lucifer-yellow and biocytin

We show here the expression, permeability and pharmacology of a voltage-gated channel in certain taste bud cells (TBCs) which is known to be permeable to Lucifer Yellow CH (LY) and known to release ATP as a neurotransmitter in response to taste substances. LY dissolved in a 200 mM K(+)-containing solution label TBCs immunoreactive to PLCβ2, a phospholipase subtype, but not the TBC subtype immunoreactive to SNAP-25, a SNARE protein. In addition to these subtypes, LY also labelled a few of the non-immunoreactive TBCs. Monovalent and divalent anion probes with molar mass less than 1200 also label PLCβ2-immunoreactive TBCs and a few non-immunoreactive TBCs, whereas a cation probe, rhodamine B, labels the cell membrane of TBCs nonselectively and K(+) independently. The number of LY-labelled TBCs is decreased by 5 μM DIDS (4,4'-diisothiocyanostilbene-2-2'disulfonate), 1mM octanol and 10(-5)M H(+), but not by 10 μM carbenoxolone, 2mM probenecid, 10mM TEA, or 30 μM flufenamic acid. PLCβ2-immunoreactive TBCs and a few non-immunoreactive TBCs generate a TEA-insensitive outwardly rectifying current. DIDS decreases this current in magnitude with IC(50) of ~0.4 μM in a voltage-independent manner. Also 10(-5)M H(+) and 1mM octanol decreases the current magnitude, but 10 μM carbenoxolone and 2mM probenecid do not. These results show that the LY-permeable channel preferably permeates anions and occurs not only on PLCβ2-immunoreactive TBCs but also on certain non-immunoreactive TBCs. Also the results show that the pharmacology of the LY-permeable channel is different from hemichannels reported. The discussion focuses on the pharmacology and the role of the LY-permeable channel.

Topics: 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Animals; Biophysics; Carbenoxolone; Electric Stimulation; Flufenamic Acid; Fluorescent Dyes; Gene Expression Regulation; Ion Channel Gating; Isoquinolines; Lysine; Membrane Potentials; Mice; Neural Inhibition; Phospholipase C beta; Potassium Channel Blockers; Sensory Receptor Cells; Synaptosomal-Associated Protein 25; Taste Buds; Tetraethylammonium; Time Factors

Dorsal root ganglia (DRG) are key elements in sensory signaling under physiological and pathological conditions. Little is known about electrical coupling among cells in these ganglia. In this study, we injected the fluorescent dye Lucifer yellow (LY) into single cells to examine dye coupling in DRG. We found no dye coupling between neurons or between neurons and their attendant satellite glial cells (SGCs). In mouse DRG, we observed that in 26.2% of the cases SGCs that surround a given neuron were dye coupled. In only 3.2% of the cases SGCs that make envelopes around different neurons were coupled. The data from mouse ganglia were very similar to those from rat and guinea pig DRG. The results obtained by injection of the tracer biocytin were very similar to those observed with LY. The coupling incidence within the envelopes increased 3.1-fold by high extracellular pH (8.0), but coupling between envelopes was not affected. Acidic pH (6.8) reduced the coupling. High extracellular K+ (9.4 mM) increased the coupling 2.4-fold and 4.7-fold within and between envelopes, respectively. Low extracellular Ca2+ (0.5, 1.0 mM) partly reversed the effect of high K+ on coupling. The results showed that SGCs in mammalian sensory ganglia are connected by gap junctions. This coupling is very sensitive to changes in pH, and can therefore be modulated under various physiological and pathological conditions. The dependence of the coupling on extracellular K+ and Ca2+ suggests that the permeability of gap junctions can be altered by physiological and pharmacological stimuli.

Topics: Animals; Calcium; Cell Communication; Extracellular Fluid; Female; Fluorescent Dyes; Ganglia, Spinal; Gap Junctions; Guinea Pigs; Hydrogen-Ion Concentration; Isoquinolines; Lysine; Male; Mice; Mice, Inbred BALB C; Neurons; Organ Culture Techniques; Potassium; Rats; Rats, Sprague-Dawley; Satellite Cells, Perineuronal; Signal Transduction; Species Specificity

Zinc is found in synaptic vesicles in a large number of glutamatergic systems. Its involvement in neurotransmission and neurological disorders has been suggested. There are methods for tracing these circuits, but they do not fill the dendritic tree. In this study, extracellular selenite injections in vivo were combined with intracellular injection of fluorochromes in fixed tissue to reveal the morphology of these zinc-rich neurones. Intraperitoneal and intracerebral injections of sodium selenite alone or intracerebral injections of selenite combined with bisbenzimide were made in the visual cortex of the rat in order to locate the somata of zinc-rich neurones. After 24 h of retrograde transport, animals were killed and fluorescent markers were injected intracellularly into fixed slices to show neuronal morphology: (a) Lucifer Yellow (LY) followed by biocytin, (b) LY coupled to biocytin or (c) micro-ruby (MR) (dextranamines bound to rhodamine and biotin). Double-labelled somata (selenite+fluorochrome) were plotted. Details of the dendritic morphology were then revealed by incubation in avidin-biotin complex and development in 3,3'-diaminobenzidine and H(2)O(2). Camera lucida drawings showed that zinc-rich neurones in layers II-III involved in cortico-cortical visual projections were typical pyramidal neurones. This technique is noteworthy for its analysis of the morphology (and connections) of zinc-rich neurones.

Topics: Animals; Biotin; Cell Count; Dextrans; Drug Administration Routes; Extracellular Space; Injections, Intraperitoneal; Injections, Jet; Iontophoresis; Isoquinolines; Lysine; Male; Microscopy, Confocal; Neural Pathways; Neuroanatomy; Neurons; Rats; Rats, Inbred WKY; Rats, Wistar; Rhodamines; Silver Staining; Sodium Selenite; Somatosensory Cortex; Visual Cortex; Zinc

Neuro-anatomical studies in the mammalian cochlea have previously identified a subpopulation of approximately 5 % of primary auditory neurones, designated type II spiral ganglion neurones (sgnII). These neurones project to outer hair cells and their supporting cells, within the 'cochlear amplifier' region. Physiological characterization of sgnII has proven elusive. Whole-cell patch clamp of spiral ganglion neurones in P7-P10 rat cochlear slices provided functional characterization of sgnII, identified by biocytin or Lucifer yellow labelling of their peripheral neurite projections (outer spiral fibres) subsequent to electrophysiological characterisation. SgnII terminal fields comprised multiple outer hair cells and supporting cells, located up to 370 mum basal to their soma. SgnII firing properties were defined by rapidly inactivating A-type-like potassium currents that suppress burst firing of action potentials. Type I spiral ganglion neurones (sgnI), had shorter radial projections to single inner hair cells and exhibited larger potassium currents with faster activation and slower inactivation kinetics, compatible with the high temporal firing fidelity seen in auditory nerve coding. Based on these findings, sgnII may be identified in future by the A-type current. Glutamate-gated somatic currents in sgnII were more potentiated by cyclothiazide than those in sgnI, suggesting differential AMPA receptor expression. ATP-activated desensitising inward currents were comparable in sgn II and sgnI. These data support a role for sgnII in providing integrated afferent feedback from the cochlear amplifier.

