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Stimulation of vestibular efferent neurons excites calyx and dimorphic (CD) afferents. This excitation consists of fast and slow components that differ >100-fold in activation kinetics and response duration. In the turtle, efferent-mediated fast excitation arises in CD afferents when the predominant efferent neurotransmitter acetylcholine (ACh) activates calyceal nicotinic ACh receptors (nAChRs); however, it is unclear whether the accompanying efferent-mediated slow excitation is also attributed to cholinergic mechanisms. To identify synaptic processes underlying efferent-mediated slow excitation, we recorded from CD afferents innervating the turtle posterior crista during electrical stimulation of efferent neurons, in combination with pharmacological probes and mechanical stimulation. Efferent-mediated slow excitation was unaffected by nAChR compounds that block efferent-mediated fast excitation, but were mimicked by muscarine and antagonized by atropine, indicating that it requires ACh and muscarinic ACh receptor (mAChR) activation. Efferent-mediated slow excitation or muscarine application enhanced the sensitivity of CD afferents to mechanical stimulation, suggesting that mAChR activation increases afferent input impedance by closing calyceal potassium channels. These observations were consistent with suppression of a muscarinic-sensitive K + -current, or M-current. Immunohistochemistry for putative M-current candidates suggested that turtle CD afferents express KCNQ3, KCNQ4, and ERG1-3 potassium channel subunits. KCNQ channels were favored as application of the selective antagonist XE991 mimicked and occluded efferent-mediated slow excitation in CD afferents. These data highlight an efferent-mediated mechanism for enhancing afferent sensitivity. They further suggest that the clinical effectiveness of mAChR antagonists in treating balance disorders may also target synaptic mechanisms in the vestibular periphery, and that KCNQ channel modulators might offer similar therapeutic value. SIGNIFICANCE STATEMENT Targeting the efferent vestibular system (EVS) pharmacologically might prove useful in ameliorating some forms of vestibular dysfunction by modifying ongoing primary vestibular input. EVS activation engages several kinetically distinct synaptic processes that profoundly alter the discharge rate and sensitivity of first-order vestibular neurons. Efferent-mediated slow excitation of vestibular afferents is of considerable interest given its ability to elevate afferent activity over an extended time course. We demonstrate for the first time that efferent-mediated slow excitation of vestibular afferents is mediated by muscarinic acetylcholine receptor (mAChR) activation and the subsequent closure of KCNQ potassium channels. The clinical effectiveness of some anti-mAChR drugs in treating motion sickness suggest that we may, in fact, already be targeting the peripheral EVS., (Copyright © 2017 the authors 0270-6474/17/371873-15$15.00/0.)

Vestibular-afferent neurons innervate hair cells from the sensory epithelia of vestibular end-organs and their action-potential discharge dynamics are driven by linear and angular accelerations of the head. The electrical activity of the vestibular-afferent neurons depends on their intrinsic properties and on the synaptic input from hair cells and from the terminals of the efferent system. Here we report that vestibular-afferent neurons of the rat are immunoreactive to RFamide-related peptides, and that the stronger signal comes from calyx-shaped neuron dendrites, with no signal detected in hair cells or supporting cells. The whole-cell voltage clamp recording of isolated afferent neurons showed that they express robust acid-sensing ionic currents (ASICs). Extracellular multiunit recordings of the vestibular nerve in a preparation in vitro of the rat inner ear showed that the perfusion of FMRFamide (a snail ortholog of this family of neuropeptides) exerts an excitatory effect on the afferent-neurons spike-discharge rate. Because the FMRFamide cannot activate the ASIC but reduces its desensitization generating a more robust current, its effect indicates that the ASIC are tonically active in the vestibular-afferent neurons and modulated by RFamide-like peptides., (Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.)

This study tested whether air-conducted sound and bone-conducted vibration activated primary vestibular afferent neurons and whether, at low levels, such stimuli are specific to particular vestibular sense organs. In response to 500 Hz bone-conducted vibration or 500 Hz air-conducted sound, primary vestibular afferent neurons in the guinea pig fall into one of two categories--some neurons show no measurable change in firing up to 2 g peak-to-peak or 140 dB SPL. These are semicircular canal neurons (regular or irregular) and regular otolith neurons. In sharp contrast, otolith irregular neurons show high sensitivity: a steep increase in firing as stimulus intensity is increased. These sensitive neurons typically, but not invariably, were activated by both bone-conducted vibration and air-conducted sound, they originate from both the utricular and saccular maculae, and their sensitivity underpins new clinical tests of otolith function.

