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Endolymphatic hydrops, increased endolymphatic fluid within the cochlea, is the key pathologic finding in patients with Meniere's disease, a disease of episodic vertigo, fluctuating hearing loss, tinnitus, and aural fullness. Endolymphatic hydrops also can occur after noise trauma and its presence correlates with cochlear synaptopathy, a form of hearing loss caused by reduced numbers of synapses between hair cells and auditory nerve fibers. Here we tested whether there is a mechanistic link between these two phenomena by using multimodal imaging techniques to analyze the cochleae of transgenic mice exposed to blast and osmotic challenge. In vivo cochlear imaging after blast exposure revealed dynamic increases in endolymph that involved hair cell mechanoelectrical transduction channel block but not the synaptic release of glutamate at the hair cell-auditory nerve synapse. In contrast, ex vivo and in vivo auditory nerve imaging revealed that synaptopathy requires glutamate release from hair cells but not endolymphatic hydrops. Thus, although endolymphatic hydrops and cochlear synaptopathy are both observed after noise exposure, one does not cause the other. They are simply co-existent sequelae that derive from the traumatic stimulation of hair cell stereociliary bundles. Importantly, these data argue that Meniere's disease derives from hair cell transduction channel blockade., (© 2024. The Author(s).)

Novel stimulation methods are needed to overcome the limitations of contemporary cochlear implants. Optogenetics is a technique that confers light sensitivity to neurons via the genetic introduction of light-sensitive ion channels. By controlling neural activity with light, auditory neurons can be activated with higher spatial precision. Understanding the behaviour of opsins at high stimulation rates is an important step towards their translation. To elucidate this, we compared the temporal characteristics of auditory nerve and inferior colliculus responses to optogenetic, electrical, and combined optogenetic-electrical stimulation in virally transduced mice expressing one of two channelrhodopsins, ChR2-H134R or ChIEF, at stimulation rates up to 400 pulses per second (pps). At 100 pps, optogenetic responses in ChIEF mice demonstrated higher fidelity, less change in latency, and greater response stability compared to responses in ChR2-H134R mice, but not at higher rates. Combined stimulation improved the response characteristics in both cohorts at 400 pps, although there was no consistent facilitation of electrical responses. Despite these results, day-long stimulation (up to 13 h) led to severe and non-recoverable deterioration of the optogenetic responses. The results of this study have significant implications for the translation of optogenetic-only and combined stimulation techniques for hearing loss., (© 2024. The Author(s).)

Cholinergic signaling is essential to mediate the auditory prepulse inhibition (PPI), an operational measure of sensorimotor gating, that refers to the reduction of the acoustic startle reflex (ASR) when a low-intensity, non-startling acoustic stimulus (the prepulse) is presented just before the onset of the acoustic startle stimulus. The cochlear root neurons (CRNs) are the first cells of the ASR circuit to receive cholinergic inputs from non-olivocochlear neurons of the ventral nucleus of the trapezoid body (VNTB) and subsequently decrease their neuronal activity in response to auditory prepulses. Yet, the contribution of the VNTB-CRNs pathway to the mediation of PPI has not been fully elucidated. In this study, we used the immunotoxin anti-choline acetyltransferase (ChAT)-saporin as well as electrolytic lesions of the medial olivocochlear bundle to selectively eliminate cholinergic VNTB neurons, and then assessed the ASR and PPI paradigms. Retrograde track-tracing experiments were conducted to precisely determine the site of lesioning VNTB neurons projecting to the CRNs. Additionally, the effects of VNTB lesions and the integrity of the auditory pathway were evaluated via auditory brain responses tests, ChAT- and FOS-immunohistochemistry. Consequently, we established three experimental groups: 1) intact control rats (non-lesioned), 2) rats with bilateral lesions of the olivocochlear bundle (OCB-lesioned), and 3) rats with bilateral immunolesions affecting both the olivocochlear bundle and the VNTB (OCB/VNTB-lesioned). All experimental groups underwent ASR and PPI tests at several interstimulus intervals before the lesion and 7, 14, and 21 days after it. Our results show that the ASR amplitude remained unaffected both before and after the lesion across all experimental groups, suggesting that the VNTB does not contribute to the ASR. The%PPI increased across the time points of evaluation in the control and OCB-lesioned groups but not in the OCB/VNTB-lesioned group. At the ISI of 50 ms, the OCB-lesioned group exhibited a significant increase in%PPI (p < 0.01), which did not occur in the OCB/VNTB-lesioned group. Therefore, the ablation of cholinergic non-olivocochlear neurons in the OCB/VNTB-lesioned group suggests that these neurons contribute to the mediation of auditory PPI at the 50 ms ISI through their cholinergic projections to CRNs. Our study strongly reinforces the notion that auditory PPI encompasses a complex mechanism of top-down cholinergic modulation, effectively attenuating the ASR across different interstimulus intervals within multiple pathways., Competing Interests: Declaration of competing interest All authors ensure that they have no affiliations or involvement in institutions with any financial or non-financial interest in the matters discussed in this manuscript., (Copyright © 2024 Elsevier B.V. All rights reserved.)

The degeneration of spiral ganglion neurons (SGNs), which convey auditory signals from hair cells to the brain, can be a primary cause of sensorineural hearing loss (SNHL) or can occur secondary to hair cell loss. Emerging therapies for SNHL include the replacement of damaged SGNs using stem cell-derived otic neuronal progenitors (ONPs). However, the availability of renewable, accessible, and patient-matched sources of human stem cells is a prerequisite for successful replacement of the auditory nerve. In this study, we derived ONP and SGN-like cells by a reliable and reproducible stepwise guidance differentiation procedure of self-renewing human dental pulp stem cells (hDPSCs). This in vitro differentiation protocol relies on the modulation of BMP and TGFβ pathways using a free-floating 3D neurosphere method, followed by differentiation on a Geltrex-coated surface using two culture paradigms to modulate the major factors and pathways involved in early otic neurogenesis. Gene and protein expression analyses revealed efficient induction of a comprehensive panel of known ONP and SGN-like cell markers during the time course of hDPSCs differentiation. Atomic force microscopy revealed that hDPSC-derived SGN-like cells exhibit similar nanomechanical properties as their in vivo SGN counterparts. Furthermore, spiral ganglion neurons from newborn rats come in close contact with hDPSC-derived ONPs 5 days after co-culturing. Our data demonstrate the capability of hDPSCs to generate SGN-like neurons with specific lineage marker expression, bipolar morphology, and the nanomechanical characteristics of SGNs, suggesting that the neurons could be used for next-generation cochlear implants and/or inner ear cell-based strategies for SNHL.

In mammalian hearing, type-I afferent auditory nerve fibers comprise the basis of the afferent auditory pathway. They are connected to inner hair cells of the cochlea via specialized ribbon synapses. Auditory nerve fibers of different physiological types differ subtly in their synaptic location and morphology. Low-spontaneous-rate auditory nerve fibers typically connect on the modiolar side of the inner hair cell, while high-spontaneous-rate fibers are typically found on the pillar side. In aging and noise-damaged ears, this fine-tuned balance between auditory nerve fiber populations can be disrupted and the functional consequences are currently unclear. Here, using immunofluorescent labeling of presynaptic ribbons and postsynaptic glutamate receptor patches, we investigated changes in synaptic morphology at three different tonotopic locations along the cochlea of aging gerbils compared to those of young adults. Quiet-aged gerbils showed about 20% loss of afferent ribbon synapses. While the loss was random at apical, low-frequency cochlear locations, at the basal, high-frequency location it almost exclusively affected the modiolar-located synapses. The subtle differences in volumes of pre- and postsynaptic elements located on the inner hair cell's modiolar versus pillar side were unaffected by age. This is consistent with known physiology and suggests a predominant, age-related loss in the low-spontaneous-rate auditory nerve population in the cochlear base, but not the apex.