Topics: Animals; Animals, Newborn; Cell Membrane; Cochlea; Electrophysiology; In Vitro Techniques; Isoquinolines; Kinetics; Lysine; Membrane Potentials; Neurons; Patch-Clamp Techniques; Phenotype; Rats; Spiral Ganglion

Changes in the gap junctional coupling and maturation of voltage-activated Na(+) currents during regeneration of newt retinas were examined by whole-cell patch-clamping in slice preparations. Progenitor cells in regenerating retinas did not exhibit Na(+) currents but showed prominent electrical and tracer couplings. Cells identified by LY-fills were typically slender. Na(+) currents were detected in premature ganglion cells with round somata in the 'intermediate-II' regenerating retina. No electrical and tracer couplings were observed between these cells. Mature ganglion cells did not exhibit electrical coupling, but showed tracer coupling. On average, the maximum Na(+) current amplitude recorded from premature ganglion cells was roughly 2.5-fold smaller than that of mature ganglion cells. In addition, the activation threshold of the Na(+) current was nearly 11 mV more positive than that of mature cells. We provide morphological and physiological evidence showing that loss of gap junctions between progenitor cells is associated with ganglion cell differentiation during retinal regeneration and that new gap junctions are recreated between mature ganglion cells. Also we provide evidence suggesting that the loss of gap junctions correlates with the appearance of voltage-activated Na(+) currents in ganglion cells.

Topics: Amphetamines; Animals; Cell Differentiation; Electric Conductivity; Fluorescent Dyes; Gap Junctions; Isoquinolines; Lysine; Nerve Regeneration; Patch-Clamp Techniques; Retina; Retinal Ganglion Cells; Salamandridae; Theophylline

We previously reported that gabapentin (GBP), a widely prescribed analgesic, enhances N-methyl-aspartate (NMDA) receptor mediated currents only when the intracellular level of protein kinase C is elevated. However, it is unclear how the potentiation of NMDA responses by GBP can lead to pain relief. To resolve this issue, we combined immunocytochemical and patch recording techniques to study the actions of GBP on NMDA receptors in dorsal horn cells isolated from rats with inflammation and to determine the gamma-aminobutyric acid (GABA) content in the recorded cells. We found that all GBP-responsive cells are GABA-immunoreactive and none of the GABA-negative neurons respond to GBP. Thus, GBP appears to enhance NMDA currents in GABAergic neurons. These observations suggest that GBP exerts its antinociceptive action by increasing the activity of these inhibitory neurons.

Topics: Acetates; Afferent Pathways; Amines; Analgesics; Animals; Cyclohexanecarboxylic Acids; Fluorescent Dyes; Gabapentin; gamma-Aminobutyric Acid; Immunohistochemistry; Inflammation; Ion Channels; Isoquinolines; Lysine; Membrane Potentials; Nociceptors; Pain; Patch-Clamp Techniques; Posterior Horn Cells; Protein Kinase C; Rats; Rats, Sprague-Dawley; Receptors, N-Methyl-D-Aspartate; Synaptic Transmission

We prepared living slice preparations of the peripheral retina of adult goldfish to examine electrical membrane properties of progenitor cells at the retinal margin. Cells were voltage-clamped near resting potential and then stepped to either hyperpolarizing or depolarizing test potentials using whole-cell voltage-clamp recordings. Electrophysiologically examined cells were morphologically identified by injecting both Lucifer Yellow (LY) and biocytin. All progenitor cells examined (n = 37) showed a large amount of passively flowing currents of either sign under suppression of the nonjunctional currents flowing through K(+) and Ca(2+) channels in the cell membrane. They did not exhibit any voltage-gated Na(+) currents. Cells identified by LY fills were typically slender. As the difference between the test potential and the resting potential increased, 13 out of 37 cells exhibited symmetrically voltage- and time-dependent current decline on either sign at the resting potential. The symmetric current profile suggests that the current may be driven and modulated by the junctional potential difference between the clamping cell and its neighbors. The remaining 24 cells did not exhibit voltage dependency. A gap junction channel blocker, halothane, suppressed the currents. A decrease in extracellular pH reduced coupling currents and its increase enhanced them. Dopamine, cAMP, and retinoic acid did not influence coupling currents. Injection of biocytin into single progenitor cells revealed strong tracer coupling, which was restricted in the marginal region. Immature ganglion cells closely located to the retinal margin exhibited voltage-gated Na(+) currents. They did not reveal apparent tracer coupling. These results demonstrate that the marginal progenitor cells couple with each other via gap junctions, and communicate biochemical molecules, which may subserve or interfere with cellular differentiation.

Topics: Animals; Cellular Senescence; Electrophysiology; Fluorescent Dyes; Gap Junctions; Goldfish; In Vitro Techniques; Isoquinolines; Lysine; Retina; Staining and Labeling; Stem Cells; Uncoupling Agents

Astrocytes respond to chemical, electrical and mechanical stimuli with transient increases in intracellular calcium concentration ([Ca2+]i). We now show that astrocytes in situ display intrinsic [Ca2+]i oscillations that are not driven by neuronal activity. These spontaneous astrocytic oscillations can propagate as waves to neighboring astrocytes and trigger slowly decaying NMDA receptor-mediated inward currents in neurons located along the wave path. These findings show that astrocytes in situ can act as a primary source for generating neuronal activity in the mammalian central nervous system.