In the mammalian vestibular nerve, some afferents have highly irregular interspike intervals and others have highly regular intervals. To investigate whether spike timing is determined by the afferents' ion channels, we studied spiking activity in their cell bodies, isolated from the vestibular ganglia of young rats. Whole cell recordings were made with the perforated-patch method. As previously reported, depolarizing current steps revealed distinct firing patterns. Transient neurons fired one or two onset spikes, independent of current level. Sustained neurons were more heterogeneous, firing either trains of spikes or a spike followed by large voltage oscillations. We show that the firing pattern categories are robust, occurring at different temperatures and ages, both in mice and in rats. A difference in average resting potential did not cause the difference in firing patterns, but contributed to differences in afterhyperpolarizations. A low-voltage-activated potassium current (I(LV)) was previously implicated in the transient firing pattern. We show that I(LV) grew from the first to second postnatal week and by the second week comprised Kv1 and Kv7 (KCNQ) components. Blocking I(LV) converted step-evoked firing patterns from transient to sustained. Separated from their normal synaptic inputs, the neurons did not spike spontaneously. To test whether the firing-pattern categories might correspond to afferent populations of different regularity, we injected simulated excitatory postsynaptic currents at pseudorandom intervals. Sustained neurons responded to a given pattern of input with more regular firing than did transient neurons. Pharmacological block of I(LV) made firing more regular. Thus ion channel differences that produce transient and sustained firing patterns in response to depolarizing current steps can also produce irregular and regular spike timing.

The activation of the efferent vestibular system modifies the basal discharge and the dynamic response of primary-afferent neurons to head motion and gravitational stimuli. The efferent input to afferent neurons is mediated primarily by cholinergic synapses that activate both muscarinic and nicotinic receptors. Previously we had shown that the muscarinic-acetylcholine-receptor (mAChR) activation modulates the low-voltage-activated M-type potassium current (I(K,M)) in the vestibular-afferent neurons. In this work we studied the second-messenger system mediating the inhibition of I(K,M) after mAChR activation. For this, voltage and current-clamp recordings were obtained in the cultured vestibular-afferent neurons of the rat. The I(K,M) was measured during its deactivation. Response to current-pulse injection was also studied. The use of the mAChR agonist oxotremorine-M significantly reduced the amplitude of the I(K,M) and modified the discharge response to current pulses from single spike to multiple spiking, reducing the adaptation of the electrical discharge. The intracellular perfusion of the phospholipase C (PLC) inhibitor U73122 significantly attenuated the inhibitory action of the mAChR receptor agonist oxotremorine-M. Its inactive analog U73343 produced no significant action. The use of the phosphatidylinositol 4,5 bis-phosphate (PIP(2)) scavenger poly-l-lysine also led to a significant reduction of the I(K,M). Our results show that the mAChR mediated activation of PLC and subsequent PIP(2) depletion (caused by its hydrolysis), modulates the I(K,M) in the vestibular-afferent neurons, modifying their discharge response dynamics to current-pulse injection., (Copyright 2009 Elsevier Ireland Ltd. All rights reserved.)

In the vestibular system, the electrical discharge of the afferent neurons has been found to be highly sensitive to external pH changes, and acid-sensing ionic-channels (ASIC) have been found to be functionally expressed in afferent neurons. No previous attempt to assay the ASIC function in vestibular afferent neurons has been done. In our work we studied the electrical discharge of the afferent neuron of the isolated inner ear of the axolotl (Ambystoma tigrinum) to determine the participation of proton-gated currents in the postransductional information processing in the vestibular system. Microperfusion of FMRF-amide significantly increased the resting activity of the afferent neurons of the semicircular canal indicating that ASIC currents are tonically active in the resting condition. The use of ASIC antagonists, amiloride and acetylsalicylic acid (ASA), significantly reduced the vestibular-nerve discharge, corroborating the idea that the afferent neurons of the vestibular system express ASICs that are sensitive to amiloride, ASA, and to FMRF-amide. The sensitivity of the vestibular afferent-resting discharge to the microperfusion of ASIC acting agents indicates the participation of these currents in the establishment of the afferent-resting discharge.

