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BACKGROUND AND PURPOSE Betahistine, the main histamine drug prescribed to treat vestibular disorders, is a histamine H3 receptor antagonist. Here, we explored the potential for modulation of the most recently cloned histamine receptor (H4 receptor) to influence vestibular system function, using a selective H4 receptor antagonist JNJ 7777120 and the derivate compound JNJ 10191584. EXPERIMENTAL APPROACH RT-PCR was used to assess the presence of H4 receptors in rat primary vestibular neurons. In vitro electrophysiological recordings and in vivo behavioural approaches using specific antagonists were employed to examine the effect of H4 receptor modulation in the rat vestibular system. KEY RESULTS The transcripts of H4 and H3 receptors were present in rat vestibular ganglia. Application of betahistine inhibited the evoked action potential firing starting at micromolar range, accompanied by subsequent strong neuronal depolarization at higher concentrations. Conversely, reversible inhibitory effects elicited by JNJ 10191584 and JNJ 7777120 began in the nanomolar range, without inducing neuronal depolarization. This effect was reversed by application of the selective H4 receptor agonist 4-methylhistamine. Thioperamide, a H3/H4 receptor antagonist, exerted effects similar to those of H3 and H4 receptor antagonists, namely inhibition of firing at nanomolar range and membrane depolarization above 100 µM. H4 receptor antagonists significantly alleviated the vestibular deficits induced in rats, while neither betahistine nor thioperamide had significant effects. CONCLUSIONS AND IMPLICATIONS H4 receptor antagonists have a pronounced inhibitory effect on vestibular neuron activity. This result highlights the potential role of H4 receptors as pharmacological targets for the treatment of vestibular disorders. [ABSTRACT FROM AUTHOR]

Sound-level coding in the auditory nerve is achieved through the progressive recruitment of auditory nerve fibers (ANFs) that differ in threshold of activation and in the stimulus level at which the spike rate saturates. To investigate the functional state of the ANFs, the electrophysiological tests routinely used in clinics only capture the first action potentials firing in synchrony at the onset of the acoustic stimulation. Assessment of other properties (e.g., spontaneous rate and adaptation time constants) requires single-fiber recordings directly from the nerve, which for ethical reasons is not allowed in humans. By combining neuronal activity measurements at the round window and signal-processing algorithms, we constructed a peristimulus time response (PSTR), with a waveform similar to the peristimulus time histograms (PSTHs) derived from single-fiber recordings in young adult female gerbils. Simultaneous recordings of round-window PSTR and single-fiber PSTH provided models to predict the adaptation kinetics and spontaneous rate of the ANFs tuned at the PSTR probe frequency. The predictive model derived from gerbils was then validated in female mice and finally applied to humans by recording PSTRs from the auditory nerve in normal-hearing patients who underwent cerebellopontine angle surgeries. A rapid adaptation time constant of ∼3 ms and a mean spontaneous rate of ∼22 spikes/s in the 4 kHz frequency range were found. This study offers a promising diagnostic tool to map the human auditory nerve, thus opening new avenues to better understanding auditory neuropathies, tinnitus, and hyperacusis. SIGNIFICANCE STATEMENT Neural adaptation in auditory nerve fibers corresponds to the reduction in the neuronal activity to prolonged or repeated sound stimulation. For obvious ethical reasons, single-fiber recordings from the auditory nerve are not feasible in humans, creating a critical gap in extending data obtained using animal models to humans. Using electrocochleography in rodents, we inferred adaptation kinetics and spontaneous discharge rates of the auditory nerve fibers in humans. Routinely used in basic and clinical laboratories, this tool will provide a better understanding of auditory disorders such as neuropathies, tinnitus, and hyperacusis, and will help to improve hearing-aid fittings., (Copyright © 2022 the authors.)