Topics: Animals; Animals, Newborn; Astrocytes; Biological Clocks; Calcium Channel Blockers; Calcium Signaling; Central Nervous System; Enzyme Inhibitors; Excitatory Amino Acid Antagonists; GABA Antagonists; Immunohistochemistry; Isoquinolines; Lysine; Magnesium; Membrane Potentials; Neurons; Patch-Clamp Techniques; Rats; Receptors, N-Methyl-D-Aspartate; Synaptic Transmission; Tetrodotoxin; Thalamus; Vimentin

Gap junctional coupling between progenitor cells of regenerating retina in the adult newt was examined by a slice-patch technique. Retinal slices at the early regeneration stage comprised one to two layers of cells with mitotic activity, progenitor cells. These cells were initially voltage-clamped at a holding potential of -80 mV, near their resting potentials, and stepped to either hyperpolarizing or depolarizing test potentials under suppression of voltage-gated membrane currents. About half the cells showed passively flowing currents that reversed polarity around their resting potentials. The currents often exhibited a voltage- and time-dependent decline. As the difference between the test potential and resting potential increased, the time until the current decreased to the steady-state level became shorter and the amount of steady-state current decreased. Thus, the overall current profile was almost symmetrical about the current at the resting potential. Input resistance estimated from the initial peak of the currents was significantly smaller than that expected in isolated progenitor cells. In a high-K(+) solution, which decreased the resting potential to around 0 mV, the symmetrical current profile was also obtained, but only when the membrane potential was held at 0 mV before the voltage steps. These observations suggest that the current was driven and modulated by the junctional potential difference between the clamping cell and its neighbors. In addition, we examined effects of uncoupling agents on the currents. A gap junction channel blocker, halothane, suppressed the currents almost completely, indicating that the currents are predominantly gap junctional currents. Furthermore, injection of biocytin into the current-recorded cells revealed tracer coupling. These results demonstrate that progenitor cells of regenerating retina couple with each other via gap junctions, and suggest the presence of their cytoplasmic communication during early retinal regeneration.

Topics: Anesthetics, Inhalation; Animals; Calcium; Cells, Cultured; Dopamine; Fluorescent Dyes; Gap Junctions; Halothane; In Vitro Techniques; Intracellular Fluid; Isoquinolines; Lysine; Membrane Potentials; Microinjections; Mitosis; Nerve Regeneration; Patch-Clamp Techniques; Potassium; Retina; Salamandridae; Stem Cells

The primate red nucleus consists of three main neuron subpopulations, namely, rubrospinal neurons in the magnocellular nucleus, rubroolivary cells in the parvocellular nucleus, and local circuit neurons in both subnuclei: Each subpopulation has unique cerebellar and neocortical inputs. The structural framework for the interactions of these rubral subpopulations remains poorly defined and was the focus of this study in six macaques. Somata of rubrospinal neurons, dorsolateral-spinal (DL-spinal) neurons, as defined in the accompanying paper (Burman et al. [2000] J. Comp. Neurol., this issue), and rubroolivary neurons were labeled retrogradely first with Fast Blue injected either into the cervical spinal cord or the inferior olive. The soma/dendrite profiles of selected cells (53 rubrospinal, 19 DL-spinal, and 17 rubroolivary cells) were visualized by the intracellular injection of Lucifer Yellow/biocytin in fixed slices (400 microm thick) of midbrain. The descriptive statistics of the somata and the dendritic arborization of each rubral neuron type were established. Projection neuron subpopulations had similar but differentiable soma/dendrite profiles, with four to six slender, spine-bearing dendritic trees radiating out approximately 400 microm from the soma. Twelve presumed interneurons, all in the parvocellular nucleus, differed from projection neurons in that they had smaller somata and many slender, spine-bearing segments that constituted the multibranching dendrite profile that radiated out approximately 250 microm from the soma. A tentative model of the macaque rubral microcircuitry was developed, and its functional implications were explored. It incorporated 1) the known topography of the nucleus and its connections, 2) our data specifying the soma/dendrite morphology of the three main rubral neuron types, and 3) the ultrastructure reported by other laboratories of intrarubral synaptic connections.

Topics: Animals; Cell Size; Dendrites; Female; Fluorescent Dyes; Interneurons; Isoquinolines; Lysine; Macaca fascicularis; Male; Neural Pathways; Olivary Nucleus; Red Nucleus; Spinal Cord

Subicular neurons receive direct afferent connections from the vast majority of CA1 pyramidal cells and send their axons to the various brain areas. Because of this strategic position, subicular cells can modulate output of the hippocampus and, thus, play a significant part in memory, spatial processing, and seizure amplification and propagation from the hippocampus. Despite its important role as a hippocampal interface with different brain regions, present knowledge of the subiculum and the plastic properties of the synapses on the subicular neurons is rather limited. By using IR-DIC videomicroscopy and whole-cell patch-clamp recordings in mouse hippocampal slices, I demonstrated that long-term potentiation (LTP) in CA1-subicular cell synapses can be readily induced by high-frequency stimulation (HFS) of the afferents, but not by pairing of low-frequency stimulation with depolarization of postsynaptic cells. This tetanus-induced LTP is input specific, insensitive to the N-methyl-D-aspartate (NMDA) receptor antagonist 3-[(R)-2Carboxipiperazin-4-yl]-propyl-1-phosphonic acid (R-CPP), and reduces paired-pulse facilitation in potentiated synapses. Subsequent morphologic analysis of the recorded cells, which were filled either with Lucifer Yellow or Biocytin, revealed pyramidal-shaped neurons localized predominantly in the deep layer of the subiculum, close to the CA1 border. Axons of the majority of these neurons extended to the alveus and on toward the hippocampus, probably exiting it via the fornix. These data indicate that CA1-subicular cell synapses in mice exhibit LTP, which can be expressed presynaptically, and its induction does not require NMDA-receptor activation. The observed activity-dependent plasticity might play an important role in the integrative mechanisms of the subiculum and may influence transfer of information from the hippocampus to subcortical and cortical brain areas.

Topics: Action Potentials; Animals; Excitatory Amino Acid Antagonists; Excitatory Postsynaptic Potentials; Female; Fluorescent Dyes; Hippocampus; Isoquinolines; Long-Term Potentiation; Lysine; Male; Mice; Mice, Inbred C57BL; Microscopy, Video; Organ Culture Techniques; Patch-Clamp Techniques; Periodicity; Piperazines; Receptors, N-Methyl-D-Aspartate; Synapses

A combination of biocytin back-fills of the cerebral-buccal connectives and immunocytochemistry of the cerebral ganglion demonstrated that of the 13 bilateral pairs of cerebral-buccal interneurons in the cerebral ganglion, a subpopulation of 3 are immunopositive for the peptide myomodulin. The present paper describes the properties of two of these cells, which we have termed CBI-8 and CBI-9. CBI-8 and CBI-9 were found to be dye coupled and electrically coupled. The cells have virtually identical properties, and consequently we consider them to be "twin" pairs and refer to them as CBI-8/9. CBI-8/9 were identified by electrophysiological criteria and then labeled with dye. Labeled cells were found to be immunopositive for myomodulin, and, using high pressure liquid chromatography, the cells were shown to contain authentic myomodulin. CBI-8/9 were found to receive synaptic input after mechanical stimulation of the tentacles. They also received excitatory input from C-PR, a neuron involved in neck lengthening, and received a slow inhibitory input from CC5, a cell involved in neck shortening, suggesting that CBI-8/9 may be active during forward movements of the head or buccal mass. Firing of CBI-8 or CBI-9 resulted in the activation of a relatively small number of buccal neurons as evidenced by extracellular recordings from buccal nerves. Firing also produced local movements of the buccal mass, in particular a strong contraction of the I7 muscle, which mediates radula opening. CBI-8/9 were found to produce a slow depolarization and rhythmic activity of B48, the motor neuron for the I7 muscle. The data provide continuing evidence that the small population of cerebral buccal interneurons is composed of neurons that are highly diverse in their functional roles. CBI-8/9 may function as a type of premotor neuron, or perhaps as a peptidergic modulatory neuron, the functions of which are dependent on the coactivity of other neurons.

