ABR/DPOAE publications

Understanding the molecular basis of hair cell function is essential for elucidating inner ear physiology and developing therapies for auditory–vestibular disorders. Here, we identify acyl-CoA binding domain-containing 7 ( Acbd7 ) as a hair cell-specific gene critical for sensory maintenance. Single-cell transcriptomics of mouse cochlear organoids revealed Acbd7 as a top hair cell-enriched transcript, with its spatiotemporal expression confirmed from embryonic development through adulthood in both auditory and vestibular hair cells. Acbd7 ‑deficient mice exhibited pronounced hair cell degeneration, characterized by synaptic defects and diminished calcium currents in inner hair cells and loss of outer hair cells. Transcriptomic and proteomic analyses linked Acbd7 to the regulation of Ca 2+ signaling and fatty acid metabolism pathways. Our findings establish Acbd7 as a critical regulator of Ca 2+ homeostasis and functional integrity in hair cells, thereby elucidating a key mechanism by which a fatty acid metabolism factor sustains hair cell function and providing potential therapeutic targets for inner ear disorders.

Cochlear damage triggers compensatory primary auditory cortex (A1) plasticity that amplifies responses to residual sensory inputs, thereby contributing to the restoration of both cortical responsiveness to sound and perceptual sound detection threshold. However, this adaptation can become maladaptive, producing neuronal hyperactivity that contributes to tinnitus and hyperacusis. The neuromodulatory mechanisms governing these adaptive and maladaptive changes remain unknown. Here, we demonstrate that noise-induced cochlear injury triggers bidirectional synaptic zinc signaling plasticity that potentiates activity in excitatory principal neurons and parvalbumin-expressing interneurons, while suppressing activity in somatostatin-expressing interneurons. These cell-type-specific effects of synaptic zinc plasticity contribute to restoring A1 responsiveness to sound and perceptual detection thresholds, while being necessary for neural hyperactivity. Together, our findings establish synaptic zinc as a pivotal neuromodulator that shapes both adaptive and maladaptive cortical plasticity and identify a promising therapeutic target for improving perceptual recovery after cochlear damage and mitigating tinnitus and hyperacusis.

Electromotility in mammalian outer hair cells (OHC) is the mechanism underlying cochlear amplification. It is brought about by the piezoelectric-like property of the membrane protein prestin (Slc26a5) that lies in the OHCs lateral plasma membrane. Prestin connects to an underlying cytoskeletal network of circumferential actin filaments that bridge longitudinal spectrin filaments. This network, in turn, lies between the plasma membrane and a closely apposed ER-like tubular array of subsurface cisternae (SSC). Two previous papers examining spectrin knockouts in embryonic hair cells were confined to analyzing the effects on the apical cuticular plate and overlying stereocilia. In this paper, we examine the effects of conditional knockouts of alpha2 spectrin in postnatal OHCs. We find a significant auditory phenotype likely due to the novel disassociation of prestin's gating charge movement from OHC electromotility. In addition, OHCs show enlargement in their SSC and plasma membrane-SSC space with preserved cuticular plates and overlying stereocilia, the latter contrasts with the findings in embryonic knockouts.

Prairie voles ( Microtus ochrogaster ) are a semi-fossorial rodent that are an emerging model in social neuroscience. Comparing laboratory-reared and wild-caught individuals is essential for understanding how environmental history shapes neural and sensory traits and for assessing the ecological validity of laboratory findings. Despite this, relatively few studies have taken this approach. We used auditory brainstem responses (ABRs) to compare ABR thresholds and ABR wave characteristics between laboratory-reared and wild-caught prairie voles. ABR recordings show that, similar to other semi-fossorial rodents, M. ochrogaster exhibit a hearing range of 1–46 kHz with peak sensitivity between 8–24 kHz in wild-caught and 8–32 kHz in laboratory-reared voles. However, wild-caught prairie voles displayed significantly lower ABR thresholds at 1, 4, 8, 16, and 24 kHz compared to laboratory-reared prairie voles. There were significant differences in interpeak latency between both tested groups, with laboratory-reared prairie voles showing faster interpeak latency responses than wild-caught voles. However, there were no differences in amplitude ratios between groups. Laboratory-reared prairie voles showed faster normalized latencies and higher relative amplitude of the binaural interaction component (BIC) of the ABR than wild-caught voles. There were no significant differences in ABR thresholds, interpeak latency, amplitude ratio, normalized latency, and relative amplitude between the sexes. These differences in auditory processing support the importance of integrating both wild and captive populations to advance comparative auditory research.

