganglion cells were identified morphologically. There are two distinct morphologic types of midget cells which differ from each other in the pattern of dendritic branching. Particular emphasis was given to analyze the dendritic geometry of the large parasol cells. A new finding in the human retina is the frequent presence of large parasol ganglion cells, the axons of which course along aberrant intraretinal paths, especially in the extreme periphery of the retina.

Topics: Axons; Carbocyanines; Dendrites; Female; Fluorescent Dyes; Humans; Image Processing, Computer-Assisted; Male; Microscopy, Fluorescence; Organ Preservation; Retinal Ganglion Cells

Receptors for low-density lipoprotein are necessary for high-affinity uptake of lipid and protein essential to cell structure and function. Distinct receptors for acetoacetylated low-density lipoprotein internalize oxidized or enzymatically modified low-density lipoprotein and extracellular matrix components. We identified low-density lipoprotein receptors on cultured human and monkey corneal endothelial cells by the avid incorporation of fluorescently labeled low-density lipoprotein that was competitively inhibited by excess unlabeled low-density lipoprotein but not by unlabeled acetoacetylated low-density lipoprotein. Specific uptake of labeled low-density lipoprotein was greatest in nonconfluent, growing cells and increased after low-density lipoprotein deprivation. Intact endothelial monolayers of whole human cornea also incorporated low-density lipoprotein but not acetoacetylated low-density lipoprotein. After scratch injury of human corneas, spreading endothelium adjacent to areas of cell loss internalized more fluorescent low-density lipoprotein than cells distant from the injury. Blood-aqueous barrier breakdown occurring in ocular diseases and after surgical and nonsurgical trauma may allow leakage of circulating low-density lipoprotein, which provides a rich supply of lipid and protein for endothelial use. Efficient, receptor-mediated, low-density lipoprotein uptake may facilitate repair of damaged corneal endothelial membranes and regeneration of intact, functional cell monolayers.

Topics: Acetoacetates; Animals; Carbocyanines; Cell Division; Cell Survival; Cells, Cultured; Child, Preschool; Cornea; Corneal Injuries; Endothelium, Corneal; Fluorescent Dyes; Humans; Lipoproteins, LDL; Macaca mulatta; Rabbits; Receptors, LDL; Wound Healing

The gastrointestinal territories innervated by the gastric, celiac, and hepatic abdominal vagi were identified in rats with selective branch vagotomies by means of 1) anterograde tracing with the carbocyanine dye DiI injected into the dorsal motor nucleus and 2) measurement of cervical vagal stimulation-induced motility responses throughout the gut axis. Presence of DiI-labeled vagal terminals in the myenteric plexus and evoked motility responses were well correlated across the sampled gastrointestinal (GI) sites. In animals with only the two gastric branches intact, the entire stomach and the most proximal duodenum showed significant motility responses and were densely innervated, having DiI-labeled vagal terminals in almost every ganglion. The hepatic branch was found to primarily innervate the duodenum, with minor projections to the distal antral stomach and the intestines. The two celiac branches were found to almost exclusively innervate the jejunum, ileum, cecum and entire colon, and, together with the other vagal branches, the duodenum. Therefore, while there is some degree of specific innervation by the abdominal vagal branches of the oral-to-anal gut axis, which could be called "viscerotopic," the considerably overlapping innervation of the duodenum does not satisfy a viscerotopy criterion and needs further functional analysis.

Topics: Animals; Carbocyanines; Digestive System; Digestive System Physiological Phenomena; Electric Stimulation; Gastrointestinal Motility; Male; Microscopy, Fluorescence; Rats; Rats, Inbred Strains; Vagotomy; Vagus Nerve

Selective axonal growth at a series of choice points along pathways is essential for the establishment of precise motoneuron projections. To reveal some of the molecules responsible for this selective growth of motoneuron axons, this study investigates the phenomenon of why motoneurons extend axons outside the spinal cord, whereas interneurons do not. Axonal growth in the chick embryonic spinal cord at early stages of development was examined immunohistochemically. MAb SC1 staining of serial sections selectively revealed the entire distribution of motoneuron axons in the embryo. In the cervical segments, some motoneurons, called dorsal motoneurons, extended axons outside the cord via the dorsal root entry zone. The axons of both dorsal and ventral motoneurons were distributed only in the anterior half of the sclerotome; inside the cord motoneuron cell bodies and axons were distributed evenly along the anterior-posterior axis. Motoneurons and interneurons extended axons during the same period, and although the growth cones of both were intermixed in the same locations, only motoneuron axons grew out from the cord. The outgrowth of all st 19 motoneurons from the cord through either the dorsal or ventral exit zone strongly suggests that extension of their axons outside the cord is a selective rather than random process.

Topics: Animals; Axons; Carbocyanines; Chick Embryo; Fluorescent Dyes; Immunoenzyme Techniques; Interneurons; Motor Neurons; Spinal Cord

Using the DiI and intracellular Lucifer Yellow labeling techniques in the rat, we have demonstrated that the unilateral neonatal thalamotomy does not result in retention of transient dendritic spines of ipsilaterally projecting retinal ganglion cells (IPRGCs), although the thalamotomy is known to retain the normally transient IPRGCs (Chan et al., Dev. Brain Res., 49 (1989) 265-274). These results suggest that the process of elimination or retraction of transient dendritic spines occurs in retinal ganglion cells during development regardless of whether they make connections with appropriate or inappropriate loci in the visual targets, and/or a decrease in interactions with neighboring retinal ganglion cells.

Topics: Aging; Animals; Animals, Newborn; Carbocyanines; Dendrites; Isoquinolines; Rats; Rats, Inbred Strains; Retinal Ganglion Cells; Thalamus

We have studied in rats the topographic targeting of retinocollicular axons anterogradely labeled by focal retinal injections of the axon tracer DiI. We find that developing retinal axons widely mistarget along both the medial-lateral and the rostral-caudal axes of the superior colliculus (SC). In neonatal rats, labeled axons originating from injection sites in the temporal periphery covering less than 1% of the retina grow over most of the contralateral SC, suggesting that the growth cones of many axons initially fail to recognize their appropriate target region at the rostral SC border. Some of these axons correct their targeting errors and are retained; most do not and are eliminated. In neonates, peripheral nasal axons transiently develop branches throughout the SC. Branches formed by nasal axons are later restricted to a discrete terminal zone at the topographically appropriate, caudal SC border. At the neonatal stage, injections in temporal or nasal retina do result in a zone of increased labeling in the topographically correct region of the SC, but this zone is considerably larger than that labeled by a similar injection at a later stage. Thus, although the early projection is very diffuse, there is some bias for the correct region of the SC. Our findings indicate that in rats, developing retinal axons show only a limited specificity in their topographic targeting and branching. We conclude that mechanisms in addition to directed axon growth are required to establish the order characteristic of mature mammalian retinal projections.

Topics: Aging; Animals; Axonal Transport; Axons; Carbocyanines; Fluorescent Dyes; Functional Laterality; Optic Nerve; Rats; Retina; Superior Colliculi; Visual Pathways

After injections of the posterior part of the lateral zone of the area dorsalis telencephalic (Dlp) with either horseradish peroxidase or the newly available carbocyanine dye DiI, efferent cells were labeled in the valvula cerebelli of the mormyrid fish, Gnathonemus petersii. This may be a unique connection for this group of electrosensory teleosts, since no other vertebrate has ever been reported before to have a direct cerebello-telencephalic projection.

Topics: Animals; Axonal Transport; Brain; Carbocyanines; Cerebellum; Efferent Pathways; Electric Fish; Fluorescent Dyes; Horseradish Peroxidase; Telencephalon

The entire process of normal development of a muscle nerve to a muscle (middle interradial muscle) in the tail region of the medaka (Oryzias latipes) is briefly reported. The nerve was stained immunohistochemically by using anti-neurofilament protein antibodies or stained by HRP and DiI labeling methods. The muscle was stained immunohistochemically by using anti-troponin T and anti-desmin antibodies. The smallness and transparency of the medaka embryos provide us with an opportunity to examine nerve-muscle development in whole-mount specimens. Our observations suggest that prior to the appearance of the middle interradial muscle a neural pathway has established, extending from the starting point to the 'door step' of the muscle.