Topics: Animals; Aplysia; Central Nervous System; Ganglia, Invertebrate; Immunohistochemistry; In Vitro Techniques; Interneurons; Isoquinolines; Lysine; Membrane Potentials; Motor Neurons; Neural Pathways; Neuropeptides; Periodicity; Synaptic Transmission

Neurons in most peripheral ganglia are surrounded by satellite glial cells (SCs), but these cells have so far received little attention. We used immunohistochemistry and intracellular injections of tracers to characterize SCs in the intramural ganglia of the guinea-pig urinary bladder, which are part of the parasympathetic system. Intracellular injections of horseradish peroxidase (HRP) revealed two morphological types: cells that surrounded neurons and are SCs proper, and bipolar cells with processes that projected into the nerve fiber bundles connecting the ganglia. SCs were immunopositive for glutamine synthetase (GS) and S100beta and immunonegative for glial fibrillary acidic protein (GFAP). Injections of Lucifer yellow (LY) or biocytin (molecules known to cross gap junctions) into single SCs showed that these cells have a very low degree of intercellular coupling. A mean of 0.31 and 0.71 cells were coupled to the injected cells, using LY and biocytin, respectively. It appears that SCs in the bladder ganglia are distinct from central and enteric glial cells in the small degree of their coupling and in the absence of GFAP immunostaining.

Topics: Animals; Cell Communication; Fluorescent Dyes; Ganglia, Parasympathetic; Glial Fibrillary Acidic Protein; Glutamate-Ammonia Ligase; Guinea Pigs; Immunohistochemistry; Isoquinolines; Lysine; Male; Microscopy, Confocal; Neuroglia; S100 Proteins; Urinary Bladder

We define a morphologic type of ganglion cell in cat retina by using intracellular staining in vitro. The eta cell has a small soma, slender axon, and delicate, highly branched dendritic arbor. Dendritic fields are intermediate in size among cat ganglion cells, with diameters typically two to three times those of beta cells. Fields increase in size as a function of distance from the area centralis, ranging in diameter from 90 microm to 200 microm centrally to a maximum of 600 microm in the periphery. This increase is unusually radially symmetric. By contrast with other cat ganglion cell types, eta cells do not have markedly smaller dendritic fields within the visual streak than above or below it nor much smaller fields nasally than temporally. Dendrites ramify broadly throughout sublamina a (OFF sublayer) of the inner plexiform layer. They arborize most densely in S2, where they costratify with dendrites of OFF alpha cells. There is apparently no matching ON variety of eta cell. Experiments combining retrograde labeling with intracellular staining indicate that eta cells project to the superior colliculus and to two components of the dorsal lateral geniculate nucleus (the C laminae and medial interlaminar nucleus). Eta cells apparently project contralaterally from the nasal retina and ipsilaterally from the temporal retina. The morphology and projection patterns of the eta cell suggest that its physiologic counterpart is a type of sluggish or W-cell with an OFF center, an ON surround, and possibly a transient light response.

Topics: Animals; Axons; Biotin; Cats; Dendrites; Ferrets; Fluorescent Dyes; Isoquinolines; Lysine; Retinal Ganglion Cells; Species Specificity

Identified neurones F76 and D1 of the suboesophageal ganglia of Helix aspersa were studied in the isolated ganglia in vitro and in culture. The neurones were examined electrophysiologically with current clamp and morphologically either with intracellular injections of Lucifer Yellow or biocytin. These nerve cells had very similar resting membrane potentials and responses to injected current. The projections of D1 and F76 have been characterised, with both neurones having two main axons. The F76 neurones project to the left pallial, right pallial, anal, and visceral nerves as well as to the left and right pleural ganglia. The D1 neurones have similar projections except that they do not project to the anal and visceral nerves. The bilateral symmetry to the pallial nerves and pleural ganglia is discussed. These cells were also studied electrophysiologically after mechanical isolation and culture. F76 and D1 neurones were separated by dissection (no enzymes) and cultured in three ways. In normal snail Ringer they remained viable for up to two weeks with no development. In Ringer preincubated with a ganglia or containing endothelial growth factor, neurite outgrowths were seen. Membrane potentials were significantly lower in cultured neurones than in vitro and the after hyperpolarization never went below resting in cultured cells but it did in vitro.

Topics: Action Potentials; Animals; Cells, Cultured; Electrophysiology; Esophagus; Excitatory Postsynaptic Potentials; Ganglia, Invertebrate; Helix, Snails; Iontophoresis; Isoquinolines; Lysine; Neurons

The mechanisms underlying resealing of transected myelinated rat dorsal root axons were investigated in vivo using an assay based on exclusion of a hydrophilic dye (Lucifer Yellow-biocytin conjugate). Smaller caliber axons (<5 microm outer diameter) resealed faster than larger axons. Resealing was Ca2+ dependent, requiring micromolar levels of extracellular [Ca2+] to proceed, and further accelerated in 1 mM Ca2+. Two hours after transection, 84% of axons had resealed in saline containing 2 mM Ca2+, 28% had resealed in saline containing no added Ca2+ and only 3% had resealed in the Ca2+ buffer BAPTA (3 mM). The enhancing effect of Ca2+ could be overcome by both non-specific cysteine protease inhibitors (e.g., leupeptin) and inhibitors specific for the calpain family of Ca2+ -activated proteases. Resealing in 2 mM Ca2+ was not inhibited by an inhibitor of phospholipase A2. Resealing in low [Ca2+] was not enhanced by agents which disrupt microtubules, but was enhanced by dimethylsulfoxide (0.5-5%). These results suggest that activation of endogenous calpain-like proteases by elevated intra-axonal [Ca2+] contributes importantly to membrane resealing in transected myelinated mammalian axons in vivo.