Introduction Spectral information of speech sounds is spatially arranged from low to high frequencies in the auditory cortex, known as tonotopic maps. Recently, we identified an envelope steepness map encoding the rise-ramp steepness of sounds, which is located orthogonally to the tonotopic map. Temporal components, particularly the sound envelope defined by the rise-ramp steepness, play a crucial role in speech perception. We hypothesized that the ability to sense temporal components of sounds, such as envelope steepness, would be diminished in aged mice. Methods Responses to variations in rise-ramp time were investigated using transcranial macroscale calcium imaging of the auditory cortices of GCaMP6f-expressing mice. Normalized distance relative to the rise-ramp time of 0.01 ms was plotted for each rise-ramp time (0.1, 1, 10, and 100 ms) in the logarithmic scale graph: the steeper the slope of the fitted regression lines, the greater the distance between the peaks of the rise-ramp time of 0.01 ms and 100 ms. The slope of this regression line was compared between the different age groups: 1/3 months and 6/9/12 months after birth. Results The slope of the fitted regression line for 5 kHz in the bilateral anterior auditory field and 20 kHz in the bilateral primary auditory field was significantly less steep in mice at 6/9/12 months after birth than at 1/3 months after birth. Conclusion The shorter distance from the peak of 0.01 ms to 100 ms in animals 6/9/12 months after birth suggests difficulty in the separation of the rise-ramp time in aged animals. These findings may support the cortical mechanisms underlying age-related decline in speech perception.

Williams syndrome is a developmental disorder caused by a microdeletion entailing the loss of a single copy of 25–27 genes on chromosome 7q11.23. Patients suffer from cardiovascular and neuropsychological symptoms. Structural abnormalities of the cardiovascular system in Williams syndrome have been attributed to the hemizygous loss of the elastin (ELN) gene. In contrast, the neuropsychological consequences of Williams syndrome, including sensorimotor deficits, hypersociability, and cognitive impairments, have been mainly attributed to altered expression of transcription factors, like LIMK1, GTF2I, and GTF2IRD1, while the potential impact of altered cerebrovascular function has been largely overlooked. To study the relationship between Williams syndrome mutations and vascularization of both the heart and brain, we generated a mouse model carrying a relatively long microdeletion Del(5Ncf1-Fkbp6). Heterozygous Del(5Ncf1-Fkbp6) mice had elongated and tortuous aortas but, unlike Eln haploinsufficient mice, showed no signs of structural cardiac hypertrophy. Remarkably, we also observed structural abnormalities in coronary and brain vessels, including disorganized extracellular matrices. Importantly, the mutant mice faithfully replicated both cardiovascular and neuropsychological symptoms observed in patients. The phenotype was even more comprehensive than in former models, with structure–function correlations evident in aberrant auditory and motor behaviors resembling those in patients with Williams syndrome. Together, our findings suggest that not only cardiovascular but also neuropsychological symptoms in Williams syndrome may be driven in part by vascular abnormalities affecting both heart and brain.

Negative feedback of the cochlear efferent system plays a critical role in control of hearing sensitivity and protection from noise trauma. Type II auditory nerves (ANs) innervate outer hair cells (OHCs) in the cochlea and provide an input to the cochlear efferent system to achieve hearing sensitivity controlling and protection; in particular, medial olivocochlear efferent nerves innervate OHCs to control OHC electromotility, which is an active cochlear amplifier in mammals. However, little is known about channel information underlying type II AN activity and consequent function. Here, we report that ATP-gated P2x7 receptor had a predominant expression at type II spiral ganglion (SG) neurons and the synaptic areas under inner hair cells and OHCs with lateral and medial olivocochlear efferent nerves. Knockout (KO) of P2x7 increased hearing sensitivity with enhanced acoustic startle response, auditory brainstem response, and cochlear microphonics by increasing OHC electromotility. P2x7 KO also increased susceptibility to noise and exacerbated ribbon synapse degeneration. Middle-level noise exposure could impair active cochlear mechanics resulting in hearing loss in P2x7 KO mice. These data demonstrate that P2x7 receptors have a critical role in type II SG neuron’s function and the cochlear efferent system to control hearing sensitivity; deficiency of P2x7 receptors can impair type II SG neuron’s function and the cochlear efferent suppression leading to increase of active cochlear amplification and hearing oversensitivity, i.e., hyperacusis, and susceptibility to noise, which may also associate with other hearing disorders, such as tinnitus.