There is consensus that muscarinic and nicotinic receptors expressed in vestibular hair cells and afferent neurons are involved in the efferent modulation of the electrical activity of the afferent neurons. However the underlying mechanisms of postsynaptic control in neurons are not well understood. In our work we show that the activation of muscarinic receptors in the vestibular neurons modulates the potassium M-current modifying the activity of afferent neurons. Whole-cell patch-clamp recordings were made on vestibular-afferent neurons isolated from Wistar rats (postnatal days 7-10) and held in primary culture (18-24 h). The M-current was studied during its deactivation after depolarizing voltage-clamp pulses. In 68% of the cells studied, those of larger capacitance, the M-current antagonists linopirdine and XE-991 reduced the amplitude of the M-current by 54%+/-7% and 50%+/-3%. The muscarinic-receptor agonist oxotremorine-M also significantly reduced the M-current by 58%+/-12% in the cells. The action of oxotremorine-M was blocked by atropine, thus indicating its cholinergic nature. The erg-channel blocker E-4031 did not significantly modify the M-current amplitude. In current-clamp experiments, linopirdine, XE-991, and oxotremorine-M modified the discharge response to current pulses from single spike to multiple spiking, reducing the adaptation of the electrical discharge. Our results indicate that large soma-size cultured vestibular-afferent neurons (most probably calyx-bearing neurons) express the M-current and that the modulation of this current by activation of muscarinic-receptor reduces its spike-frequency adaptation.

The otic placode generates the auditory and vestibular sense organs and their afferent neurons; however, how auditory and vestibular fates are specified is unknown. We have generated a fate map of the otic placode and show that precursors for vestibular and auditory cells are regionally segregated in the otic epithelium. The anterior-lateral portion of the otic placode generates vestibular neurons, whereas the posterior-medial region gives rise to auditory neurons. Precursors for vestibular and auditory sense organs show the same distribution. Thus, different regions of the otic placode correspond to particular sense organs and their innervating neurons. Neurons from contiguous domains rarely intermingle suggesting that the regional organisation of the otic placode dictates positional cues to otic neurons. But, in addition, vestibular and cochlear neurogenesis also follows a stereotyped temporal pattern. Precursors from the anterior-lateral otic placode delaminate earlier than those from its medial-posterior portion. The expression of the proneural genes NeuroM and NeuroD reflects the sequence of neuroblast formation and differentiation. Both genes are transiently expressed in vestibular and then in cochlear neuroblasts, while differentiated neurons express Islet1, Tuj1 and TrkC, but not NeuroM or NeuroD. Together, our results indicate that the position of precursors within the otic placode confers identity to sensory organs and to the corresponding otic neurons. In addition, positional information is integrated with temporal cues that coordinate neurogenesis and sensory differentiation.

Sensory signal transduction of the inner ear afferent neurons and hair cells (HCs) requires numerous ionic conductances. The KCNQ4 voltage-gated M-type potassium channel is thought to set the resting membrane potential in cochlear HCs. Here we describe the spatiotemporal expression patterns of Kcnq4 and the associated alternative splice forms in the HCs of vestibular labyrinth. Whole mount immunodetection, qualitative and quantitative RT-PCR were performed to characterize the expression patterns of Kcnq4 transcripts and proteins. A topographical expression and upregulation of Kcnq4 during development was observed and indicated that Kcnq4 is not restricted to either a specific vestibular structure or cell type, but is present in afferent calyxes, vestibular ganglion neurons, and both type I and type II HCs. Of the four alternative splice variants, Kcnq4_v1 transcripts were the predominant form in the HCs, while Kcnq4_v3 was the major variant in the vestibular neurons. Differential quantitative expression of Kcnq4_v1 and Kcnq4_v3 were respectively detected in the striolar and extra-striolar regions of the utricle and saccule. Analysis of gerbils and rats yielded results similar to those obtained in mice, suggesting that the spatiotemporal expression pattern of Kcnq4 in the vestibular system is conserved among rodents. Analyses of vestibular HCs of Bdnf conditional mutant mice, which are devoid of any innervation, demonstrate that regulation of Kcnq4 expression in vestibular HCs is independent of innervation.