Topics: Animals; Axons; Calcium; Calpain; Cell Size; Dimethyl Sulfoxide; Enzyme Inhibitors; Female; Fluorescent Dyes; Ganglia, Spinal; Image Processing, Computer-Assisted; Isoquinolines; Kinetics; Lysine; Male; Microscopy, Confocal; Microtubules; Myelin Sheath; Phospholipases A; Phospholipases A2; Rats; Rats, Sprague-Dawley

Local collateral projections from the medial superior olivary nucleus in the gerbil auditory brainstem were examined to study the possible communication of this nucleus with periolivary cell groups. The projections were investigated using intracellular and extracellular labeling with Biocytin in the medial superior olive (MSO) in brainstem tissue slices. Collateral axons were found to branch from the main axons of the central cells of the MSO as the latter passed through a dorsally neighboring periolivary nucleus, the superior paraolivary nucleus (SPN), toward the ipsilateral inferior colliculus (IC), their traditionally accepted target. Bouton-like endings and en passant varicosities of these collaterals appeared to contact the somata and proximal dendrites of cells within the SPN. Furthermore, close observation revealed that these collaterals terminate on at least two types of SPN cells. Intracellular labeling of the collateral axons of the MSO neurons combined with retrograde prelabeling of their target cells, however, revealed that the collaterals selectively contact the cells of the SPN that project to the ipsilateral IC. A link between the MSO and SPN has not been reported previously. This connection is of interest since SPN cells themselves project either to the cochlear nuclei (CN) or the IC. The MSO-SPN projection identified here raises the possibility that the latter may serve as an ancillary channel to convey MSO information to the IC.

Topics: Animals; Auditory Pathways; Axons; Coloring Agents; Female; Fluorescent Dyes; Gerbillinae; Isoquinolines; Lysine; Male; Olivary Nucleus; Organ Culture Techniques; Phenothiazines; Sound Localization; Stilbamidines

This paper provides the first detailed description of the trigeminal innervation of the inner ear vasculature. This system provides a newly discovered neural substrate for rapid vasodilatatory responses of the inner ear to high levels of activity and sensory input. Moreover, this discovery may provide an alternative mechanism for a set of clinical disturbances (imbalance, hearing loss, tinnitus and headache) for which a central neural basis has been speculated. Iontophoretic injections of biocytin were made via a glass microelectrode into the trigeminal ganglion in guinea-pigs. Tissue for histological sections was obtained 24 h later. Labeled fibers from the injection site were observed as bundles around the ipsilateral spiral modiolar blood vessels, as individual labeled fibers in the interscala septae, and in the ipsilateral stria vascularis. The dark cell region of the cristae ampullaris in the vestibular labyrinth was also intensively labeled. No labeled fibers were observed in the neuroepithelium of the cristae ampullaris or the semicircular canals. These results confirm and localize an earlier indirect observation of the trigeminal ganglion projection to the cochlea. This innervation may play a role in normal vascular tone and in some inner ear disturbances, e.g., sudden hearing loss may reflect an abnormal activity of trigeminal ganglion projections to the cochlear blood vessels.

Topics: Animals; Axonal Transport; Axons; Basilar Artery; Blood Vessels; Cerebral Arteries; Cochlea; Functional Laterality; Guinea Pigs; Horseradish Peroxidase; Isoquinolines; Lysine; Nerve Fibers; Neurons; Trigeminal Ganglion; Trigeminal Nerve; Vasodilation

Cells infected with the intracellular parasite Eimeria nieschulzi were microinjected with lucifer yellow (457 Da), biocytin lucifer yellow (850 Da) and dextranrhodamine (10,000 Da). Immediately after injection of a mixture of the markers into the host cell cytoplasm, a differential diffusion pattern was observed in trophozoites and schizonts. Lucifer yellow and biotin lucifer yellow were seen to enter the parasitophorous vacuole, whereas the dextran was excluded. Since these markers cannot permeate cell membranes, this suggests that the Eimerian parasitophorous vacuole acts as a molecular sieve. This method allows the visualization and further characterization of the parasitophorous vacuole in living cells.

Topics: Animals; Cell Line; Dextrans; Diffusion; Eimeria; Fluorescent Dyes; Indicators and Reagents; Isoquinolines; Lysine; Microinjections; Rats; Rhodamines; Vacuoles

Dye tracers were chosen, based on net charge, chemical structure, and reactive groups, to test for the existence of and to provide novel insight into channel selectivities of junctional pathways connecting smooth muscle and endothelial cells of the arteriolar wall. Dyes were injected into individual smooth muscle or endothelial cells of hamster cheek pouch arterioles using microiontophoresis. Coupling, independent of tracer net charge, was seen both within and between cell layers. Endothelial cells were well coupled by all of the tested dyes. Smooth muscle junctions appeared less effective in dye transfer than endothelial junctions. Lucifer yellow was confirmed to be a poor tracer of smooth muscle gap junctions, and remarkably this dye and other related sulfate-containing molecules interfered with dye movement through smooth muscle but not endothelial junctions. Myoendothelial junctions showed a striking polarity of dye movement, with dye transfer from endothelial to smooth muscle cells but little or no transfer in the reverse direction. Because the dyes have size and charge characteristics similar to those of known cellular second messengers, these findings have important implications for cell-cell signaling in the vessel wall.

Topics: Animals; Arterioles; Cell Communication; Cricetinae; Endothelium, Vascular; Gap Junctions; Isoquinolines; Lysine; Male; Mesocricetus; Muscle, Smooth, Vascular

The present study characterizes the morphology and tracer coupling patterns of oligodendrocytes in the myelinated band of the rabbit retina, as revealed by intracellular injection of biocytin or Lucifer yellow in an isolated superfused preparation. Based on the observed heterogeneity in morphology, we have grouped the presumptive oligodendrocytes into three categories termed 'parallel', 'stratified' and 'radial'. Most parallel oligodendrocytes were tracer coupled to nearby oligodendrocytes and astrocytes, whereas the stratified and radial oligodendrocytes rarely showed coupling. We conclude that the different categories of oligodendrocytes may be stages in a developmental series, with radial oligodendrocytes being premyelinating cells, parallel oligodendrocytes being mature myelinating cells and the stratified cells representing a transition between these categories.