Aminoglycoside antibiotics remain indispensable despite their ototoxicity. Like other sensorineural forms, aminoglycoside-induced hearing loss (AGIHL) has no effective pharmacotherapy. Oxidative stress, apoptosis, excitotoxicity and inflammation are key pathological factors of the disease. We hypothesised that selegiline, an irreversible monoamine oxidase-B (MAO-B) inhibitor used in Parkinson’s disease, could be repurposed as an otoprotective agent against AGIHL and its effect on dopamine (DA) release from lateral olivocochlear (LOC) fibres, the efferent division of a protective feedback loop plays a major role in the protection against excitotoxicity. Selegiline mitigated AGIHL in BALB/c mice in a dose-dependent manner at different auditory brainstem response frequencies, including 16 kHz, the hearing sensitivity optimum of the animals. It also enhanced the action potential-evoked DA release from LOC efferents in mouse cochlear preparation dose-dependently. Inhibition of DA reuptake contributed to its basic effect of saving DA from metabolism. Among four selegiline analogues tested, the one that increased LOC DA release also provided otoprotection. In contrast, neither safinamide (a reversible MAO-B inhibitor) nor LJP-1207 (a selective semicarbazide-sensitive amine oxidase/vascular-adhesion protein 1 (SSAO/VAP1) inhibitor) prevented AGIHL, despite their antioxidant and anti-inflammatory properties. The reversibility or lack of MAO-B inhibition in safinamide and LJP-1207, respectively, as well as the absence of the propargylamine moiety with known intrinsic neuroprotective activity in both molecules, may explain their ineffectiveness. Selegiline, or certain propargylamine analogues of it, offer a promising therapy against AGIHL by addressing its multifactorial pathology through antioxidant, antiapoptotic, neuroprotective, and anti-inflammatory actions, while enhancing endogenous DAergic protective mechanisms.

Molecular profiling of inner ear neurons has broadened the classification of the primary afferents that support neural coding for hearing and balance. To extend spatiotemporal characterization of auditory and vestibular neuron diversity, we established a transgenic reporter mouse model (Prphp-mCherry), where elements of the peripherin promoter (Prphp) drive expression of the mCherry fluorescent reporter. Type III intermediate filament protein peripherin expression is a marker for type II spiral ganglion neurons (SGN) that innervate the cochlear outer hair cells, and the small diameter 'bouton' vestibular ganglion neurons (VGN) innervating the type II vestibular hair cells. Using Nanopore genome sequencing, the integration site of the transgene construct was identified within the class III metabotropic glutamate receptor 8 gene (Grm8, chromosome 6). Use of CUBIC / PEGASOS clearing of early postnatal to adult inner ear tissues enabled in situ 3D spatial localization of a dispersed population of cochlear mCherry + ve SGN, with highest expression and density in the hook (high frequency encoding) basal region. Of these mCherry + ve SGN, type II SGN (peripherin-immunopositive) were all co-labeled in the basal region, but the majority of the overall mCherry-delineated SGN auditory subpopulation were type I SGN innervating inner hair cells. In the VGN, mCherry + ve neurons represented ~ 15% of the adult population, dispersed as a small diameter subpopulation throughout both the inferior and superior VGN regions. These findings resolve heterogeneous type I and type II cochlear SGN subpopulations, particularly in the structurally complex hook region, and further differentiate vestibular primary afferents across postnatal development.

Auditory neuropathies affect the spiral ganglion neurons of the auditory nerve or their synapses with the sensory hair cells, distorting the sound information transmitted from the ear to the brain. Deciphering the underlying pathophysiological mechanisms remains challenging owing to the diversity of spiral ganglion neuron subtypes and associated central auditory circuits. An auditory neuropathy mechanism is unraveled by investigating the origin of auditory hyperexcitability in a mouse model for hereditary congenital deafness. Otogl encodes the large Otogelin‐like protein, which is related to secreted epithelial mucins and is implicated in the mechanical stimulation of cochlear outer hair cells. Heterozygous Otogl +/− mutant mice display auditory hyperexcitability, highlighted by their susceptibility to audiogenic seizures induced by loud sounds. It is shown that Otogl is transiently expressed in a subpopulation of spiral ganglion neurons during cochlear development. Despite their apparently normal hearing, Otogl +/− mice display poor activation of the spiral ganglion neurons processing loud sounds and an elevation of the activation threshold of the middle the ear muscle reflex that attenuates loud sounds. The findings reveal how a neuropathy affecting spiral ganglion neurons specialized in loud sound processing and associated with the middle the ear muscle reflex can manifest itself as auditory hyperexcitability.