P2X 7 receptors (P2X 7 Rs) are associated with numerous pathophysiological mechanisms, and this promotes them as therapeutic targets for certain neurodegenerative conditions. However, the identity of P2X 7 R-expressing cells in the nervous system remains contentious. Here, we examined P2X 7 R functionality in auditory nerve cells from rodents of either sex, and determined their functional and anatomic expression pattern. In whole-cell recordings from rat spiral ganglion cultures, the purinergic agonist 2',3'-O-(4-benzoylbenzoyl)-ATP (BzATP) activated desensitizing currents in spiral ganglion neurons (SGNs) but non-desensitizing currents in glia that were blocked by P2X 7 R-specific antagonists. In imaging experiments, BzATP gated sustained Ca 2+ entry into glial cells. BzATP-gated uptake of the fluorescent dye YO-PRO-1 was reduced and slowed by P2X 7 R-specific antagonists. In rats, P2X 7 Rs were immuno-localized predominantly within satellite glial cells (SGCs) and Schwann cells (SCs). P2X 7 R expression was not detected in the portion of the auditory nerve within the central nervous system. Mouse models allowed further exploration of the distribution of cochlear P2X 7 Rs. In GENSAT reporter mice, EGFP expression driven via the P2rx7 promoter was evident in SGCs and SCs but was undetectable in SGNs. A second transgenic model showed a comparable cellular distribution of EGFP-tagged P2X 7 Rs. In wild-type mice the discrete glial expression was confirmed using a P2X 7 -specific nanobody construct. Our study shows that P2X 7 Rs are expressed by peripheral glial cells, rather than by afferent neurons. Description of functional signatures and cellular distributions of these enigmatic proteins in the peripheral nervous system (PNS) will help our understanding of ATP-dependent effects contributing to hearing loss and other sensory neuropathies. SIGNIFICANCE STATEMENT P2X 7 receptors (P2X 7 Rs) have been the subject of much scrutiny in recent years. They have been promoted as therapeutic targets in a number of diseases of the nervous system, yet the specific cellular location of these receptors remains the subject of intense debate. In the auditory nerve, connecting the inner ear to the brainstem, we show these multimodal ATP-gated channels localize exclusively to peripheral glial cells rather than the sensory neurons, and are not evident in central glia. Physiologic responses in the peripheral glia display classical hallmarks of P2X 7 R activation, including the formation of ion-permeable and also macromolecule-permeable pores. These qualities suggest these proteins could contribute to glial-mediated inflammatory processes in the auditory periphery under pathologic disease states., (Copyright © 2021 the authors.)

Background: The SCN11A gene, encoded Nav1.9 TTX resistant sodium channels, is a main effector in peripheral inflammation related pain in nociceptive neurons. The role of SCN11A gene in the auditory system has not been well characterized. We therefore examined the expression of SCN11A in the murine cochlea, the morphological and physiological features of Nav1.9 knockout (KO) ICR mice., Results: Nav1.9 expression was found in the primary afferent endings beneath the inner hair cells (IHCs). The relative quantitative expression of Nav1.9 mRNA in modiolus of wild-type (WT) mice remains unchanged from P0 to P60. The number of presynaptic CtBP2 puncta in Nav1.9 KO mice was significantly lower than WT. In addition, the number of SGNs in Nav1.9 KO mice was also less than WT in the basal turn, but not in the apical and middle turns. There was no lesion in the somas and stereocilia of hair cells in Nav1.9 KO mice. Furthermore, Nav1.9 KO mice showed higher and progressive elevated ABR threshold at 16 kHz, and a significant increase in CAP thresholds., Conclusions: These data suggest a role of Nav1.9 in regulating the function of ribbon synapses and the auditory nerves. The impairment induced by Nav1.9 gene deletion mimics the characters of cochlear synaptopathy.

Growth hormone (GH) plays an important role in auditory development during the embryonic stage. Exogenous agents such as sound, noise, drugs or trauma, can induce the release of this hormone to perform a protective function and stimulate other mediators that protect the auditory pathway. In addition, GH deficiency conditions hearing loss or central auditory processing disorders. There are promising animal studies that reflect a possible regenerative role when exogenous GH is used in hearing impairments, demonstrated in in vivo and in vitro studies, and also, even a few studies show beneficial effects in humans presented and substantiated in the main text, although they should not exaggerate the main conclusions.

Neurons exhibit diverse intrinsic dynamics, which govern how they integrate synaptic inputs to produce spikes. Intrinsic dynamics are often plastic during development and learning, but the effects of these changes on stimulus encoding properties are not well known. To examine this relationship, we simulated auditory responses to zebra finch song using a linear-dynamical cascade model, which combines a linear spectrotemporal receptive field with a dynamical, conductance-based neuron model, then used generalized linear models to estimate encoding properties from the resulting spike trains. We focused on the effects of a low-threshold potassium current (KLT) that is present in a subset of cells in the zebra finch caudal mesopallium and is affected by early auditory experience. We found that KLT affects both spike adaptation and the temporal filtering properties of the receptive field. The direction of the effects depended on the temporal modulation tuning of the linear (input) stage of the cascade model, indicating a strongly nonlinear relationship. These results suggest that small changes in intrinsic dynamics in tandem with differences in synaptic connectivity can have dramatic effects on the tuning of auditory neurons., Competing Interests: The authors have declared that no competing interests exist.

Afferent activity dynamically regulates neuronal properties and connectivity in the central nervous system. The Fragile X mental retardation protein (FMRP) is an RNA-binding protein that regulates cellular and synaptic properties in an activity-dependent manner. Whether and how FMRP level and localization are regulated by afferent input remains sparsely examined and how such regulation is associated with neuronal response to changes in sensory input is unknown. We characterized changes in FMRP level and localization in the chicken nucleus magnocellularis (NM), a primary cochlear nucleus, following afferent deprivation by unilateral cochlea removal. We observed rapid (within 2 hr) aggregation of FMRP immunoreactivity into large granular structures in a subset of deafferented NM neurons. Neurons that exhibited persistent FMRP aggregation at 12-24 hr eventually lost cytoplasmic Nissl substance, indicating cell death. A week later, FMRP expression in surviving neurons regained its homeostasis, with a slightly reduced immunostaining intensity and enhanced heterogeneity. Correlation analyses under the homeostatic status (7-14 days) revealed that neurons expressing relatively more FMRP had a higher capability of maintaining cell body size and ribosomal activity, as well as a better ability to detach inactive presynaptic terminals. Additionally, the intensity of an inhibitory postsynaptic protein, gephyrin, was reduced following deafferentation and was positively correlated with FMRP intensity, implicating an involvement of FMRP in synaptic dynamics in response to reduced afferent inputs. Collectively, this study demonstrates that afferent input regulates FMRP expression and localization in ways associated with multiple types of neuronal responses and synaptic rearrangements., (© 2020 Wiley Periodicals LLC.)

Inner hair cells (IHCs) are the primary receptors for hearing. They are housed in the cochlea and convey sound information to the brain via synapses with the auditory nerve. IHCs have been thought to be electrically and metabolically independent from each other. We report that, upon developmental maturation, in mice 30% of the IHCs are electrochemically coupled in 'mini-syncytia'. This coupling permits transfer of fluorescently-labeled metabolites and macromolecular tracers. The membrane capacitance, Ca 2+ -current, and resting current increase with the number of dye-coupled IHCs. Dual voltage-clamp experiments substantiate low resistance electrical coupling. Pharmacology and tracer permeability rule out coupling by gap junctions and purinoceptors. 3D electron microscopy indicates instead that IHCs are coupled by membrane fusion sites. Consequently, depolarization of one IHC triggers presynaptic Ca 2+ -influx at active zones in the entire mini-syncytium. Based on our findings and modeling, we propose that IHC-mini-syncytia enhance sensitivity and reliability of cochlear sound encoding.

