Presynaptic GABA(B) receptors decrease neurotransmitter release in vestibular nuclei neurons during vestibular compensation.

Unilateral damage to the peripheral vestibular receptors precipitates a debilitating syndrome of oculomotor and balance deficits at rest, which extensively normalize during the first week after the lesion due to vestibular compensation. In vivo studies suggest that GABA(B) receptor activation facilitates recovery. However, the presynaptic or postsynaptic sites of action of GABA(B) receptors in vestibular nuclei neurons after lesions have not been determined. Accordingly, here presynaptic and postsynaptic GABA(B) receptor activity in principal cells of the tangential nucleus, a major avian vestibular nucleus, was investigated using patch-clamp recordings correlated with immunolabeling and confocal imaging of the GABA(B) receptor subunit-2 (GABA(B)R2) in controls and operated chickens shortly after unilateral vestibular ganglionectomy (UVG). Baclofen, a GABA(B) agonist, generated no postsynaptic currents in principal cells in controls, which correlated with weak GABA(B)R2 immunolabeling on principal cell surfaces. However, baclofen decreased miniature excitatory postsynaptic current (mEPSC) and GABAergic miniature inhibitory postsynaptic current (mIPSC) events in principal cells in controls, compensating and uncompensated chickens three days after UVG, indicating the presence of functional GABA(B) receptors on presynaptic terminals. Baclofen decreased GABAergic mIPSC frequency to the greatest extent in principal cells on the intact side of compensating chickens, with concurrent increases in GABA(B)R2 pixel brightness and percentage overlap in synaptotagmin 2-labeled terminals. In uncompensated chickens, baclofen decreased mEPSC frequency to the greatest extent in principal cells on the intact side, with concurrent increases in GABA(B)R2 pixel brightness and percentage overlap in synaptotagmin 1-labeled terminals. Altogether, these results revealed changes in presynaptic GABA(B) receptor function and expression which differed in compensating and uncompensated chickens shortly after UVG. This work supports an important role for GABA(B) autoreceptor-mediated inhibition in vestibular nuclei neurons on the intact side during early stages of vestibular compensation, and a role for GABA(B) heteroreceptor-mediated inhibition of glutamatergic terminals on the intact side in the failure to recover function.

Electrophysiological properties of morphologically-identified medial vestibular nucleus neurons projecting to the abducens nucleus in the chick embryo.

Neurons in the medial vestibular nucleus (MVN) show a wide range of axonal projection pathways, intrinsic firing properties, and responses to head movements. To determine whether MVN neurons participating in the vestibulocular reflexes (VOR) have distinctive electrophysiological properties related to their output pathways, a new preparation was devised using transverse brain slices containing the chicken MVN and abducens nucleus. Biocytin Alexa Fluor was injected extracellularly into the abducens nucleus so that MVN neurons whose axons projected to the ipsilateral (MVN/ABi) and contralateral (MVN/ABc) abducens nuclei were labeled selectively. Whole-cell, patch-clamp recordings were performed to study the active and passive membrane properties, sodium conductances, and spontaneous synaptic events in morphologically-identified MVN/AB neurons and compare them to MVN neurons whose axons could not be traced (MVN/n). Located primarily in the rostral half of the ventrolateral part of the MVN, MVN/AB neurons mainly have stellate cell bodies with diameters of 20-25 µm. Compared to MVN/n neurons, MVN/ABi and MVN/ABc neurons had lower input resistances. Compared to all other MVN neuron groups studied, MVN/ABc neurons showed unique firing properties, including type A-like waveform, silence at resting membrane potential, and failure to fire repetitively on depolarization. It is interesting that the frequency of spontaneous excitatory and inhibitory synaptic events was similar for all the MVN neurons studied. However, the ratio for miniature to spontaneous inhibitory events was significantly lower for MVN/ABi neurons compared to MVN/n neurons, suggesting that MVN/ABi neurons retained a larger number and/or more active inhibitory presynaptic neurons within the brain slices. Also, MVN/ABi neurons had miniature excitatory postsynaptic currents (mEPSCs) with slower decay time and half width compared to MVN/n neurons. Altogether, these findings underscore the diversity of electrophysiological properties of MVN neuron classes distinguished by axonal projection pathways. This represents the first study of MVN/AB neurons in brain slice preparations and supports the concept that the in vitro brain slice preparation provides an advantageous model to investigate the cellular and molecular events in vestibular signal processing.

