Hair Cell Afferent Synapse Research Articles

C1ql1 is expressed in adult outer hair cells of the cochlea in a tonotopic gradient

Hearing depends on the transduction of sounds into neural signals by the inner hair cells of the cochlea. Cochleae also have outer hair cells with unique electromotile properties that increase auditory sensitivity, but they are particularly susceptible to damage by intense noise exposure, ototoxic drugs, and aging. Although the outer hair cells have synapses on afferent neurons that project to the brain, the function of this neuronal circuit is unclear. Here, we created a novel mouse allele that inserts a fluorescent reporter at the C1ql1 locus which revealed gene expression in the outer hair cells and allowed creation of outer hair cell-specific C1ql1 knockout mice. We found that C1ql1 expression in outer hair cells corresponds to areas with the most sensitive frequencies of the mouse audiogram, and that it has an unexpected adolescence-onset developmental timing. No expression was observed in the inner hair cells. Since C1QL1 in the brain is made by neurons, transported anterogradely in axons, and functions in the synaptic cleft, C1QL1 may serve a similar function at the outer hair cell afferent synapse. Histological analyses revealed that C1ql1 conditional knockout cochleae may have reduced outer hair cell afferent synapse maintenance. However, auditory behavioral and physiological assays did not reveal a compelling phenotype. Nonetheless, this study identifies a potentially useful gene expressed in the cochlea and opens the door for future studies aimed at elucidating the function of C1QL1 and the function of the outer hair cell and its afferent neurons.

Hair Cell Afferent Synapses: Function and Dysfunction.

To provide a meaningful representation of the auditory landscape, mammalian cochlear hair cells are optimized to detect sounds over an incredibly broad range of frequencies and intensities with unparalleled accuracy. This ability is largely conferred by specialized ribbon synapses that continuously transmit acoustic information with high fidelity and sub-millisecond precision to the afferent dendrites of the spiral ganglion neurons. To achieve this extraordinary task, ribbon synapses employ a unique combination of molecules and mechanisms that are tailored to sounds of different frequencies. Here we review the current understanding of how the hair cell's presynaptic machinery and its postsynaptic afferent connections are formed, how they mature, and how their function is adapted for an accurate perception of sound.

A new model for underwater noise research in larval fishes: Biomedical and ecological implications

In humans, excessive noise exposure from occupational or recreational sources causes permanent hearing loss. Similarly, exposure to underwater anthropogenic noise can cause hearing loss in aquatic organisms, including fish. While fish can recover from noise-induced hearing loss, underwater noise exposure can cause behavioral changes that reduce organismal fitness. In all vertebrates, acoustic trauma can cause damage to sensory hair cells. To better study the effects of noise on hair cells, we have developed a noise exposure system that uses broadband sound to damage hair cells of the inner ear and lateral line of larval zebrafish. Acoustic over-exposure kills hair cells in an intensity- and time-dependent manner, with maximum hair cell damage observed 72 hours after noise exposure. This time course is consistent with mammalian studies, where hair cell death occurs days to weeks after noise exposure. Other features of acoustic trauma are also conserved between zebrafish and mammals, including activation of apoptotic signaling cascades and changes in hair cell-afferent synapses. These studies demonstrate that larval zebrafish are a tractable new model for studies of noise-induced hair cell death. However, our acoustic trauma system could also be used in other species, allowing for new studies of underwater noise in larval fishes.

The presynaptic ribbon maintains vesicle populations at the hair cell afferent fiber synapse.

The ribbon is the structural hallmark of cochlear inner hair cell (IHC) afferent synapses, yet its role in information transfer to spiral ganglion neurons (SGNs) remains unclear. We investigated the ribbon's contribution to IHC synapse formation and function using KO mice lacking RIBEYE. Despite loss of the entire ribbon structure, synapses retained their spatiotemporal development and KO mice had a mild hearing deficit. IHCs of KO had fewer synaptic vesicles and reduced exocytosis in response to brief depolarization; a high stimulus level rescued exocytosis in KO. SGNs exhibited a lack of sustained excitatory postsynaptic currents (EPSCs). We observed larger postsynaptic glutamate receptor plaques, potentially compensating for the reduced EPSC rate in KO. Surprisingly, large-amplitude EPSCs were maintained in KO, while a small population of low-amplitude slower EPSCs was increased in number. The ribbon facilitates signal transduction at physiological stimulus levels by retaining a larger residency pool of synaptic vesicles.

