Axon Cap Research Articles

Distribution of glycinergic neurons in the brain of glycine transporter‐2 transgenic Tg(glyt2:Gfp) adult zebrafish: Relationship to brain–spinal descending systems

We used a Tg(glyt2:gfp) transgenic zebrafish expressing the green fluorescent protein (GFP) under control of the glycine transporter 2 (GLYT2) regulatory sequences to study for the first time the glycinergic neurons in the brain of an adult teleost. We also performed in situ hybridization using a GLYT2 probe and glycine immunohistochemistry. This study was combined with biocytin tract tracing from the spinal cord to reveal descending glycinergic pathways. A few groups of GFP(+) /GLYT2(-) cells were observed in the midbrain and forebrain, including numerous pinealocytes. Conversely, a small nucleus of the midbrain tegmentum was GLYT2(+) but GFP(-) . Most of the GFP(+) and GLYT2(+) neurons were observed in the rhombencephalon and spinal cord, and a portion of these cells showed double GLYT2/GFP labeling. In the hindbrain, GFP/GLYT2(+) populations were observed in the medial octavolateral nucleus; the secondary, magnocellular, and descending octaval nuclei; the viscerosensory lobes; and reticular populations distributed from trigeminal to vagal levels. No glycinergic cells were observed in the cerebellum. Tract tracing revealed three conspicuous pairs of GFP/GLYT2(+) reticular neurons projecting to the spinal cord. In the spinal cord, GFP/GLYT2(+) cells were observed in the dorsal and ventral horns. GFP(+) fibers were observed from the olfactory bulbs to the spinal cord, although their density varied among regions. The Mauthner neurons received very rich GFP(+) innervation, mainly around the axon cap. Comparison of the zebrafish glycinergic system with the glycinergic systems of other adult vertebrates reveals shared patterns but also divergent traits in the evolution of this system.

GABAergic inputs and its development on the zebrafish Mauthner cells

Event Abstract Back to Event GABAergic inputs and its development on the zebrafish Mauthner cells Takanori Ikenaga1*, Takeshi Higashi1, Takayuki Sumimoto1 and Kohei Hatta1 1 University of Hyogo, Graduate School of Life Science, Japan The teleost fish show rapid escape behavior from aversive stimuli. This is triggered by Mauthner (M) cells which are identifiable neurons in the teleost hindbrain. Activity of M-cell during initial phase of escape behavior is controlled via the glycinergic inputs. In addition, some physiological and histological date indicated that M-cells receive inputs from GABA, another inhibitory neurotransmitter in the vertebrate brain. However, detail of their function in the Mauthner systems is still unknown. In the present study, immunohistochemisry with Gad1 or Gad2 antibodies, raised from partial sequence of the zebrafish Gad1 or 2 proteins respectively, were performed with the transgenic line (Tol056) which expresses GFP in the M-cells. Gad1 immunopositive clusters could be observed on the lateral and ventral dendrites from at least 3 days post fertilization (dpf). They were most prominent in the distal portion of lateral dendrite. Amount of clusters were increased according to development but distribution pattern was not changed in their larval stages. No immunoreactivity was observed around the initial segment of the axon. In Gad2 immunohistochmistry, puncta could be observed also in the lateral and ventral dendrites but amount of them was fewer compared with that of Gad1. In contrast to Gad1, scattered Gad2 immunopositive signals could be seen around the initial segment of the axon from 5dpf. In 7dpf, they showed larger cluster shape. These clusters were distributed around the axon but did not contact directly. This finding raised a possibility that these Gad2 positive clusters were associated with the axon cap which surround initial segment of M-axon. Phalloidin staining could visualize structure of axon cap including cap cells and central core. Double staining with Gad2 antibody and phalloidin showed that Gad2 clusters were localized inside of axon cap. These results suggest that there are unknown functions of GABA for controlling M-cell activity within the escape behavior in addition to the functions of glycine. Keywords: GABA, inhibition, Mauthner Cell, reticulo-spinal neurons, Zebrafish Conference: Tenth International Congress of Neuroethology, College Park. Maryland USA, United States, 5 Aug - 10 Aug, 2012. Presentation Type: Poster (but consider for Participant Symposium) Topic: Motor Systems Citation: Ikenaga T, Higashi T, Sumimoto T and Hatta K (2012). GABAergic inputs and its development on the zebrafish Mauthner cells. Conference Abstract: Tenth International Congress of Neuroethology. doi: 10.3389/conf.fnbeh.2012.27.00265 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 30 Apr 2012; Published Online: 07 Jul 2012. * Correspondence: Dr. Takanori Ikenaga, University of Hyogo, Graduate School of Life Science, Ako-gun, Hyogo, 6781297, Japan, ikenagat@sci.u-hyogo.ac.jp Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Takanori Ikenaga Takeshi Higashi Takayuki Sumimoto Kohei Hatta Google Takanori Ikenaga Takeshi Higashi Takayuki Sumimoto Kohei Hatta Google Scholar Takanori Ikenaga Takeshi Higashi Takayuki Sumimoto Kohei Hatta PubMed Takanori Ikenaga Takeshi Higashi Takayuki Sumimoto Kohei Hatta Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Axon cap morphology of the sea robin (Prionotus carolinus): mauthner cell is correlated with the presence of “signature” field potentials and a C‐Type startle response