Topics: Animals; Carbocyanines; Fluorescent Dyes; Horseradish Peroxidase; Immunohistochemistry; Motor Neurons; Muscle Development; Muscles; Oryzias; Spinal Nerves; Staining and Labeling

The vagal innervation of the different layers of the rat gastrointestinal wall was identified with the fluorescent carbocyanine dye Dil, injected into the dorsal motor nucleus of the vagus (dmnX). Multiple, bilateral injections were used to label all dmnX preganglionic motoneurons, and as a consequence, most of the vagal primary afferents that terminate in the adjacent nucleus of the solitary tract (nts) were also retrogradely and transganglionically labeled. With Fluorogold used to label the enteric nervous system completely and specifically, the Dil-labeled vagal profiles could be visualized and quantified in their anatomical relation to the neurons of the myenteric and submucous ganglia. In the myenteric plexus, vagal fibers and terminals were found throughout the gastrointestinal tract as far caudal as the descending colon, but there was a general decreasing proximodistal gradient in the density of vagal innervation. All parts of the gastric myenteric plexus (fundus, corpus, antrum), as well as the proximal duodenum, were extremely densely innervated, with vagal fibers and terminals in virtually every ganglion and connective. Further caudally, both the percentage of innervated myenteric ganglia and the average density of label within the ganglia rapidly decreased, with the exception of the cecum and proximal colon, where up to 65% of the ganglia were innervated. In the gastric and duodenal submucosa very few and in the mucosa no vagal fibers and terminals were found. With both normal epifluorescence and laser scanning confocal microscopy, highly varicose or beaded terminal structures of various size and geometry could be identified. The Dil injections, which impregnated the dmnX as well as the adjacent nts, resulted in retrograde and anterograde labeling of all the previously reported forebrain connections with the dorsal vagal complex. We conclude that the myenteric plexus is the primary target of vagal innervation throughout the gastrointestinal tract, and that its innervation is more complete than previously assumed. In contrast, vagal afferent (and efferent) innervation of mucosa and submucosa seems conspicuously sparse or absent. Furthermore, the use of more focal injections of Dil offers the prospect to simultaneously identify specific subsets of vagal preganglionics and their central nervous inputs.

Topics: Animals; Brain; Carbocyanines; Digestive System; Fluorescent Dyes; Male; Microscopy, Fluorescence; Motor Neurons; Nerve Endings; Neurons, Afferent; Neurons, Efferent; Nodose Ganglion; Rats; Rats, Inbred Strains; Staining and Labeling; Stereotaxic Techniques; Stilbamidines; Vagus Nerve

Motoneurons of adult frog spinal cord have been retrogradely labeled with the carbocyanine derivative diI. Spinal cords were then dissociated and the labeled motoneurons partially purified by centrifugation over a bovine serum albumin (BSA) density gradient. The resulting cell suspension was plated on a substrate of innervated muscle extracellular matrix (ECM) to which the motoneurons attached and extended processes. Labeled adult motoneurons survived for more than 4 weeks in a defined medium in the absence of added serum or growth factors. These cultures of adult motoneurons provide a favorable preparation for studying molecular factors that influence process outgrowth and synapse formation.

Topics: Animals; Axonal Transport; Carbocyanines; Cell Survival; Cells, Cultured; Culture Media; Culture Techniques; Extracellular Matrix; Fluorescent Dyes; Macrophages; Male; Motor Neurons; Rana pipiens; Spinal Cord

In order to study the pattern of innervation of taste buds and the surrounding epithelium, the carbocyanine dye diI was applied to the nerve stump in isolated, paraformaldehyde-fixed barbels obtained from channel catfish, Ictalurus punctatus. After a diffusion period of 7-41 days, the barbels were sectioned on a vibratome and examined with epifluorescence. Labeled axons were observed up to 1 cm from the site of application. Frequently, a fascicle of labeled axons turned outward toward the epithelium to innervate taste buds or to end apparently as free endings within the epithelium. Within 2-3 mm of the dye-application site, many taste buds contained one or at most 5-10, labeled spindle-shaped, presumed receptor, cells. In taste buds containing multiple labeled cells, the cells usually were arranged as intertwined pairs or triplets rather than being homogeneously distributed within the taste bud. In a few cases, labeled basal cells could be discerned among the labeled axons of the basal plexus. The cells of the taste bud apparently were labeled by transcellular passage of the dye from the nerve fibers into the cells. The limited number of labeled cells within each taste bud may indicate a special relationship between these cells and the nerve fibers innervating them.

Topics: Afferent Pathways; Animals; Carbocyanines; Fluorescent Dyes; Ictaluridae; Sense Organs; Taste Buds

The role of neuronal activity in the refinement of the retinotectal projection in chick embryos was studied using tetrodotoxin (TTX) which blocks sodium channels and grayanotoxin I (GTX) which opens the channels. Optic nerve fibers were traced by the fluorescent dye DiI. The toxins were injected into the eyeball during the refinement phase of retinotectal projection (either the 13th, 14th or 15th day of incubation). A tiny crystal of DiI was placed in the temporal part of the retina. In the control embryos, fibers caudally overshooting and arborizations outside the terminal zone were found before maturation of retinotectal projection. Overshooting fibers regressed linearly, and aberrant arborizations reduced quadratically, and then the precise retinotopic map is formed by stage 44 (18th day of incubation). Both TTX and GTX interfered with the regression of overshooting fibers and arborizations outside the terminal zone. The initial action of the toxins are reverse, but their final effect may be comparable, and they may interfere with synchronous firing of the neighboring fibers. Our results emphasize a role of neuronal activity in the refinement of retinotectal projection.

Topics: Action Potentials; Animals; Carbocyanines; Chick Embryo; Diterpenes; Fluorescent Dyes; Neuromuscular Depolarizing Agents; Optic Nerve; Pharmaceutical Vehicles; Retina; Retinal Ganglion Cells; Superior Colliculi; Tetrodotoxin

The fluorescent carbocyanine dye DiI can be used for retrograde and anterograde labeling of neuronal pathways. To investigate the possible neurochemical identity of DiI-labeled neuronal cell bodies and terminals, we used a procedure for double-labeling of the same tissue with antisera to specific neuroactive substances. This procedure involves visualizing the immunohistochemical label with an FITC-conjugated secondary antiserum. Both labels can be viewed in the same tissue by fluorescence microscopy, and individual cell bodies and processes double-labeled with DiI and antiserum can be identified by switching between filter sets appropriate for rhodamine (to see the DiI labeling) and for fluorescein (to see the immunhistochemical labeling). The method has been used with primary antisera to excitatory and inhibitory amino acid neurotransmitters, as well as to neuropeptides, and is likely to be useful with antibodies against a wide variety of substances. Several other immunocytochemical methods were found to be incompatible with DiI labeling.

Topics: Animals; Carbocyanines; Fluorescein-5-isothiocyanate; Fluoresceins; Fluorescent Dyes; Immune Sera; Immunohistochemistry; Microscopy, Fluorescence; Quinolines; Thiocyanates; Visual Cortex

Low-density lipoprotein (LDL) is a carrier of the cholesterol found in human plasma. Cells utilize cholesterol for membrane synthesis by taking up LDL via receptor-mediated endocytosis. In the present study, interactions of LDL with human malignant glioma cell lines (U-251 MG and KMG-5) were investigated biochemically and morphologically. The LDL, labeled with the fluorescent dyes 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine (DiI) and fluorescein isothiocyanate (FITC), was internalized by both cell processes and cell bodies. Reductive methylation of DiI-labeled LDL, which abolishes the ability of the cell to bind to the LDL receptor, prevented the internalization of the cholesterol moiety of LDL. Cellular binding of 125I-LDL to U-251 MG cells at 4 degrees C revealed the presence of a specific saturable-associated receptor (dissociation constant (Kd) approximately 38 micrograms/ml). Endocytic uptake of 125I-LDL or 3H-cholesterol oleate-labeled LDL (3H-LDL) at 37 degrees C demonstrated the cell-associated 125I-LDL and 3H-LDL increase. The intracellular degradation of protein moiety increased linearly with time. Reductive methylation of 3H-LDL led to a remarkable decrease in the cell-associated cholesterol moiety of LDL. The difference in uptake of the cholesterol moiety of LDL between U-251MG cells and KMG-5 cells showed that the U-251MG cells, which proliferate more actively than KMG-5 cells, take up more of the cholesterol moiety of LDL than do the KMG-5 cells. Thus, LDL cholesterol seems to be endocytosed predominantly via the LDL receptor present on the plasma membrane of malignant glioma cells. In addition, for growth, these cells may require large amounts of the cholesterol moiety of LDL.