Assembly of actin-based stereocilia is critical for cochlear hair cells to detect sound. To tune their mechanosensivity, stereocilia form bundles composed of graded rows of ascending height, necessitating the precise control of actin polymerization. Myosin 15 (MYO15A) drives hair bundle development by delivering critical proteins to growing stereocilia that regulate actin polymerization via an unknown mechanism. Here, we show that MYO15A is itself an actin nucleation-promoting factor. Moreover, a deafness-causing mutation in the MYO15A actin-binding interface inhibits nucleation activity but still preserves some movement on filaments in vitro and partial trafficking on stereocilia in vivo. Stereocilia fail to elongate correctly in this mutant mouse, providing evidence that MYO15A-driven actin nucleation contributes to hair bundle biogenesis. Our work shows that in addition to generating force and motility, the ATPase domain of MYO15A can directly regulate actin polymerization and that disrupting this activity can promote cytoskeletal disease, such as hearing loss.

Age-related hearing loss (ARHL) is a disease that impacts human quality of life and contributes to the progression of other neuronal problems. Various stressors induce an increase in free radicals, destroy mitochondria to further contribute to cellular malfunction, and compromise cell viability, ultimately leading to functional decline. Cisd2, a master gene for Marfan syndrome, plays an essential role in maintaining mitochondrial integrity and functions. As shown by our data, specific deletion of Cisd2 in the cochlea exacerbated the hearing impairment of ARHL in C57BL/6 mice. Increased defects in mitochondrial function, potassium homeostasis and synapse activity were observed in the Cisd2-deleted mouse models. These mechanistic phenotypes combined with oxidative stress contribute to cell death in the whole cochlea. Human patients with obviously deteriorated ARHL had low Cisd2 expression; therefore, Cisd2 may be a potential target for designing therapeutic methods to attenuate the disease progression of ARHL.

The goal of the present study was to determine, through a detailed study of the auditory brainstem response (ABR) waves, the possible existence of an early functional marker for the onset of presbycusis in an animal model. Toward this goal, Wistar rats were divided into four age groups: 3-month-old (3M, n = 6, control), 9-month-old (9M, n = 6), 14-month-old (14M, n = 6), and 20-month-old (20M, n = 6). ABR recordings were performed at 0.5, 1, 2, 4, 8, 16, and 32 kHz. The novel result reported here is that wave amplitudes, particularly wave II, were significantly diminished in the 9M group, even though there was no evidence of significant age-related threshold shift at that age. A significant increase in auditory thresholds with age was first detected at 14M, which further progressed at 20M, confirming our previous findings. These findings suggest that measurable alterations in ABR waves may precede age-related threshold shift and could serve as early markers to detect the onset of age-related hearing loss. Upon translation to humans, they could be used to implement early objective diagnosis, crucial to prevent or mitigate the negative consequences of presbycusis, a common, progressive, and irreversible neurodegenerative age-related disorder. This may allow, for instance, a better preservation of residual hearing, thus delaying the progression of the disease and minimizing the impact of hearing loss, ultimately improving the quality of life for those who suffer from this neurodegenerative condition.

Hearing mediates many behaviours critical for survival in echolocating bats, including foraging and navigation. Although most mammals are susceptible to progressive age-related hearing loss, the evolution of biosonar, which requires the ability to hear low-intensity echoes from outgoing sonar signals, may have selected against the development of hearing deficits in bats. Many echolocating bats exhibit exceptional longevity and rely on acoustic behaviours for survival to old age; however, relatively little is known about the ageing bat auditory system. In this study, we used DNA methylation to estimate the ages of wild-caught big brown bats ( Eptesicus fuscus ) and measured hearing sensitivity in young and ageing bats using auditory brainstem responses (ABRs) and distortion product otoacoustic emissions (DPOAEs). We found no evidence for hearing deficits in bats up to 12.5 years of age, demonstrated by comparable thresholds and similar ABR and DPOAE amplitudes across age groups. We additionally found no significant histological evidence for cochlear ageing, with similar hair cell counts, afferent and efferent innervation patterns in young and ageing bats. Here, we demonstrate that big brown bats show minimal evidence for age-related hearing loss and therefore represent informative models for investigating mechanisms that may preserve hearing function over a long lifetime.