Studies on the Mauthner cell (M-cell) of goldfish, Carassius auratus, have facilitated our understanding of how sensory information is integrated in the hindbrain to initiate C-type fast startle responses (C-starts). The goldfish M-cell initial segment/axon hillock is surrounded by a composite axon cap consisting of a central core and a peripheral zone covered by a glial cell layer. The high resistivity of the axon cap results in "signature" field potentials recorded on activation of the M-cell, allowing unequivocal physiological identification of the M-cell and of its feedback and reciprocal inhibitory networks that are crucial in ensuring that only one M-cell is active and that it fires only once. Phylogenetic mapping of axon cap morphology to muscle activity patterns and behavior predicts that teleost fishes that have a composite axon cap, like that of the goldfish, will perform C-start behavior with primarily unilateral muscle activity. We have chosen to study these predictions in the northern sea robin, Prionotus carolinus, a percomorph fish. Although sea robins have a very different phylogenetic position, body form, and habitat compared with the goldfish, they display the correlation of axon cap morphology to physiology and C-start behavior. Differences in response parameters suggest some evolutionary trade-offs in sea robin C-start behavior compared with that of the goldfish, but the correlations in morphology, physiology, and behavior are common features of both otophysan and nonotophysan teleosts. The M-cell will continue to provide an unprecedented opportunity to study the evolution of a neural circuit in the context of behavior.

Reticulospinal neurons in anamniotic vertebrates: A celebration of Alberto Stefanelli's contributions to comparative neuroscience

Over the past 76 years Alberto Stefanelli has successfully used a comparative approach to study the nervous system. His main research focus during that time has been on identifiable reticulospinal neurons including Müller and Mauthner neurons found in anamniotic vertebrates. Born in Venice, Italy in 1908, Professor Stefanelli pursued most of his academic career at the University of Rome, where he retired as Chair of Comparative Anatomy in 1978.His seminal work on the constancy in number and position of giant identifiable reticulospinal neurons in the brains of larval and adult lampreys, and his assertion that only a subset of these neurons were Müller cells, provided the framework in which subsequent authors have refined our understanding of the cellular anatomy, axonal projections, physiology, and function of Müller cells in the control of movement. Stefanelli has also provided the most comprehensive study to date of the Mauthner cell and its axon cap. His description of the differences in axon cap structure among many fishes and amphibians and his use of the “morpho-ecological” approach to determine Mauthner cell function has provided the basis for future studies on the neuronal basis of behavior and its evolution.As Professor Stefanelli approaches his 100th birthday, we celebrate his scientific contributions to comparative neuroscience with a biographical sketch of his life, an overview of his scientific accomplishments, and our view of how his comparative studies continue to contribute to our understanding of the nervous system.