Auditory nerve fibers (ANFs) encode pure tones through two modes of coding, spike time and spike rate, depending on the tone frequency. In response to a low-frequency tone, ANF firing is phase locked to the sinusoidal waveform. Because time coding vanishes with an increase in the tone frequency, high-frequency tone coding relies on the spike rate of the ANFs. Adding a continuous broadband noise to a tone compresses the rate intensity function of ANFs and shifts its dynamic range toward higher intensities. Therefore, the ANFs with high-threshold/low-spontaneous rate (SR) are thought to contribute to behavioral tone detection in noise. However, this theory relies on the discharge rate of the ANFs. The direct comparison with the masking threshold through spike timing, irrespective of the spontaneous rate, has not so far been investigated. Taking advantage of a unique proxy to quantify the spike synchrony (i.e., the shuffle autocorrelogram), we show in female gerbils that high-SR ANFs are more adapted to encode low-frequency thresholds through temporal code, giving them a strong robustness in noise. By comparing behavioral thresholds measured using prepulse inhibition of the acoustical startle reflex with population thresholds calculated from ANFs pooled per octave band, we show that threshold-based spike timing provides a better estimate of behavioral thresholds in the low-frequency range, whereas the high-frequency behavioral thresholds rely on the spiking rate, particularly in noise. This emphasizes the complementarity of temporal and rate modes to code tone-in-noise thresholds over a large range of frequencies. SIGNIFICANCE STATEMENT There is a general agreement that high-threshold/low-spontaneous rate (SR) auditory nerve fibers (ANFs) are of prime importance for tone detection in noise. However, this theory is based on the discharge rate of the fibers. Comparing the behavioral thresholds and single ANF thresholds shows that this is only true in the high-frequency range of tone stimulations. In the low-frequency range of tones (up to 2.7 kHz in the gerbil), the most sensitive ANFs (high-SR fibers) carry neural information through a spike-timing mode, even for noise in which tones do not induce a noticeable increment in the spike rate. This emphasizes the interplay between spike-time and spike-rate modes in the auditory nerve to encode tone-in-noise threshold over a large range of tone frequencies., (Copyright © 2018 the authors 0270-6474/18/385727-12$15.00/0.)

Auditory nerve fibers (ANFs) transmit acoustic information from the sensory hair cells to the cochlear nuclei. In experimental and clinical audiology, probing the whole ANF population remains a difficult task, as the ANFs differ greatly in their threshold and onset response to sound. Thus, low spontaneous rate (SR) fibers, which have rather higher thresholds, delay and larger jitter in their first spike latency are not detectable in the far-field compound action potential of the auditory nerve. Here, we developed a new protocol of acoustic stimulation together with electrophysiological signal processing to track the steady state activity of ANFs. Mass potentials at the round window were recorded in response to repetitive 300-ms bursts of 1/3 octave band noise centered on a frequency probe. Analysis was assessed during the last 200-ms of the response to capture the steady-state response of ANFs. To eliminate the microphonic component reflecting the sensory cells activity, repetitive pairs of sounds of opposite polarities were used. The spectral analysis was calculated on the average of two consecutive responses, and the neural gain was calculated by dividing point-by-point the spectrum to sound over unstimulated condition. In response to low-sound-level stimulation, neural gain predominated in the low-frequency cochlear regions, while a second component of responses centered on higher cochlear frequency regions appeared beyond 30 dB SPL. At 60 dB SPL, neural gain showed a bimodal shape, with a notch near 5.6 kHz. In addition to correlate with the functional mapping of ANFs along the tonotopic axis, the deletion of low-SR fibers leads to a reduction in the high-frequency response, where the low-SR fibers are preferentially located. Thus, mass potentials at the round window may provide a useful tool to probe the SR-based distribution of ANFs in humans and in other species in which direct single-unit recordings are difficult to achieve or not feasible., Competing Interests: The authors have declared that no competing interests exist.