Topics: Animals; Antibody Specificity; Astrocytes; Cell Size; Female; Fluorescent Dyes; Glial Fibrillary Acidic Protein; Isoquinolines; Lysine; Male; Microinjections; Microscopy, Confocal; Oligodendroglia; Rabbits; Retina

Horizontal and amacrine cells in the isolated carp retina were impaled with micropipette electrode, identified by their characteristic light responses, and injected iontophoretically with markers for morphological study. Both Lucifer Yellow CH and biocytin were injected simultaneously. Lucifer Yellow was seen by its own fluorescence while biocytin was visualized by binding with Texas Red-linked or horseradish peroxidase-conjugated avidin. For cone-connected horizontal cells, biocytin-coupled cells were found to be approximately five-times more numerous than Lucifer Yellow-coupled cells. Coupling for both tracers was consistently hampered by intravitreally applied dopamine. In untreated retinas, the injected Lucifer Yellow was restricted within one rod-connected horizontal cell, while biocytin revealed several coupled neighbors. Amacrine cells, labeled by the tracers, were morphologically grouped into eight types, based on our earlier classification. Among them, amacrine cells, belonging to three types (Fnd, Pmb or Pma), were confirmed to be Lucifer Yellow-coupled, and the number of biocytin-coupled cells was more numerous (about 2.5 times) than that of Lucifer Yellow-coupled cells. Most amacrine cells (i.e. Pwd, Fnb and Fna) showed biocytin-coupling with no Lucifer Yellow-coupling. A few classified (i.e. Pwb and Fwa) and unclassified cells did not show any coupling. Since the tracer coupling takes place via gap junctions, the majority of amacrine cells, belonging to certain homologous types, appear to be functionally coupled with each other in the inner plexiform layer. However, dopamine did not influence the range of tracer coupling between amacrine cells in the carp retina under the present experimental conditions.

Topics: Animals; Carps; Diffusion; Dopamine; Fluorescent Dyes; Horseradish Peroxidase; Isoquinolines; Lysine; Microscopy, Fluorescence; Retina; Staining and Labeling

The avidin-biotin-complex method is a popular immunocytochemical technique. This method labels consistently a group of neurons in the lobster ventral nerve cord in the absence of primary antibodies. The specific staining is due to a relatively high level of endogenous biotin (or biocytin) in these neurons. These biotin-positive neurons are located in the supraesophageal, thoracic, and abdominal ganglia. Intraaxonal injection of Lucifer yellow followed by Texas red-conjugated streptavidin staining reveals that the neurons are members of the medial giant (MG) and lateral giant (LG) systems, which are important in mediating rapid tail flipping during escape maneuvers. In neuronal somata, staining is restricted to the cytoplasm. Within MG axons, staining appears as punctate, subaxolemmal structures. Preincubating nerve cords in biocytin or direct intraaxonal injection of biocytin enhances staining of these punctate organelles. In LG axons, staining is localized to fragments of braided filamentous structures that also appear to be associated with the axolemma. Preincubation of ventral nerve cords in various concentrations of biocytin results in the appearance of additional groups of stained neurons, suggesting that there are subsets of neurons with specific biocytin-uptake or -retention mechanisms. In the crayfish, biotin-positive staining is confined to the MG neurons; the LG neurons are not stained. In the earthworm, no staining is observed in the MG and LG axon escape systems. In the goldfish, no biotin-staining is seen in the Mauthner neurons and their axons. The significance of specific localization of biotin or biocytin to subsets of neurons is unclear. It may reflect the presence of high levels of biocytin moieties on biotin-dependent enzymes. Biotin is an important cofactor in the catalytic functions of several decarboxylases crucial in energy production and lipogenesis. Axons of the giant fiber systems in lobsters and crayfish may have high energy and fatty acid synthesis requirements. Increased levels of biotin accumulation may also be related to other functions of the giant axon systems, such as the formation of electrical synapses among themselves and with phasic motoneurons.

Topics: Animals; Astacoidea; Avidin; Bacterial Proteins; Biotin; Fluorescent Dyes; Ganglia, Invertebrate; Goldfish; Isoquinolines; Lysine; Nephropidae; Nerve Fibers; Oligochaeta; Species Specificity; Staining and Labeling; Streptavidin

We have examined in vitro the morphology and visual response properties of retinal ganglion cells innervating a component of the cat's lateral geniculate nucleus known as the geniculate wing (or retinorecipient zone of the pulvinar). Ganglion cells were first labeled in situ by retrograde transport of fluorescent microspheres from the geniculate wing. Labeled cells were injected intracellular with Lucifer yellow and biocytin in the isolated retina and visualized immunohistochemically. With one exception, stained cells appeared to belong to a single morphological class that corresponded closely to the epsilon cell of earlier descriptions (Leventhal et al., 1980; Rodieck and Watanabe, 1986). They had somas comparable in size to those of beta cells and large, sparse dendritic trees that ramified in the inner (ON) sublayer of the inner plexiform layer. Dendritic fields increased in size with eccentricity, but only within the central retina, and were among the largest so far reported for cat ganglion cells, exceeding those of alpha cells at most eccentricities. Dendritic profiles were typically elliptical with long axes pointing toward the area centralis. Axons were about as thick as those of beta cells and thicker than those of other varieties of non-alpha, non-beta ganglion cells. We recorded extracellularly from microsphere-labeled wing-projecting ganglion cells in a superfused, flattened eyecup preparation. All such cells exhibited sustained responses to standing contrast and had very large, concentric receptive fields with ON-centers and OFF-surrounds. Their response to gratings showed that they have relatively poor spatial resolution and a moderate amount of nonlinearity of spatial summation. These cells thus have many physiological response properties in common with ganglion cells previously termed "on-center tonic W-cells," "on-center sluggish sustained cells," and "Q-cells." These findings indicate that ganglion cells innervating the cat's geniculate wing form a structurally and functionally homogeneous class. Their large dendritic and receptive fields and low-pass spatial frequency tuning suggest that fine spatial resolution is not required for the execution of their functional role(s).

Topics: Animals; Cats; Electrophysiology; Fluorescent Dyes; Geniculate Bodies; In Vitro Techniques; Isoquinolines; Lysine; Microspheres; Retinal Ganglion Cells

This paper describes the early morphological and physiological development of pyramidal neurons in layer 5 of the rat visual cortex in relation to the targets chosen by their axons. Cells were prelabeled by retrograde transport from the superior colliculus or the contralateral visual cortex and intracellularly injected either in fixed slices or after recording in living slices. In the adult, corticotectal cells have thick apical dendrites with an extensive terminal arborization extending into layer 1, and fire characteristic bursts of action potentials when injected with a depolarizing current; interhemispheric cells have slender apical dendrites that terminate without a terminal tuft, usually in layer 2/3, and they display a more regular firing pattern (Kasper et al.: J Comp Neurol, this issue, 339:459-474). At embryonic day E18 (when axons of the two classes of cells are already taking different routes towards their targets) and E21, pyramidal-like cells throughout the cortical plate all have similar soma-dendritic morphology, with spindle-shaped cell bodies and few, short basal dendrites but apical dendrites that all end in distinct tufts in the marginal zone. At postnatal day P3, after the axons of both cell classes have reached their targets, all pyramidal neurons in layer 5 still have distinct terminal arborizations in layer 1, though they vary in complexity and extent. The somata are now more mature (round to ovoid in shape), and the basal dendritic tree has extended. As early as P5, all cells studied could be clearly classified as tufted or untufted (considerably earlier than previously reported; Koester and O'Leary: J Neurosci 12:1382, '92), and these features correlated precisely with the projection target, as in the adult. Measurement showed that although interhemispheric cells lose their terminal tufts, in general the trunks of their apical dendrites do not withdraw but continue to grow, at roughly the same rate as those of corticotectal cells. The two classes of layer 5 pyramidal neurons differentiate from each other in three distinct phases: pathway selection by axons precedes the loss of the apical tuft by interhemispheric cells, and these morphological characteristics are established 10 days before the onset of burst-firing in corticotectal cells. These three steps may be guided by different molecular signals.