Physiological properties of the mauthner system in the adult zebrafish

We investigated the morphological and electrophysiological properties of the Mauthner (M-) cell and its networks in the adult zebrafish (Danio rerio) in comparison with those in the goldfish (Carassius auratus). The zebrafish M-cell has an axon cap, a high resistivity structure which surrounds the initial segment of the M-axon, and accounts for an unusual amplification of the fields generated within and around it. Second, extra- and intracellular recordings were performed with microelectrodes. The resting potential was approximately -80 mV with an input resistance of approximately 0.42 M omega. The M-cell extracellular field was large (10-20 mV), close to the axon hillock, and the latency of antidromic spikes short (approximately 0.4 milliseconds), confirming a high conduction velocity in the M-axon. The extrinsic hyperpolarizing potential (EHP), which signals firing of presynaptic cells and collateral inhibition, was markedly lower at frequencies of spinal stimulation > approximately 5/second, suggesting an organization of the recurrent collateral network similar to that in the goldfish. Inhibitory postsynaptic potentials (IPSPs) were highly voltage-dependent; their decay time constant was increased by depolarizations. The presynaptic neurons which are numerous could be identified by their passive hyperpolarizing potential (PHP) produced by the M-spike current. Auditory responses, mediated via mixed synapses (electrical and chemical), had short delays and hence are well suited to trigger the escape reaction. The similarities of their properties indicate that the wealth of information generated over decades in the goldfish can be extrapolated to the zebrafish.

Co-localization of calretinin and parvalbumin with nicotinamide adenine dinucleotide phosphate-diaphorase in tench Mauthner cells

The co-localization of calretinin (CR) and parvalbumin (PV) immunoreactivity with nicotinamide adenine dinucleotide phosphate-diaphorase (ND) activity was analyzed in the Mauthner cells of the tench. Mauthner cells were ND active, and ND staining was observed in the soma, axon cap region, and axon of these neurons. CR co-localized with ND in the axon of the Mauthner cells but not in the cell body or in the dendrites, whereas PV immunoreactivity co-localized with ND in the soma, axon and dendrites. The presence of two different calcium-binding proteins in the Mauthner cells indicates that these neurons need complex calcium-buffering systems. The co-localization of these calcium-binding proteins with ND might suggests their involvement in nitric oxide-related events.

Mauthner cell-initiated abrupt increase of the electric organ discharge in the weakly electric fish Gymnotus carapo

Stimulation of the spinal cord of the electric fish Gymnotus carapo, evoked an abrupt increase in the discharge rate of the electric organ. At the maximum of this response, the rate increased an average of 26 ± 11.8%. The duration of the response was 4.9 ± 2.12 s; its latency was 10.4 ± 1.1 ms. Activation of the Mauthner axon played a decisive role in this phenomenon as indicated by the following: (1) recordings from the axon cap of the Mauthner cell demonstrated that the response was evoked if the Mauthner axon was antidromically activated and (2) a response that was similar to that produced by spinal cord stimulation, was elicited by intracellular stimulation of either Mauthner cell. Stimulation of the eighth nerve could also increase the discharge rate of the electric organ. The effect was greater if a Mauthner cell action potential was elicited. The findings described in the present report, indicate the existence of a functional connection between the Mauthner cell and the electromotor system in Gymnotus carapo. This connection may function to enhance the electrolocative sampling of the environment during Mauthner-cell mediated behaviors. This is a novel function for the Mauthner cell.

Morphology of the release site of inhibitory synapses on the soma and dendrite of an identified neuron