Gerbils possess a very specialized cochlea in which the low-frequency inner hair cells (IHCs) are contacted by auditory nerve fibers (ANFs) having a high spontaneous rate (SR), whereas high frequency IHCs are innervated by ANFs with a greater SR-based diversity. This specificity makes this animal a unique model to investigate, in the same cochlea, the functional role of different pools of ANFs. The distribution of the characteristic frequencies of fibers shows a clear bimodal shape (with a first mode around 1.5 kHz and a second around 12 kHz) and a notch in the histogram near 3.5 kHz. Whereas the mean thresholds did not significantly differ in the two frequency regions, the shape of the rate-intensity functions does vary significantly with the fiber characteristic frequency. Above 3.5 kHz, the sound-driven rate is greater and the slope of the rate-intensity function is steeper. Interestingly, high-SR fibers show a very good synchronized onset response in quiet (small first-spike latency jitter) but a weak response under noisy conditions. The low-SR fibers exhibit the opposite behavior, with poor onset synchronization in quiet but a robust response in noise. Finally, the greater vulnerability of low-SR fibers to various injuries including noise- and age-related hearing loss is discussed with regard to patients with poor speech intelligibility in noisy environments. Together, these results emphasize the need to perform relevant clinical tests to probe the distribution of ANFs in humans, and develop appropriate techniques of rehabilitation. This article is part of a Special Issue entitled ., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Previous experimental data indicates the hyperpolarization-activated cation (Ih) current, in the inner ear, consists of two components [different hyperpolarization-activated cyclic nucleotide-gated (HCN) subunits] which are impossible to pharmacologically isolate. To confirm the presence of these two components in vestibular ganglion neurons we have applied a parameter identification algorithm which is able to discriminate the parameters of the two components from experimental data. Using simulated data we have shown that this algorithm is able to identify the parameters of two populations of non-inactivated ionic channels more accurately than a classical method. Moreover, the algorithm was demonstrated to be insensitive to the key parameter variations. We then applied this algorithm to Ih current recordings from mouse vestibular ganglion neurons. The algorithm revealed the presence of a high-voltage-activated slow component and a low-voltage-activated fast component. Finally, the electrophysiological significance of these two Ih components was tested individually in computational vestibular ganglion neuron models (sustained and transient), in the control case and in the presence of cAMP, an intracellular cyclic nucleotide that modulates HCN channel activity. The results suggest that, first, the fast and slow components modulate differently the action potential excitability and the excitatory postsynaptic potentials in both sustained and transient vestibular neurons and, second, the fast and slow components, in the control case, provide different information about characteristics of the stimulation and this information is significantly modified after modulation by cAMP., (© 2015 Federation of European Neuroscience Societies and John Wiley & Sons Ltd.)

Sound-evoked compound action potential (CAP), which captures the synchronous activation of the auditory nerve fibers (ANFs), is commonly used to probe deafness in experimental and clinical settings. All ANFs are believed to contribute to CAP threshold and amplitude: low sound pressure levels activate the high-spontaneous rate (SR) fibers, and increasing levels gradually recruit medium- and then low-SR fibers. In this study, we quantitatively analyze the contribution of the ANFs to CAP 6 days after 30-min infusion of ouabain into the round window niche. Anatomic examination showed a progressive ablation of ANFs following increasing concentration of ouabain. CAP amplitude and threshold plotted against loss of ANFs revealed three ANF pools: 1) a highly ouabain-sensitive pool, which does not participate in either CAP threshold or amplitude, 2) a less sensitive pool, which only encoded CAP amplitude, and 3) a ouabain-resistant pool, required for CAP threshold and amplitude. Remarkably, distribution of the three pools was similar to the SR-based ANF distribution (low-, medium-, and high-SR fibers), suggesting that the low-SR fiber loss leaves the CAP unaffected. Single-unit recordings from the auditory nerve confirmed this hypothesis and further showed that it is due to the delayed and broad first spike latency distribution of low-SR fibers. In addition to unraveling the neural mechanisms that encode CAP, our computational simulation of an assembly of guinea pig ANFs generalizes and extends our experimental findings to different species of mammals. Altogether, our data demonstrate that substantial ANF loss can coexist with normal hearing threshold and even unchanged CAP amplitude., (Copyright © 2014 the American Physiological Society.)