The main objective of this study was to determine whether bone-conducted vibration (BCV) is equally effective in activating both semicircular canal and otolith afferents in the guinea pig or whether there is preferential activation of one of these classes of vestibular afferents. To answer this question a large number (346) of single primary vestibular neurons were recorded extracellularly in anesthetized guinea pigs and were identified by their location in the vestibular nerve and classed as regular or irregular on the basis of the variability of their spontaneous discharge. If a neuron responded to angular acceleration it was classed as a semicircular canal neuron, if it responded to maintained roll or pitch tilts it was classified as an otolith neuron. Each neuron was then tested by BCV stimuli-either clicks, continuous pure tones (200-1,500 Hz) or short tone bursts (500 Hz lasting 7 ms)-delivered by a B-71 clinical bone-conduction oscillator cemented to the guinea pig's skull. All stimulus intensities were referred to that animal's own auditory brainstem response (ABR) threshold to BCV clicks, and the maximum intensity used was within the animal's physiological range and was usually around 70 dB above BCV threshold. In addition two sensitive single axis linear accelerometers cemented to the skull gave absolute values of the stimulus acceleration in the rostro-caudal direction. The criterion for a neuron being classed as activated was an audible, stimulus-locked increase in firing rate (a 10% change was easily detectable) in response to the BCV stimulus. At the stimulus levels used in this study, semicircular canal neurons, both regular and irregular, were insensitive to BCV stimuli and very few responded: only nine of 189 semicircular canal neurons tested (4.7%) showed a detectable increase in firing in response to BCV stimuli up to the maximum 2 V peak-to-peak level we delivered to the B-71 oscillator (which produced a peak-to-peak skull acceleration of around 6-8 g and was usually around 60-70 dB above the animal's own ABR threshold for BCV clicks). Regular otolithic afferents likewise had a poor response; only 14 of 99 tested (14.1%) showed any increase in firing rate up to the maximum BCV stimulus level. However, most irregular otolithic afferents (82.8%) showed a clear increase in firing rate in response to BCV stimuli: of the 58 irregular otolith neurons tested, 48 were activated, with some being activated at very low intensities (only about 10 dB above the animal's ABR threshold to BCV clicks). Most of the activated otolith afferents were in the superior division of the vestibular nerve and were probably utricular afferents. That was confirmed by evidence using juxtacellular injection of neurobiotin near BCV activated neurons to trace their site of origin to the utricular macula. We conclude there is a very clear preference for irregular otolith afferents to be activated selectively by BCV stimuli at low stimulus levels and that BCV stimuli activate some utricular irregular afferent neurons. The BCV generates compressional and shear waves, which travel through the skull and constitute head accelerations, which are sufficient to stimulate the most sensitive otolithic receptor cells.

Vestibular-afferent neurons (VANs) transmit information about linear and angular accelerations during head movements from vestibular end organs to vestibular nuclei. In situ, these neurons show heterogeneous discharge patterns that may be produced by differences in their intrinsic properties. However, little is known about the ionic currents underlying their different firing patterns. Using the whole cell patch-clamp technique, we analyzed the expression of Ca(2+) and Ca(2+)-activated K(+) currents (I(KCa)) in primary cultured neurons isolated from young rats (p7-p10). We found two overlapping subpopulations of VANs classified according to low-threshold Ca(2+)-current [low-voltage-activated (LVA)] expression; LVA (-) neurons, formed by small cells, and LVA (+) neurons composed of medium to large cells. The I(KCa) in both cell-groups was carried through channels of high (BK), intermediate (IK), and low conductance (SK), besides a resistant channel to classical blockers (IR). BK was expressed preferentially in LVA (+) cells, whereas IR expression was preferentially in LVA (-) cells. No correlation between SK and IK expression with the soma size was found. Current-clamp experiments showed that BK participates in the adaptation of discharge and in the duration of the action potential, whereas SK and IK did not show a significant contribution to electrical discharge of cultured VANs. However, because of the low number of VANs in culture with repetitive firing it is difficult to interpret our results in terms of discharge patterns. Our results demonstrate that vestibular-afferent neurons possess different Ca(2+)-activated K(+) (K(Ca)) channels and that their expression, heterogeneous among the cells, would contribute to explain some of the differences in the electrical-firing properties of these neurons.