Proper functioning of the auditory nerve is of critical importance for auditory rehabilitation by cochlear implants. Here we used the Cldn14 -/- mouse to study in detail the effects of Claudin 14 loss on auditory synapses and the auditory nerve. Mutations in the tight junction protein Claudin 14 cause autosomal recessive non-syndromic hearing loss (DFNB29) in humans and mice, due to extensive degeneration of outer and inner hair cells. Here we show that massive inner hair cell loss in Cldn14 -/- mice starts after the third postnatal week. Immunohistochemical analysis, using presynaptic Ribeye and postsynaptic GluR2 or PSD 95 as markers, revealed the degeneration of full ribbon synapses in inner hair cells from apical cochlear regions already at postnatal day 12 (P12). At P20, significant reduction in number of ribbon synapses has been observed for all cochlear regions and the loss of synaptic ribbons becomes even more prominent in residual inner hair cells from middle and apical cochlear regions at P45, which by then lost more than 40% of all ribbon synapses. In contrast to excessive noise exposure, loss of Claudin 14 does not cause an increase in "orphan" ribbons with no postsynaptic counterpart due to a reduction of postsynaptic structures. Hair cell loss in Cldn14 -/- mice is associated with regression of peripheral auditory nerve processes, especially of outer radial fibers, which normally innervate the outer hair cells. The number of spiral ganglion neurons per area, however, was unchanged between the genotypes. Different effects were observed in the cochlear nucleus complex (CNC), the central projection area of the auditory nerve. While the dorsal cochlear nucleus (DCN) showed a significant 19.7% volume reduction, VGLUT-1 input was reduced by 34.4% in the ventral cochlear nucleus (VCN) but not in the DCN of Cldn14 -/- mice. Taken together, massive inner hair cell loss starts after the third postnatal week in Cldn14 -/- mice, but is preceded by the loss of ribbon synapses, which may be a first sign of an ongoing degeneration process in otherwise morphologically inconspicuously inner hair cells. In addition to the regression of peripheral nerve processes, reduced levels of VGLUT-1 in the VCN of Cldn14 -/- mice suggests that Claudin 14 loss does not only cause hair cell loss but also affects peripheral and central connectivity of the auditory nerve., Competing Interests: Declaration of competing interest The authors declare no competing financial interests., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Hearing loss (HL) is one of the most common sensory impairments and etiologically and genetically heterogeneous disorders in humans. Muscular dystrophies (MDs) are neuromuscular disorders characterized by progressive degeneration of skeletal muscle accompanied by non-muscular symptoms. Aberrant glycosylation of α-dystroglycan causes at least eighteen subtypes of MD, now categorized as MD-dystroglycanopathy (MD-DG), with a wide spectrum of non-muscular symptoms. Despite a growing number of MD-DG subtypes and increasing evidence regarding their molecular pathogeneses, no comprehensive study has investigated sensorineural HL (SNHL) in MD-DG. Here, we found that two mouse models of MD-DG, Largemyd/myd and POMGnT1-KO mice, exhibited congenital, non-progressive, and mild-to-moderate SNHL in auditory brainstem response (ABR) accompanied by extended latency of wave I. Profoundly abnormal myelination was found at the peripheral segment of the cochlear nerve, which is rich in the glycosylated α-dystroglycan-laminin complex and demarcated by "the glial dome." In addition, patients with Fukuyama congenital MD, a type of MD-DG, also had latent SNHL with extended latency of wave I in ABR. Collectively, these findings indicate that hearing impairment associated with impaired Schwann cell-mediated myelination at the peripheral segment of the cochlear nerve is a notable symptom of MD-DG., Competing Interests: The authors have declared that no competing interests exist.

In different animal models, auditory nerve fibers display variation in spontaneous activity and response threshold. Functional and structural differences among inner hair cell ribbon synapses are believed to contribute to this variation. The relative volumes of synaptic proteins at individual synapses might be one such difference. This idea is based on the observation of opposing volume gradients of the presynaptic ribbons and associated postsynaptic glutamate receptor patches in mice along the pillar modiolar axis of the inner hair cell, the same axis along which fibers were shown to vary in their physiological properties. However, it is unclear whether these opposing gradients are expressed consistently across animal models. In addition, such volume gradients observed for separate populations of presynaptic ribbons and postsynaptic glutamate receptor patches suggest different relative volumes of these synaptic structures at individual synapses; however, these differences have not been examined in mice. Furthermore, it is unclear whether such gradients are limited to these synaptic proteins. Therefore, we analyzed organs of Corti isolated from CBA/CaJ, C57BL/6, and FVB/NJ mice using immunofluorescence, confocal microscopy, and quantitative image analysis. We find consistent expression of presynaptic volume gradients across strains of mice and inconsistent expression of postsynaptic volume gradients. We find differences in the relative volume of synaptic proteins, but these are different between CBA/CaJ mice, and C57BL/6 and FVB/NJ mice. We find similar results in C57BL/6 and FVB/NJ mice when using other postsynaptic density proteins (Shank1, Homer, and PSD95). These results have implications for the mechanisms by which volumes of synaptic proteins contribute to variations in the physiology of individual auditory nerve fibers and their vulnerability to excitotoxicity., Competing Interests: Declaration of competing interest The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2020 The Authors. Published by Elsevier B.V. All rights reserved.)

Lateral olivocochlear (LOC) efferent neurons modulate auditory nerve fiber (ANF) activity using a large repertoire of neurotransmitters, including dopamine (DA) and acetylcholine (ACh). Little is known about how individual neurotransmitter systems are differentially utilized in response to the ever-changing acoustic environment. Here we present quantitative evidence in rodents that the dopaminergic LOC input to ANFs is dynamically regulated according to the animal's recent acoustic experience. Sound exposure upregulates tyrosine hydroxylase, an enzyme responsible for dopamine synthesis, in cholinergic LOC intrinsic neurons, suggesting that individual LOC neurons might at times co-release ACh and DA. We further demonstrate that dopamine down-regulates ANF firing rates by reducing both the hair cell release rate and the size of synaptic events. Collectively, our results suggest that LOC intrinsic neurons can undergo on-demand neurotransmitter re-specification to re-calibrate ANF activity, adjust the gain at hair cell/ANF synapses, and possibly to protect these synapses from noise damage., Competing Interests: JW, EY, MM, HJ, AL, EG No competing interests declared, (© 2020, Wu et al.)

Sensory systems exploit parallel processing of stimulus features to enable rapid, simultaneous extraction of information. Mechanisms that facilitate this differential extraction of stimulus features can be intrinsic or synaptic in origin. A subdivision of the avian cochlear nucleus, nucleus angularis (NA), extracts sound intensity information from the auditory nerve and contains neurons that exhibit diverse responses to sound and current injection. NA neurons project to multiple regions ascending the auditory brain stem including the superior olivary nucleus, lateral lemniscus, and avian inferior colliculus, with functional implications for inhibitory gain control and sound localization. Here we investigated whether the diversity of auditory response patterns in NA can be accounted for by variation in intrinsic physiological features. Modeled sound-evoked auditory nerve input was applied to NA neurons with dynamic clamp during in vitro whole cell recording at room temperature. Temporal responses to auditory nerve input depended on variation in intrinsic properties, and the low-threshold K + current was implicated as a major contributor to temporal response diversity and neuronal input-output functions. An auditory nerve model of acoustic amplitude modulation produced synchrony coding of modulation frequency that depended on the intrinsic physiology of the individual neuron. In Primary-Like neurons, varying low-threshold K + conductance with dynamic clamp altered temporal modulation tuning bidirectionally. Taken together, these data suggest that intrinsic physiological properties play a key role in shaping auditory response diversity to both simple and more naturalistic auditory stimuli in the avian cochlear nucleus. NEW & NOTEWORTHY This article addresses the question of how the nervous system extracts different information in sounds. Neurons in the cochlear nucleus show diverse responses to acoustic stimuli that may allow for parallel processing of acoustic features. The present studies suggest that diversity in intrinsic physiological features of individual neurons, including levels of a low voltage-activated K + current, play a major role in regulating the diversity of auditory responses.

Cochlear implantation, a surgical method to bypass cochlear hair cells and directly stimulate the spiral ganglion, is the standard treatment for severe-to-profound hearing loss. Changes in cochlear implant electrode array design and surgical approach now allow for preservation of acoustic hearing in the implanted ear. Electrocochleography (ECochG) was performed in eight hearing preservation subjects to assess hair cell and neural function and elucidate underlying genetic hearing loss. Three subjects had pathogenic variants in TMPRSS3 and five had pathogenic variants in genes known to affect the cochlear sensory partition. The mechanism by which variants in TMPRSS3 cause genetic hearing loss is unknown. We used a 500-Hz tone burst to record ECochG responses from an intracochlear electrode. Responses consist of a cochlear microphonic (hair cell) and an auditory nerve neurophonic. Cochlear microphonics did not differ between groups. Auditory nerve neurophonics were smaller, on average, in subjects with TMPRSS3 deafness. Results of this proof-of-concept study provide evidence that pathogenic variants in TMPRSS3 may impact function of the spiral ganglion. While ECochG as a clinical and research tool has been around for decades, this study illustrates a new application of ECochG in the study of genetic hearing and deafness in vivo.

CaBPs are a family of Ca 2+ binding proteins related to calmodulin. Two CaBP family members, CaBP1 and CaBP2, are highly expressed in the cochlea. Here, we investigated the significance of CaBP1 and CaBP2 for hearing in mice lacking expression of these proteins (CaBP1 KO and CaBP2 KO) using auditory brain responses (ABRs) and distortion product otoacoustic emissions (DPOAEs). In CaBP1 KO mice, ABR wave I was larger in amplitude, and shorter in latency and faster in decay, suggestive of enhanced synchrony of auditory nerve fibers. This interpretation was supported by the greater excitability of CaBP1 KO than WT neurons in whole-cell patch clamp recordings of spiral ganglion neurons in culture, and normal presynaptic function of CaBP1 KO IHCs. DPOAEs and ABR thresholds were normal in 4-week old CaBP1 KO mice, but elevated ABR thresholds became evident at 32 kHz at 9 weeks, and at 8 and 16 kHz by 6 months of age. In contrast, CaBP2 KO mice exhibited significant ABR threshold elevations at 4 weeks of age that became more severe in the mid-frequency range by 9 weeks. Though normal at 4 weeks, DPOAEs in CaBP2 KO mice were significantly reduced in the mid-frequency range by 9 weeks. Our results reveal requirements for CaBP1 and CaBP2 in the peripheral auditory system and highlight the diverse modes by which CaBPs influence sensory processing., (Copyright © 2018. Published by Elsevier B.V.)