Wbp2 is required for normal glutamatergic synapses in the cochlea and is crucial for hearing.

WBP2 encodes the WW domain-binding protein 2 that acts as a transcriptional coactivator for estrogen receptor α (ESR1) and progesterone receptor (PGR). We reported that the loss of Wbp2 expression leads to progressive high-frequency hearing loss in mouse, as well as in two deaf children, each carrying two different variants in the WBP2 gene. The earliest abnormality we detect in Wbp2-deficient mice is a primary defect at inner hair cell afferent synapses. This study defines a new gene involved in the molecular pathway linking hearing impairment to hormonal signalling and provides new therapeutic targets.

Hair Cell Ribbon Synapse Function - Differently Optimized for Hearing and Balance

In the cochlea, inner hair cells transmit the sound signal via type I auditory nerve fibers to the brain, and outer hair cells mainly operate as local amplifiers of the sound signal in the periphery. However, outer hair cells also contact a small group of afferent type II fibers, of unknown function. In the vestibular organs in the inner ear, hair cells transmit head rotation signals to the brain, via vestibular nerve fibers. All of these different hair cell types release glutamate onto their afferent contacts via ribbon synapses. Interestingly, synaptic transmission at these individual hair cell ribbon synapses operates differently in many respects, most likely optimized for their specific function.For example, at the inner hair cell synapse, known for its high reliability and precision, EPSC are fast and amplitudes are quite variable in size and unusually large, with distributions from 20 pA to 1 nA (holding potential −94 mV). In comparison, at outer and vestibular type I hair cells, EPSCs show rather ‘conventional’ sizes of 20 - 50 pA. At inner hair cell ribbon synapses, EPSC amplitude distributions are quite diverse for individual nerve fibers, and we propose that specific distributions underlie ‘high’ and ‘low spontaneous rate fibers carrying different aspects of the sound signal. Interestingly, EPSC distributions are calcium-independent, as hair cell depolarizations do not change the distributions. At the vestibular type I hair cell afferent synapse, hair cell depolarization shifts the amplitude distribution, suggesting a calcium-dependence. Postsynaptically, EPSCs are unusually slow, causing slow, summed depolarizations of the postsynaptic membrane and thereby a change in firing rate.In summary, different mechanisms of ribbon synapse transmission and their function at different hair cells will be compared and discussed.Supported by NIDCD R01DC006476 and R01DC012957 to EG.

Vesicular glutamate transporter 3 is strongly upregulated in cochlear inner hair cells and spiral ganglion cells of developing circling mice

Vesicular glutamate transporter 3 (VGLUT3) plays a major role in hearing, and mice lacking the VGLUT3 are congenitally deaf due to absence of glutamate release at the inner hair cell afferent synapses. However, whether VGLUT3 is expressed normally in the cochleae of developing circling mice (homozygous (cir/cir) mice), the animal model for human deafness type DFNB6, has not been established. In this study, we investigated the developmental expression of VGLUT3 in cochlear inner hair cells (IHCs) and spiral ganglion cells (SGCs) of homozygous (cir/cir) mice from postnatal day (P)1 to P14 using immunofluorescence (IF) staining and Western blot. VGLUT3 immunoreactivity (IR) and protein expression increased progressively with age in homozygous (cir/cir) and control mice (heterozygous (+/cir) mice and ICR mice). The rank order of VGLUT3 IR in IHCs and SGCs in P14 mice was homozygous (cir/cir) mice=heterozygous (+/cir) mice>ICR mice. The rank order of total protein expression was homozygous (cir/cir) mice>heterozygous (+/cir) mice=ICR mice at P14. IF staining and Western blot analysis indicated that developmental VGLUT3 expression in cochleae was most prominent in homozygous (cir/cir) mice. The possible contribution of VGLUT3 upregulation in the cochlear degeneration is discussed.

Mechanotransduction and hyperpolarization-activated currents contribute to spontaneous activity in mouse vestibular ganglion neurons.