The function of the zinc finger transcription factor GATA3 was studied in a newly established, conditionally immortal cell line derived to represent auditory sensory neuroblasts migrating from the mouse otic vesicle at embryonic day E10.5. The cell line, US/VOT-33, expressed GATA3, the bHLH transcription factor NeuroD and the POU-domain transcription factor Brn3a, as do auditory neuroblasts in vivo. When GATA3 was knocked down reversibly with antisense oligonucleotides, NeuroD was reversibly down-regulated. Auditory and vestibular neurons form from neuroblasts that express NeuroD and that migrate from the antero-ventral, otic epithelium at E9.5-10.5. On the medial side, neuroblasts and epithelial cells express GATA3 but on the lateral side they do not. At E13.5 most auditory neurons express GATA3 but no longer express NeuroD, whereas vestibular neurons express NeuroD but not GATA3. Neuroblasts expressing NeuroD and GATA3 were located in the ventral, otic epithelium, the adjacent mesenchyme and the developing auditory ganglion. The results suggest that auditory and vestibular neurons arise from different, otic epithelial domains and that they gain their identity prior to migration. In auditory neuroblasts, NeuroD appears to be dependent on the expression of GATA3.

Neurotrophins have multiple functions during peripheral nervous system development such as controlling neuronal survival, target innervation and synaptogenesis. Neurotrophin specificity has been attributed to the selective expression of the Trk tyrosine kinase receptors in different neuronal subpopulations. However, despite overlapping expression of TrkB and TrkC in many sensory ganglia, brain-derived neurotrophic factor (BDNF) and neurotrophin 3 (NT3) null mutant mice display selective losses in neuronal subpopulations. In the present study we have replaced the coding part of the BDNF gene in mice with that of NT3 (BDNF(NT3/NT3)) to analyse the specificity and selective roles of BDNF and NT3 during development. Analysis of BDNF(NT3/NT3) mice showed striking differences in the ability of NT3 to promote survival, short-range innervation and synaptogenesis in different sensory systems. In the cochlea, specificity is achieved by a tightly controlled spatial and temporal ligand expression. In the vestibular system TrkB or TrkC activation is sufficient to promote vestibular ganglion neuron survival, while TrkB activation is required to promote proper innervation and synaptogenesis. In the gustatory system, NT3 is unable to replace the actions of BDNF possibly because of a temporally selective expression of TrkB in taste neurons. We conclude that there is no general mechanism by which neurotrophin specificity is attained and that specificity is achieved by (i) a tightly controlled spatial and temporal expression of ligands, (ii) different Trk receptors playing distinct roles within the same neuronal subpopulation, or (iii) selective receptor expression in sensory neuron subpopulations.

Betahistine has been used to treat several vestibular disorders of both central and peripheral origin. The objective of this work was to study the action of betahistine in the vestibular endorgans. Experiments were done in wild larval axolotl (Ambystoma tigrinum). Multiunit extracellular recordings were obtained from the semicircular canal nerve using a suction electrode. Betahistine (10 microM to 10 mM; n = 32) inhibited the basal spike discharge of the vestibular afferent neurons with an IC50 of 600 microM. To define the site of action of betahistine, its interactions with the nitric oxide synthase inhibitor NG-nitro-L-arginine (3 microM) and with the cholinergic antagonists atropine (10 microM; n = 3) and d-tubocurarine (10 microM; n = 3) were studied. The action of betahistine when co-administered with these drugs was the same as that in control experiments, indicating that its effects did not include nitric oxide production or the activation of cholinergic receptors. In contrast, 0.01-1 mM betahistine reduced the excitatory action of kainic acid (10 microM; n = 6) and quiscualic acid (1 microM; n = 13). These results indicate that the action of betahistine on the spike discharge of afferent neurons seems to be due to a post-synaptic inhibitory action on the primary afferent neuron response to the hair cell neurotransmitter.