Cochlear outer hair cells (OHCs) play a fundamental role in the hearing sensitivity and frequency selectivity of mammalian hearing and are especially vulnerable to noise-induced damage. The OHCs depend on Ca 2+ homeostasis, which is a balance between Ca 2+ influx and extrusion, as well as Ca 2+ buffering by proteins and organelles. Alterations in OHC Ca 2+ homeostasis is not only an immediate response to noise, but also associated with impaired auditory function. However, there is little known about the contribution of Ca 2+ buffering proteins and organelles to the vulnerability of OHCs to noise. In this study, we used a knockout (KO) mouse model where oncomodulin ( Ocm ), the major Ca 2+ binding protein preferentially expressed in OHCs, is deleted. We show that Ocm KO mice were more susceptible to noise induced hearing loss compared to wildtype (WT) mice. Following noise exposure (106 dB SPL, 2 h), Ocm KO mice had higher threshold shifts and increased OHC loss and TUNEL staining, compared to age-matched WT mice. Mitochondrial morphology was significantly altered in Ocm KO OHCs compared to WT OHCs. Before noise exposure, Ocm KO OHCs showed decreased mitochondrial abundance, volume, and branching compared to WT OHCs, as measured by immunocytochemical staining of outer mitochondrial membrane protein, TOM20. Following noise exposure, mitochondrial proteins were barely visible in Ocm KO OHCs. Using a mammalian cell culture model of prolonged cytosolic Ca 2+ overload, we show that OCM has protective effects against changes in mitochondrial morphology and apoptosis. These experiments suggest that disruption of Ca 2+ buffering leads to an increase in noise vulnerability and mitochondrial-associated changes in OHCs.

Ribbon synapses of inner hair cells are uniquely designed for ultrafast and indefatigable neurotransmission of the sound. The molecular machinery ensuring the efficient, compensatory recycling of the synaptic vesicles, however, remains elusive. This study showed that hair cell knockout of murine Dmxl2 , whose human homolog is responsible for non-syndromic sensorineural hearing loss DFNA71, resulted in auditory synaptopathy by impairing synaptic endocytosis and recycling. The mutant mice in the C57BL/6J background of either sex had mild hearing loss with severely diminished wave-I amplitude of the auditory brainstem response. Membrane capacitance measurements of the inner hair cells revealed deficiency in sustained synaptic exocytosis and endocytic membrane retrieval. Consistent with the electrophysiological findings, 3D electron microscopy reconstruction showed reduced reserve pool of synaptic vesicles and endocytic compartments, while the membrane-proximal and ribbon-associated vesicles remain intact. Our results propose an important role of DMXL2 in hair cell endocytosis and recycling of the synaptic vesicles. Significance Statement The molecular basis underlying efficient recycling of the ribbon synaptic vesicles in cochlear hair cells remains elusive. In this study, investigation of a hair cell-specific knockout mouse for Dmxl2 identifies its import roles in endocytosis and recycling of the auditory synaptic vesicles. The mutant mice display auditory synaptopathy, a common feature for noise-induced hearing loss and age-related hearing loss. The inner hair cells show deficiency in sustained synaptic exocytosis and endocytic membrane retrieval with reduced reserve pool of synaptic vesicles and endocytic compartments. Our results provide new insights into the molecular machinery ensuring ultrafast and indefatigable neurotransmission of the sound.

