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

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Abstract
Previous 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.