Topics: Brain Neoplasms; Carbocyanines; Cholesterol, LDL; Endocytosis; Fluorescein-5-isothiocyanate; Fluoresceins; Glioma; Humans; Microscopy, Fluorescence; Radioligand Assay; Receptors, LDL; Thiocyanates; Tumor Cells, Cultured

Retinotectal projection is precisely organized in a retinotopic manner. In normal projection, temporal retinal axons project to the rostral part of the tectum, and nasal axons to the caudal part of the tectum. The two-dimensional relationship between the retina and the tectum offers a useful experimental system for analysis of neuronal target recognition. We carried out rotation of the tectal primordium in birds at an early stage of development, around the 10-somite stage, to achieve a better understanding of the characteristics of target recognition, especially the rostrocaudal specificity of the tectum. Our results showed that temporal retinal axons projected to the rostral part of the rotated tectum, which was originally caudal, and that nasal axons projected to the caudal part of the rotated tectum, which was originally rostral. Therefore, the tectum that had been rotated at the 10-somite stage received normal topographic projection from the retinal ganglion cells. Rostrocaudal specificity of the tectum for target recognition is not determined by the 10-somite stage and is acquired through interactions between the tectal primordium and its surrounding structures.

Topics: Animals; Brain; Carbocyanines; Chick Embryo; Chimera; Coturnix; Neural Pathways; Neuronal Plasticity; Retina; Transplantation, Heterologous

The membrane potential in Entamoeba is an important driving force for the uptake of substrates. In Entamoeba invadens PZ a membrane potential of -36 mV was obtained when Nernst equation was applied to the distribution at equilibrium of 86Rb+ in the presence of valinomycin. This could explain the levels of accumulation of up to 4 times found for positively charged substrates. Membrane potential was diminished by depolarizing conditions (high external K+ concentration in the presence of valinomycin). Moreover, we recorded continuously the membrane potential of Entamoeba invadens PZ and Entamoeba histolytica HM1 using the fluorescent lipophilic cation diisopropylthiodicarbocyanine. It was found that the uptake of this cation by the amoebae was fast in both species, conditions that modify the membrane potential (hyperpolarization and depolarization) produced changes in the fluorescence of the dye in agreement with its reported capability to detect variations in membrane potential. It can be concluded that these microorganisms have a membrane potential negative inside them.

Topics: Animals; Carbocyanines; Entamoeba; Entamoeba histolytica; Membrane Potentials; Potassium; Rubidium; Valinomycin

Experiments were done in order to test the hypothesis that neurons in the bowel send axonal projections to the pancreas and can modify pancreatic activity. pancreatic injections of the retrograde tracer, Fluoro-Gold, labeled neurons in the myenteric plexus of the antrum of the stomach and in the first 6 cm of the duodenum. this labeling was not due to the diffusion of Fluoro-Gold from the pancreas, because the injections did not label longitudinal muscle cells overlying labeled ganglia in the bowel or neurons in the phrenic nerve nucleus or nucleus ambiguous; nor were enteric neurons labeled if insufficient time was allotted for retrograde transport. More Fluoro-Gold labeled neurons were found in the stomach (9.2 +/- 0.9/ganglion) than in the duodenum (3.8 +/- 0.3/ganglion; p less than 0.001). Neurons were found in myenteric ganglia of both duodenum and stomach that were doubly labeled by retrograde transport of Fluoro-Gold and anti-serotonin (5-HT) sera. In addition, thick bundles of 5-HT immunoreactive nerve trunks were found to run between the duodenum and the pancreas. Most 5-HT immunoreactive axons in the pancreas terminated in ganglia, although some fibers were also observed near acini, ducts, vessels, and islet cells. The B subunit of cholera toxin (B-CT) was microinjected into single myenteric ganglia in order to determine if axon terminals in the pancreas would become labeled by anterograde transport in the pancreas. B-CT labeled bundles of axons in the pancreatic stroma. Branches of these bundles entered the pancreatic parenchyma and varicose B-CT labeled terminal axons were found in pancreatic ganglia and in proximity to acinar and insulin immunoreactive cells. The intercalating fluorochrome 1, 1', dioctadecyl-3,3,3',3'-tetramethylcarbocyanine perchlorate (Dil), which moves by lateral diffusion to outline entire cells, was introduced by microinjection into individual myenteric ganglia of fixed preparations. Fluorescence was seen in sequential observations to move away from the injected ganglion along connectives of the myenteric plexus. After about a month, neurons in ganglia at some distance from the injection site displayed Dil fluorescence as did nerve bundles that exited from the myenteric plexus and pierced the longitudinal muscle in the direction of the pancreas. Varicose Dil fluorescent terminal varicosities were also observed int he pancreas. These observations indicate that there is an extensive entero-pancreatic innervation.(ABSTRACT

Topics: Animals; Axons; Carbocyanines; Cholera Toxin; Electron Transport Complex IV; Female; Fluorescent Dyes; Injections; Intestines; Neurons; Pancreas; Rats; Serotonin; Stilbamidines; Synaptic Transmission; Tetrodotoxin; Veratridine

During development of the mammalian CNS, axons encounter multiple pathway choices on their way to central target structures. A major pathway branch point in the visual system occurs at the optic chiasm, where retinal ganglion cell axons may either enter the ipsilateral or the contralateral optic tract. To investigate whether embryonic mouse retinal ganglion cell axons, upon reaching the optic chiasm, selectively grow into the correct pathway, developing retinal ganglion cells were retrogradely labeled using either 1,1'-dioctadecyl- 3,3,3',3'-tetramethylindocarbocyanine perchlorate (Dil) or fluorescent microspheres placed into the optic tract on one side. The distribution of ipsilaterally and contralaterally projecting ganglion cells in the embryo was then examined and compared to that of the adult animal. Results show that axon routing at the chiasm is already extremely adult-like as early as embryonic day 15 (E15), shortly after retinal axons arrive at the chiasm. [Retinal ganglion cell neurogenesis = E11-E18 (Drager, 1985); birth = E21.] Throughout the development of this pathway, routing errors are infrequent and are on the order of only about 3-8/1000 retinal ganglion cells. Thus, embryonic retinal ganglion cell axons do not project randomly at the optic chiasm but instead appear to be highly specific in their choice of pathway. To learn how correct pathway choices are made, retinal axons were retrogradely labeled with Dil and their trajectories at the optic chiasm were reconstructed. Results show that ipsilaterally and contralaterally projecting axons are highly intermixed as they enter the chiasm region but selectively grow into the correct pathway. For example, a contralaterally projecting axon near the entrance of the ipsilateral optic tract will turn and bypass this pathway and grow towards the midline to head into the contralateral optic tract. Similarly, axons far away from the ipsilateral optic tract frequently turn abruptly at right angles to enter the ipsilateral tract, directly crossing over contralaterally projecting axons heading to the opposite side. The sorting out of intermixed ipsilaterally and contralaterally projecting retinal axons into the appropriate optic tracts strongly suggests the presence of specific guidance cues at the optic chiasm during embryonic development. Together, results from this study demonstrate that the pattern of axon projection at the adult mammalian optic chiasm is gradually built upon a highly specific patt

Topics: Animals; Axons; Carbocyanines; Embryonic and Fetal Development; Fluorescence; Fluorescent Dyes; Mice; Mice, Inbred C3H; Microspheres; Optic Chiasm; Retina; Retinal Ganglion Cells; Synaptic Transmission

The lipophilic carbocyanine fluorescent label DiI was injected in one eye of aldehyde-fixed embryonic or postnatal hamsters and the brains were examined using flat-mounts of the chiasm region, of the lateral surface of the brainstem, or of the midbrain tectum. Single axons could be discerned within the optic nerves and along the optic tract. Many fibers were tipped by growth cones, ending at various levels of the brainstem. Fine details of retinofugal axon morphology, including varicosities, branch-points and filopodial extensions on growth cones were visible in the flat-mounts. Such preparations allow a high-resolution view of labeled axons which course near the surface of the brain. It is possible, with this method, to simultaneously examine the morphogenesis of multiple collateral arbors on single fibers which project to more than one terminal zone.