The hyperpolarization-activated, cyclic nucleotide-sensitive current, Ih, is present in vestibular hair cells and vestibular ganglion neurons, and is required for normal balance function. We sought to identify the molecular correlates and functional relevance of Ih in vestibular ganglion neurons. Ih is carried by channels consisting of homo- or heteromeric assemblies of four protein subunits from the Hcn gene family. The relative expression of Hcn1-4 mRNA was examined using a quantitative reverse transcription PCR (RT-PCR) screen. Hcn2 was the most highly expressed subunit in vestibular neuron cell bodies. Immunolocalization of HCN2 revealed robust expression in cell bodies of all vestibular ganglion neurons. To characterize Ih in vestibular neuron cell bodies and at hair cell-afferent synapses, we developed an intact, ex vivo preparation. We found robust physiological expression of Ih in 89% of cell bodies and 100% of calyx terminals. Ih was significantly larger in calyx terminals than in cell bodies; however, other biophysical characteristics were similar. Ih was absent in calyces lacking Hcn1 and Hcn2, but small Ih was still present in cell bodies, which suggests expression of an additional subunit, perhaps Hcn4. To determine the contributions of hair cell mechanotransduction and Ih to the firing patterns of calyx terminals, we recorded action potentials in current-clamp mode. Mechanotransduction currents were modulated by hair bundle defection and application of calcium chelators to disrupt tip links. Ih activity was modulated using ZD7288 and cAMP. We found that both hair cell transduction and Ih contribute to the rate and regularity of spontaneous action potentials in the vestibular afferent neurons. We propose that modulation of Ih in vestibular ganglion neurons may provide a mechanism for modulation of spontaneous activity in the vestibular periphery.

Disruption of the Presynaptic Cytomatrix Protein Bassoon Degrades Ribbon Anchorage, Multiquantal Release, and Sound Encoding at the Hair Cell Afferent Synapse

Inner hair cells (IHCs) of the cochlea use ribbon synapses to transmit auditory information faithfully to spiral ganglion neurons (SGNs). In the present study, we used genetic disruption of the presynaptic scaffold protein bassoon in mice to manipulate the morphology and function of the IHC synapse. Although partial-deletion mutants lacking functional bassoon (Bsn(ΔEx4/5)) had a near-complete loss of ribbons from the synapses (up to 88% ribbonless synapses), gene-trap mutants (Bsn(gt)) showed weak residual expression of bassoon and 56% ribbonless synapses, whereas the remaining 44% had a loosely anchored ribbon. Patch-clamp recordings and synaptic CaV1.3 immunolabeling indicated a larger number of Ca(2+) channels for Bsn(gt) IHCs compared with Bsn(ΔEx4/5) IHCs and for Bsn(gt) ribbon-occupied versus Bsn(gt) ribbonless synapses. An intermediate phenotype of Bsn(gt) IHCs was also found by membrane capacitance measurements for sustained exocytosis, but not for the size of the readily releasable vesicle pool. The frequency and amplitude of EPSCs were reduced in Bsn(ΔEx4/5) mouse SGNs, whereas their postsynaptic AMPA receptor clusters were largely unaltered. Sound coding in SGN, assessed by recordings of single auditory nerve fibers and their population responses in vivo, was similarly affected in Bsn(gt) and Bsn(ΔEx4/5) mice. Both genotypes showed impaired sound onset coding and reduced evoked and spontaneous spike rates. In summary, reduced bassoon expression or complete lack of full-length bassoon impaired sound encoding to a similar extent, which is consistent with the comparable reduction of the readily releasable vesicle pool. This suggests that the remaining loosely anchored ribbons in Bsn(gt) IHCs were functionally inadequate or that ribbon independent mechanisms dominated the coding deficit.

Transmitter Release from Cochlear Hair Cells Is Phase Locked to Cyclic Stimuli of Different Intensities and Frequencies

The auditory system processes time and intensity through separate brainstem pathways to derive spatial location as well as other salient features of sound. The independent coding of time and intensity begins in the cochlea, where afferent neurons can fire action potentials at constant phase throughout a wide range of stimulus intensities. We have investigated time and intensity coding by simultaneous presynaptic and postsynaptic recording at the hair cell-afferent synapse from rats. Trains of depolarizing steps to the hair cell were used to elicit postsynaptic currents that occurred at constant phase for a range of membrane potentials over which release probability varied significantly. To probe the underlying mechanisms, release was examined using single steps to various command voltages. As expected for vesicular release, first synaptic events occurred earlier as presynaptic calcium influx grew larger. However, synaptic depression produced smaller responses with longer first latencies. Thus, during repetitive hair cell stimulation, as the hair cell is more strongly depolarized, increased calcium channel gating hurries transmitter release, but the resulting vesicular depletion produces a compensatory slowing. Quantitative simulation of ribbon function shows that these two factors varied reciprocally with hair cell depolarization (stimulus intensity) to produce constant synaptic phase. Finally, we propose that the observed rapid vesicle replenishment would help maintain the vesicle pool, which in turn would equilibrate with the stimulus intensity (and therefore the number of open Ca(2+) channels), so that for trains of different levels the average phase will be conserved.