GABA and glycine immunolabeling in the chicken tangential nucleus.

In the vestibular nuclei, GABAergic and glycinergic neurons play important roles in signal processing for normal function, during development, and after peripheral vestibular lesions. The chicken tangential nucleus is a major avian vestibular nucleus, whose principal cells are projection neurons with axons transmitting signals to the oculomotor nuclei and/or cervical spinal cord. Antibodies against GABA, glycine and glutamate were applied to study immunolabeling in the tangential nucleus of 5-7 days old chicken using fluorescence detection and confocal imaging. All the principal cells and primary vestibular fibers were negative for GABA and glycine, but positive for glutamate. GABA is the predominant inhibitory neurotransmitter in the tangential nucleus, labeling most of the longitudinal fibers in transverse tissue sections and more than 50% of all synaptic terminals. A large fraction of GABAergic terminals were derived from the longitudinal fibers, with fewer horizontal GABAergic fibers detected. GABA synapses terminated mainly on dendrites in the tangential nucleus. In contrast, glycine labeling represented about one-third of all synaptic terminals, and originated from horizontally-coursing fibers. A distinct pool of glycine-positive terminals was found consistently around the principal cell bodies. While no GABA or glycine-positive neuron cell bodies were found in the tangential nucleus, several pools of immunopositive neurons were present in the neighboring vestibular nuclei, mainly in the descending vestibular and superior vestibular nuclei. GABA and glycine double-labeling experiments revealed little colocalization of these two neurotransmitters in synaptic terminals or fibers in the tangential nucleus. Our data support the concept of GABA and glycine playing critical roles as inhibitory neurotransmitters in the tangential nucleus. The two inhibitory neurotransmitters have distinct and separate origins and display contrasting subcellular termination patterns, which underscore their discrete roles in vestibular signal processing.

Adaptation of chicken vestibular nucleus neurons to unilateral vestibular ganglionectomy.

Vestibular compensation refers to the behavioral recovery after a unilateral peripheral vestibular lesion. In chickens, posture and balance deficits are present immediately following unilateral vestibular ganglionectomy (UVG). After three days, most operated chickens begin to recover, but severe deficits persist in others. The tangential nucleus is a major avian vestibular nucleus whose principal cells are vestibular reflex projection neurons. From patch-clamp recordings on brain slices, the percentage of spontaneous spike firing principal cells, spike discharge rate, ionic conductances, and spontaneous excitatory postsynaptic currents (sEPSCs) were investigated one and three days after UVG. Already by one day after UVG, sEPSC frequency increased significantly on the lesion side, although no differences were detected in the percentage of spontaneous spike firing cells or discharge rate. In compensated chickens three days after UVG, the percentage of spontaneous spike firing cells increased on the lesion side and the discharge rate increased bilaterally. In uncompensated chickens three days after UVG, principal cells on the lesion side showed increased discharge rate and increased sEPSC frequency, whereas principal cells on the intact side were silent. Typically, silent principal cells exhibited smaller persistent sodium conductances and higher activation thresholds for the fast sodium channel than spiking cells. In addition, silent principal cells on the intact side of uncompensated chickens had larger dendrotoxin-sensitive potassium conductance, with a higher ratio of Kv1.1 surface/cytoplasmic expression. Increased sEPSC frequency in principal cells on the lesion side of uncompensated chickens was accompanied by decreased Kv1.2 immunolabeling of presynaptic terminals on principal cell bodies. Thus, both intrinsic ionic conductances and excitatory synaptic inputs play crucial roles at early stages after lesions. Unlike the principal cells in compensated chickens which showed similar percentages of spontaneous spike firing cells, discharge rates, and sEPSC frequencies bilaterally, principal cells in uncompensated chickens displayed gross asymmetry in these properties bilaterally.

Dye coupling in developing vestibular nuclei.