Topics: Animals; Animals, Newborn; Axons; Brain Stem; Carbocyanines; Cricetinae; Fetus; Histological Techniques; Retina; Synaptic Transmission; Visual Pathways

The molecule 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI) is a fluorescent dye which diffuses within cell membranes. The properties of DiI diffusion and fluorescence are maintained in aldehyde-fixed tissue, thereby allowing selective neuronal tracing post mortem. We describe three modifications of this tracing method. First, while DiL diffuses along neuronal membranes the tissue can be decalcified in EDTA at 37 degrees C. Tracing in decalcified tissue extends the possible application of the DiI technique to the investigation of neuronal tissue enclosed in bony structures. Second, we describe a protocol that allows sectioning of DiI-injected tissue on a cryostat with minimal subsequent spread of DiI in dried sections. Third, we demonstrate that DiI label of fluorescent neurons in cryosections as well as Vibratome sections can be photo-oxidated and converted to a stable diaminobenzidine reaction product. The photo-converted DiI label is electron dense and allows analysis of labeled cell bodies and processes at the electron microscopic level. DiI does not stay confined to the surface cell membrane in fixed tissue but reaches internal organelles, presumably via membranes of the endoplasmic reticulum, and concentrates in microsomal structures adjacent to mitochondria. Photoconversion of DiI label is compatible with gold immunocytochemistry. Long-term incubation and subsequent photoconversion of post-mortem DiI-labeled neurons provides remarkable tissue preservation at the ultrastructural level.

Topics: Animals; Carbocyanines; Decalcification Technique; Evaluation Studies as Topic; Fluorescent Dyes; Freezing; Histological Techniques; Light; Microscopy, Electron; Neurons; Quinolines; Time Factors

Centrifugal fibers to the retinas of chick embryos and hatched chicks have been examined and traced following staining by diffusion along their axonal membranes of the carbocyanine dye DiI in fixed tissue. In the older embryos and hatched chicks, the report of Dogiel (Arch. Mikrosk. Anat. 44:622-648, 1895) has been confirmed that there are two very different morphological types of centrifugal fiber. The restricted type ends as a relatively thick fiber, lacking varicosities, that runs for a short distance in the most sclerad level of the inner plexiform layer before terminating in a pericellular nest overlying the flask-shaped body of a single amacrine cell. Thin filaments occasionally leave the pericellular net, apparently to terminate on adjacent cells. The widespread type also runs in the most sclerad level of the inner plexiform layer, but it is thin, varicose, and highly branched, and its terminal arbor may span more than 1 mm, remaining at the same level. Both types of terminal arbor issue from parent axons in the optic fiber layer of the retina. A single parent axon gives either a single terminal fiber of the restricted type or several terminals of the widespread type, but never a mixture of the two. It is argued that the restricted and widespread types originate respectively from the neurons of the contralateral isthmo-optic nucleus and from the "ectopic" neurons scattered outside the isthmo-optic nucleus. In development, the centrifugal fibers reach the retina between E9 and E10 and initially run radially in the optic fiber layer, parallel to the retinofugal fibers but avoiding the dorsal retina. They dive into the inner plexiform layer at about E12. By E13, the terminal arbors are forming, and the widespread and restricted types can already be distinguished. The widespread type continues to increase its territory until about E18, and then appears to remain stable, whereas the restricted type attains its maximum ramification between E13 and E15 and then contracts. Prior to the retraction, the terminal territories of the restricted type fibers overlap, which may provide the anatomical basis for the interaxonal competition that apparently contributes to neuronal death in the isthmo-optic nucleus between E13 and E16. Axons of ganglion cells exhibit transient side branches between E11 and E13; these never reach as deep as the level where the centrifugal fibers run.

Topics: Animals; Axons; Carbocyanines; Cell Survival; Chick Embryo; Coloring Agents; Dendrites; Efferent Pathways; Kainic Acid; Nerve Endings; Neurons, Efferent; Retina; Retinal Ganglion Cells

We analyzed the pattern and development of the earliest tracts and followed pathfinding by the growth cones of an identified cluster of neurons in the brain of zebrafish embryos. Neurons were labeled with an antibody which labels many embryonic neurons, a lipophilic axonal tracer dye, and intracellular dye injections. The embryonic brain is extremely simple, and at 28 hr of development, the forebrain and midbrain consist of 8 main axonal tracts which are arranged as a set of longitudinal tracts connected by commissures. Each tract is established by identified clusters of approximately 2-12 neurons found in discrete regions of the brain. Many identified clusters of neurons project axons in a defined direction appropriate for the cluster and have axons with stereotyped trajectories, suggesting that their growth cones follow cell-specific routes. This was confirmed with intracellular dye injections for neurons of the nucleus of the posterior commissure. The growth cones of these neurons arrive at a site in the anterior tegmentum where 4 tracts meet. At this site, they could, in principle, turn in a number of directions but always extend posteriorly into one of the tracts. The pattern of pathfinding by these growth cones suggests the testable hypothesis that the growth cones of identified clusters of neurons establish the simple set of early tracts by selecting cluster-specific pathways at such intersections in order to reach their targets in the brain.

Topics: Acetylation; Animals; Antibodies, Monoclonal; Axons; Brain; Carbocyanines; Cyprinidae; Diencephalon; Fluorescent Dyes; Isoquinolines; Microscopy, Electron; Neurons; Tegmentum Mesencephali; Telencephalon; Tubulin; Zebrafish

We have examined the migration of murine macrophages from the vascular compartment to normal and inflammatory tissues by the adoptive transfer of resident peritoneal macrophages (RPM phi) fluorescently labeled with the hydrophobic dye 1,1'-dioctadecyl 3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI). After initial labeling of the plasma membrane of RPM phi, the dye accumulated stably in intracellular vesicles of low density (rho = 1.042-1.045 kg/l) and cells remained viable in culture for 4 weeks. Like the normal monocyte, DiI-RPM phi, but not exudate-derived or fixed cells, migrated to peritoneal exudates, following i.v. adoptive transfer, by a mechanism inhibitable by an antibody to the type 3 complement receptor. In the absence of an inflammatory stimulus there was no migration to the peritoneal cavity, and DiI-RPM phi accumulated within 4 h in the red pulp and marginal zone of the spleen. By day 6 these cells still formed a tight ring of fluorescence in the marginal zone alone, outside the marginal metallophil cells. DiI-RPM phi injected into the peritoneal cavity migrated to the parathymic lymph nodes where they were found in the subcapsular sinus and in the medullary cords, whereas very few fluorescent cells could be found in the T cell areas. The migration of RPM phi to lymphoid organs required viable cells but, unlike the recruitment of cells to peritoneal exudates, was not inhibitable by antibodies to CR3. We conclude that the RPM phi is a useful surrogate for the analysis of constitutive and induced monocyte migration to secondary lymphoid and inflammatory sites, respectively.