Background and Objective: The optical stimulation of neurons from pulsed infrared lasers has appeared over the last years as an alternative to classical electric stimulations based on conventional electrodes. Laser stimulation could provide a better spatial selectivity allowing single-cell stimulation without prerequisite contact. In this work we present relevant physical characteristics of a non-lethal stimulation of cultured mouse vestibular and retinal ganglion neurons by single infrared laser pulses., Study Design/materials and Methods: Vestibular and retinal ganglion neurons were stimulated by a 100-400 mW pulsed laser diode beam (wavelengths at 1,470, 1,535, 1,875 nm) launched into a multimode optical fiber positioned at a few hundred micrometers away from the neurons. Ionic exchange measurements at the neuron membrane were achieved by whole-cell patch-clamp recordings. Stimulation and damage thresholds, duration and repetition rate of stimulation and temperature were investigated., Results: All three lasers induced safe and reproducible action potentials (APs) on both types of neurons. The radiant exposure thresholds required to elicit APs range from 15 ± 5 to 100 ± 5 J cm(-2) depending on the laser power and on the pulse duration. The damage thresholds, observed by a vital dye, were significantly greater than the stimulation thresholds. In the pulse duration range of our study (2-30 milliseconds), similar effects were observed for the three lasers. Measurements of the local temperature of the neuron area show that radiant exposures required for reliable stimulations at various pulse durations or laser powers correspond to a temperature increase from 22 °C (room temperature) to 55-60 °C. Stimulations by laser pulses at repetition rate of 1, 2, and 10 Hz during 10 minutes confirmed that the neurons were not damaged and were able to survive such temperatures., Conclusion: These results show that infrared laser radiations provide a possible way to safely stimulate retinal and vestibular ganglion neurons. A similar temperature threshold is required to trigger neurons independently of variable energy thresholds, suggesting that an absolute temperature is required., (Copyright © 2012 Wiley Periodicals, Inc.)

Glutamate is the neurotransmitter released from hair cells. Its clearance from the synaptic cleft can shape neurotransmission and prevent excitotoxicity. This may be particularly important in the inner ear and in other sensory organs where there is a continually high rate of neurotransmitter release. In the case of most cochlear and type II vestibular hair cells, clearance involves the diffusion of glutamate to supporting cells, where it is taken up by EAAT1 (GLAST), a glutamate transporter. A similar mechanism cannot work in vestibular type I hair cells as the presence of calyx endings separates supporting cells from hair-cell synapses. Because of this arrangement, it has been conjectured that a glutamate transporter must be present in the type I hair cell, the calyx ending, or both. Using whole-cell patch-clamp recordings, we demonstrate that a glutamate-activated anion current, attributable to a high-affinity glutamate transporter and blocked by DL-TBOA, is expressed in type I, but not in type II hair cells. Molecular investigations reveal that EAAT4 and EAAT5, two glutamate transporters that could underlie the anion current, are expressed in both type I and type II hair cells and in calyx endings. EAAT4 has been thought to be expressed almost exclusively in the cerebellum and EAAT5 in the retina. Our results show that these two transporters have a wider distribution in mice. This is the first demonstration of the presence of transporters in hair cells and provides one of the few examples of EAATs in presynaptic elements.