The chick tangential nucleus is a major vestibular nucleus whose principal cells participate in the vestibular reflexes. During development, most mature vestibular nucleus neurons must acquire repetitive firing of action potentials on depolarization and spontaneous spike activity to process signals effectively. In the chicken, these properties emerge gradually in the embryo, starting the week before (E13, E16) and continuing through the first week after hatching (H7). Since gap junction-mediated cell coupling may influence the emergence of neuronal excitability, we investigated whether neuron-neuron and neuron-glia coupling are present in this morphologically distinctive vestibular nucleus during the period for establishing signal processing. In brain slices, principal cells were injected with biocytin in the whole-cell configuration and visualized via confocal imaging at E13, E16, and H7. The incidence of dye coupling between the injected principal cell and neurons was 42% at E13, 75% at E16, and 7% at H7, whereas the incidence of dye coupling with glia was 100% at both embryonic ages but decreased to 27% by H7. For each injected principal cell at E13, one coupled neuron and 35 coupled glia were detected, whereas three coupled neurons and 12 coupled glia were observed at E16, and few if any coupled neurons and glia were detected at H7. These results suggest that neuron-neuron and neuron-glia coupling are developmentally regulated and present before, but not after, the onset of mature signal processing by these neurons. Thus, transient neuron-neuron and neuron-glia coupling may both play roles in establishing excitability in vestibular nucleus neurons during development.

Maturation of firing pattern in chick vestibular nucleus neurons.

The principal cells of the chick tangential nucleus are vestibular nucleus neurons participating in the vestibuloocular and vestibulocollic reflexes. In birds and mammals, spontaneous and stimulus-evoked firing of action potentials is essential for vestibular nucleus neurons to generate mature vestibular reflex activity. The emergence of spike-firing pattern and the underlying ion channels were studied in morphologically-identified principal cells using whole-cell patch-clamp recordings from brain slices of late-term embryos (embryonic day 16) and hatchling chickens (hatching day 1 and hatching day 5). Spontaneous spike activity emerged around the perinatal period, since at embryonic day 16 none of the principal cells generated spontaneous action potentials. However, at hatching day 1, 50% of the cells fired spontaneously (range, 3 to 32 spikes/s), which depended on synaptic transmission in most cells. By hatching day 5, 80% of the principal cells could fire action potentials spontaneously (range, 5 to 80 spikes/s), and this activity was independent of synaptic transmission and showed faster kinetics than at hatching day 1. Repetitive firing in response to depolarizing pulses appeared in the principal cells starting around embryonic day 16, when <20% of the neurons fired repetitively. However, almost 90% of the principal cells exhibited repetitive firing on depolarization at hatching day 1, and 100% by hatching day 5. From embryonic day 16 to hatching day 5, the gain for evoked spike firing increased almost 10-fold. At hatching day 5, a persistent sodium channel was essential for the generation of spontaneous spike activity, while a small conductance, calcium-dependent potassium current modulated both the spontaneous and evoked spike firing activity. Altogether, these in vitro studies showed that during the perinatal period, the principal cells switched from displaying no spontaneous spike activity at resting membrane potential and generating one spike on depolarization to the tonic firing of spontaneous and evoked action potentials.

Spontaneous synaptic activity in chick vestibular nucleus neurons during the perinatal period.

The principal cells of the chick tangential nucleus are second-order vestibular neurons involved in the vestibuloocular and vestibulocollic reflexes. The spontaneous synaptic activity of morphologically identified principal cells was characterized in brain slices from 1-day-old hatchlings (H1) using whole-cell voltage-clamp recordings and Cs-gluconate pipet solution. The frequency was 1.45 Hz for spontaneous excitatory postsynaptic currents (sEPSCs) and 1.47 Hz for spontaneous inhibitory postsynaptic currents (sIPSCs). Using specific neurotransmitter receptor antagonists, all of the sEPSCs were identified as AMPA receptor-mediated events, whereas 56% of the sIPSCs were glycine and 44% were GABA(A) receptor-mediated events. On exposure to TTX, the frequency of EPSCs decreased by 68%, while the frequency of IPSCs decreased by 33%, indicating greater EPSC dependency on presynaptic action potentials. These data on spontaneous synaptic activity at H1 were compared with those obtained in previous studies of 16-day old embryos (E16). After birth, the spontaneous synaptic activity exhibited increased EPSC frequency, increased ratio for excitatory to inhibitory events, increased percentage of TTX-dependent EPSCs, and faster kinetics. In addition, the ratio for glycine/GABA receptor-mediated events increased significantly. Altogether, these data indicate that at hatching spontaneous synaptic activity of vestibular nucleus neurons in brain slices of the chick tangential nucleus undergoes appreciable changes, with increased frequency of EPSCs and glycinergic activity playing more important roles compared with the late-term chick embryo when GABAergic activity prevailed. The definition of this developmental pattern of synaptic activity in vestibular nucleus neurons should contribute to understanding how vestibular reflex activity is established in the hatchling chick.