The existence of voltage-sensitive Ca2+ channels in type I vestibular hair cells of mammals has not been conclusively proven. Furthermore, Ca2+ channels present in type II vestibular hair cells of mammals have not been pharmacologically identified. Fura-2 fluorescence was used to estimate, in both cell types, intracellular Ca2+ concentration ([Ca2+]i) variations induced by K+ depolarization and modified by specific Ca2+ channel agonists and antagonists. At rest, [Ca2+]i was 90 +/- 20 nM in both cell types. Microperifusion of high-K+ solution (50 mM) for 1 s increased [Ca2+]i to 290 +/- 50 nM in type I (n = 20) and to 440 +/- 50 nM in type II cells (n = 10). In Ca2+-free medium, K+ did not alter [Ca2+]i. The specific L-type Ca2+ channel agonist, Bay K, and antagonist, nitrendipine, modified in a dose-dependent manner the K+-induced [Ca2+]i increase in both cell types with maximum effect at 2 microM and 400 nM, respectively. Ni2+, a T-type Ca2+ channel blocker, reduced K+-evoked Ca2+ responses in a dose-dependent manner. For elevated Ni2+ concentrations, the response was differently affected by Ni2+ alone, or combined to nitrendipine (500 nM). In optimal conditions, nitrendipine and Ni2+ strongly depressed by 95% the [Ca2+]i increases. By contrast, neither omega-agatoxin IVA (1 microM), a specific P- and Q-type blocker, nor omega-conotoxin GVIA (1 microM), a specific N-type blocker, affected K+-evoked Ca2+i responses. These results provide the first direct evidence that L- and probably T-type channels control the K+-induced Ca2+ influx in both types of sensory cells.

We have reinvestigated the embryonic development of the vestibulocochlear system in mice using anterograde and retrograde tracing techniques. Our studies reveal that rhombomeres 4 and 5 include five motor neuron populations. One of these, the abducens nucleus, will not be dealt with here. Rhombomere 4 gives rise to three of the remaining populations: the facial branchial motor neurons; the vestibular efferents; and the cochlear efferents. The migration of the facial branchial motor neurons away from the otic efferents is completed by 13.5 days post coitum (dpc). Subsequently the otic efferents separate into the vestibular and cochlear efferents, and complete their migration by 14.5 dpc. In addition to their common origin, all three populations have perikarya that migrate via translocation through secondary processes, form a continuous column upon completion of their migrations, and form axonal tracts that run in the internal facial genu. Some otic efferent axons travel with the facial branchial motor nerve from the internal facial genu and exit the brain with that nerve. These data suggest that facial branchial motor neurons and otic efferents are derived from a common precursor population and use similar cues for pathway recognition within the brain. In contrast, rhombomere 5 gives rise to the fourth population to be considered here, the superior salivatory nucleus, a visceral motor neuron group. Other differences between this group and those derived from rhombomere 4 include perikaryal migration as a result of translocation first through primary processes and only then through secondary processes, a final location lateral to the branchial motor/otic efferent column, and axonal tracts that are completely segregated from those of the facial branchial and otic efferents throughout their course inside the brain. Analysis of the peripheral distribution of the cochlear efferents and afferents show that efferents reach the spiral ganglion at 12.5 dpc when postmitotic ganglion cells are migrating away from the cochlear anlage. The efferents begin to form the intraganglionic spiral bundle by 14.5 dpc and the inner spiral bundle by 16.5 dpc in the basal turn. They have extensive collaterals among supporting cells of the greater epithelial ridge from 16.5 dpc onwards. Afferents and efferents in the basal turn of the cochlea extend through all three rows of outer hair cells by 18.5 dpc. Selective labeling of afferent fibers at 20.5 dpc (postnatal day 1) shows that although some afferents are still in early developmental stages, some type II spiral ganglion cells already extend for long distances along the outer hair cells, and some type I spiral ganglion cells end on a single inner hair cell. These data support previous evidence that in mice the early outgrowth of afferent and efferent fibers is essentially achieved by birth.