The Mongolian gerbil is a classic animal model for age-related hearing loss. As a prerequisite for studying age-related changes, we characterized cochlear afferent synaptic morphology in young adult gerbils, using immunolabeling and quantitative analysis of confocal microscopic images. Cochlear wholemounts were triple-labeled with a hair-cell marker, a marker of presynaptic ribbons, and a marker of postsynaptic AMPA-type glutamate receptors. Seven cochlear positions covering an equivalent frequency range from 0.5 - 32 kHz were evaluated. The spatial positions of synapses were determined in a coordinate system with reference to their individual inner hair cell. Synapse numbers confirmed previous reports for gerbils (on average, 20-22 afferents per inner hair cell). The volumes of presynaptic ribbons and postsynaptic glutamate receptor patches were positively correlated: larger ribbons associated with larger receptor patches and smaller ribbons with smaller patches. Furthermore, the volumes of both presynaptic ribbons and postsynaptic receptor patches co-varied along the modiolar-pillar and the longitudinal axes of their hair cell. The gradients in ribbon volume are consistent with previous findings in cat, guinea pig, mouse and rat and further support a role in differentiating the physiological properties of type I afferents. However, the positive correlation between the volumes of pre- and postsynaptic elements in the gerbil is different to the opposing gradients found in the mouse, suggesting species-specific differences in the postsynaptic AMPA receptors that are unrelated to the fundamental classes of type I afferents., (Copyright © 2018 Elsevier B.V. All rights reserved.)

The basolateral membrane of the mammalian inner hair cell (IHC) expresses large voltage and Ca 2+ gated outward K + currents. To quantify how the voltage-dependent activation of the K + channels affects the functionality of the auditory nerve innervating the IHC, this study adopts a model of mechanical-to-neural transduction in which the basolateral K + conductances of the IHC can be made voltage-dependent or not. The model shows that the voltage-dependent activation of the K + channels (i) enhances the phase-locking properties of the auditory fiber (AF) responses; (ii) enables the auditory nerve to encode a large dynamic range of sound levels; (iii) enables the AF responses to synchronize precisely with the envelope of amplitude modulated stimuli; and (iv), is responsible for the steep offset responses of the AFs. These results suggest that the basolateral K + channels play a major role in determining the well-known response properties of the AFs and challenge the classical view that describes the IHC membrane as an electrical low-pass filter. In contrast to previous models of the IHC-AF complex, this study ascribes many of the AF response properties to fairly basic mechanisms in the IHC membrane rather than to complex mechanisms in the synapse., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Cochlear synaptopathy, i.e. the loss of auditory-nerve connections with cochlear hair cells, is seen in aging, noise damage, and other types of acquired sensorineural hearing loss. Because the subset of auditory-nerve fibers with high thresholds and low spontaneous rates (SRs) is disproportionately affected, audiometric thresholds are relatively insensitive to this primary neural degeneration. Although suprathreshold amplitudes of wave I of the auditory brainstem response (ABR) are attenuated in synaptopathic mice, there is not yet a robust diagnostic in humans. The middle-ear muscle reflex (MEMR) might be a sensitive metric (Valero et al., 2016), because low-SR fibers may be important drivers of the MEMR (Liberman and Kiang, 1984; Kobler et al., 1992). Here, to test the hypothesis that narrowband reflex elicitors can identify synaptopathic cochlear regions, we measured reflex growth functions in unanesthetized mice with varying degrees of noise-induced synaptopathy and in unexposed controls. To separate effects of the MEMR from those of the medial olivocochlear reflex, the other sound-evoked cochlear feedback loop, we used a mutant mouse strain with deletion of the acetylcholine receptor required for olivocochlear function. We demonstrate that the MEMR is normal when activated from non-synaptopathic cochlear regions, is greatly weakened in synaptopathic regions, and is a more sensitive indicator of moderate synaptopathy than the suprathreshold amplitude of ABR wave I., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Systemic corticosteroids have been the mainstay of treatment for various hearing disorders for more than 30 yr. Accordingly, numerous studies have described glucocorticoids (GCs) and stressors to be protective in the auditory organ against damage associated with a variety of health conditions, including noise exposure. Conversely, stressors are also predictive risk factors for hearing disorders. How both of these contrasting stress actions are linked has remained elusive. Here, we demonstrate that higher corticosterone levels during acoustic trauma in female rats is highly correlated with a decline of auditory fiber responses in high-frequency cochlear regions, and that hearing thresholds and the outer hair cell functions (distortion products of otoacoustic emissions) are left unaffected. Moreover, when GC receptor (GR) or mineralocorticoid receptor (MR) activation was antagonized by mifepristone or spironolactone, respectively, GR, but not MR, inhibition significantly and permanently attenuated trauma-induced effects on auditory fiber responses, including inner hair cell ribbon loss and related reductions of early and late auditory brainstem responses. These findings strongly imply that higher corticosterone stress levels profoundly impair auditory nerve processing, which may influence central auditory acuity. These changes are likely GR mediated as they are prevented by mifepristone.-Singer, W., Kasini, K., Manthey, M., Eckert, P., Armbruster, P., Vogt, M. A., Jaumann, M., Dotta, M., Yamahara, K., Harasztosi, C., Zimmermann, U., Knipper, M., Rüttiger, L. The glucocorticoid antagonist mifepristone attenuates sound-induced long-term deficits in auditory nerve response and central auditory processing in female rats.

Higher stages of central auditory processing compensate for a loss of cochlear nerve synapses by increasing the gain on remaining afferent inputs, thereby restoring firing rate codes for rudimentary sound features. The benefits of this compensatory plasticity are limited, as the recovery of precise temporal coding is comparatively modest. We reasoned that persistent temporal coding deficits could be ameliorated through modulation of voltage-gated potassium (Kv) channels that regulate temporal firing patterns. Here, we characterize AUT00063, a pharmacological compound that modulates Kv3.1, a high-threshold channel expressed in fast-spiking neurons throughout the central auditory pathway. Patch clamp recordings from auditory brainstem neurons and in silico modeling revealed that application of AUT00063 reduced action potential timing variability and improved temporal coding precision. Systemic injections of AUT00063 in vivo improved auditory synchronization and supported more accurate decoding of temporal sound features in the inferior colliculus and auditory cortex in adult mice with a near-complete loss of auditory nerve afferent synapses in the contralateral ear. These findings suggest modulating Kv3.1 in central neurons could be a promising therapeutic approach to mitigate temporal processing deficits that commonly accompany aging, tinnitus, ototoxic drug exposure or noise damage.

The neurotransmitter receptor subtype, number, density, and distribution relative to the location of transmitter release sites are key determinants of signal transmission. AMPA-type ionotropic glutamate receptors (AMPARs) containing GluA3 and GluA4 subunits are prominently expressed in subsets of neurons capable of firing action potentials at high frequencies, such as auditory relay neurons. The auditory nerve (AN) forms glutamatergic synapses on two types of relay neurons, bushy cells (BCs) and fusiform cells (FCs) of the cochlear nucleus. AN-BC and AN-FC synapses have distinct kinetics; thus, we investigated whether the number, density, and localization of GluA3 and GluA4 subunits in these synapses are differentially organized using quantitative freeze-fracture replica immunogold labeling. We identify a positive correlation between the number of AMPARs and the size of AN-BC and AN-FC synapses. Both types of AN synapses have similar numbers of AMPARs; however, the AN-BC have a higher density of AMPARs than AN-FC synapses, because the AN-BC synapses are smaller. A higher number and density of GluA3 subunits are observed at AN-BC synapses, whereas a higher number and density of GluA4 subunits are observed at AN-FC synapses. The intrasynaptic distribution of immunogold labeling revealed that AMPAR subunits, particularly GluA3, are concentrated at the center of the AN-BC synapses. The central distribution of AMPARs is absent in GluA3-knockout mice, and gold particles are evenly distributed along the postsynaptic density. GluA4 gold labeling was homogenously distributed along both synapse types. Thus, GluA3 and GluA4 subunits are distributed at AN synapses in a target-cell-dependent manner.