Trimetazidine (1[2,3,4-trimethoxy-benzyl] piperazine, 2 HCl) is an anti-ischemic agent frequently administered as a prophylactic treatment for episodes of angina pectoris and chorioretinal disturbances. It is also employed as a symptomatic treatment of vertigo but its mechanism of action is yet to be defined. Using Fura-2 fluorescence photometry and whole-cell patch-clamp recordings we investigated the effect of trimetazidine on the [Ca(2+)](i) and current responses induced by the application of non-N-methyl-D-aspartate (NMDA) receptor agonists on low density vestibular ganglion neuronal cultures explanted from 3 day s postnatal rats. Trimetazidine blocked the [Ca(2+)](i) and current responses induced by 100 microM applications of both kainate and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA). These responses were dependent on external Ca(2+) and were blocked by the voltage-dependent Ca(2+) channel blockers Ni(2+) and Cd(2+) . Trimetazidine only acts on the AMPA/kainate receptors and had no effect on K(+)-induced depolarizations. Dose-dependent curves were obtained for the inhibition by 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and trimetazidine (IC(50) 7 microM and 0.7 microM) of kainate stimulations. After AMPA stimulation, dose-response inhibition curves showed an IC(50) of 3 microM for CNQX and 25 microM for trimetazidine. These results indicate that trimetazidine could be a potent antagonist of AMPA/kainate receptors in vestibular ganglion neurons. This may explain the protective role of trimetazidine in the inner ear suggesting an anti-excitotoxic activity.

Glutamate is thought to be the main neurotransmitter at the synapse between the type I vestibular hair cell and its cognate calyx afferent. The present study was designed to identify the type of glutamate receptors involved in neurotransmission at this unusual synapse. Immunocytochemistry showed that AMPA GluR2, NMDA NR1 and NR2A/B subunits of the glutamate receptors were confined to the synaptic contact. We then examined the electrical activity at calyx terminals using direct electrophysiological recordings from intact dendritic terminals in explanted turtle posterior crista. We found that sodium-based action potentials support a background discharge that could be modulated by the mechanical stimulation of the hair bundle of the sensory cells. These activities were prevented by blocking both the mechano-electrical transduction channels and L-type voltage-gated Ca(2+) channels involved in synaptic transmission. Although pharmacological analysis revealed that NMDA receptors could operate, our results show that AMPA receptors are mainly involved in synaptic neurotransmission. We conclude that although both AMPA and NMDA glutamate receptor subunits are present at the calyx synapse, only AMPA receptors appear to be involved in the synaptic transmission between the type I vestibular hair cell and the calyx afferent.

Ca2+ influx through voltage-gated calcium channels probably influences neuronal ontogenesis. Many developing neurones transiently express T-type/Cav3 calcium channels that contribute to their electrical activity and potentially to their morphological differentiation. Here we have characterized the electrophysiological properties and the functional role of a large T-type calcium current that is present in mouse developing primary vestibular neurones at embryonic day E17. This T-type current showed fast activation and inactivation, as well as slow deactivation kinetics. The overlap of activation and inactivation parameters produced a window current between -65 and -45 mV. Recovery from short-term inactivation was slow suggesting the presence of the Cav3.2 subunit. This T-type current was blocked by micromolar concentrations of Ni2+ and was inhibited by fast perfusion velocities in a similar fashion to recombinant Cav3.2 T-type channels expressed in HEK-293 cells. More importantly, current clamp experiments have revealed that the T-current could elicit afterdepolarization potentials during the repolarization phase of action potentials, and occasionally generate calcium spikes. Taken together, we demonstrate that the Cav3.2 subunit is likely to be the main T-type calcium channel subunit expressed in embryonic vestibular neurones and should play a key role in the excitability of these neurones during the ontogenesis of vestibular afferentation.

Different subsets of dorsal root ganglion (DRG) mechanoreceptors transduce low- and high-intensity mechanical stimuli. It was shown recently that, in vivo, neurotrophin-4 (NT-4)-dependent D-hair mechanoreceptors specifically express a voltage-activated T-type calcium channel (Ca(v)3.2) that may be required for their mechanoreceptive function. Here we show that D-hair mechanoreceptors can be identified in vitro by a rosette-like morphology in the presence of NT-4 and that these rosette neurons are almost all absent in DRG cultures taken from NT-4 knock-out mice. In vitro identification of the D-hair mechanoreceptor allowed us to explore the electrophysiological properties of these cells. We demonstrate that the T-type Ca(v)3.2 channel induced slow membrane depolarization that contributes to lower the voltage threshold for action potential generation and controls spike latency after stimulation of D-hair mechanoreceptors. Indeed, the properties of the T-type amplifier are particularly well suited to explain the high sensitivity of D-hair mechanoreceptors to slowly moving stimuli.

