Delayed hearing loss after cochlear implantation: Re-evaluating the role of hair cell degeneration.

Delayed loss of residual acoustic hearing after cochlear implantation is a common but poorly understood phenomenon due to the scarcity of relevant temporal bone tissues. Prior histopathological analysis of one case of post-implantation hearing loss suggested there were no interaural differences in hair cell or neural degeneration to explain the profound loss of low-frequency hearing on the implanted side (Quesnel et al., 2016) and attributed the threshold elevation to neo-ossification and fibrosis around the implant. Here we re-evaluated the histopathology in this case, applying immunostaining and improved microscopic techniques for differentiating surviving hair cells from supporting cells. The new analysis revealed dramatic interaural differences, with a > 80 % loss of inner hair cells in the cochlear apex on the implanted side, which can account for the post-implantation loss of residual hearing. Apical degeneration of the stria further contributed to threshold elevation on the implanted side. In contrast, spiral ganglion cell survival was reduced in the region of the electrode on the implanted side, but apical counts in the two ears were similar to that seen in age-matched unimplanted control ears. Almost none of the surviving auditory neurons retained peripheral axons throughout the basal half of the cochlea. Relevance to cochlear implant performance is discussed.

BAI1 localizes AMPA receptors at the cochlear afferent post-synaptic density and is essential for hearing.

Type I spiral ganglion neurons (SGNs) convey sound information to the central auditory pathway by forming synapses with inner hair cells (IHCs) in the mammalian cochlea. The molecular mechanisms regulating the formation of the post-synaptic density (PSD) in the SGN afferent terminals are still unclear. Here, we demonstrate that brain-specific angiogenesis inhibitor 1 (BAI1) is required for the clustering of AMPA receptors GluR2-4 (glutamate receptors 2-4) at the PSD. Adult Bai1-deficient mice have functional IHCs but fail to transmit information to the SGNs, leading to highly raised hearing thresholds. Despite the almost complete absence of AMPA receptor subunits, the SGN fibers innervating the IHCs do not degenerate. Furthermore, we show that AMPA receptors are still expressed in the cochlea of Bai1-deficient mice, highlighting a role for BAI1 in trafficking or anchoring GluR2-4 to the PSDs. These findings identify molecular and functional mechanisms required for sound encoding at cochlear ribbon synapses.

Extrasynaptic distribution of NMDA receptors in cochlear inner hair cell afferent signaling complex.

OBJECTIVE: The distribution and role of NMDA receptors is unclear in the afferent signaling complex of the cochlea. The present study aimed to examine the distribution of NMDA receptors in cochlear afferent signaling complex of the adult mouse, and their relationship with ribbon synapses of inner hair cells (IHCs) and GABAergic efferent terminals of the lateral olivocochlear (LOC). METHODS: Immunofluorescence staining in combination with confocal microscopy was used to investigate the distribution of glutamatergic NMDA and AMPA receptors in afferent terminals of SGNs, and their relationship with ribbon synapses of IHCs and GABAergic efferent terminals of LOC. RESULTS: Terminals with AMPA receptors along with Ribbons of IHC formed afferent synapses in the basal pole of IHCs, and those with NMDA receptors were mainly distributed longitudinally in the IHCs nuclei region. Significant difference was found in the distribution of NMDA and AMPA receptors in IHC afferent signaling complex (P<0.05). Some GABAergic terminals colocalized with NMDA receptors at the IHC nucleus region (P>0.05). CONCLUSION: There is significant difference in the distribution of NMDA and AMPA receptors in cochlear afferent signaling complex. NMDA receptors are present in the extra-synaptic region of ribbon synapses of IHCs, and they are related to GABA efferent terminals of the afferent signaling complex.

Cochlear Ribbon Synapses in Aged Gerbils.

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.

AIF translocation into nucleus caused by Aifm1 R450Q mutation: generation and characterization of a mouse model for AUNX1.