Development of the gravity sensing system.

The utricle and saccule contain hair cells, which are the peripheral sensors of change in gravity that transmit signals regarding these changes to the neural components of the vestibular system. Although the fundamental neural pathways, especially the vestibular reflex pathways, have been investigated extensively, the principals underlying the functional development of this system are under study at present. The objective of this review is to identify the gravity-sensing components of the vestibular system and to present an overview of the research performed on their development. The second part of this review is focused on one important aspect of development, the emergence of electrical excitability using the chick tangential vestibular nucleus as a model. The importance of this research to understanding vestibular compensation and vestibular disturbance during spaceflight is considered. Because there is a conservation of the fundamental pathways and function in vertebrate phylogeny from birds through mammals, findings from studies on avians should contribute significantly to understanding the mechanisms operating in mammals. Also, we expect that as the events and basic mechanisms underlying normal vestibular development are revealed, these will provide practical tools to investigate the pattern of recovery from dysfunction of the vestibular system. This is related to the evidence suggesting that recovery of function in different systems and cell lines, including neurons, involves repeating certain patterns established during development.

Firing properties and dendrotoxin-sensitive sustained potassium current in vestibular nuclei neurons of the hatchling chick.

To understand the emergence of excitability in vestibular nuclei neurons, we performed patch-clamp recordings on brain slices to characterize the firing pattern on depolarization and the underlying currents in principal cells of the chick tangential nucleus. This study, on 0- to 3-day-old hatchlings, distinguishes electrophysiologically one main group of principal cells based on their response to depolarizing current pulses (300-400 ms) in current-clamp recordings. This group (90%; n=29) displayed nonaccommodating, repetitive firing on depolarization. The remaining cells fired one action potential at the beginning of the current pulse and then accommodated. In voltage-clamp recordings, a low-threshold, sustained, dendrotoxin-sensitive (DTX; 200 nM) potassium current, I(DS), was identified in both cell groups. In the repetitively firing principal cells, the mean proportion of the DTX-sensitive sustained current contributing to the total outward current was less than 20%. This percentage is significantly less than that reported (45%) in a previous study performed in late chick embryos (E16), in which most of the cells (83%; n=89) were accommodating neurons. Tonic firing is an important electrophysiological feature characterizing most mature, second-order vestibular neurons, since it allows the neurons to process signals from behaviorally relevant inputs. Accordingly, this study contributes toward defining the emergence of the mature pattern of neuronal excitability and the ionic currents involved.

Potassium currents and excitability in second-order auditory and vestibular neurons.

Potassium channels are involved in the control of neuronal excitability by fixing the membrane potential, shaping the action potential, and setting firing rates. Recently, attention has been focused on identifying the factors influencing excitability in second-order auditory and vestibular neurons. Located in the brainstem, second-order auditory and vestibular neurons are sites for convergence of inputs from first-order auditory or vestibular ganglionic cells with other sensory systems and also motor areas. Typically, second-order auditory neurons exhibit two distinct firing patterns in response to depolarization: tonic, with a repetitive firing of action potentials, and phasic, characterized by only one or a few action potentials. In contrast, all mature vestibular second-order neurons fire tonically on depolarization. Already, certain fundamental roles have emerged for potassium currents in these neurons. In mature auditory and vestibular neurons, I(K), the delayed rectifier, is required for the fast repolarization of action potentials. In tonically firing auditory neurons, I(A), the transient outward rectifier, defines the discharge pattern. I(DS), a delayed rectifier-like current distinguished by its low threshold of activation, is found in phasically firing auditory and some developing vestibular neurons where it limits firing to one or a few spikes, and also may contribute to forming short-duration excitatory postsynaptic potential (EPSPs). Also, I(DS) sets the threshold for action potential generation rather high, which may prevent spontaneous discharge in phasically firing cells. During development, there is a gradual acquisition and loss of some potassium conductances, suggesting developmental regulation. As there are similarities in membrane properties of second-order auditory and vestibular neurons, investigations on firing pattern and its underlying mechanisms in one system should help to uncover fundamental properties of the other.

The differential expression of low-threshold sustained potassium current contributes to the distinct firing patterns in embryonic central vestibular neurons.