Topics: Animals; Carbocyanines; Cell Movement; Female; Fluorescent Dyes; Immunization, Passive; Inflammation; Lymphoid Tissue; Macrophages; Male; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Peritoneal Cavity

Analysis of neural crest cell migration in the mouse has been difficult due to the lack of reliable cell markers. Recently, we found that injection of DiI into the chick neural tube marks premigratory neural crest cells whose endfeet are in contact with the lumen of the neural tube (Serbedzija et al. Development 106, 809-819 (1989)). In the present study, this technique was applied to study neural crest cell migratory pathways in the trunk of the mouse embryo. Embryos were removed from the mother between the 8th and the 10th days of development and DiI was injected into the lumen of the neural tube. The embryos were then cultured for 12 to 24 h, and analyzed at the level of the forelimb. We observed two predominant pathways of neural crest cell migration: (1) a ventral pathway through the rostral portion of the somite and (2) a dorsolateral pathway between the dermamyotome and the epidermis. Neural crest cells were observed along the dorsolateral pathway throughout the period of migration. The distribution of labelled cells along the ventral pathway suggested that there were two overlapping phases of migration. An early ventrolateral phase began before E9 and ended by E9.5; this pathway consisted of a stream of cells within the rostral sclerotome, adjacent to the dermamyotome, that extended ventrally to the region of the sympathetic ganglia and the dorsal aorta.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Animals; Carbocyanines; Cell Movement; Culture Techniques; Female; Fluorescent Dyes; Male; Mice; Neural Crest

Hypercholesterolemic rabbit beta-VLDL and human LDL are both internalized by mouse peritoneal macrophages by receptor-mediated endocytosis. However, only beta-VLDL (which binds to the cells with a much higher affinity than LDL) markedly stimulates acyl-CoA/cholesterol acyl transferase (ACAT) and induces foam cell formation in these cells. As an initial step to test whether the two lipoproteins might be targeted to different organelles (which might differ in their ability to deliver cholesterol to microsomal ACAT), we studied the endocytic pathways of beta-VLDL and LDL. Lipoproteins were labeled with the non-transferable fluorescent label, DiI. When the macrophages were incubated with DiI-LDL for 10 min at 37 degrees C, the fluorescence was concentrated near the center of the cell both in heavily labeled vesicles and in a diffuse pattern. The pattern with DiI-beta-VLDL was quite different: an array of bright vesicles throughout the cytoplasm was the predominant feature. Differences in distribution were seen as early as 2 min of incubation and persisted throughout a 10-min chase period. By using a procedure in which photobleaching of DiI fluorescence converts diaminobenzidine into an electron-dense marker, we were able to identify at the ultrastructural level vesicles containing electron-dense material in cells incubated with DiI-beta-VLDL. Human E2/E2 beta-VLDL (from a patient with familial dysbetalipoproteinemia), which has a binding affinity and ACAT-stimulatory potential similar to LDL, gave a pattern of fluorescence virtually identical to LDL. Pulse-chase studies with 125I-labeled and [3H]cholesteryl ester-labeled lipoproteins disclosed that both protein degradation and cholesteryl ester hydrolysis were markedly retarded in beta-VLDL compared with LDL. Thus, in mouse peritoneal macrophages, endocytosed beta-VLDL appears in a distinct set of widely-distributed vesicles not seen with LDL (or with E2-beta-VLDL) and, compared with LDL, has a markedly diminished rate of protein degradation and cholesteryl ester hydrolysis. The differential routing of LDL and beta-VLDL may provide a mechanism for differences in ACAT-stimulatory potential between the two lipoproteins.

Topics: Animals; Carbocyanines; Cholesterol; Endocytosis; Female; Fluorescent Dyes; Lipoproteins, LDL; Lipoproteins, VLDL; Macrophages; Male; Mice; Microscopy, Electron; Microscopy, Fluorescence; Organelles; Peritoneal Cavity; Sterol O-Acyltransferase; Time Factors; Video Recording

Clinical and psychophysical observations indicate that the visual cortex is critical for the perception of color, form, depth, and movement. Little, however, is known about the cortical circuitry that underlies these functions in humans. In an attempt to learn more about these connections, we have traced projections of primary (V1) and secondary (V2) visual cortex in the postmortem, fixed human brain, using the fluorescent dye 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate as an axonal marker. The results show that V1 makes a forward projection to layers 3 and 4 of V2, and V2 projects back to layers 1, 2, 3, 5, and 6 of V1. Some V2 injections also show an input to layer 4B of V1. Projections to 4B probably originate from cytochrome oxidase (CO)-reactive stripes that we have identified in V2. Differential connections between CO-rich (blobs) and CO-poor regions (interblobs) also exist within V1; blobs are connected to blobs and interblobs are connected to interblobs. The results show that the connections in human visual cortex are similar to those of nonhuman primates and that their organization is consistent with the concept of multiple processing streams in the visual system.

Topics: Adult; Aged; Aged, 80 and over; Axonal Transport; Carbocyanines; Electron Transport Complex IV; Fluorescent Dyes; Humans; Middle Aged; Neurons; Visual Cortex

We have utilized the carbocyanine dye, DiI, to label suspensions of dissociated ciliary ganglion cells removed from 6 to 12 day old quail embryos. Some of the cells were injected into the trunk somites of 2.5-3 day old chick embryos along pathways where neural crest cells migrate to form sensory and sympathetic ganglia, aortic plexuses and the adrenal medulla; the remainder of the cells were cultured to check their viability and the persistence of the DiI label. Embryos were incubated for 1-8 days post-injection, fixed in 4% paraformaldehyde/0.25% glutaraldehyde and processed for cryostat sectioning. DiI-labelled cells were readily identifiable in culture and in sections of embryos at all stages examined. Several cell types were identified, based on their morphology and soma size. These included cells with large cell bodies and bright DiI-labelling that appeared to be neurons and smaller, more weakly labelled cells that appeared non-neuronal. The latter presumably had divided several times, accounting for their reduced levels of dye. Many of the DiI-labelled cells were found in and around neural crest-derived sympathetic ganglia, aortic plexuses and adrenomedullary cords, but were rarely observed in dorsal root ganglia. The aldehyde fixative (Faglu mixture) used in this study reacts with catecholamines to form a bright reaction product in adrenergic cells including those in the sympathetic ganglia and the adrenal medulla. The catecholamine biproduct and the DiI in the same cell can easily be viewed with different fluorescent filter sets. A variable number of the DiI-labelled cells in these adrenergic sites contained catecholamines. Cells derived from younger 6 day ciliary ganglion dissociates exhibited detectable catecholamine neurotransmitters earlier and more frequently than those derived from 8 day embryos. The presence of cells exhibiting both bright DiI and catecholamine fluorescence is consistent with previous indications that post-mitotic ciliary ganglion neurons can undergo phenotypic conversion from cholinergic to adrenergic when transplanted to the trunk environment.

Topics: Animals; Carbocyanines; Catecholamines; Cells, Cultured; Chick Embryo; Coturnix; Embryo, Nonmammalian; Fetal Tissue Transplantation; Fluorescent Dyes; Ganglia, Parasympathetic

To enable the study of glomerular endothelial cell functions and interactions with other glomerular cells, bovine glomerular capillary endothelial cells were established in culture. Selective media were used to facilitate endothelial cell proliferation and to suppress glomerular mesangial cell growth. Glomerular endothelial cells were separated from other cell types by fluorescence-activated cell sorting or, alternatively, by cloning. Glomerular endothelial cells expressed angiotensin I-converting enzyme and factor VIII activity and acetylated LDL uptake, properties generally held to be specific for endothelial cells. Proliferation of subconfluent glomerular endothelial cells was stimulated by basic fibroblast growth factor and, in the presence of heparin sodium, by acidic fibroblast growth factor. Platelet-derived growth factor was without effect on glomerular endothelial cell proliferation. Coculture with mesangial cells markedly inhibited proliferation of subconfluent glomerular endothelial cells. By contrast, medium conditioned by confluent glomerular endothelial cells markedly enhanced proliferation of subconfluent glomerular endothelial cells. These findings suggest that glomerular endothelial cell growth is under autocrine and paracrine control.

Topics: Animals; Carbocyanines; Cattle; Cell Division; Cell Separation; Cells, Cultured; Clone Cells; Culture Media; Endocytosis; Endothelium; Fibroblast Growth Factors; Flow Cytometry; Fluorescent Dyes; Glomerular Mesangium; Growth Substances; Heparin; Hydrogen-Ion Concentration; Kidney Glomerulus; Lipoproteins, LDL; Microscopy, Fluorescence; Peptidyl-Dipeptidase A; Platelet-Derived Growth Factor

The binding and uptake of native low density lipoproteins (LDL) and malondialdehyde treated (MDA) LDL by human hepatocytes in primary culture has been analyzed. Indirect immunofluorescent technique and lipoproteins labeled with fluorescent dye 3.3 dioctadecylindocarbocyanine (Dil) were used. Practically all culture cells have binding sites for native LDL which visualized in a form of separate granules on the cell surface. The binding sites for MDA LDL were found only on some (5%) cells culture that differed from hepatocytes in shape and size. Like other cells of culture hepatocytes internalized native Dil-LDL and acquired brightly specific fluorescence.