Effect of Different Gentamicin Dose on the Plasticity of the Ribbon Synapses in Cochlear Inner Hair Cells of C57BL/6J Mice

Faithful information transfer at the hair cell afferent synapse requires synaptic transmission to be both reliable and temporally precise. The release of neurotransmitter must exhibit both rapid on and off kinetics to accurately follow acoustic stimuli with a periodicity of 1 ms or less. To ensure such remarkable temporal fidelity, the cochlear hair cell afferent synapse undoubtedly relies on unique cellular and molecular specializations. To study effects of different doses of gentamicin on the changes of synaptic ribbons of cochlear inner hair cells (IHCs) in mice, the availability of genetic information, transgenic and knock-out animals make the C57BL/6J mouse a primary model in biomedical research. Aminoglycoside ototoxicity, however, has rarely been studied in mature mice because they are considered highly resistant to the drugs. This study presents models for gentamicin ototoxicity in adult C57BL/6J mouse strains. Five-week-old mice were injected intraperitoneally once daily with 50-300 mg gentamicin base/kg body weight for 7 days. Higher doses of gentamicin appear to be associated with earlier hearing damage in C57BL/6J mice, although not necessarily with more severe damage. At 200 mg/kg, gentamicin appears to induce significant hearing damage while not significantly affect the animal's general condition. Therefore, 200 mg/kg may be an ideal dose for ototoxicity modeling in C57BL/6J mice using gentamicin. In the early period of different dose of gentamicin effect, when the number of hair cells had not changed, the number changes of IHC ribbon synapses had taken place. Through the number of ribbon synapses changing, IHCs increased or decreased connections with spiral ganglion nerves (SGNs). The ribbon synapses played a compensatory role for gentamicin ototoxicity, while this effect was not sufficient to maintain the normal threshold of hearing.

Restoration of Hearing in the VGLUT3 Knockout Mouse Using Virally Mediated Gene Therapy

Mice lacking the vesicular glutamate transporter-3 (VGLUT3) are congenitally deaf due to loss of glutamate release at the inner hair cell afferent synapse. Cochlear delivery of VGLUT3 using adeno-associated virus type 1 (AAV1) leads to transgene expression in only inner hair cells (IHCs), despite broader viral uptake. Within 2weeks of AAV1-VGLUT3 delivery, auditory brainstem response (ABR) thresholds normalize, along with partial rescue of the startle response. Lastly, we demonstrate partial reversal of the morphologic changes seen within the afferent IHC ribbon synapse. These findings representa successful restoration of hearing by gene replacement in mice, which is a significant advance toward gene therapy of human deafness.

Chemical neuroprotection in the cochlea: The modulation of dopamine release from lateral olivocochlear efferents

The prevalence of sensorineural hearing loss is increasing worldwide, mainly due to ageing, increased noise exposure and cardiovascular risk factors. Several papers dealt with the mechanisms underlying the primary causes of impaired hearing and eventual deafness, including the damage and loss of auditory hair cells; however, very little is known about the protective mechanisms that exist for hearing. Several recent investigations have implicated dopamine (DA) in a neuroprotective circuit for the cochlea. The lateral olivocochlear (LOC) efferents provide axonal innervation of the inner hair cell afferent synapses and release DA and other substances in response to different stimuli. Under ischemic conditions or during noise exposure, DA has been proven to play a neuroprotective role against glutamate excitotoxicity. This review summarises what is currently known about the modulation of DA release in the cochlea, using primarily in vitro experimental data. Based on recent knowledge, there could be two functional subgroups within the LOC fibres, i.e., the DA- and GABA-containing projections. In this review, we attempt to show the neurochemical interactions between these two subsystems. Other aspects of cochlear neurotransmission are also discussed to provide a complete picture of cochlear dopaminergic function in physiological and pathophysiological cases with particular reference to excitotoxicity.