The principal cells of the chick tangential nucleus are second-order sensory neurons that participate in the three-neuron vestibulo-ocular and vestibulocollic reflexes. In postnatal animals, second-order vestibular neurons fire repetitively on depolarization. Previous studies have shown that, although this is an important feature for normal reflex function, it is only acquired gradually during embryonic development. Whereas at 13 embryonic days (E13) the principal cells accommodate after firing a single spike, at E16 a few principal cells repetitively can fire multiple action potentials on depolarization. Finally, in the hatchling, the vast majority of principal cells is capable of nonaccommodating firing on depolarization. As a first step in understanding the mechanisms underlying developmental change in excitability of these second-order vestibular neurons, we analyzed the outward potassium currents and their role in accommodation, using brainstem slices at E16. The principal cells exhibited transient and sustained potassium currents, with both of these containing calcium-dependent components. Further, both high- and low-threshold sustained potassium currents have been distinguished. The low-threshold dendrotoxin-sensitive sustained potassium current (IDS) is associated with principal cells that accommodate and is not expressed in those that fire repetitively. Finally, blocking of IDS transforms accommodating cells into neurons capable of firing trains of action potentials on depolarization. These findings indicate that suppression of IDS during development is sufficient to transform accommodating principal cells into nonaccommodating firing neurons and suggests that developmental regulation of this current is necessary for the establishment of normal vestibular function.

Ontogeny of electrophysiological properties and dendritic pattern in second-order chick vestibular neurons.

The pattern of development of several subpopulations of second-order vestibular neurons was investigated by using intracellular recordings from chicken brain slices to define the timing of morphological and electrophysiological changes occurring at 3 critical ages. Two embryonic stages, embryonic day 13 (E13) and E15-16, and also newborn chicks were selected according to previous anatomical findings showing the differentiation of primary vestibular afferents and their synapses within a distinctive brainstem vestibular nucleus, the tangential nucleus. The responses of these cells to depolarizing and hyperpolarizing current pulses and their postsynaptic responses to vestibular nerve stimulation were recorded, while simultaneously biocytin was injected for subsequent morphogenetic analysis. From this study, developmental schedules of membrane properties, synaptic responses, and dendritic differentiation were established for the 2 cell populations of the tangential nucleus and other neurons located in the surrounding vestibular nuclei. Compared with all other second-order vestibular neurons, the principal cells of the tangential nucleus exhibited a distinctive schedule. Mainly, this includes their pattern of firing on depolarization, the shape and duration of the vestibular-evoked excitatory postsynaptic potential, and the time of onset of dendritic outgrowth. In regard to these observations, E15-16 appears to be a turning point in principal cell ontogeny, whereas these features occur earlier in development for other second-order vestibular neurons. These findings, which indicate that the principal cells may have distinct membrane properties at specific ages, are discussed in light of their unique vestibular innervation and the possible consequences for vestibular signal processing.

Lack of biocytin transfer at gap junctions in the chicken vestibular nuclei.

In vivo experiments were designed to test for functional gap junctions at 'mixed' synapses that were morphologically characterized between the large-diameter, primary vestibular fibers and second-order vestibular neurons in the chicken, Gallus gallus. In previous intracellular recordings and dye injections into these neurons from brain slice preparations of chick embryos (E15/16) and also newborn hatchlings (HI-2), no evidence was obtained for functional gap junctions. Therefore, biocytin, a low molecular weight tracer that permeates gap junction channels, was extracellularly applied to either the ampullary nerves or to the vestibular ganglion of 3-6 day old hatchlings and adult chickens (9 months). This procedure resulted in the uptake of the dye and heavy staining of both the thick and thin fibers composing the vestibular nerve and in loading of vestibular efferent neurons. However, no dye transfer was observed between the large-diameter, primary vestibular fibers and second-order vestibular neurons. This observation, which was performed using a relatively non-invasive approach on intact animals, suggests that the gap junctions at these mixed synapses are probably not functional under the conditions of these experiments.

The first developing "mixed" synapses between vestibular sensory neurons mediate glutamate chemical transmission.