Synapses are complex arrangements of pre- and postsynaptic differentiations involved in neural communication. A key element in this synaptic transmission is the presynaptic active zone where the release of neurotransmitter occurs. Active zones can be visualized and analyzed after staining with ethanolic phosphotungstic acid (EPTA) on semithin (0.5 micron) sections. This staining has been used in association with postembedding immunogold labeling for the neurotransmitters glycine or GABA, to investigate the organization of chemically defined inhibitory active zones, viewed in their full extent, on different regions of the goldfish Mauthner (M-) cell. With this approach, a marked variability in size and shape was observed for the release sites contacting the different parts of the postsynaptic neuron. In the axon cap and on the soma, glycinergic afferent terminals have small presynaptic grids (0.066 +/- 0.029 micron2, n = 30 and 0.076 +/- 0.037 micron2, n = 46, respectively). These grids are quite circular and they include 12 to 13 presynaptic dense projections (PDPs). The situation is different on the lateral dendrite, where glycinergic and GABAergic active zones display a greater variability in their surface areas (mean = 0.147 +/- 0.100 micron2, n = 115 and 0.139 +/- 0.080 micron2, n = 125, respectively), and their number of PDPs (mean = 19 +/- 9) per individual grid. Similarly, the shape of the release sites over the dendrite is more complex (annular, horseshoe-shaped) when compared to those on the soma. These differences of dendritic versus somatic release sites could represent a structural basis to maximize the shunting effect of glycinergic and GABAergic inhibitory junctions, i.e., close to excitatory inputs. We also observed that the proportion of endings containing 1 or more active zones also varies. More precisely, 96% and 82% of glycinergic terminals in the axon cap and on the soma, respectively, display only one active zone. On the dendrite, their proportion falls to 65.5% for both glycine- and GABA-containing boutons. The remaining inhibitory terminals contain 2 (30%) and 3 to 4 (4.5%) presynaptic grids. These results reveal a greater variability of morphology and organization of the inhibitory release sites at dendritic versus somatic locations. The functional significance of this observation for the synaptic transmission is discussed.

Origin and function of spiral fibers projecting to the goldfish Mauthner cell.

Two neuron types contact the Mauthner cell (M cell) in the axon cap, a specialized region of high electrical resistance surrounding the initial segment of the M cell axon. One type produces a mixed electrical and chemical inhibition of the M cell. The second sends axons into the central core of the axon cap, where they spiral around the initial segment making both conventional synapses and gap junction contacts. The origin and synaptic effects of these spiral fibers have not been studied previously. When goldfish M cells were filled with Lucifer yellow, presynaptic spiral fibers were seen in the axon cap. These fibers could be traced back through the medial longitudinal fasciculus to their somata, near the contralateral fifth nerve motor nucleus. The same somata were labeled by horseradish peroxidase injected extracellularly into the axon cap. Recordings were made in the axon cap and the M cell after stimulation of hindbrain areas near the spiral fiber somata and axons. Extracellularly, a negative potential was observed close to the termination of the spiral fibers and termed the spiral fiber potential (SFP). Intracellularly, a graded, short latency depolarization of the M cell corresponded to the SFP and could cause the M cell to spike. This depolarization did not shunt the membrane, indicating that it may be produced through gap junctions. Intracellular responses to hindbrain stimulation also had a chloride-dependent, second component that shunted the membrane during paired-pulse testing. This inhibitory second component was probably evoked by cells other than the spiral fiber cells themselves.

GABAergic innervation of the Mauthner cell and other reticulospinal neurons in the goldfish

The Mauthner cells are pair of identifiable hindbrain neurons that participate in the escape response of fishes. Membrane excitability in these cells is regulated by inhibitory neurons that use glycine as a transmitter. We examined the possibility that the inhibitory transmitter gamma-amino butyric acid (GABA) may also act on the Mauthner cells. We used immunocytochemical methods involving an antibody against glutamic acid decarboxylase (GAD), the synthesizing enzyme for GABA. Our study revealed dense GAD immunoreactive terminals surrounding the Mauthner cells. Puncta counts showed that the distribution of GAD immunoreactivity was densest at the distal lateral dendrite of the Mauthner cells; the distribution of puncta tapers gradually in regions closer to the soma. The axon cap was devoid of GABAergic immunoreactivity. We also performed unilateral lesions of the octaval nuclei to evaluate the origin of the GAD immunoreactive terminals. Following the lesions, we found marked decreases in GAD immunoreactive terminals on the proximal lateral dendrite, soma, and proximal ventral dendrite of both Mauthner cells. These results suggest that the octaval region contributes to bilateral inhibition of the Mauthner cells. The distal lateral dendrite of the ipsilateral Mauthner cell also showed a reduction in GAD immunoreactive terminals. This suggests that GABA mediates remote dendritic inhibition of this cell. GAD immunoreactive puncta also surrounded other large reticulospinal neurons, some of which are serially reiterated along the anterior-posterior axis of the hindbrain. Thus, GABA may also exert an influence not only on the Mauthner cells, but also on other reticulospinal neurons.