A key feature of age-related hearing loss is a reduction in the expression of inhibitory neurotransmitters in the central auditory system. This loss is partially responsible for changes in central auditory processing, as inhibitory receptive fields play a critical role in shaping neural responses to sound stimuli. Vigabatrin (VGB), an antiepileptic agent that irreversibly inhibits γ-amino butyric acid (GABA) transaminase, leads to increased availability of GABA throughout the brain. This study used multi-channel electrophysiology measurements to assess the excitatory frequency response areas in old CBA mice to which VGB had been administered. We found a significant post-VGB reduction in the proportion of V-type shapes, and an increase in primary-like excitatory frequency response areas. There was also a significant increase in the mean maximum driven spike rates across the tonotopic frequency range of all treated animals, consistent with observations that GABA buildup within the central auditory system increases spike counts of neural receptive fields. This increased spiking is also seen in the rate-level functions and seems to explain the improved low-frequency thresholds., (Copyright © 2017 Elsevier Inc. All rights reserved.)

In vitro cultures provide a valuable tool in studies examining the survival, morphology and function of cells in the auditory system. Primary cultures of primary auditory neurons have most notably provided critical insights into the role of neurotrophins in cell survival and morphology. Functional studies have also utilized in vitro models to study neuronal physiology and the ion channels that dictate these patterns of activity. Here we examine what influence time-in-culture has on the activity of primary auditory neurons, and how this affects our interpretation of neurotrophin and antibiotic-mediated effects in this population. Using dissociated cell culture we analyzed whole-cell patch-clamp recordings of spiral ganglion neurons grown in the presence or absence of neurotrophins and/or penicillin and streptomycin for 1-3 days in vitro. Firing threshold decreased, and both action potential number and latency increased over time regardless of treatment, whilst input resistance was lowest where neurotrophins were present. Differences in firing properties were seen with neurotrophin concentration but were not consistently maintained over the 3 days in vitro. The exclusion of antibiotics from culture media influenced most firing properties at 1 day in vitro in both untreated and neurotrophin-treated conditions. The only difference still present at 3 days was an increase in input resistance in neurotrophin-treated neurons. These results highlight the potential of neurotrophins and antibiotics to influence neural firing patterns in vitro in a time-dependent manner, and advise the careful consideration of their impact on SGN function in future studies., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Common causes of hearing loss in humans - exposure to loud noise or ototoxic drugs and aging - often damage sensory hair cells, reflected as elevated thresholds on the clinical audiogram. Recent studies in animal models suggest, however, that well before this overt hearing loss can be seen, a more insidious, but likely more common, process is taking place that permanently interrupts synaptic communication between sensory inner hair cells and subsets of cochlear nerve fibers. The silencing of affected neurons alters auditory information processing, whether accompanied by threshold elevations or not, and is a likely contributor to a variety of perceptual abnormalities, including speech-in-noise difficulties, tinnitus and hyperacusis. Work described here will review structural and functional manifestations of this cochlear synaptopathy and will consider possible mechanisms underlying its appearance and progression in ears with and without traditional 'hearing loss' arising from several common causes in humans., (Copyright © 2017 Elsevier B.V. All rights reserved.)

An understanding of the molecular pathology that underlies noise induced neurotoxicity is a prerequisite to the design of targeted therapies. The objective of the current experiment was to determine whether or not DNA damage is part of the pathophysiologic sequela of noise induced neurotoxicity. The experiment consisted of 41 hooded Long-Evans rats (2 month old males) that were randomized into control and noise exposed groups. Both the control and the noise group followed the same time schedule and therefore started and ended the experiment together. The noise dose consisted of a 6000 Hz noise band at 105 dB SPL. Temporal bones from both groups were harvested, and immunohistochemistry was used to identify neurons with DNA damage. Quantitative morphometric analyses was then employed to determine the level of DNA damage. Neural action potentials were recorded to assess the functional impact of noise induced DNA damage. Immunohistochemical reactions revealed that the noise exposure precipitated DNA damage within the nucleus of auditory neurons. Quantitative morphometry confirmed the noise induced increase in DNA damage levels and the precipitation of DNA damage was associated with a significant loss of nerve sensitivity. Therefore, DNA damage is part of the molecular pathology that drives noise induced neurotoxicity. Anat Rec, 300:520-526, 2017. © 2016 Wiley Periodicals, Inc., (© 2016 Wiley Periodicals, Inc.)

Key Points: Following the genetically controlled formation of neuronal circuits, early firing activity guides the development of sensory maps in the auditory, visual and somatosensory system. However, it is not clear whether the activity of central auditory neurons is specifically regulated depending on the position within the sensory map. In the ventral cochlear nucleus, the first central station along the auditory pathway, we describe a mechanism through which paracrine ATP signalling enhances firing in a cell-specific and tonotopically-determined manner. Developmental down-regulation of P2X2/3R currents along the tonotopic axis occurs simultaneously with an increase in AMPA receptor currents, suggesting a high-to-low frequency maturation pattern. Facilitated action potential (AP) generation, measured as higher firing rate, shorter EPSP-AP delay in vivo and shorter AP latency in slice experiments, is consistent with increased synaptic efficacy caused by ATP. The long lasting change in intrinsic neuronal excitability is mediated by the heteromeric P2X2/3 receptors., Abstract: Synaptic refinement and strengthening are activity-dependent processes that establish orderly arranged cochleotopic maps throughout the central auditory system. The maturation of auditory brainstem circuits is guided by action potentials (APs) arising from the inner hair cells in the developing cochlea. The AP firing of developing central auditory neurons can be modulated by paracrine ATP signalling, as shown for the cochlear nucleus bushy cells and principal neurons in the medial nucleus of the trapezoid body. However, it is not clear whether neuronal activity may be specifically regulated with respect to the nuclear tonotopic position (i.e. sound frequency selectivity). Using slice recordings before hearing onset and in vivo recordings with iontophoretic drug applications after hearing onset, we show that cell-specific purinergic modulation follows a precise tonotopic pattern in the ventral cochlear nucleus of developing gerbils. In high-frequency regions, ATP responsiveness diminished before hearing onset. In low-to-mid frequency regions, ATP modulation persisted after hearing onset in a subset of low-frequency bushy cells (characteristic frequency< 10 kHz). Down-regulation of P2X2/3R currents along the tonotopic axis occurs simultaneously with an increase in AMPA receptor currents, thus suggesting a high-to-low frequency maturation pattern. Facilitated AP generation, measured as higher firing frequency, shorter EPSP-AP delay in vivo, and shorter AP latency in slice experiments, is consistent with increased synaptic efficacy caused by ATP. Finally, by combining recordings and pharmacology in vivo, in slices, and in human embryonic kidney 293 cells, it was shown that the long lasting change in intrinsic neuronal excitability is mediated by the P2X2/3R., (© 2016 The Authors. The Journal of Physiology © 2016 The Physiological Society.)

AMPA glutamate receptor complexes with fast kinetics conferred by subunits like GluA3 and GluA4 are essential for temporal precision of synaptic transmission. The specific role of GluA3 in auditory processing and experience related changes in the auditory brainstem remain unknown. We investigated the role of the GluA3 in auditory processing by using wild type (WT) and GluA3 knockout (GluA3-KO) mice. We recorded auditory brainstem responses (ABR) to assess auditory function and used electron microscopy to evaluate the ultrastructure of the auditory nerve synapse on bushy cells (AN-BC synapse). Since labeling for GluA3 subunit increases on auditory nerve synapses within the cochlear nucleus in response to transient sound reduction, we investigated the role of GluA3 in experience-dependent changes in auditory processing. We induced transient sound reduction by plugging one ear and evaluated ABR threshold and peak amplitude recovery for up to 60 days after ear plug removal in WT and GluA3-KO mice. We found that the deletion of GluA3 leads to impaired auditory signaling that is reflected in decreased ABR peak amplitudes, an increased latency of peak 2, early onset hearing loss and reduced numbers and sizes of postsynaptic densities (PSDs) of AN-BC synapses. Additionally, the lack of GluA3 hampers ABR threshold recovery after transient ear plugging. We conclude that GluA3 is required for normal auditory signaling, normal ultrastructure of AN-BC synapses in the cochlear nucleus and normal experience-dependent changes in auditory processing after transient sound reduction., Competing Interests: statement The authors declare no competing financial interests., (Copyright © 2016 Elsevier B.V. All rights reserved.)