Calcium-Dependent Synaptic Vesicle Trafficking Underlies Indefatigable Release at the Hair Cell Afferent Fiber Synapse

Sensory hair cell ribbon synapses respond to gradedstimulation in a linear, indefatigable manner, requiring that vesicle trafficking to synapses be rapid and nonrate-limiting. Real-time monitoring of vesicle fusion identified two release components. The first was saturable with both release rate and magnitude varying linearly with Ca(2+), however the magnitude was too small to account for sustained afferent firing rates. A second superlinear release component required recruitment, in a Ca(2+)-dependent manner, of vesicles not in the immediate vicinity of the synapse. The superlinear component had a constant rate with its onset varying with Ca(2+) load. High-speed Ca(2+) imaging revealed a nonlinear increase in internal Ca(2+) correlating with the superlinear capacitance change, implicating release of stored Ca(2+) in driving vesicle recruitment. These data, supported by a mass action model, suggest sustained release at hair cell afferent fiber synapse is dictated by Ca(2+)-dependent vesicle recruitment from a reserve pool.

Structure and function of cochlear afferent innervation

For the perception of sound, acoustic signals need to be encoded into a neuronal code. This takes place at the inner hair cells of the organ of Corti and the afferent fibres of the auditory nerve. We will review the current knowledge of the anatomy and function of these elements as well as their connection - formed by the afferent inner hair cell synapse. Depending on their tonotopic location, inner hair cells are innervated by 5-30 dendrites of spiral ganglion neurons. Electrophysiological recordings from single fibres demonstrate - apart from a high-frequency selectivity - a pronounced heterogeneity in their response to sound of varying intensity. The source as well as the function of this heterogeneity is not well understood, but recent publications have suggested several mechanisms, including variations in the presynaptic Ca2+ influx and subsequent transmitter release, the postsynaptic sensitivity to neurotransmitter and electrical as well as anatomical variability of single fibres. These mechanisms might act together to expand the dynamic range of sound that can be encoded. Classical studies as well as recent publications demonstrate that sound encoding at the inner hair cell afferent synapse involves mechanisms leading to tonotopic frequency separation and distribution of intensity coding over many neuronal channels.

Dendritic HCN Channels Shape Excitatory Postsynaptic Potentials at the Inner Hair Cell Afferent Synapse in the Mammalian Cochlea

Synaptic transmission at the inner hair cell (IHC) afferent synapse, the first synapse in the auditory pathway, is specialized for rapid and reliable signaling. Here we investigated the properties of a hyperpolarization-activated current (I(h)), expressed in the afferent dendrite of auditory nerve fibers, and its role in shaping postsynaptic activity. We used whole cell patch-clamp recordings from afferent dendrites directly where they contact the IHC in excised postnatal rat cochlear turns. Excitatory postsynaptic potentials (EPSPs) of variable amplitude (1-35 mV) were found with 10-90% rise times of about 1 ms and time constants of decay of about 5 ms at room temperature. Current-voltage relations recorded in afferent dendrites revealed I(h). The pharmacological profile and reversal potential (-45 mV) indicated that I(h) is mediated by hyperpolarization-activated cyclic nucleotide-gated cation (HCN) channels. The HCN channel subunits HCN1, HCN2, and HCN4 were found to be expressed in afferent dendrites using immunolabeling. Raising intracellular cAMP levels sped up the activation kinetics, increased the magnitude of I(h) and shifted the half activation voltage (V(half)) to more positive values (-104 +/- 3 to -91 +/- 2 mV). Blocking I(h) with 50 microM ZD7288 resulted in hyperpolarization of the resting membrane potential (approximately 4 mV) and slowing the decay of the EPSP by 47%, suggesting that I(h) is active at rest and shortens EPSPs, thereby potentially improving rapid and reliable signaling at this first synapse in the auditory pathway.