Heterogeneous distribution of glycinergic and GABAergic afferents on an identified central neuron.

Immunocytochemical methods were used on serial sections to study the glycine- and gamma-amino butyric acid (GABA)ergic innervations of the teleost Mauthner (M) cell. We found different distributions for the boutons containing the two amino acids. Endings filled with GABA predominate on the distal portion of the lateral dendrite (LD) while glycine-positive profiles are more abundant on the soma and within the axon cap (AC), a specialized neuropil surrounding the M-cell initial segment. A few endings containing both transmitters are present on the soma and on the small dendrites issuing ventrally from it. At this level some glutamic acid decarboxylase (GAD)-containing boutons face glycine receptor-93 kD-associated protein, an observation suggesting that the associated glycine functions as a neurotransmitter. Elsewhere on the M-cell, where glycine and GABA are not colocalized, GAD-positive profiles were never observed in front of postsynaptic differentiations with 93 kD labelling. GABA was detected in the small vesicle boutons (SVBs), most of them, following the classification of Tuttle et al., J. Comp. Neurol. 265:254-274, 1987, belonging to the A-type, while glycine was found in the unmyelinated club endings in the AC, and in C- and B-type SVBs, outside this region. All terminals established symmetrical synapses and were filled with a population of pleiomorphic vesicles. Boutons with GABA also contained numerous dense-core vesicles suggesting the presence of an associated peptide(s). A quantitative study of the transmitter content based on the number of the gold particles revealed a variable intensity of the labelling over certain profiles. For GABA, it was maximum at the tip of the LD and it decreased proximally. In contrast, the staining density was constant for glycine along all parts of the cell, except for the ventral dendrite (VD) where it decreased progressively. Taken together, these data suggest that the amino acid content varies, depending upon the location of the synapses on their target neuron.

Activation of Mauthner neurons during prey capture

The Mauthner (M-) cells, a bilateral pair of medullary neurons in fish, initiate the characteristic “C-start” predatory escape response of teleosts. Similar movements have been described during hatching, social interactions, and feeding. M-cell firing, however, has not been correlated directly with these other behaviors. The objective of this study was to determine whether the M-cell, in addition to escape, plays a role in feeding. 1. Goldfish were chronically implanted with electrodes positioned near the axon cap of one of the two M-cells. Subsequently, M-cell activity was monitored for up to 8 days while fish were surface feeding on live crickets. 2. The M-cell fires and the fish performs a C-shaped flexion in association with the terminal phase of prey capture. Thus, the M-cell is active in the context of at least two behaviors, predator escape and prey capture, and may be considered a part of behaviorally shared neural circuitry. 3. For the goldfish, Mauthner-initiated flexions during feeding rapidly remove the prey from the water's surface and minimizes the fish's own susceptibility to surface predation. Other species may possess a diverse repertoire of Mauthner-mediated feeding behaviors that depend on their adaptive specializations for predation. Moreover, group competition between predators and their prey may have facilitated a “neural arms race” for M-cell morphology and physiology.