Vestibular nerve afferents innervating the bullfrog utriculus differ in their response dynamics and sensitivity to natural stimulation. They also supply hair cells that differ markedly in hair bundle morphology. To examine the peripheral innervation patterns of individual utricular afferents more closely, afferent fibers were labeled by the extracellular injection of horseradish peroxidase (HRP) into the vestibular nerve after sectioning the vestibular nerve medial to Scarpa's ganglion to allow the degeneration of sympathetic and efferent fibers. The peripheral arborizations of individual afferents were then correlated with the diameters of their parent axons, the regions of the macula they innervate, and the number and type of hair cells they supply. The utriculus is divided by the striola, a narrow zone of distinctive morphology, into medial and lateral parts. Utricular afferents were classified as striolar or extrastriolar according to the epithelial entrance of their parent axons and the location of their terminal fields. In general, striolar afferents had thicker parent axons, fewer subepithelial bifurcations, larger terminal fields, and more synaptic endings than afferents in extrastriolar regions. Afferents in a juxtastriolar zone, immediately adjacent to the medial striola, had innervation patterns transitional between those in the striola and more peripheral parts of the medial extrastriola. Most afferents innervated only a single macular zone. The terminal fields of striolar afferents, with the notable exception of a few afferents with thin parent axons, were generally confined to one side of the striola. Hair cells in the bullfrog utriculus have previously been classified into four types based on hair bundle morphology (Lewis and Li: Brain Res. 83:35-50, 1975). Afferents in the extrastriolar and juxtastriolar zones largely or exclusively innervated Type B hair cells, the predominant hair cell type in the utricular macula. Striolar afferents supplied a mixture of four hair cell types, but largely contacted Type B and Type C hair cells, particularly on the outer rows of the medial striola. Afferents supplying more central striolar regions innervated fewer Type B and large numbers of Type E and Type F hair cells. Striolar afferents with thin parent axons largely supplied Type E hair cells with bulbed kinocilia in the innermost striolar rows.

This study aimed to define the pharmacology and physiological role of the N-methyl-D-aspartate (NMDA) receptor in the synapse between the hair cells and primary afferent neurons in the vestibular system. The spontaneous and mechanically evoked spike discharges of vestibular nerve fibers were extracellularly recorded in isolated inner ear from the axolotl (Ambystoma tigrinum). Pressure ejection of NMDA (10(-6) to 10(-3) M) elicited a dose-dependent increase of the basal spike discharge from the vestibular nerve fibers. Extracellular magnesium antagonized the NMDA effect in a dose-dependent manner. D(-)-2-amino-5-phosphonovaleric acid (AP5, 10(-5) to 10(-3) M) and 7-chloro-kynurenic acid (7ClKyn, 10(-6) to 10(-3) M) inhibited the basal activity of the vestibular nerve fibers. 7ClKyn also diminished the responses elicited by the mechanical stimulation of the preparation. Glycine (10(-9) to 10(-6) M) applied by bath substitution enhanced the NMDA response, and the glycine agonist D-serine partially reversed the 7ClKyn inhibitory action. These results suggest that NMDA receptors participate in the generation of the basal spike discharge of vestibular system primary afferent neurons, but its activation is not critical for the response to brief mechanical stimuli.

Synaptic connections between physiologically classified primary vestibular afferents (PVAs) and their target vestibular nucleus (VN) neurons were examined by a combination of intra-axonal staining and electron microscopic techniques. PVAs originating from the horizontal semicircular canal were electrophysiologically classified as either regular- or irregular-type based on the regularity of their spontaneous discharge patterns, and were intra-axonally labeled with horseradish peroxidase (HRP). HRP-labeled PVAs of both types had many swellings along their course that contacted VN neurons. These swellings contained spherical synaptic vesicles and showed asymmetric postsynaptic specialization. Target VN neurons of both types of PVAs were distributed primarily in the superior, medial, and inferior VN. Irregular-type PVAs made more axosomatic contacts than did regular-type PVAs. The soma size of target VN neurons and the number of terminal boutons per target VN neuron were larger for irregular-type PVAs than for regular-type PVAs. Large VN neurons (presumably kinetic neurons) were innervated exclusively by irregular-type PVAs. Small VN neurons were innervated by PVAs of the regular-type and the irregular-type. These results demonstrate that there is a correlation between the physiological properties and morphological characteristics of PVAs and their target VN neurons.