More than 100 genes have been associated with deafness. However, SMAD4 is rarely considered a contributor to deafness in humans, except for its well-defined role in cell differentiation and regeneration. Here, we report that a SMAD4 defect in mice can cause auditory neuropathy, which was defined as a mysterious hearing and speech perception disorder in human for which the genetic background remains unclear. Our study showed that a SMAD4 defect induces failed formation of cochlear ribbon synapse during the earlier stage of auditory development in mice. Further investigation found that there are nearly normal morphology of outer hair cells (OHCs) and post-synapse spiral ganglion nerves (SGNs) in SMAD4 conditional knockout mice (cKO); however, a preserved distortion product of otoacoustic emission (DPOAE) and cochlear microphonic (CM) still can be evoked in cKO mice. Moreover, a partial restoration of hearing detected by electric auditory brainstem response (eABR) has been obtained in the cKO mice using electrode stimuli toward auditory nerves. Additionally, the ribbon synapses in retina are not affected by this SMAD4 defect. Thus, our findings suggest that this SMAD4 defect causes auditory neuropathy via specialized disruption of cochlear ribbon synapses.

We evaluated the role of iron deficiency (ID) without anemia on hearing function and cochlear pathophysiology of young rats before and after noise exposure. We used rats at developmental stages as an animal model to induce ID without anemia by dietary iron restriction. We have established this dietary restriction model in the rat that should enable us to study the effects of iron deficiency in the absence of severe anemia on hearing and ribbon synapses. Hearing function was measured on Postnatal Day (PND) 21 after induction of ID using auditory brainstem response (ABR). Then, the young rats were exposed to loud noise on PND 21. After noise exposure, hearing function was again measured. We observed the morphology of ribbon synapses, hair cells and spiral ganglion cells (SGCs), and assessed the expression of myosin VIIa, vesicular glutamate transporter 3 and prestin in the cochlea. ID without anemia did not elevate ABR threshold shifts, but reduced ABR wave I peak amplitude of young rats. At 70, 80, and 90 dB SPL, amplitudes of wave I (3.11 ± 0.96 µV, 3.52 ± 1.31 µV, and 4.37 ± 1.08 µV, respectively) in pups from the ID group were decreased compared to the control (5.92 ± 1.67 µV, 6.53 ± 1.70 µV, and 6.90 ± 1.76 µV, respectively) (p < 0.05). Moreover, ID without anemia did not impair the morphology hair cells and SGCs, but decreased the number of ribbon synapses. Before noise exposure, the mean number of ribbon synapses per inner hair cell (IHC) was significantly lower in the ID group (8.44 ± 1.21) compared to that seen in the control (13.08 ± 1.36) (p < 0.05). In addition, the numbers of ribbon synapses per IHC of young rats in the control (ID group) were 6.61 ± 1.59, 3.07 ± 0.83, 5.85 ± 1.63 and 12.25 ± 1.97 (3.75 ± 1.45, 2.03 ± 1.08, 3.81 ± 1.70 and 4.01 ± 1.65) at 1, 4, 7 and 14 days after noise exposure, respectively. Moreover, ABR thresholds at 4 and 8 kHz in young rats from the ID group were significantly elevated at 7 and 14 days after noise exposure compared to control (p < 0.05). The average number of young rat SGCs from the ID group were significantly decreased in the basal turn of the cochlea compared to the control (p < 0.05). Therefore, ID without anemia delayed the recovery from noise-induced hearing loss and ribbon synapses damage, increased SGCs loss, and upregulated prestin after noise exposure. Thus, the cochleae in rat pups with ID without anemia were potentially susceptible to loud noise exposure, and this deficit may be attributed to the reduction of ribbon synapses and SGCs.

In adult animals, the neuropeptide calcitonin gene-related peptide (CGRP) is contained in cochlear efferent fibers projecting out to the cochlea, and contributes to increased suprathreshold sound-evoked activity in the adult auditory nerve. Similarly, CGRP applied to the lateral-line organ (hair cell organ) increases afferent nerve activity in adult frogs (post-metamorphic day 30), yet this increase is developmentally delayed from post-metamorphic day 4-30. In this study, we discovered that there was also a developmental delay in increased suprathreshold sound-evoked activity auditory nerve between juvenile and adult mice similar to what had been observed previously in frog. Moreover, juvenile mice with a targeted deletion of the αCGRP gene [CGRP null (-/-)] did not show a similar developmental increase in nerve activity, suggesting CGRP signaling is involved. This developmental delay is not due to a delay in CGRP expression, but instead is due to a delay in receptor formation. We observed that the increase in sound-evoked nerve activity is correlated with increased formation of cochlear CGRP receptors, which require three complexed proteins (CLR, RAMP1, RCP) to be functional. CGRP receptor formation in the cochlea was incomplete at 1 month of age (juvenile), but complete by 3 months (adult), which corresponded to the onset of suprathreshold enhancement of sound-evoked activity in wild-type animals. Taken together, these data support a model for cochlear function that is enhanced by maturation of CGRP receptor complexes., (© 2016 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of the American Physiological Society and The Physiological Society.)

Background and Objective: The auditory efferent system is a complex network of descending pathways, which mainly originate in the primary auditory cortex and are directed to several auditory subcortical nuclei. These descending pathways are connected to olivocochlear neurons, which in turn make synapses with auditory nerve neurons and outer hair cells (OHC) of the cochlea. The olivocochlear function can be studied using contralateral acoustic stimulation, which suppresses auditory nerve and cochlear responses. In the present work, we tested the proposal that the corticofugal effects that modulate the strength of the olivocochlear reflex on auditory nerve responses are produced through cholinergic synapses between medial olivocochlear (MOC) neurons and OHCs via alpha-9/10 nicotinic receptors., Methods: We used wild type (WT) and alpha-9 nicotinic receptor knock-out (KO) mice, which lack cholinergic transmission between MOC neurons and OHC, to record auditory cortex evoked potentials and to evaluate the consequences of auditory cortex electrical microstimulation in the effects produced by contralateral acoustic stimulation on auditory brainstem responses (ABR)., Results: Auditory cortex evoked potentials at 15 kHz were similar in WT and KO mice. We found that auditory cortex microstimulation produces an enhancement of contralateral noise suppression of ABR waves I and III in WT mice but not in KO mice. On the other hand, corticofugal modulations of wave V amplitudes were significant in both genotypes., Conclusion: These findings show that the corticofugal modulation of contralateral acoustic suppressions of auditory nerve (ABR wave I) and superior olivary complex (ABR wave III) responses are mediated through MOC synapses.

Sensorineural hearing impairment is the most common form of hearing loss, and encompasses pathologies of the cochlea and the auditory nerve. Hearing impairment caused by abnormal neural encoding of sound stimuli despite preservation of sensory transduction and amplification by outer hair cells is known as 'auditory neuropathy'. This term was originally coined for a specific type of hearing impairment affecting speech comprehension beyond changes in audibility: patients with this condition report that they "can hear but cannot understand". This type of hearing impairment can be caused by damage to the sensory inner hair cells (IHCs), IHC ribbon synapses or spiral ganglion neurons. Human genetic and physiological studies, as well as research on animal models, have recently shown that disrupted IHC ribbon synapse function--resulting from genetic alterations that affect presynaptic glutamate loading of synaptic vesicles, Ca(2+) influx, or synaptic vesicle exocytosis--leads to hearing impairment termed 'auditory synaptopathy'. Moreover, animal studies have demonstrated that sound overexposure causes excitotoxic loss of IHC ribbon synapses. This mechanism probably contributes to hearing disorders caused by noise exposure or age-related hearing loss. This Review provides an update on recently elucidated sensory, synaptic and neural mechanisms of hearing impairment, their corresponding clinical findings, and discusses current rehabilitation strategies as well as future therapies.

Improving the electrode-neuron interface to reduce current spread between individual electrodes has been identified as one of the main objectives in the search for future improvements in cochlear-implant performance. Here, we address this problem by presenting a novel stimulation strategy that takes account of the biophysical properties of the auditory neurons (spiral ganglion neurons, SGNs) stimulated in electrical hearing. This new strategy employs a ramped pulse shape, where the maximum amplitude is achieved through a linear slope in the injected current. We present the theoretical framework that supports this new strategy and that suggests it will improve the modulation of SGNs' activity by exploiting their sensitivity to the rising slope of current pulses. The theoretical consequence of this sensitivity to the slope is a reduction in the spread of excitation within the cochlea and, consequently, an increase in the neural dynamic range. To explore the impact of the novel stimulation method on neural activity, we performed in vitro recordings of SGNs in culture. We show that the stimulus efficacy required to evoke action potentials in SGNs falls as the stimulus slope decreases. This work lays the foundation for a novel, and more biomimetic, stimulation strategy with considerable potential for implementation in cochlear-implant technology., (© The Author(s) 2015.)