Differential distribution of GABA- and serotonin-containing afferents on an identified central neuron

The distributions of γ-aminobutyric acid (GABA)- and serotonin (5-HT)-containing terminals impinging on the surface of the Mauthner (M-) cell were studied at the light microscopic level using double immunofluorescent labeling and were compared with that of the glycine receptor. The latter was visualized indirectly, using a monoclonal mouse antibody which recognizes its 93-kDa associated protein. This neuron has two large principal dendrites: one extending ventrorostrally (ventral dendrite) and the other dorsolaterally (lateral dendrite). There are also two other classes of smaller processes: one that projects ventrally (small ventral dendrites) and one penetrating in the axons cap (cap dendrites), a peculiar neuropil surrounding the initial segment of the M-cell axon. A cellular regionalization of these afferent systems was found: GABA boutons, labeled for glutamic acid decar☐ylase (GAD), were localized preferentially on the lateral dendrite while 5-HT-filled endings predominated on the ventral one. The density of these two classes of inputs was comparable in the other areas of the M-cell: less of their terminals were in contact with the soma outside the axon cap, and more numerous boutons, which presented either GABA or 5-HT immunoreactivities, were apposed to the small ventral dendrites. This preferential pattern of innervation differed with the ubiquitous presence of glycine receptor clusters on the M-cell membrane. Finally no evidence of a colocalization of GABA and 5-HT in afferent endings was detected at any portion of the M-cell. Since the studied networks are involved in the inhibitory control of this neuron, and in its modulation, our results are discussed in relation with possible heterosynaptic interactions between them, and between other similarly segregated excitatory inputs.

Amphibian Mauthner cells.

Presently available data on anatomical, electrophysiological, and behavioral aspects of the Mauthner cell in amphibians are reviewed. Urodelian Mauthner cells appear morphologically distinct from those in anurans with respect to their abundant somatic dendrites, axon cap structures, lack of nodes of Ranvier, and size of axon collaterals. The presence of Mauthner cells does not seem to correlate with the presence of a tail or with an aquatic life-style in either larvae or adults, as they are also found in terrestrial adults, as well as in species without an aquatic larval stage. Electrophysiological data on Mauthner cell circuits are available for only two anuran species and indicate some interspecific differences. Special attention is given to a comparison of startle responses in larval and adult anurans and their possible relationship to Mauthner cell activities. Because of specific differences in Mauthner cell activities, as well as in startle response performance, it is argued that amongst the various ichthyopsid species Mauthner cells may have been adapted to modified functions due to the diverse morphoecological situations in which they are found.

Amphibian Mauthner Cells (Part 2 of 2)

Presently available data on anatomical, electrophysiological, and behavioral aspects of the Mauthner cell in amphibians are reviewed. Urodelian Mauthner cells appear morphologically distinct from those in anurans with respect to their abundant somatic dendrites, axon cap structures, lack of nodes of Ranvier, and size of axon collaterals. The presence of Mauthner cells does not seem to correlate with the presence of a tail or with an aquatic life-style in either larvae or adults, as they are also found in terrestrial adults, as well as in species without an aquatic larval stage. Electrophysiological data on Mauthner cell circuits are available for only two anuran species and indicate some interspecific differences. Special attention is given to a comparison of startle responses in larval and adult anurans and their possible relationship to Mauthner cell activities. Because of specific differences in Mauthner cell activities, as well as in startle response performance, it is argued that amongst the various ichthyopsid species Mauthner cells may have been adapted to modified functions due to the diverse morphoecological situations in which they are found.

Morphology of afferent synapses in the Mauthner cell of larval Xenopus laevis.

The fine structure of the afferent synapses on the Mauthner cell of larval Xenopus laevis has been studied as a first step toward comparing the fine structure of the afferent synaptic apparatus before and after metamorphosis. There are various types of afferent endings on this cell, some of which are confined to specific cellular regions, while others are distributed over most of the large surface of the neuron. Four different main types of endings have been observed: club endings, round-vesicle end bulbs, flattened-vesicle end bulbs and spiral fibers endings. While the myelinated club endings and the spiral fibers endings are located at the distal end of the lateral dendrite and in the axon cap, respectively, the end bulbs are widely distributed over the whole cell. A further type of ending has been observed, although rarely, on the Mauthner cell soma and dendrites: end bulbs characterized by an unusually dense presynaptic substance. Results obtained in the present research suggest that, as in fish, different endings on the anuran Mauthner neuron correspond to different synaptic inputs. The possible origin of some of these inputs is discussed.