Topics: Binding Sites; Carbocyanines; Cells, Cultured; Fluorescent Antibody Technique; Humans; Lipoproteins, LDL; Liver; Malondialdehyde; Membrane Proteins; Receptors, Cell Surface; Receptors, Immunologic; Receptors, Lipoprotein; Receptors, Scavenger; Scavenger Receptors, Class B

To permit a more detailed analysis of neural crest cell migratory pathways in the chick embryo, neural crest cells were labelled with a nondeleterious membrane intercalating vital dye, DiI. All neural tube cells with endfeet in contact with the lumen, including premigratory neural crest cells, were labelled by pressure injecting a solution of DiI into the lumen of the neural tube. When assayed one to three days later, migrating neural crest cells, motor axons, and ventral root cells were the only cells types external to the neural tube labelled with DiI. During the neural crest cell migratory phase, distinctly labelled cells were found along: (1) a dorsolateral pathway, under the epidermis, as well adjacent to and intercalating through the dermamyotome; and (2) a ventral pathway, through the rostral portion of each sclerotome and around the dorsal aorta as described previously. In contrast to those cells migrating through the sclerotome, labelled cells on the dorsolateral pathway were not segmentally arranged along the rostrocaudal axis. DiI-labelled cells were observed in all truncal neural crest derivatives, including subepidermal presumptive pigment cells, dorsal root ganglia, and sympathetic ganglia. By varying the stage at which the injection was performed, neural crest cell emigration at the level of the wing bud was shown to occur from stage 13 through stage 22. In addition, neural crest cells were found to populate their derivatives in a ventral-to-dorsal order, with the latest emigrating cells migrating exclusively along the dorsolateral pathway.

Topics: Animals; Carbocyanines; Cell Movement; Chick Embryo; Fluorescent Dyes; Neural Crest; Time Factors

Topics: Carbocyanines; Fibroblasts; Fluorescent Dyes; Humans; Image Enhancement; Microscopy, Fluorescence; Receptors, LDL

Developmental changes in the phosphorylation state of neurofilament proteins (NFPs) in the chick embryonic optic nerve were histochemically and biochemically studied using monoclonal antibody (MAb) 82E10 specific to the highly phosphorylated components of high (180K)- and middle (160K)-molecular-weight subunits of neurofilament (NF) in the chicken. Cross sections of developing embryonic optic nerve were studied by enzyme immunohistochemistry using this MAb. The staining pattern showed marked changes with the developmental stage. In 6-day embryos (E6) the entire cross section was stained, whereas in E10 only about a ventroposterior half of the cross section was stained. In E14 nearly the entire area of the cross section became unstained. Thereafter, the immunoreactivity reappeared and gradually increased, such that in E20 the entire cross section became immunopositive again. Electrophoretic and immunoblot analyses were made on optic nerves dissected out of embryos of various stages. The 82E10 immunoreactivity at the position of NF-M underwent a transient loss in E14 in parallel with the time course of histochemical change. Two-dimensional gels stained for protein further showed that the highly phosphorylated form of NF-M is transiently lost from embryonic optic nerve in E14, while the less phosphorylated form persists throughout the embryonic developmental stages. In order to understand the orderly loss of the 82E10 immunoreactivity in relation to retinotopic and chronotopic organizations of the fibers in the embryonic optic nerve, retinal injection of a fluorescent dye DiI as an anterograde tracing marker for selected fibers was utilized. An ordered arrangement of the fibers was present within the embryonic optic pathway, suggesting that the orderly loss of the 82E10 immunoreactivity in the embryonic optic nerve reflects the chronological order of the optic axons. These changes in the phosphorylation state of NFPs in the embryonic optic nerve presumably reflect dynamic changes of the neuronal cytoskeleton at certain stages during development.

Topics: Alkaline Phosphatase; Animals; Antibodies, Monoclonal; Axons; Carbocyanines; Chick Embryo; Electrophoresis, Gel, Two-Dimensional; Electrophoresis, Polyacrylamide Gel; Escherichia coli; Fluorescent Dyes; Histocytochemistry; Immunoblotting; Immunoenzyme Techniques; Intermediate Filament Proteins; Molecular Weight; Optic Nerve; Phosphorylation; Time Factors

To study the retrograde labeling of intact and axotomized retinal ganglion cells (RGCs) over long periods of time, we applied the carbocyanine dye diI to the superior colliculi (SC) and dorsal lateral geniculate nuclei (dLGN) in adult albino rats and examined the retinas by fluorescence microscopy after different periods of survival. Retrogradely labeled RGCs, which were observed in the retinas as early as 3 days after application of the dye, gradually increased in density so that by 7 days more than 80% of the RGCs were labeled and by 30 days diI-labeled cell densities were similar to those observed after short applications of other tracers. Using short-term retrograde labeling with fast blue (FB) as an independent marker of RGCs, it was determined that these neurons remained labeled with diI for periods of up to 9 months without apparent leakage of the tracer to other retinal cells. In addition, diI labeling persisted in the somata of more than 80% of axotomized RGCs whose contact with the source of label had been interrupted for 3 months. Thus, we propose that retrogradely transported diI is a useful label for quantitative studies of neuronal populations, even after axotomy.

Topics: Amidines; Animals; Axons; Carbocyanines; Fluorescent Dyes; Nerve Crush; Neurons; Optic Nerve; Quinolines; Rats; Rats, Inbred Strains; Retina; Retinal Ganglion Cells

To characterize the lipoprotein metabolism of lipid-filled cells of atherosclerotic lesions, uptake of 3,3'-dioctadecylindocarbocyanine (DiI)-labelled low density lipoprotein (LDL), acetylated LDL (Ac-LDL) and beta-very low density lipoprotein (beta-VLDL) was studied by fluorescence microscopy and flow cytometry in primary cultures of enzymatically dispersed aortic cells from cholesterol-fed rabbits. Most of the foam cells were identified as macrophages on the basis of Fc-receptors and high activities of nonspecific esterase and acid lipase, although cholesteryl ester (CE) inclusions were found by filipin staining also in smooth muscle cells (SMCs). During the culture only SMCs proliferated and were confluent in about 1 week. After incubation with DiI-Ac-LDL most macrophage foam cells were brightly fluorescent, but also many SMCs accumulated fluorescence. In SMCs, an excess of LDL inhibited the uptake of DiI-beta-VLDL and DiI-LDL, indicating that these lipoproteins were taken up by the apoB,E receptor; the activity of this receptor was low 2 days after cell isolation but increased considerably during SMC proliferation. DiI-beta-VLDL was not taken up by the macrophage foam cells until after 7 days' culture, when their CE content had decreased, reflecting a feed-back regulation of these receptors as well. Our results indicate that, in primary cultures of enzyme-dispersed cells from rabbit atherosclerotic lesions, most of the foam cells have lipoprotein receptors resembling those described in macrophages and that also many SMCs accumulate Ac-LDL.

Topics: Animals; Aorta; Arteriosclerosis; Carbocyanines; Cells, Cultured; Cholesterol Esters; Cholesterol, Dietary; Flow Cytometry; Fluorescent Dyes; Foam Cells; Lipoproteins, LDL; Lipoproteins, VLDL; Male; Microscopy, Fluorescence; Rabbits

We describe a technique to visualize substrate-attached materials (SAM) of polymorphonuclear leukocytes (PMN) using the fluorescent lipid analog 1,1'-dioctadecyl-3,3,3',3',-tetramethylindocarbocyanine-perchlorate (DiC18Icc). DiC18Icc was incorporated into the membranes of living cells or SAMs. Since cell preparation does not require fixation, SAMs can be rapidly visualized by fluorescence microscopy. SAMs are generated by subjecting attached cells to a shearing force by rinsing with phosphate-buffered saline (PBS). The SAM-labeling protocol identified a membrane compartment as shown by detergent extraction. The SAMs of PMN leukocytes observed with this technique display complex patterns of interconnecting filaments, foci with radiating filaments, and smooth membranous areas with interconnecting filaments. The sensitivity and nondestructive nature of the DiC18Icc-labeling procedure have allowed us to observe filopodia of motile cells. The results are consistent with the hypothesis that locomotion involves a series of attachment and detachment steps. After 60 minutes of locomotion, these trailing filopodia have been measured at lengths up to 100 micron. The amount of membrane associated with these filopodia accounts for roughly 10% of the total membrane area of resting cells. These data set limits for models of membrane flow during chemotaxis.