Hearing impairment is the most common human sensory deficit. Considering the sophisticated anatomy and physiology of the auditory system, disease-related failures frequently occur. To meet the demands of the neuronal circuits responsible for processing auditory information, the synapses of the lower auditory pathway are anatomically and functionally specialized to process acoustic information indefatigably with utmost temporal precision. Despite sharing some functional properties, the afferent synapses of the cochlea and of auditory brainstem differ greatly in their morphology and employ distinct molecular mechanisms for regulating synaptic vesicle release. Calyceal synapses of the endbulb of Held and the calyx of Held profit from a large number of release sites that project onto one principal cell. Cochlear inner hair cell ribbon synapses exhibit a unique one-to-one relation of the presynaptic active zone to the postsynaptic cell and use hair-cell-specific proteins such as otoferlin for vesicle release. The understanding of the molecular physiology of the hair cell ribbon synapse has been advanced by human genetics studies of sensorineural hearing impairment, revealing human auditory synaptopathy as a new nosological entity., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Mutations in the OTOF gene encoding otoferlin result in a disrupted function of the ribbon synapses with impairment of the multivesicular glutamate release. Most affected subjects present with congenital hearing loss and abnormal auditory brainstem potentials associated with preserved cochlear hair cell activities (otoacoustic emissions, cochlear microphonics [CMs]). Transtympanic electrocochleography (ECochG) has recently been proposed for defining the details of potentials arising in both the cochlea and auditory nerve in this disorder, and with a view to shedding light on the pathophysiological mechanisms underlying auditory dysfunction. We review the audiological and electrophysiological findings in children with congenital profound deafness carrying two mutant alleles of the OTOF gene. We show that cochlear microphonic (CM) amplitude and summating potential (SP) amplitude and latency are normal, consistently with a preserved outer and inner hair cell function. In the majority of OTOF children, the SP component is followed by a markedly prolonged low-amplitude negative potential replacing the compound action potential (CAP) recorded in normally-hearing children. This potential is identified at intensities as low as 90 dB below the behavioral threshold. In some ears, a synchronized CAP is superimposed on the prolonged responses at high intensity. Stimulation at high rates reduces the amplitude and duration of the prolonged potentials, consistently with their neural generation. In some children, however, the ECochG response only consists of the SP, with no prolonged potential. Cochlear implants restore hearing sensitivity, speech perception and neural CAP by electrically stimulating the auditory nerve fibers. These findings indicate that an impaired multivesicular glutamate release in OTOF-related disorders leads to abnormal auditory nerve fiber activation and a consequent impairment of spike generation. The magnitude of these effects seems to vary, ranging from no auditory nerve fiber activation to an abnormal generation of EPSPs that occasionally trigger a synchronized electrical activity, resulting in high-threshold CAPs., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Sound signal is detected by sensory hair cells located in the cochlear region of the inner ear, and transmitted to the central nervous system by the spiral ganglion neurons (SGNs). These bipolar neurons develop long peripheral processes to connect hair cells, forming ribbon synapses, specialised for the precision and speed required to process auditory information. The establishment of a complex innervation pattern relies on specific signals, intrinsic to SGNs or provided by neighbouring cells, which are tightly controlled in time and space. In this paper, we review recent advances about stepwise development of afferent auditory neuronal circuitries, from neuron specification within the early otic vesicle to definitive synaptic connections with target cells. We especially focus on the cellular and molecular developmental changes involved in fibre outgrowth and extension to the sensory epithelium, specific afferent targeting to hair cells, and synaptic pruning., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Information processing in the brain requires reliable synaptic transmission. High reliability at specialized auditory nerve synapses in the cochlear nucleus results from many release sites (N), high probability of neurotransmitter release (Pr), and large quantal size (Q). However, high Pr also causes auditory nerve synapses to depress strongly when activated at normal rates for a prolonged period, which reduces fidelity. We studied how synapses are influenced by prolonged activity by exposing mice to constant, nondamaging noise and found that auditory nerve synapses changed to facilitating, reflecting low Pr. For mice returned to quiet, synapses recovered to normal depression, suggesting that these changes are a homeostatic response to activity. Two additional properties, Q and average excitatory postsynaptic current (EPSC) amplitude, were unaffected by noise rearing, suggesting that the number of release sites (N) must increase to compensate for decreased Pr. These changes in N and Pr were confirmed physiologically using the integration method. Furthermore, consistent with increased N, endbulbs in noise-reared animals had larger VGlut1-positive puncta, larger profiles in electron micrographs, and more release sites per profile. In current-clamp recordings, noise-reared BCs had greater spike fidelity even during high rates of synaptic activity. Thus, auditory nerve synapses regulate excitability through an activity-dependent, homeostatic mechanism, which could have major effects on all downstream processing. Our results also suggest that noise-exposed bushy cells would remain hyperexcitable for a period after returning to normal quiet conditions, which could have perceptual consequences.

We examined the synaptic structure, quantity, and distribution of α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA)- and N-methyl-D-aspartate (NMDA)-type glutamate receptors (AMPARs and NMDARs, respectively) in rat cochlear nuclei by a highly sensitive freeze-fracture replica labeling technique. Four excitatory synapses formed by two distinct inputs, auditory nerve (AN) and parallel fibers (PF), on different cell types were analyzed. These excitatory synapse types included AN synapses on bushy cells (AN-BC synapses) and fusiform cells (AN-FC synapses) and PF synapses on FC (PF-FC synapses) and cartwheel cell spines (PF-CwC synapses). Immunogold labeling revealed differences in synaptic structure as well as AMPAR and NMDAR number and/or density in both AN and PF synapses, indicating a target-dependent organization. The immunogold receptor labeling also identified differences in the synaptic organization of FCs based on AN or PF connections, indicating an input-dependent organization in FCs. Among the four excitatory synapse types, the AN-BC synapses were the smallest and had the most densely packed intramembrane particles (IMPs), whereas the PF-CwC synapses were the largest and had sparsely packed IMPs. All four synapse types showed positive correlations between the IMP-cluster area and the AMPAR number, indicating a common intrasynapse-type relationship for glutamatergic synapses. Immunogold particles for AMPARs were distributed over the entire area of individual AN synapses; PF synapses often showed synaptic areas devoid of labeling. The gold-labeling for NMDARs occurred in a mosaic fashion, with less positive correlations between the IMP-cluster area and the NMDAR number. Our observations reveal target- and input-dependent features in the structure, number, and organization of AMPARs and NMDARs in AN and PF synapses., (© 2014 Wiley Periodicals, Inc.)

The brain stem auditory system of the chick is an advantageous model for examining changes that occur as a result of deafness. Elimination of acoustic input through cochlear ablation results in the eventual death of approximately 30% of neurons in the chick cochlear nucleus, nucleus magnocellularis (NM). One early change following deafness is an alteration in NM ribosomes, evidenced both by a decrease in protein synthesis and reduction in antigenicity for Y10B, a monoclonal antibody that recognizes a ribosomal epitope. Previous studies have shown that mGluR activation is necessary to maintain Y10B antigenicity and NM viability. What is still unclear, however, is whether or not mGluR activation is sufficient to prevent deafness-induced changes in these neurons, or if other activity-dependent factors are also necessary. The current study investigated the ability of mGluR activation to regulate cochlear nucleus ribosomes in the absence of auditory nerve input. In vitro methods were employed to periodically pressure eject glutamate or mGluR agonists over neurons on one side of a slice preparation leaving the opposite side of the same slice untreated. Immunohistochemistry was then performed using Y10B in order to assess ribosomal changes. Application of glutamate and both group I and II selective mGluR agonists effectively rescued ribosomal antigenicity on the treated side of the slice in comparison to ribosomes on the untreated side. These findings suggest that administration of mGluR agonists is sufficient to reduce the early interruption of normal ribosomal integrity that is typically seen following loss of auditory nerve activity.