Medium sized dorsal root ganglion neurones are involved in tactile sensation and responsible for allodynia following nerve injury. We examined the effects of sciatic nerve injury on the expression of low and high voltage-gated calcium currents in medium sized neurones isolated from lumbar dorsal root ganglia of adult mice. Based on the relative expression of these calcium channel types, three populations of medium sized neurones were identified in controls. Type I, II and III populations were characterised respectively by small, predominant and no low voltage-gated current compared to the high voltage-gated current. Five days after nerve injury, calcium current expression was differentially affected by axotomy in these three subsets of medium neurones. Altogether, these results suggest that calcium channels are heterogeneously distributed among the medium sized neurones. This heterogeneity should provide specificity not only to sensory functions but also to sensory responses following nerve injury., (Copyright 2003 Lippincott Williams & Wilkins)

The presence of a hyperpolarization-activated inward current (Ih) was investigated in mouse vestibular primary neurons using the whole-cell patch-clamp technique. In current-clamp configuration, injection of hyperpolarizing currents induced variations of membrane voltage with prominent time-dependent rectification increasing with current amplitudes. This effect was abolished by 2 mM Cs+ or 100 microM ZD7288. In voltage-clamp configuration, hyperpolarization pulses from -60 mV to -140 mV triggered a slow activating and non inactivating inward current that was sensitive to the two blockers, but insensitive to 5 mM Ba2+. Changing Na+ and K+ concentrations demonstrated that Ih current is carried by both these monovalent cations. This is the first demonstration of a Ih current in vestibular primary neurons.

Voltage-activated sodium currents (INa) in vestibular ganglion neurones acutely isolated from postnatal mice were investigated using the whole-cell configuration of the patch-clamp technique. Under recording conditions designed to allow the complete isolation of INa depolarizations from a holding potential of -80 mV revealed a fast inactivating inward current which was activated around -60 mV and exhibited maximum peak current around -30 mV. This current was eliminated when the cells were perifused with a Na(+)-free solution and almost totally blocked by application of 100 nM tetrodotoxin (TTX). These properties identify this inward current as TTX-sensitive INa. The half-maximum activation potential of INa was -46 mV and its half-maximum inactivation potential was -69 mV. This is the first report of voltage-activated sodium currents in vestibular primary neurones.

Calretinin is a calcium-binding protein of the EF-hand family. It has been previously identified in particular cell types of adult guinea pig, rat, and chinchilla inner ear. Development of calretinin immunoreactivity in the mouse inner ear was investigated from embryonic day 13 (E13) to the adult stage. In the adult mouse vestibule, calretinin immunoreactivity was present in the same structures as described for the rat and guinea pig: the population of afferent fibers forming calyx units and a small number of ganglion neurons. The earliest immunoreactivity was found at E17 in vestibular hair cells (VHCs), then, at E19, in afferent fibers entering the sensory epithelia and in rare ganglion neurons. At postnatal day 4 (P4), a few vestibular nerve fibers and ganglion neurons were reactive. From this stage until P14, immunoreactivity developed in the calyx units and disappeared from VHCs. At P14, immunostaining was adult-like. In the adult mouse cochlea, immunoreactivity was present in the same cell populations as described in the rat: the inner hair cells (IHCs) and most of Corti's ganglion neurons. Calretinin immunoreactivity appeared at E19-P0 in IHCs and ganglion neurons of the basal turn. At P1, outer hair cells (OHCs) of the basal turn were positive. Calretinin immunoreactivity then appeared in IHCs, OHCs, and ganglion neurons of the medial turn, then of the apical turn. At P4, all IHCs and OHCs and most of the ganglion neurons were immunostained. Immunoreactivity gradually disappeared from the OHCs starting at P10 and, at P22, only IHCs and ganglion neurons were positive. The sequences of appearance of calretinin were specific to each cell type of the inner ear and paralleled their respective maturation. Calretinin was transiently expressed in VHCs and OHCs.