Why Do Hair Cells Have Ribbons? Focus on “Synaptic Ribbon Enables Temporal Precision of Hair Cell Afferent Synapse by Increasing the Number of Readily Releasable Vesicles: A Modeling Study”

Hair cells, like retinal photoreceptors and bipolar cells, encode stimuli with graded changes in membrane potential and transmit that signal through ribbon synapses to drive action potentials in associated afferent neurons ([Sterling and Matthews 2005][1]). Thus one would expect transmitter release

Synaptic Ribbon Enables Temporal Precision of Hair Cell Afferent Synapse by Increasing the Number of Readily Releasable Vesicles: A Modeling Study

Synaptic ribbons are classically associated with mediating indefatigable neurotransmitter release by sensory neurons that encode persistent stimuli. Yet when hair cells lack anchored ribbons, the temporal precision of vesicle fusion and auditory nerve discharges are degraded. A rarified statistical model predicted increasing precision of first-exocytosis latency with the number of readily releasable vesicles. We developed an experimentally constrained biophysical model to test the hypothesis that ribbons enable temporally precise exocytosis by increasing the readily releasable pool size. Simulations of calcium influx, buffered calcium diffusion, and synaptic vesicle exocytosis were stochastic (Monte Carlo) and yielded spatiotemporal distributions of vesicle fusion consistent with experimental measurements of exocytosis magnitude and first-spike latency of nerve fibers. No single vesicle could drive the auditory nerve with requisite precision, indicating a requirement for multiple readily releasable vesicles. However, plasmalemma-docked vesicles alone did not account for the nerve's precision--the synaptic ribbon was required to retain a pool of readily releasable vesicles sufficiently large to statistically ensure first-exocytosis latency was both short and reproducible. The model predicted that at least 16 readily releasable vesicles were necessary to match the nerve's precision and provided insight into interspecies differences in synaptic anatomy and physiology. We confirmed that ribbon-associated vesicles were required in disparate calcium buffer conditions, irrespective of the number of vesicles required to trigger an action potential. We conclude that one of the simplest functions ascribable to the ribbon--the ability to hold docked vesicles at an active zone--accounts for the synapse's temporal precision.

Time course and calcium dependence of transmitter release at a single ribbon synapse

At the first synapse in the auditory pathway, the receptor potential of mechanosensory hair cells is converted into a firing pattern in auditory nerve fibers. For the accurate coding of timing and intensity of sound signals, transmitter release at this synapse must occur with the highest precision. To measure directly the transfer characteristics of the hair cell afferent synapse, we implemented simultaneous whole-cell recordings from mammalian inner hair cells (IHCs) and auditory nerve fiber terminals that typically receive input from a single ribbon synapse. During a 1-s IHC depolarization, the synaptic response depressed >90%, representing the main source for adaptation in the auditory nerve. Synaptic depression was slightly affected by alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor desensitization; however, it was mostly caused by reduced vesicular release. When the transfer function between transmitter release and Ca(2+) influx was tested at constant open probability for Ca(2+) channels (potentials >0 mV), a super linear relation was found. This relation is presumed to result from the cooperative binding of three to four Ca(2+) ions at the Ca(2+) sensor. However, in the physiological range for receptor potentials (-50 to -30 mV), the relation between Ca(2+) influx and afferent activity was linear, assuring minimal distortion in the coding of sound intensity. Changes in Ca(2+) influx caused an increase in release probability, but not in the average size of multivesicular synaptic events. By varying Ca(2+) buffering in the IHC, we further investigate how Ca(2+) channel and Ca(2+) sensor at this synapse might relate.

A missense mutation in the conserved C2B domain of otoferlin causes deafness in a new mouse model of DFNB9

Mutations of the otoferlin gene have been shown to underlie deafness disorders in humans and mice. Analyses of genetically engineered mice lacking otoferlin have demonstrated an essential role for this protein in vesicle exocytosis at the inner hair cell afferent synapse. Here, we report on the molecular and phenotypic characterization of a new ENU-induced missense mutation of the mouse otoferlin gene designated Otof deaf5Jcs . The mutation is a single T to A base substitution in exon 10 of Otof that causes a non-conservative amino acid change of isoleucine to asparagine in the C2B domain of the protein. Although strong immunoreactivity with an otoferlin-specific antibody was detected in cochlear hair cells of wildtype mice, no expression was detected in mutant mice, indicating that the missense mutation has a severe effect on the stability of the protein and potentially its localization. Auditory brainstem response (ABR) analysis demonstrated that mice homozygous for the missense mutation are profoundly deaf, consistent with an essential role for otoferlin in inner hair cell neurotransmission. Vestibular-evoked potentials (VsEPs) of mutant mice, however, were equivalent to those of wildtype mice, indicating that otoferlin is unnecessary for vestibular function even though it is highly expressed in both vestibular and cochlear hair cells.