Topics: Carbocyanines; Cell Adhesion; Cell Membrane; Chemotaxis, Leukocyte; Fluorescent Dyes; Humans; Microscopy, Fluorescence; N-Formylmethionine Leucyl-Phenylalanine; Neutrophils

The use of high order autocorrelation in fluorescence correlation spectroscopy for investigating aggregation in a sample that contains fluorescent molecules is described. Theoretical expressions for the fluorescence fluctuation autocorrelation functions defined by gm,n(tau) = [(delta fm(t + tau)delta fm(t] - (delta Fm(t] (delta Fn(t]]/(F)m+n, where delta F(t) is the fluorescence fluctuation at time t, (F) is the average fluorescence, and m and n are integers less than or equal to 3, are derived. Methods for determining the number densities and relative fluorescence yields of aggregates of different sizes from a series of Gm,n(0) values are outlined. The method is applied to 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate suspended in solutions of water and ethyl alcohol. The technique presented may prove useful in detecting and characterizing aggregates of fluorescent-labeled biological molecules such as cell surface receptors.

Topics: Carbocyanines; Fluorescent Dyes; Macromolecular Substances; Mathematics; Models, Theoretical; Molecular Conformation; Quinolines; Spectrometry, Fluorescence

The major feature of sickle cell anemia is the tendency of erythrocytes to sickle when exposed to decreased oxygen tension and to unsickle when reoxygenated. Irreversible sickle cells (ISCs) are sickle erythrocytes which retain bipolar elongated shapes despite reoxygenation. ISCs are believed to owe their biophysical abnormalities to acquired membrane alterations which decrease membrane deformability. While increased membrane surface viscosity has been measured in ISCs, the lateral dynamics of membrane lipids in these cells have not heretofore been examined. We have measured the lateral diffusion of the lipid analog 3,3'-dioctadecylindocyanine iodide (DiI) in the plasma membrane of intact normal erythrocytes, reversible sickle cells (RSCs), and irreversible sickle cells by fluorescence photobleaching recovery (FPR). The diffusion coefficients +/- standard errors of the mean of DiI in intact normal red blood cells (RBCs), RSCs, and ISCs at 37 degrees C are (8.06 +/- 0.29) X 10(-9) cm2 X s-1, (7.74 +/- 0.22) X 10(-9) cm2 X s-1, and (7.29 +/- 0.24) X 10(-9) cm2 X s-1, respectively. A similar decrease in the diffusion coefficient of DiI in the plasma membranes of the three cell types was observed at 4, 10, 17, 23, and 30 degrees C. ANOVA analysis of the changes in DiI diffusion showed significant differences between the RBC and ISC membranes at all temperatures examined. The characteristic breaks in Arrhenius plots of the diffusion coefficients for the RBCs, RSCs, and ISCs occurred at 20, 19, and 18.6 degrees C, respectively. Photobleaching recovery data were used to estimate (Boullier, J.A., Melnykovich, G. and Barisas, B.G. (1982) Biochim. Biophys. Acta 692, 278-286) the microviscosities of the plasma membranes of the three cell types at 25 degrees C. We find significant differences between our microviscosity values and those obtained in previous fluorescence depolarization studies. However, both methods indicate qualitatively similar differences in membrane microviscosity among the various cell types.

Topics: Anemia, Sickle Cell; Carbocyanines; Diffusion; Erythrocyte Membrane; Fluorescent Dyes; Humans; Membrane Fluidity; Quinolines; Spectrometry, Fluorescence; Thermodynamics; Viscosity

Small unilamellar liposomes with an average external diameter of approximately 550 A were prepared by high pressure extrusion in a French press. Liposomes, composed of phosphatidylcholine, phosphatidylserine, and cholesterol at a molar ratio of 7:1:2, were incubated with suspensions of bovine adrenal chromaffin cells. The cell-liposome interactions were characterized using fluorescence and radiotracer techniques. Transfer of the liposomal contents into the cytoplasm was visualized by fluorescence microscopy, using fluorescence-labeled macromolecules, and further documented by flow cytometry with liposome-entrapped 5,6-carboxy-fluorescein. The dose dependence, time course, and temperature dependence of the cell-liposome association, as determined by radioactive labeling both the liposomal membranes and their contents, indicate saturable interaction of the cells with intact liposomes (KappM approximately 5 X 10(-7) M lipid/10(6) cells at 37 degrees C). Using nonexchangeable fluorescent phospholipid analogs, the cell-liposome interactions were characterized by fluorescence resonance energy transfer and by fluorescence recovery after photobleaching. From these latter experiments we conclude that after 1-h incubation of 10(6) cells with 1 microM lipid at 37 degrees C, 30% of the cell-associated liposomes will have fused with the plasma membranes, resulting in the delivery of the contents of approximately 1.25 X 10(5) liposomes into each cell. Thus, liposomal delivery is an effective means to gain access to the cytoplasm and can be exploited to modulate physiological responses from within intact chromaffin cells.

Topics: Adrenal Glands; Animals; Carbocyanines; Cattle; Chromaffin System; Cytoplasm; Energy Transfer; Flow Cytometry; Fluorescent Dyes; Kinetics; Liposomes; Membrane Fusion; Microscopy, Fluorescence; Phospholipids; Pressure; Spectrometry, Fluorescence

Uptake of low density lipoprotein (LDL) and of acetyl LDL was compared in skin fibroblasts, smooth muscle cells, and peritoneal macrophages with the use of lipoproteins labeled with either 125I or the fluorescent probe 3,3'-dioctadecylindocarbocyanine (DiI). The uptake of DiI-labeled lipoproteins was assessed by quantitative spectrofluorometry and by fluorescence microscopy. The DiI was quantitatively retained by the cells, while the 125I-LDL was degraded and 125I-labeled degradation products were excreted from the cells. In smooth muscle cells and fibroblasts the uptake of LDL was virtually the same whether measured with the use of the DiI or 125I-label (sum of cell-associated and degraded 125I). The labeling of acetyl LDL with DiI enhanced its uptake in peritoneal macrophages by an average of 18%. With the DiI label, lipoprotein uptake (DiI-LDL for smooth muscle cells and skin fibroblasts and DiI-acetyl-LDL for mouse peritoneal macrophages) could be determined after as little as 10 minutes of incubation at 37 C. The pattern of uptake of the DiI-labeled lipoproteins was consistent with binding to specific receptors, because no DiI could be detected in mutant cells without LDL receptors, and uptake was competitively inhibited by addition of excess unlabeled lipoprotein. When the DiI-labeled lipoproteins were removed from the medium, there was a 5-15% loss of DiI from all cell types studied over the first 24 hours. Thereafter, DiI loss from cells was dependent on cell type and culture medium. No further loss of DiI occurred from skin fibroblasts for up to 96 hours of incubation in medium supplemented with either lipoprotein-deficient serum (LPDS) or 10% fetal bovine serum. During this same time period there was a 40-60% loss of DiI from smooth muscle cells and macrophages incubated in medium supplemented with LPDS. Most of the DiI lost from the cells (60-70%) could be recovered in the culture medium but was not the result of cell death, as was indicated by the relatively constant protein concentrations per dish. The loss of DiI was markedly reduced in smooth muscle cells and macrophages when 10% fetal bovine serum was substituted for the LPDS in the culture medium. This suggests that some cells incubated with LPDS undergo changes, perhaps in the plasma membrane, that alter their ability to retain the DiI. In the presence of 10% fetal bovine serum, however, the DiI label is quantitatively retained by all cells tested for up to 96 hours.(ABSTRACT TRUNCATED A