In the present study, the nature of the synaptic transmission responsible for a monophasic potential generated by vestibular nerve stimulation of the principal cells in the chick tangential nucleus was established. This work was performed in slice preparations at the critical embryonic age of 15-16 days, the time of first observation of morphologically mixed (chemical and electrical) synapses at the axosomatic endings called spoon endings. The spoon endings are formed by the primary vestibular fibers with the largest diameters, the colossal vestibular fibers. This monophasic potential fits the criteria for chemical rather than electrical transmission due to the following responses in most cases: (i) the absence of collision between a direct spike initiated by depolarization in the principal cell and a vestibular-evoked action potential; (ii) failure to follow high frequency stimulation (up to 50 Hz); (iii) sensitivity to low calcium solution (0.1 mM). These tests indicate that strong electrical coupling between spoon endings and principal cells does not prevail at this stage. The recordings were obtained from principal cells injected intracellularly with biocytin, allowing their identification by morphological criteria. The lack of tracer coupling between the stained principal cells and their innervating vestibular fibers (n = 17) is consistent with the absence of electrical coupling. Identification of the neurotransmitter involved in this vestibular response was achieved by bath application of glutamate receptor antagonists, DL-2-amino-5-phosphonovaleric acid (40 microM) and 6-cyano-7-nitro-quinoxaline-2,3-dione (10 microM), which blocked transmission reversibly. These results suggest that at the onset of formation of these "mixed" vestibular synapses, the gap junctions identified morphologically are likely not functional, and that the main response of the principal cells to vestibular nerve stimulation is mediated by glutamate.

Ultrastructural evidence that early synapse formation on central vestibular sensory neurons is independent of peripheral vestibular influences.

Migration and early differentiation of neurons of the tangential vestibular nucleus of the chick take place between embryonic days 5 and 8. In the absence of primary vestibular afferents (otocyst-ablation), a previous light microscope study documented that early developmental events still occurred, but the neurons failed to complete differentiation and to survive. In order to understand why these neurons undergo normal early development, we have repeated the vestibular deafferentation paradigm followed by ultrastructural observations on these neurons. We found that the ultrastructural events associated with migration and differentiation in the deafferented tangential nucleus were essentially normal from 5 to 8 days. Most important, longitudinal fibers, presumably of central, nonvestibular origins, formed the first synapses at the same time and sequence as observed in normal embryos. Thus vestibular sensory neurons receive their first input from central fibers, initiating events in the formation of a central vestibular circuitry without the influence of peripheral vestibular fibers or endorgan.

The earliest ultrastructural development of the tangential vestibular nucleus in the chick embryo.

The tangential nucleus is a primary vestibular nucleus located where the vestibular fibers enter the medulla. It is composed of neurons that migrate between 5 and 8 days in the chick embryo. Although primary vestibular fibers enter the medulla at 3 days, the first synapses are formed at 5 days on the processes of neuron precursors by longitudinally coursing fibers. Since the major components, or their precursors, are present at 3 days within the presumptive nucleus, we are interested in determining what cellular interactions occur among these structures following their entry and during the time leading up to synapse formation. At 2 days, prior to the appearance of VIIth and VIIIth nerve fibers in the medulla, the tangential nucleus anlage contained processes and endfeet of primitive epithelial cells, separated from each other by enlarged extracellular spaces. Longitudinal fibers first appeared within these spaces coincident with the appearance of root fibers, including some identified VIIth motor axons, associated with the primordial VII/VIIIth ganglia. By 3 days, some vestibular and VIIth nerve fibers could be identified by their ultrastructure and relative positions within the marginal zone and nerve roots. However, it was not until 4 days that the presumptive tangential nucleus acquired its orderly, characteristic organization. Although synapses were rare from 2 to 4 days, attachment plaques and coated pits were observed commonly between structures, especially between future synaptic structures. Thus, we confirm that synapse formation begins at 5 days. This represents the first detailed ultrastructural study of cranial sensory nerve ingrowth into the medulla.

Horseradish peroxidase labeling of the central pathways in the medulla of the ampullary nerves in the chicken, Gallus gallus.