Altered excitability of goldfish Mauthner cell following axotomy. II. Localization and ionic basis

The ionic basis and spatial localizations of spike generation were examined in normal and axotomized goldfish Mauthner (M-) cells using intra- and extracellular recordings and pharmacological manipulation of ionic conductances, including localized iontophoretic drug applications. Tetrodotoxin (TTX) abolished both the initial segment (IS) spike in normal cells and the larger, two-component action potential in axotomized cells, whereas calcium (Ca2+) blockers did not. Thus, sodium (Na+) appears to be the major inward current carrier in both cases. A shoulder or plateau following the fast-rising Na+-dependent action potential was unmasked in both normal and axotomized M-cells by intracellular injections of tetraethylammonium (TEA), either alone or in conjunction with 4-aminopyridine (4-AP) or cesium (Cs+). This plateau potential was abolished by superfusing with saline containing the Ca2 antagonists, Co2+, Mn2+, or Cd2+. However, barium (Ba2+), which normally substitutes for Ca2+ and also blocks K+ conductances, did not produce a plateau spike, and no action potentials could be evoked in the presence of TTX. Simultaneous extra- and intracellular recordings from the soma and lateral dendrite revealed that both the full-sized axotomized spike and its individual labile components were always maximal at the soma. These data support the earlier suggestion that the axotomy-induced electrogenicity is primarily localized to that region. Iontophoretic application of TTX inside the axon cap, a distinctive neuropil surrounding the initial segment and the axon hillock and circumscribed by a glial border, and at various positions along the lateral dendrite confirmed the Na+-dependency of the action potentials recorded in normal and axotomized cells and further demonstrated that the soma generates the additional spike component in the latter. The results suggest that axotomy causes a persistent change in voltage-gated Na+ channel distribution in the M-cell, with Na+ channels appearing or becoming more numerous in the soma while becoming less concentrated in the initial segment-axon hillock. Possible related shifts in other voltage-dependent conductances are also discussed. Finally, these are the first detailed studies of the ionic basis of axotomy-induced electrogenicity in a vertebrate neuron, central or peripheral, and the similarity to the results obtained with invertebrate neurons suggests common mechanisms underlying the axon reaction.

Development of the axon cap neuropil of the Mauthner cell in the goldfish.

Development of the axon cap neuropil of the Mauthner neuron in post-hatching larval goldfish brains was observed electron-microscopically. The axonal initial segment of newly hatched (day-4) larvae is completely covered with synaptic terminals containing clear spherical synaptic vesicles. Profiles of thin terminal axons, the spiral fibers, containing similar synaptic vesicles, rapidly increase in number around the initial segment and form glomerular neuropil similar to the central core of the adult axon cap by day 7. Three types of synapses are formed in the core neuropil. Bouton-type synapses contacting the initial segment are most abundant in day-4 to -14 larvae; they decrease thereafter and are rare on the distal half of the initial segment of day-40 larvae. Asymmetric axo-axonic synapses are commonly observed between spiral fibers in the core neuropil of day-7 to -19 larvae, but become fewer by day 40. Unique symmetrical axo-axonic synapses showing accumulation of synaptic vesicles on either side of apposed membrane thickenings first appear in day-14 core neuropil, gradually increase in number, and become the predominant type in day-40 core neuropil. Thick myelinated axons, which lose their myelin sheaths in the glial cap cell layer, start to penetrate into the axon cap on day 10. They gradually increase in number and form the peripheral part of the axon cap together with the cap dendrites, which finally grow into the axon cap from the axon hillock region of the Mauthner cell by day 40.