Cisplatin is an election chemotherapeutic agent used for many cancer treatments. Its cytotoxicity against neoplastic cells is mirrored by that taking place in healthy cells and tissues, resulting in serious adverse events. A very frequent one is ototoxicity, causing hearing loss which may permanently affect quality of life after successful oncologic treatments. Exacerbated oxidative stress is a main cytotoxic mechanism of cisplatin, including ototoxicity. Previous reports have shown antioxidant protection against cisplatin ototoxicity, but there is a lack of comparative studies on the otoprotectant activity and mechanism of antioxidant formulations. Here, we show evidence that a cocktail of vitamins A, C, and E along with Mg++ (ACEMg), previously shown to protect against noise-induced hearing loss, reverses auditory threshold shifts, promotes outer hair cell survival, and attenuates oxidative stress in the cochlea after cisplatin treatment, thus protecting against extreme cisplatin ototoxicity in rats. The addition of 500 mg N-acetylcysteine (NAC), which, administered individually, also shows significant attenuation of cisplatin ototoxicity, to the ACEMg formulation results in functional degradation of ACEMg otoprotection. Mg++ administered alone, as MgSO4, also prevents cisplatin ototoxicity, but in combination with 500 mg NAC, otoprotection is also greatly degraded. Increasing the dose of NAC to 1000 mg also results in dramatic loss of otoprotection activity compared with 500 mg NAC. These findings support that single antioxidants or antioxidant combinations, particularly ACEMg in this experimental series, have significant otoprotection efficacy against cisplatin ototoxicity. However, an excess of combined antioxidants and/or elevated doses, above a yet-to-be-defined “antioxidation threshold”, results in unrecoverable redox imbalance with loss of otoprotectant activity.

The hearing abilities of mammals are impacted by factors such as social cues, habitat, and physical characteristics. Despite being used commonly to study social behaviors, hearing of the monogamous prairie vole (Microtus ochrogaster) has never been characterized. In this study, anatomical features are measured and auditory brainstem responses (ABRs) are used to measure auditory capabilities of prairie voles, characterizing monaural and binaural hearing and hearing range. Sexually naive male and female voles were measured to characterize differences due to sex. It was found that prairie voles show a hearing range with greatest sensitivity between 8 and 32 kHz, binaural hearing across interaural time difference ranges appropriate for their head sizes. No differences are shown between the sexes in binaural hearing or hearing range (except at 1 kHz), however, female voles have increased amplitude of peripheral ABR waves I and II and longer latency of waves III and IV compared to males. The results confirm that prairie voles have a broad hearing range, binaural hearing consistent with rodents of similar size, and differences in amplitudes and thresholds of monaural physiological measures between the sexes. These data further highlight the necessity to understand sex-specific differences in neural processing that may underly variability in responses between sexes.

The auditory brainstem response (ABR) to tone burst stimuli of thirteen frequencies ranging from 0.5 to 48 kHz was recorded in the nine-banded armadillo (Dasypus novemcinctus ), the only extant member of the placental mammal superorder Xenarthra in North America. The armadillo ABR consisted of five main peaks that were visible within the first 10 ms when stimuli were presented at high intensities. The latency of peak I of the armadillo ABR increased as stimulus intensity decreased by an average of 20 μs/dB. Estimated frequency-specific thresholds identified by the ABR were used to construct an estimate of the armadillo audiogram describing the mean thresholds of the eight animals tested. The majority of animals tested (six out of eight) exhibited clear responses to stimuli from 0.5 to 38 kHz, and two animals exhibited responses to stimuli of 48 kHz. Across all cases, the lowest thresholds were observed for frequencies from 8 to 12 kHz. Overall, we observed that the armadillo estimated audiogram bears a similar pattern as those observed using ABR in members of other mammalian clades, including marsupials and later-derived placental mammals.

Afferent synapses of cochlear inner hair cells (IHCs) employ a unique molecular machinery. Otoferlin is a key player in this machinery, and its genetic defects cause human auditory synaptopathy. We employed site-directed mutagenesis in mice to investigate the role of Ca 2+ binding to the C 2 F domain of otoferlin. Substituting two aspartate residues of the C 2 F top loops, which are thought to coordinate Ca 2+ -ions, by alanines ( Otof D1841/1842A ) abolished Ca 2+ -influx-triggered IHC exocytosis and synchronous signaling in the auditory pathway despite substantial expression (~60%) of the mutant otoferlin in the basolateral IHC pole. Ca 2+ influx of IHCs and their resting membrane capacitance, reflecting IHC size, as well as the number of IHC synapses were maintained. The mutant otoferlin showed a strong apex-to-base abundance gradient in IHCs, suggesting impaired protein targeting. Our results indicate a role of the C 2 F domain in otoferlin targeting and of Ca 2+ binding by the C 2 F domain for IHC exocytosis and hearing.