Vestibular neurons were studied by cytochrome oxidase (CO) histochemistry and by immunocytochemistry using antibodies against parvalbumin (PV), calbindin (CaBP), calretinin (CaR) and 160 KD neurofilament protein (NF). All the neurons present a high level of CO activity and a high content of PV. CaBP and CaR are restricted to a specific population of about 16% of the neurons and are among the largest ones. The latter neurons also have a high density of NF 160 KD protein. In conclusion the biochemical characteristics of the vestibular ganglion neurons are discussed in relation to their morphological and physiological properties.

The contribution of short-latency evoked potentials to the exploration of auditory, visual and somatosensory neurosensory pathways is capital. The determination of short-latency vestibular evoked potentials has encountered many difficulties, mainly due to the necessity to find a specific, very short stimulation in order to select and synchronize the vestibular nerve fibers. Recent studies in animals have demonstrated short-latency potentials evoked by angular accelerations, linear accelerations or electric shocks on the round window. The generators of these potentials seem to be located in the vestibular nerve and nuclei. Electric stimulations of the internal ear have the advantage of providing a study of the peripheral vestibular pathways separately on both sides, while accelerations involve both vestibula, which probably makes the interpretation of data very difficult.

Immunohistochemical investigations with calretinin, a neuronal calcium binding protein, were made in the vestibular end organs of five guinea pigs and one chinchilla. A specific pattern of immunoreactivity of afferent nerve fibers was found. Immunostaining was restricted to thick fibers innervating the apex of the cristae or the striola of the utricular macula. A study of serial sections revealed that the stained afferents gave rise to calyx endings, but not to collaterals containing bouton endings. The results are consistent with the conclusion that, of the three classes of fibers defined by Fernández et al. (1988, 1990), only calyx units are calretinin immunoreactive. A count of the number of labelled fibers in the chinchilla crista suggests that the entire population of calyx units is immunoreactive. The conclusion is surprising since the physiology of calyx units does not differ qualitatively from that of other afferents (Baird et al. 1988; Goldberg et al. 1990). The presence of this protein in the calyx neurons may be related to specific post-synaptic functions of this type of afferents.

The ontogenesis of vestibular primary neuron sensitivity to depolarisation produced by galvanic current stimulations was studied in mouse inner ear explants maintained in vitro. Cathodal galvanic stimulations, which elicit an increase of the discharge frequencies, are assumed to act on the spike initiation site by depolarizing the neuron. The responses of neurons to galvanic currents at various developmental stages were recorded. The pattern of responses reflected the sensitivities of the neurons to depolarization. At birth, about 75% of the vestibular neurons responded weakly to high intensity galvanic currents thus indicating that they were able to generate action potentials. However, the very low gain of the response to the stimulation revealed the immaturity of the neurons at the spike generation site. Between the day of birth and the ninth postnatal day, an increase in the gain of the responses was observed, indicating the enhancement of the sensitivity of the vestibular neurons to the galvanic currents. This increase in sensitivity was more pronounced from the fourth postnatal day. The response of the neurons to galvanic stimulation increased gradually during postnatal development without reaching a plateau at postnatal day 9 indicating that a further physiological maturation occurs after this stage. These results are consistent with the morphological maturation of the vestibular primary afferents and with previous studies showing that the physiological maturation parallels myelination of the afferent fibers.

