lucifer-yellow and 3-3--dioctadecylindocarbocyanine

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

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

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

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

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

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

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

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

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

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 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

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 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