Although neural stem cell (NSC) transplantation is widely expected to become a therapy for nervous system degenerative diseases and injuries, the low neuronal differentiation rate of NSCs transplanted into the inner ear is a major obstacle for the successful treatment of spiral ganglion neuron (SGN) degeneration. In this study, we validated whether the local microenvironment influences the neuronal differentiation of transplanted NSCs in the inner ear. Using a rat SGN degeneration model, we demonstrated that transplanted NSCs were more likely to differentiate into microtubule-associated protein 2 (MAP2)-positive neurons in SGN-degenerated cochleae than in control cochleae. Using real-time quantitative PCR and an immunofluorescence assay, we also proved that the expression of Wnt1 (a ligand of Wnt signaling) increases significantly in Schwann cells in the SGN-degenerated cochlea. We further verified that NSC cultures express receptors and signaling components for Wnts. Based on these expression patterns, we hypothesized that Schwann cell-derived Wnt1 and Wnt signaling might be involved in the regulation of the neuronal differentiation of transplanted NSCs. We verified our hypothesis in vitro using a coculture system. We transduced a lentiviral vector expressing Wnt1 into cochlear Schwann cell cultures and cocultured them with NSC cultures. The coculture with Wnt1-expressing Schwann cells resulted in a significant increase in the percentage of NSCs that differentiated into MAP2-positive neurons, whereas this differentiation-enhancing effect was prevented by Dkk1 (an inhibitor of the Wnt signaling pathway). These results suggested that Wnt1 derived from cochlear Schwann cells enhanced the neuronal differentiation of transplanted NSCs through Wnt signaling pathway activation. Alterations of the microenvironment deserve detailed investigation because they may help us to conceive effective strategies to overcome the barrier of the low differentiation rate of transplanted NSCs.

The auxiliary subunit α2δ3 modulates the expression and function of voltage-gated calcium channels. Here we show that α2δ3 mRNA is expressed in spiral ganglion neurons and auditory brainstem nuclei and that the protein is required for normal acoustic responses. Genetic deletion of α2δ3 led to impaired auditory processing, with reduced acoustic startle and distorted auditory brainstem responses. α2δ3(-/-) mice learned to discriminate pure tones, but they failed to discriminate temporally structured amplitude-modulated tones. Light and electron microscopy analyses revealed reduced levels of presynaptic Ca(2+) channels and smaller auditory nerve fiber terminals contacting cochlear nucleus bushy cells. Juxtacellular in vivo recordings of sound-evoked activity in α2δ3(-/-) mice demonstrated impaired transmission at these synapses. Together, our results identify a novel role for the α2δ3 auxiliary subunit in the structure and function of specific synapses in the mammalian auditory pathway and in auditory processing disorders.

Prior to the "onset of hearing," developing cochlear inner hair cells (IHCs) and primary auditory neurons undergo experience-independent activity, which is thought to be important in retaining and refining neural connections in the absence of sound. One of the major hypotheses regarding the origin of such activity involves a group of columnar epithelial supporting cells forming Kölliker's organ, which is only present during this critical period of auditory development. There is strong evidence for a purinergic signalling mechanism underlying such activity. ATP released through connexin hemichannels may activate P2 purinergic receptors in both Kölliker's organ and the adjacent IHCs, leading to generation of electrical activity throughout the auditory system. However, recent work has suggested an alternative origin, by demonstrating the ability of IHCs to generate this spontaneous activity without activation by ATP. Regardless, developmental abnormalities of Kölliker's organ may lead to congenital hearing loss, considering that mutations in ion channels (hemichannels, gap junctions, and calcium channels) involved in Kölliker's organ activity share strong links with such types of deafness.

In the presented model, electrical, acoustical, and mechanical elements of the cochlea are explicitly integrated into a signal transmission line where these elements convey physiological interpretations of the human cochlear structures. As a result, this physiologically-motivated model enables simulation of specific cochlear lesions such as presbyacusis. The hypothesis is that high-frequency hearing loss in older adults may be due to metabolic presbyacusis whereby age-related cellular/chemical degenerations in the lateral wall of the cochlea cause a reduction in the endocochlear potential. The simulations quantitatively confirm this hypothesis and emphasize that even if the outer and inner hair cells are totally active and intact, metabolic presbyacusis alone can significantly deteriorate the cochlear functionality. Specifically, in the model, as the endocochlear potential decreases, the transduction mechanism produces less receptor current such that there is a reduction in the battery of the somatic motor. This leads to a drastic decrease in cochlear amplification and frequency sensitivity, as well as changes in position-frequency map (tuning pattern) of the cochlea. In addition, the simulations show that the age-related reduction of the endocochlear potential significantly inhibits the firing rate of the auditory nerve which might contribute to the decline of temporal resolution in the aging auditory system.

Osteoprotegerin (OPG) is a key regulator of bone remodeling. Mutations and variations in the OPG gene cause many human diseases that are characterized by not only skeletal abnormalities but also poorly understood hearing loss: Paget's disease, osteoporosis, and celiac disease. To gain insight into the mechanisms of hearing loss in OPG deficiency, we studied OPG knockout (Opg(-/-)) mice. We show that they develop sensorineural hearing loss, in addition to conductive hearing loss due to abnormal middle-ear bones. OPG deficiency caused demyelination and degeneration of the cochlear nerve in vivo. It also activated ERK, sensitized spiral ganglion cells (SGC) to apoptosis, and inhibited proliferation and survival of cochlear stem cells in vitro, which could be rescued by treatment with exogenous OPG, an ERK inhibitor, or bisphosphonate. Our results demonstrate a novel role for OPG in the regulation of SGC survival, and suggest a mechanism for sensorineural hearing loss in OPG deficiency., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Inner ear damage leads to nerve fiber growth and synaptogenesis in the ventral cochlear nucleus (VCN). In this study, we documented the relationship between hair cell loss patterns and synaptic plasticity in the chinchilla VCN using immunolabeling of the growth associated protein-43 (GAP-43), a protein associated with axon outgrowth and modification of presynaptic endings. Unilateral round window application of carboplatin caused hair cell degeneration in which inner hair cells (IHC) were more vulnerable than outer hair cells (OHC). One month after carboplatin treatment (0.5-5 mg/ml), we observed varying patterns of cochlear hair cell loss and GAP-43 expression in VCN. Both IHC loss and OHC loss were strongly correlated with increased GAP-43 immunolabeling in the ipsilateral VCN. We speculate that two factors might promote the expression of GAP-43 in the VCN; one is the loss of afferent input through IHC or the associated type I auditory nerve fibers. The other occurs when the medial olivocochlear efferent neurons lose their cochlear targets, the OHC, and may as compensation increase their synapse numbers in the VCN., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Congenital or acquired hearing loss is often associated with a progressive degeneration of the auditory nerve (AN) in the inner ear. The AN is composed of processes and axons of the bipolar spiral ganglion neurons (SGN), forming the connection between the hair cells in the inner ear cochlea and the cochlear nuclei (CN) in the brainstem (BS). Therefore, replacement of SGNs for restoring the AN to improve hearing function in patients who receive a cochlear implantation or have severe AN malfunctions is an attractive idea. A human neural precursor cell (HNPC) is an appropriate donor cell to investigate, as it can be isolated and expanded in vitro with maintained potential to form neurons and glia. We recently developed a post-natal rodent in vitro auditory BS slice culture model including the CN and the central part of the AN for initial studies of candidate cells. Here we characterized the survival, distribution, phenotypic differentiation, and integration capacity of HNPCs into the auditory circuitry in vitro. HNPC aggregates (spheres) were deposited adjacent to or on top of the BS slices or as a monoculture (control). The results demonstrate that co-cultured HNPCs compared to monocultures (1) survive better, (2) distribute over a larger area, (3) to a larger extent and in a shorter time-frame form mature neuronal and glial phenotypes. HNPC showed the ability to extend neurites into host tissue. Our findings suggest that the HNPC-BS slice co-culture is appropriate for further investigations on the integration capacity of HNPCs into the auditory circuitry.

In the common frog Rana temporaria, the neuronal firing evoked by long amplitude-modulated tones was recorded in auditory regions of medulla (dorsal nucleus) and of midbrain (torus semicircularis). We recorded firing rate, synchronization with modulation, and phase of response every 2 or 4 s. After adaptation of neuronal response to the acting stimulus (30-60 s after its onset) the mean level of signal sharply changed (by 20-40 dB), whereas frequency and modulation depth remained constant. Changes of firing density and of degree of its synchronization with modulation period were recorded, as well as the phase of maximum of reaction at the period of modulation. At low modulation depths at the initial site, we observed an adaptation decrease of impulsation density accompanied by an improvement of the response synchronization. A sharp increase (by 20-40 dB) in the mean level led to a rise in the implication density, which could be accompanied either by a continued increase of synchronicity (the more typical effect for the dorsal nucleus) or by a sharp fall of synchronicity with its subsequent slow recovery (the more typical effect for the torus semicircularis). The character of changes in reaction after replacement of intensity could also depend on the signal parameters (the initial level, the jump value, the frequency and depth of modulation). The connection of the revealed physiological effects with psychophysics of perception of small amplitude modulations is discussed.