The development of vestibular afferent innervation patterns was studied by labeling the peripheral terminations between gestation day 17 and postnatal day 10. Extracellular injections of horseradish peroxidase were performed into vestibular ganglia in mouse otocysts maintained in vitro for several hours. At gestation days 17 and 18, the afferent innervation patterns were characterized by the presence of a few collaterals that arose from the parent fiber and sometimes ended by swellings or by enlargement extended by filopods rising to the epithelium surface. At the 20th gestation day the first endings differentiated into boutons or calyces were seen. At birth, the afferent innervation consisted of collaterals that could terminate either in boutons or in incomplete thin calyces. Starting on postnatal days 1 and 2, boutons and calyces became more pronounced and 3 afferent innervation patterns could be distinguished, i.e., calyx, dimorphic, and bouton. By postnatal day 5, the filopods had disappeared and the characteristic endings on type I or II cells were clearly individualized. On postnatal day 10, the afferent innervation patterns were comparable to those in the adult. These results are discussed in relation to ultrastructural data concerning the synaptogenesis and to the physiological development that have been described during the first postnatal days.

The cytogenesis in the vestibular sensory epithelium of mouse embryo otocysts grown in organ culture was examined using tritiated thymidine autoradiography. Pulse-labeling with [(3)H]-thymidine was applied either in vivo, at various intervals before explantation, or in vitro at specific stages of development. Observations of the development in vitro showed that the cytogenesis and cytodifferentiation of vestibular sensory cells were disturbed by explantation. By varying the intervals between the [(3)H]thymidine exposure and the date of explantation, we were able to demonstrate that explantation itself caused a significant decrease in the mitotic and the postmitotic phases of the hair cell precursors. Labeling of the expiants excised on day 13 of gestation revealed that precursors of the hair cells were progressively losing their mitotic capacity in vitro. In contrast with this finding, precursors of the supporting cells were less affected by explantation and culture conditions and they retained the capacity for survival, proliferation and differentiation., (Copyright © 1985. Published by Elsevier Ltd.)

Unitary recordings of spontaneous activity were performed from vestibular ganglion perikaria and axons of Mouse inner ear explants. The latter were from 1-11 day postnatal mice and were maintained in vitro at 37 degrees C, in a defined bathing medium. Spontaneous action potentials were obtained at each developmental stage and presented the distinctive features of the in vivo recorded activities during normal development. On the 3rd day post partum the recorded activities were of the immature type, with slow irregular discharge frequencies. In ganglia 3 to 9 days post partum the spike frequencies increased and the discharges exhibited 3 distinct firing patterns. This electrophysiological maturation of vestibular receptors is discussed in comparison with the morphological maturation of the vestibular organ during the same postnatal period.

Spontaneous discharges from vestibular ganglion neurons have been studied in mice between 0 and 10 days in freshly isolated in vitro preparations. The vestibular ganglion neurons were electrophysiologically active at birth with irregular activities. From the 3rd day. the activities could be divided into irregular and regular discharging units based on the coefficient of variation of their interspike intervals. This study provides two criteria for maturation of vestibular activities: a decreasing number of irregular units with age and an increase of the spontaneous discharge frequencies.

Myelination of mouse vestibular ganglion cells and preganglionic fibres was examined by light and electron microscopy from gestation day 16 to postnatal day 23. Myelination of the preganglionic fibres occurred essentially during the first 2 days after birth and was almost complete by postnatal day 7. Myelination of the ganglion cells occurred later and lasted longer than that of the fibres. Its onset was observed on postnatal day 9 and the myelin sheaths had fully developed structures 2 weeks later. Thus, myelination of vestibular ganglion neurons appeared to be the latest event to take place in the maturation of the peripheral vestibular system. The sequences of myelination are discussed with respect to those of electrophysiological maturation.

The cytogenesis of the two cell populations of the vestibular sensory epithelium-hair cells and supporting cells-was investigated in explanted Mouse embryo otocysts using tritiated thymidine autoradiography. The otocysts were explanted on the 13th day of gestation and grown in vitro for 7 days. A close in vitro and in vivo correspondence was found in the cytodifferentiation of the sensory epithelium. At different times after explanation, a single administration of tritiated thymidine was added to the otocysts. The time schedule of the hair cell generation in the explants was essentially different from that observed during normal development. In contrast, the time schedule of the supporting cell generation was only mildly altered. The data suggest that the generative processes of the two cell populations are influenced differently, depending on their later specific differentiation.