Topics: Action Potentials; Animals; Axons; Dendrites; Electrophysiology; Female; Fluoresceins; Histocytochemistry; In Vitro Techniques; Isoquinolines; Lysine; Membrane Potentials; Pregnancy; Pyramidal Cells; Rats; Visual Cortex; Visual Pathways

We have assessed the properties of three intracellular markers, horseradish peroxidase, biocytin/Neurobiotin, and Lucifer Yellow, and have compared their usefulness as neuronal markers for light and electron microscopic visualization. Neurons in the acute slice preparation of rat hippocampus were filled with one of these markers, and the marker was converted to an optical and electron-dense reaction product. Dimethylsulfoxide (DMSO) greatly facilitated penetration of recognition reagents while preserving membrane integrity. The markers were compared with respect to injection parameters, mobility and recognition, stability and visibility, and ultrastructural clarity. Horseradish peroxidase (HRP)-labeled neurons, recognized histochemically with diaminobenzedine (DAB), were easily visualized by the density of the DAB reaction product; however, the electron density was often so great as to obscure ultrastructural details. Biocytin (BC)-/Neurobiotin (NB)-labeled neurons were recognized by avidin-HRP, followed by histochemical localization of HRP with DAB. The optically dense reaction product gave complete visualization of the soma and processes at the light microscopic level. The electron density was homogeneously distributed throughout the cell, so that ultrastructural features were easily identified. Lucifer Yellow (LY), a fluorescent marker, was converted to an optical and electron-dense reaction product via immunocytochemical staining with a rabbit anti-LY antibody, followed by goat anti-rabbit IgG-HRP and DAB histochemical localization. Similar to BC/NB, the reaction product was evenly dispersed, providing good light microscopic and ultrastructural clarity. Under our experimental conditions, BC/NB and LY were superior markers that could be used routinely to label neurons, and give excellent visualization not only at the light but also at the electron microscopic level.

Topics: Animals; Antigens, Ly; Female; Hippocampus; Horseradish Peroxidase; Isoquinolines; Lysine; Male; Microscopy, Electron; Neurons; Rats; Rats, Sprague-Dawley; Staining and Labeling

Gap junctions permit the passage of ions and small molecules between cells, thereby providing a basis for direct intercellular communication. In the rabbit retina, the low molecular weight dyes Lucifer yellow and biocytin passed readily from astrocytes into adjacent astrocytes, oligodendrocytes, and Müller cells. However, the dyes rarely passed from either oligodendrocytes or Müller cells into astrocytes. Unidirectional passage of dye suggests the presence of an asymmetric barrier to the movement of molecules through heterologous gap junctions and indicates the potential for a hierarchy of command between interconnected cells.

Topics: Animals; Astrocytes; Cell Communication; Connexins; Diffusion; Gap Junctions; Isoquinolines; Lysine; Models, Neurological; Neuroglia; Oligodendroglia; Permeability; Rabbits; Retina

Vertebrate retinal amacrine cells produce transient or sustained responses. Sustained depolarizing amacrine cells in the dace retina were identified by their intracellularly recorded responses to light flashes. The response amplitude produced a notable spatial summation which exceeded that of individual dendritic arbors. When sustained type amacrine cells were intracellularly injected with Lucifer Yellow and biocytin, there was extensive transfer of biocytin, but not Lucifer Yellow, to surrounding cells with similar cellular morphology. Ultrastructural analysis of the interconnections by electron microscopy revealed the presence of gap junctions at the contact area, which did not include conventional synapses. Present results demonstrate that sustained response amacrine cells make direct electrical connections between the cells of the same type and electrical coupling may contribute to extension of their receptive fields.

Topics: Animals; Cyprinidae; Electric Conductivity; Electric Stimulation; Fluorescent Dyes; Gap Junctions; Isoquinolines; Lysine; Microscopy, Electron; Photic Stimulation; Retina

We have developed a method for reliable, permanent, high-resolution intracellular staining of ganglion cells in mammalian retinas. Living ganglion cells in the isolated retina are impaled in vitro and injected intracellularly with both Lucifer Yellow (LY) and biocytin. After fixation and aggressive pretreatment of the retina with detergents, the LY is tagged immunohistochemically with biotin using a commercially available anti-LY antibody and a biotinylated secondary antibody. A conventional avidin-biotin procedure is then used to visualize both the biocytin and the biotinylated bridge antibody, yielding complete Golgi-like filling of the soma, dendrites and axon. Advantages of the method include the ease and speed of dye injection, the reliable recovery of stained cells, the large number of cells which can be stained in single retinas, and the high resolution and permanence of the stain, which permit prolonged examination and quantitative analysis.

Topics: Animals; Axons; Cats; Dendrites; Immunohistochemistry; Isoquinolines; Lysine; Rabbits; Rats; Retinal Ganglion Cells; Staining and Labeling

A circadian rhythm in the frequency of compound action potentials (CAPs) in the optic nerve of the mollusc Bulla gouldiana is believed to be generated by the basal retinal neurons (BRNs) of the eye. Along with the CAPs, which are about 100 microV in amplitude, there are 10- to 40-microV impulses from an undetermined cell type in records from the optic nerve. These impulses, called "small spikes," are generated spontaneously in darkness and show a circadian rhythm in frequency that is about 12 hr out of phase with the CAP rhythm. To enable us to determine the origin of the small spikes, intracellular recordings were made from retinal cells while optic nerve activity was monitored. The cells were identified by their light responses and then injected with the fluorescent dye Lucifer Yellow CH or the tracer biocytin. It was found that the large photoreceptors of the distal retina generated graded depolarizations in response to light, and had axons in the optic nerve, but did not show impulses at the level of the photoreceptor layer. By contrast, the spiking retinal cells of the photoreceptor layer generated depolarizations and impulses in response to light. In addition, the spiking cells were found to be dye-coupled to a series of retinal cells approximately 7 microns in diameter, connected to a single axon in the optic nerve. Impulses from the spiking cells occurred spontaneously and correspond with the small spikes in the optic nerve. The BRNs appear to inhibit the retinal cells that generate the small spikes. Hyperpolarization of the BRNs, through constant-current injection, increased the number of small spikes in the optic nerve. Release from hyperpolarization led to a decrease in small spikes. This could explain how circadian changes in BRN membrane potential might modulate spontaneous firing of the spiking cells, resulting in the circadian rhythm in small-spike frequency.