Morphologically distinct classes of inhibitory synapses arise from the same neurons: Ultrastructural identification from crossed vestibular interneurons intracellularly stained with HRP

AbstractPrevious studies have demonstrated that in goldfish and tench inhibitory interneurons mediating both electrical and chemical inhibition of the Mauthner cell can be identified by a passive hyperpolarizing potential (PHP) which is recorded intracellularly when the latter is firing; some of them are crossed second‐order vestibular cells, the axons of which project ipsi‐laterally and contralaterally to their cell's body in several medullary nuclei. Since PHP‐exhibiting neurons and their target Mauthner cells can be simultaneously impaled, commissural interneurons were intracellularly stained with HRP contained in presynaptic microelectrodes and their synaptic features were correlated with electrophysiological findings. In this study we were able to confirm that commissural neurons are monosynaptically activated by VIIIth nerve afferents and that spikes selectively evoked in these cells produce monosynaptic Cl−‐dependent unitary IPSPs in their adjacent Mauthner cell; in addition to observations related to their general morphology, including their mode of termination within vestibular and reticular nuclei, new data were obtained, most of them with EM techniques. (1) The Mauthner cell is covered by well‐segregated synaptic endings of different shapes; among them, the unmyelinated club endings (which mediate field effect inhibition) penetrate within the peripheral part of the axon cap and terminate on this cell's soma and fine cap dendrites, while the small vesicle boutons which remain outside the axon cap (and can therefore only carry chemical inhibition) impinge on the Mauthner cell's soma and the proximal part of its large dendrites. An important finding was that endings of the two groups were stained by HRP, indicating that most commissural interneurons issue synaptic knobs of these two distinct classes. (2) The fine structure of labeled terminals was investigated: They exhibit the typical aspect of Gray type II synapses, with well‐differentiated presynaptic and postsynaptic differentiations and they contain pleiomorphic synaptic vesicles (mean diameter 40–46 nm; elongation coefficient 1.4–1.5; surface area 13–15.102 nm2). A comparison with adjacent unstained equivalent terminals has revealed that HRP allows a reliable analysis of synaptic organelles with, however, a slight tendency of vesicles to be more spherical in presence of the enzyme. (3) Spikes do not propagate actively in unmyelinated club endings but the remaining passively transmitted depolarization is nevertheless above threshold for inhibitory transmitter release, as indicated by unitary IPSPs evoked by fibers terminating exclusively within the axon cap. Thus, club endings have a dual action, and they mediate part of the M‐cell's chemical inhibition. (4) Synapses established by commissural cells on vestibular neurons contain a pleiomorphic population of vesicles; they can be compared to those which were identified in the vestibular nuclei of other species and which were also postulated to have inhibitory functions. (5) The synaptic investment of HRP‐stained commissural neuronal cell bodies included mixed synapses associating gap junctions and active zones established by presumably VIIIth nerve afferents. This observation can be related to the fast‐occurring inhibition produced in both M‐cells by vestibular nerve stimulation. (6) Axons of commissural fibers are myelinated and the largest of them have an internal diameter of no less than 10 μm; the ratio g (axon/total fiber) is close to the optimal value for maximum conduction velocity. Thus, again, these cells are likely to carry the nearly simultaneous ipsi‐lateral and contralateral inhibition of the M‐cell following VIIIth nerve excitation and their widespread distribution suggests a diffuse and synchronous ipsi‐lateral and contralateral inhibitory action on other nuclei as well. (7) In contradiction to electrophysiological results suggesting that some of these neurons were efferent to the labyrinth, a stained process was only observed once in the vestibular tract; this case confirms that commissural cells can project to the labyrinth but its rarity raises questions about the criteria commonly used for efferent identification.

Electron microscopic findings on mauthner cell cap dendrites in rainbow trout

Electron microscopic investigations were made on the cap dendrites within the Mauthner cell axon cap of the trout. The surface of the cap dendrites is covered nearly completely by synapses of four morphologically different types, chemical synapses containing round or elongated vesicles, as well as mixed synapses containing round or elongated vesicles. The myelinated axons forming these synapses frequently produce bulbs before entering the axon cap. They make contact with the cap dendrites in a different manner: end terminals or en passant-synapses exhibiting one or more synapses occur. Sometimes collaterals of the myelinated axons are seen. Because of the complex structure of their synaptic contacts the cap dendrites should consequently be regarded as a further prominent feature not only of the axon cap but also of the whole Mauthner cell. It seems likely that even the cap dendrites are one of the target elements of ipsilaterally-functioning PHP neurons and collaterals of contralateral vestibular inter-neurons.