The purpose of the present study was to place horseradish peroxidase on the distal processes of the three ampullary nerves in 6-8-week-old chickens so that we could identify the ganglion cells associated with each nerve and trace the specific central pathways taken by each nerve in the brainstem. We are especially interested in the pathways of the colossal vestibular fibers, which may play a role in a fast reflex pathway as suggested by their large caliber and electrotonic mode of transmission in the tangential vestibular nucleus. The cells of origin of each ampullary nerve occupy discrete portions of the vestibular ganglion. Those vestibular ganglion cells giving rise to the posterior ampullary nerve (PAN) occupy the posterior portion of Scarpa's ganglion; the ganglion cells producing the anterior (AAN) and lateral (LAN) ampullary nerves occupy the anterior ganglion, within the dorsal and ventral portions, respectively. Centrally the vestibular fibers occupy discrete portions of the tangential vestibular nucleus before bifurcating into ascending and descending tracts. The tangential nucleus receives afferents from the colossal fibers, which form spoon endings, and also from the fine vestibular fibers, which form small terminals. The ascending fibers of the posterior ampullary nerve are associated with the nucleus piriformis; the ascending fibers of the anterior and lateral ampullary nerves occupy discrete cell groups of the vestibulo-cerebellar nucleus. All three ampullary nerves have descending branches that course through the retrotangential nucleus into the descending vestibular nucleus (DVN). Within the descending vestibular nucleus, the descending fibers of the posterior ampullary nerve run dorsally and centrally, whereas fibers of the anterior ampullary nerve course ventromedially, and the lateral ampullary nerve fibers take a ventrolateral course until all three fiber bundles converge in the posterior tip of the descending vestibular nucleus. The ascending and descending fibers of each ampullary nerve form collaterals that pass to the ventrolateral and dorsomedial parts of the medial vestibular nucleus. These collaterals are derived exclusively from the fine and medium diameter vestibular fibers. Some of these ascending fibers form a distinctive tract that courses posteriorly within medial regions of the dorsomedial part of the medial vestibular nucleus. The colossal vestibular fibers, which are found within all three ampullary nerves, conform to the ampullary pathways as described, excluding the innervation of the medial vestibular nucleus.

Horseradish peroxidase labeling of the efferent and afferent pathways of the avian tangential vestibular nucleus.

The efferent and afferent pathways of the chick tangential nucleus were studied by using horseradish peroxidase (HRP: Sigma type VI) to label nerve cell bodies and fibers. Depositions of HRP into the tangential nucleus, as well as into the second cervical level of the spinal cord, show that the axons of tangential neurons on leaving the nucleus form an anteriorly coursing tract that passes through the ventrolateral vestibular nucleus without branching and then to the contralateral medial longitudinal fasciculus (MLF). Within the MLF, the tangential axons course posteriorly, forming collaterals that innervate the abducens nucleus, and then proceed to the cervical spinal cord. This pathway was demonstrated for the axons of the two main neurons, the principal and elongate cells, in 1-day, 1-week, and 7-week-old animals. In addition, we propose the existence of an unidentified, ipsilateral pathway to the spinal cord for the tangential axons, since HRP injections into one side of the spinal cord resulted in the bilateral labeling of tangential neurons. No labeled cells were found in the tangential nucleus following HRP depositions into the uvula, flocculus, pontine reticular formation, nucleus piriformis, nucleus jumeaux, vestibulocerebellar nucleus, retrotangential nucleus, or the dorsomedial part of the medial vestibular nucleus. The tangential nucleus receives afferents from the colossal vestibular fibers (spoon endings), small collaterals of fine vestibular ampullary fibers, flocculus, and high cervical levels of the spinal cord. From our small sample, it appears that the spinal cord fibers form most of the afferent terminals in the tangential nucleus in 1-day, 1-week, and 7-week-old animals.

Ultrastructural study of synapses at the time of neuronal migration and early differentiation in the tangential vestibular nucleus of the chick embryo in vivo.

The chick tangential nucleus is a primary vestibular nucleus whose two main neuron types migrate and begin to differentiate between 5 and 8 days in the embryo (gestation takes 20-21 days). Based on rapid Golgi impregnations of developing tangential neurons and growing fibers, we have identified ultrastructural counterparts and characterized interactions in the nucleus from 5 to 8 days. Developing tangential neurons received the earliest synapses at 5 days on their primitive processes and subsequently on their cell bodies by longitudinal fibers of unknown origins. In contrast, the primary vestibular afferents did not form identified synapses on the developing tangential neurons until 7 1/2 days. In conclusion, the earliest synapses in the tangential nucleus are formed by longitudinal fibers, which are probably not primary vestibular afferents. Since a specific class of fibers forms particular synapses on the tangential neuron precursors at predictable times prior to and during neuronal migration and also at the onset of differentiation, the role of these synapses in developmental events should be explored.