Topics: Animals; Circadian Rhythm; Histocytochemistry; In Vitro Techniques; Isoquinolines; Lysine; Membrane Potentials; Neurons; Optic Nerve; Photoreceptor Cells; Retina; Snails; Staining and Labeling

Here we describe a method for intracellularly injecting mixtures of the fluorescent dye Lucifer Yellow and the permanent tracers HRP or biocytin into aldehyde-fixed slices of the dorsal lateral geniculate nucleus in young postnatal cats. Lucifer Yellow was used for visual control in the injection procedure and the inclusion of HRP or biocytin allowed the subsequent use of simple histochemical processing to give a permanent record of the injected cells. Both tracer mixtures revealed the dendritic morphology of injected cells. However, HRP was found to be superior to biocytin, in that dendrites were better defined and fine details of cellular morphology such as spines were consistently revealed. Using this technique we were able to demonstrate that the dendritic morphology of geniculate cells is much more mature between birth and 2 weeks than was thought from previous studies using Golgi methods.

Topics: Animals; Animals, Newborn; Cats; Dendrites; Electrodes; Electrophysiology; Geniculate Bodies; Histocytochemistry; Horseradish Peroxidase; Isoquinolines; Lysine; Neurons; Tissue Fixation

Uniglomerular projection neurons in the antennal lobe of Periplaneta americana, the axons of which connect the lobe to the protocerebrum, were labeled by intracellular injection of Lucifer Yellow or biocytin. The fine structure of individual neurons within the antennal lobe was examined after the injected substances had been converted (by immunohistochemical or histochemical treatment) to electron microscopically visible reaction products. Seven projection neurons were investigated, including attractant neurons, with dendritic arbors in the macroglomerulus, and projection neurons of normal-sized glomeruli. From reconstructions of thin serial sections and examination of additional processes present at various places in the arborization regions, the distribution of synapses within the glomeruli was inferred. Although the projection neurons differ from one another in their glomerular arborization patterns, they are very similar in the spatial segregation of their input and output synapses within the arborization. Output synapses are found on the thick part of the fiber near its site of entry into the glomerulus, as well as in regions within the glomerulus where the neuron has begun to ramify into thinner fibers. In the latter regions, the many output synapses are accompanied by occasional input synapses; hence these are regarded as transitional regions. At the terminal arbors only input synapses were found. This suggests that neurons with dense terminal arborizations receive particularly numerous inputs in these regions. The large number of input synapses reflects the high degree of convergence of afferents onto projection neurons previously demonstrated physiologically. However, the presence of numerous output synapses indicates that projection neurons not only transport sensory information into the protocerebrum but are also a major component of the neuronal circuitry within the antennal lobe.

Topics: Animals; Axonal Transport; Cockroaches; Fluorescent Dyes; Isoquinolines; Lysine; Microscopy, Electron; Nerve Fibers; Neurons; Olfactory Pathways; Synapses

Hypothalamic paraventricular and supraoptic neurons were recorded intracellularly in coronal slices and injected with Lucifer yellow, ethidium bromide or biocytin. Electrical properties, morphological staining and neurophysin immunohistochemistry were compared among the 3 markers. Lucifer yellow electrodes had a high resistance and frequently blocked during experiments. Neurons recorded with Lucifer yellow electrodes had low input resistances and low-amplitude, broad spikes. Lucifer yellow labeling in whole mount was highly fluorescent, revealing distal dendrites and axons. Of cells injected with Lucifer yellow, 64% were recovered but were faint after immunohistochemical processing. Recordings with ethidium bromide electrodes were similar to controls, although electrode blockage sometimes occurred. Only somata and proximal dendrites of ethidium bromide-filled neurons were visible in whole-mount. Forty percent of cells injected with ethidium bromide were recovered after immunohistochemical processing; these were invariably faint. Recordings with biocytin-filled electrodes were similar to control recordings. Biocytin-filled, HRP-labeled cells showed distal dendrites and often dendritic spines and axons in 50-75-microns sections. Seventy percent of biocytin-injected cells labeled with fluorescent markers were recovered and remained strongly labeled after immunohistochemical processing. Biocytin had the best electrical and staining properties for combined electrophysiological and anatomical studies.

Topics: Animals; Brain; Brain Mapping; Ethidium; Fluorescent Antibody Technique; Immunoenzyme Techniques; Isoquinolines; Lysine; Membrane Potentials; Microelectrodes; Neurons; Neurophysiology; Rats; Rats, Inbred Strains; Staining and Labeling

The use of in vitro preparations such as brain slices poses difficulties in determining the correct identity of cells under study. To circumvent this problem, we first used a fluorescence pre-labeling technique (rhodamine-dextran-lysine) to identify cranial motoneurons projecting to the tongue (hypoglossal motoneurons) in the guinea-pig. Following preparation of slices, cells were recorded intracellularly and their electrophysiological properties determined. The cells were then intracellularly stained with both a fluorescence label (Lucifer Yellow) and with the stable, non-fading label biocytin. Under fluorescent illumination, the great majority of recorded cells within the hypoglossal nucleus were double-labeled (rhodamine and Lucifer Yellow) suggesting that most are indeed motoneurons. Biocytin injected into the same motoneurons provided permanent and detailed images of their morphology. Intracellularly stained cells surrounding the hypoglossal nucleus were not labeled with rhodamine and had distinct electro-physiological properties. The use of the retrogradely transported marker rhodamine-dextran-lysine allows the unambiguous identification of motoneurons in a brainstem slice. The combined intracellular injection of Lucifer Yellow and biocytin provides a simple means of melding the advantages of a fluorescent label (compatible with other fluorescence labels and with immunocytochemistry) with the benefits of a stable, non-fading, electron-dense marker. Application of this technique should prove useful in establishing morphological and functional correlates in other areas of the CNS.

Topics: Animals; Brain Stem; Coloring Agents; Dextrans; Fluorescent Dyes; Guinea Pigs; Hypoglossal Nerve; In Vitro Techniques; Isoquinolines; Lysine; Motor Neurons; Neurons; Rhodamines; Synaptic Transmission; Tongue