In order to determine the nature of the transmitter in the synapse between hair cells and primary afferent fibers, both resting and evoked spike activity of vestibular system afferents were recorded. Excitatory amino acid agonists and antagonists were applied by micro perfusion. Excitatory amino acid agonists consistently increased the firing rate of these afferents. The rank order in potencies of the agonists tested was: kainate greater than or equal to quisqualate greater than D-aspartate greater than or equal to L-glutamate greater than or equal to L-aspartate greater than N-methyl D-aspartate. Blockade of synaptic transmission with high-Mg2+ and low-Ca2+ solutions did not seem to affect the responses to the excitatory amino acid agonists indicating their postsynaptic action. Excitatory amino acid antagonists inhibit both resting and physiologically evoked activity. The rank order of inhibitory potency was: kynurenate greater than L-glutamate diethyl ester greater than D,L-2-amino-4-phosphono-butyrate greater than D-alpha-amino adipate greater than D,L-2-amino-5-phosphonovalerate. These findings suggest that an amino acid-related compound may be the transmitter at this synapse. The relative potencies of agonists and antagonists tested provide evidence that the transmitter released from the hair cells' basal pole in the axolotl vestibular system interacts with postsynaptic kainic/quisqualic type receptors.

The central distribution of afferents that innervate the macula of the saccule and the crista of the posterior canal was assessed in the gerbil following the direct injection of horseradish peroxidase (HRP) separately into the sensory neuroepithelia of each peripheral receptor organ. Ganglion cells innervating the posterior canal were located in the caudal part of the inferior ganglion, while those cells innervating the saccule were located in the rostral part of the inferior ganglion, scattered in the superior ganglion, and concentrated at the junction (isthmus) between the two. The paths of the central axons of these two groups of ganglion cells through the vestibular root and their division into ascending or descending pathways were similar. However, the distributions of their terminals were different. The posterior canal projected to medial parts of the vestibular nuclear complex. Terminals were found in the medial and superior vestibular nuclei. The posterior canal also projected to the uvula of the cerebellum. The saccule projected to more lateral-lying brainstem areas. Terminal fields were located in the lateral and descending vestibular nuclei and cell group y. Saccule projections outside the vestibular complex were observed to the lateral cuneate nucleus, the N. gigantocellularis, and the cerebellar cortex. Of the eight areas receiving primary afferent projections from these two organs, only within the medial and descending vestibular nuclei and the cerebellar cortex were overlapping projections observed.

A stochastic version of Kernell's (1968, 1972) model with cumulative afterhyperpolarization (AHP) was simulated. A characteristic of the model is that the AHP is the result of an increased potassium conductance (g K) that is time-dependent but not voltage-dependent. Quantal synaptic inputs are assumed to be the only source of interspike interval variability. The model reproduces many features of the steady-state discharge of peripheral vestibular afferents, provided that firing rates are higher than 40 spikes/s. Among the results accounted for are the interspike interval statistics occurring during natural stimulation, their alteration by externally applied galvanic currents and the increase in the interspike interval following an interposed shock. Empirical studies show that some vestibular afferents have a regular spacing of action potentials, others an irregular spacing (Goldberg and Fernández 1971b; Fernández and Goldberg 1976). Irregularly discharging afferents have a higher sensitivity to externally applied galvanic currents than do regular afferents (Goldberg et al. 1984). To explain the relation between galvanic sensitivity and discharge regularity requires the assumption that neurons differ in both their synaptic noise (sigma v) and the slopes of their postspike voltage trajectories (d mu v/dt). The more irregular the neuron's discharge at a given firing frequency, the greater is sigma v and the smaller is d mu v/dt. Of the two factors, d mu v/dt is estimated to be four times more influential in determining discharge regularity across the afferent population. The shortcomings of the model are considered, as are possible remedies. Our conclusions are compared to previous discussions of mechanisms responsible for differences in the discharge regularity of vestibular afferents.

The central projection of vestibular nerve fibers was investigated with anterograde axonal transport of wheatgerm agglutinin-horseradish peroxidase (WGA-HRP) and tritiated leucine following injection in the vestibular ganglion. Labeled fibers and terminal ramifications were observed throughout the vestibular complex, but absent from the lateral vestibular nucleus. Termination in the cortex was restricted to the vermis. Small numbers of mossy fiber terminals were present bilaterally, close to the midline in lobules I and II, and in the depth of the main fissures separating lobules II-VI. In the posterior vermis labeled mossy fiber terminals were found in lobule X and the ventral aspect of lobule IXd. Here, the entire ipsilateral hemivermis contained a large number of terminals, while contralaterally the medial one-third hemivermis contained fewer terminals. Labeled mossy fibers and terminals were absent in the flocculus and adjacent ventral paraflocculus.