Topics: Animals; Aorta; Carbocyanines; Cells, Cultured; Female; Fibroblasts; Fluorescent Dyes; Humans; Iodine Radioisotopes; Lipoproteins; Lipoproteins, LDL; Macaca mulatta; Macrophages; Mice; Microscopy, Fluorescence; Muscle, Smooth; Peritoneum; Receptors, LDL; Skin; Spectrometry, Fluorescence; Time Factors

Topics: Animals; Arteriosclerosis; Carbocyanines; Cells, Cultured; Fibroblasts; Fluorescent Dyes; Haplorhini; Humans; Lipoproteins; Lipoproteins, LDL; Mice; Muscle, Smooth, Vascular; Quinolines; Skin

Sinusoidal endothelial cells from adult rat liver have been isolated by centrifugal elutriation and been established in primary culture. Their identification has made use of a novel process of these cells, the sequestration of human acetoacetylated low-density lipoprotein. After its administration in vivo, labeled acetoacetylated low-density lipoprotein within the liver was associated solely with sinusoidal endothelial cells and, when labeled with the stable fluorescent compound 3,3'-dioctadecylindocarbocyanine, provided a means of identifying isolated cells by fluorescence microscopy. In endothelial cell cultures, 90% of the cells were fluorescent and exhibited fenestrae. The collagen phenotype of cultures was assessed by an immunofluorescent approach, which revealed cell-associated type IV collagen only; types I, III, and V were undetectable. In other studies, it was found that these cells lacked factor VIII-R antigen and Weibel-Palade bodies, adding to the evidence that they differ substantially from large-vessel and other capillary endothelia.

Topics: Animals; Carbocyanines; Cell Separation; Cells, Cultured; Collagen; Endothelium; Fluorescent Dyes; Iodine Radioisotopes; Lipoproteins; Lipoproteins, LDL; Liver; Male; Microbial Collagenase; Microscopy, Electron, Scanning; Microscopy, Fluorescence; Phenotype; Pronase; Rats; Rats, Inbred Strains

Macrophages were labeled in sections of rabbit lung with acetoacetylated low density lipoprotein (LDL), a marker internalized by cultured macrophages but not by other connective tissue cells. Using a modified technique, thin slices of fresh rabbit lung were incubated in 3,3'-dioctadecylindocarbocyanine (DiI)-labeled, acetoacetylated LDL, fixed in paraformaldehyde, and sectioned. Alveolar macrophages incorporated the fluorescently labeled, modified LDL, but surrounding stroma and parenchyma did not stain. Our results indicate that DiI-labeled, acetoacetylated LDL may be used to identify mononuclear phagocytes in tissue sections.

Topics: Animals; Carbocyanines; Lipoproteins, LDL; Lung; Macrophages; Male; Muscle, Smooth, Vascular; Pulmonary Alveoli; Quinolines; Rabbits

The visible wavelength excited fluorophore 3,3'-dioctadecylindocarbocyanine iodide (Dil[3]) was incorporated into human low density lipoprotein (LDL) to form the highly fluorescent LDL derivative dil(3)-LDL. Dil(3)-LDL binds to normal human fibroblasts and to human fibroblasts defective in LDL receptor internalization but does not bind to LDL receptor-negative human fibroblasts at 4 degrees C or 37 degrees C. It is internalized rapidly at 37 degrees C by normal fibroblasts and depresses the activity of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG-CoA reductase) in a manner similar to that of LDL. It is prevented from binding to the LDL receptor by an excess of unlabeled LDL or by heparin sulfate. Identical distributions of dil(3)-LDL are observed on cells by either indirect immunofluorescence with fluorescein-labeled antibody or directly by dil(3) fluorescence. Upwards of 45 molecules of dil(3) are incorporated per molecule of LDL without affecting binding to the receptor. This labeling renders individual molecules visible by their fluorescence and enables the derivative to be used in dynamic studies of LDL-receptor motion on living fibroblasts by standard fluorescence techniques at low LDL receptor density. Observations with this derivative indicate that the LDL-receptor complex is immobilized on the surface of human fibroblasts but, when free of this linkage, undergoes a Brownian motion consistent with theory.

Topics: Carbocyanines; Cell Line; Fibroblasts; Fluorescent Dyes; Heparin; Humans; Hydroxymethylglutaryl CoA Reductases; Lipoproteins, LDL; Microscopy, Fluorescence; Quinolines; Receptors, Cell Surface; Receptors, LDL; Temperature

The surfaces of most cells bear a net negative charge. The imposition of an electric field parallel to the surface of the cell should produce, therefore, an electro-osmotic flow of fluid towards the cathodal side of the cell. Our analysis of a simple model of the cell surface indicates that a negatively charged mobile macromolecule will be swept by this electro-osmotic flow of fluid to the cathodal side of the cell if its zeta potential, zeta 1, is less negative than the zeta potential of the cell surface, zeta 2. Conversely, if zeta 2 is less negative than zeta 1, the negatively charged macromolecule will accumulate at the anodal side of the cell. Our experimental results demonstrate that concanavalin A (Con A) receptors on embryonic muscle cells normally accumulate at the cathodal side of the cell, but that they can be induced to accumulate at the anodal side of the cell by preincubating the myotubes either with neuraminidase, a treatment that removes negatively charged sialic acid residues, or with the lipid diI, a treatment that adds positive charges to the surface of the cell. Addition of the negatively charged lipid monosialoganglioside (GM1), on the other hand, enhances the accumulation of Con A receptors at the cathodal side of the cell.

Topics: Animals; Carbocyanines; Cell Membrane; Electric Stimulation; Fluorescent Dyes; Microscopy, Fluorescence; Models, Biological; Muscles; Neuraminidase; Osmosis; Receptors, Concanavalin A; Surface Properties; Xenopus

The effect of cholesterol depletion of the human erythrocyte membrane on the lateral diffusion rate of a fluorescent lipid probe is reported. At low temperature (-5 to 5 degrees C), the diffusion of the probe is 50% slower in the cholesterol-depleted membrane than in non-depleted membrane. At high temperatures (30 to 40 degrees C), probe mobility is not affected by cholesterol depletion. These results suggest that cholesterol suppresses aspects of phospholipid phase changes in animal cells in a manner consistent with its behavior in artificial bilayers and multilayers. Whole erythrocytes were depleted of 30--50% of their cholesterol by incubation with a sonicated dispersion of dipalmitoyl phosphatidylcholine. Cells were then labeled with 3,3'-dioctadecylindocarbocyanine (diI), a phospholipid-like fluorescent dye, and hemolyzed into spherical ghosts. The rate of lateral motion of diI was measured by observing the fluorescence recovery after local photobleaching with a focused laser spot. The diffusion rate of the lipid probe in both control and cholesterol-depleted erythrocyte membrane is substantially smaller than in any cell or model membrane previously measured.

Topics: Carbocyanines; Cholesterol; Diffusion; Erythrocyte Membrane; Erythrocytes; Fatty Acids; Humans; Lasers; Liposomes; Membrane Lipids; Photolysis; Pulmonary Surfactants; Spectrometry, Fluorescence

The orientation of an amphipathic, long acyl chain fluorescent carbocyanine dye [diI-C18-(3)] in a biological membrane is examined by steady-state fluorescence polarization microscopy on portions of single erythrocyte ghosts. The thermodynamically plausible orientation model most consistent with the experimental data is one in which the diI-C18-(3) conjugated bridge chromophore is parallel to the surface of the cell and the acyl chains are imbedded in the bilayer parallel to the phospholipid acyl chains. Comparison of the predictions of this model with the experimental data yields information on the intramolecular orientations of the dye's transition dipoles and on the dye's rate of rotation in the membrane around an axis normal to the membrane. To interpret the experimental data, formulae are derived to account for the effect of high aperture observation on fluorescence polarization ratios. These formulae are generally applicable to any high aperture polarization studied on microscopic samples, such as portions of single cells.

Topics: Carbocyanines; Erythrocyte Membrane; Erythrocytes; Fluorescent Dyes; Humans; Mathematics; Microscopy, Fluorescence; Quinolines