Mutations in AIFM1, encoding for apoptosis-inducing factor (AIF), cause AUNX1, an X-linked neurologic disorder with late-onset auditory neuropathy (AN) and peripheral neuropathy. Despite significant research on AIF, there are limited animal models with the disrupted AIFM1 representing the corresponding phenotype of human AUNX1, characterized by late-onset hearing loss and impaired auditory pathways. Here, we generated an Aifm1 p.R450Q knock-in mouse model (KI) based on the human AIFM1 p.R451Q mutation. Hemizygote KI male mice exhibited progressive hearing loss from P30 onward, with greater severity at P60 and stabilization until P210. Additionally, muscle atrophy was observed at P210. These phenotypic changes were accompanied by a gradual reduction in the number of spiral ganglion neuron cells (SGNs) at P30 and ribbons at P60, which coincided with the translocation of AIF into the nucleus starting from P21 and P30, respectively. The SGNs of KI mice at P210 displayed loss of cytomembrane integrity, abnormal nuclear morphology, and dendritic and axonal demyelination. Furthermore, the inner hair cells and myelin sheath displayed abnormal mitochondrial morphology, while fibroblasts from KI mice showed impaired mitochondrial function. In conclusion, we successfully generated a mouse model recapitulating AUNX1. Our findings indicate that disruption of Aifm1 induced the nuclear translocation of AIF, resulting in the impairment in the auditory pathway.

Disruption of Cdh23 exon 68 splicing leads to progressive hearing loss in mice by affecting tip-link stability.

Inner ear hair cells are characterized by the F-actin-based stereocilia that are arranged into a staircase-like pattern on the apical surface of each hair cell. The tips of shorter-row stereocilia are connected with the shafts of their neighboring taller-row stereocilia through extracellular links named tip links, which gate mechano-electrical transduction (MET) channels in hair cells. Cadherin 23 (CDH23) forms the upper part of tip links, and its cytoplasmic tail is inserted into the so-called upper tip-link density (UTLD) that contains other proteins such as harmonin. The Cdh23 gene is composed of 69 exons, and we show here that exon 68 is subjected to hair cell-specific alternative splicing. Tip-link formation is not affected in genetically modified mutant mice lacking Cdh23 exon 68. Instead, the stability of tip links is compromised in the mutants, which also suffer from progressive and noise-induced hearing loss. Moreover, we show that the cytoplasmic tail of CDH23(+68) but not CDH23(-68) cooperates with harmonin in phase separation-mediated condensate formation. In conclusion, our work provides evidence that inclusion of Cdh23 exon 68 is critical for the stability of tip links through regulating condensate formation of UTLD components.

Neural Fluctuation Contrast as a Code for Complex Sounds: The Role and Control of Peripheral Nonlinearities.

The nonlinearities of the inner ear are often considered to be obstacles that the central nervous system has to overcome to decode neural responses to sounds. This review describes how peripheral nonlinearities, such as saturation of the inner-hair-cell response and of the IHC-auditory-nerve synapse, are instead beneficial to the neural encoding of complex sounds such as speech. These nonlinearities set up contrast in the depth of neural-fluctuations in auditory-nerve responses along the tonotopic axis, referred to here as neural fluctuation contrast (NFC). Physiological support for the NFC coding hypothesis is reviewed, and predictions of several psychophysical phenomena, including masked detection and speech intelligibility, are presented. Lastly, a framework based on the NFC code for understanding how the medial olivocochlear (MOC) efferent system contributes to the coding of complex sounds is presented. By modulating cochlear gain control in response to both sound energy and fluctuations in neural responses, the MOC system is hypothesized to function not as a simple feedback gain-control device, but rather as a mechanism for enhancing NFC along the tonotopic axis, enabling robust encoding of complex sounds across a wide range of sound levels and in the presence of background noise. Effects of sensorineural hearing loss on the NFC code and on the MOC feedback system are presented and discussed.

Letting the calcium flow.

Two calcium-binding proteins, CaBP1 and CaBP2, cooperate to keep calcium channels in the hair cells of the inner ear open.

The Piezo channel is a mechano-sensitive complex component in the mammalian inner ear hair cell.

The inner ear is the hub where hair cells (HCs) transduce sound, gravity, and head acceleration stimuli to the brain. Hearing and balance rely on mechanosensation, the fastest sensory signals transmitted to the brain. The mechanoelectrical transducer (MET) channel is the entryway for the sound-balance-brain interface, but the channel-complex composition is not entirely known. Here, we report that the mouse utilizes Piezo1 (Pz1) and Piezo2 (Pz2) isoforms as MET-complex components. The Pz channels, expressed in HC stereocilia, and cell lines are co-localized and co-assembled with MET complex partners. Mice expressing non-functional Pz1 and Pz2 at the ROSA26 locus have impaired auditory and vestibular traits that can only be explained if the Pzs are integral to the MET complex. We suggest that Pz subunits constitute part of the MET complex and that interactions with other MET complex components yield functional MET units to generate HC MET currents.

Stem Cell-Based Hair Cell Regeneration and Therapy in the Inner Ear.

Hearing loss has become increasingly prevalent and causes considerable disability, thus gravely burdening the global economy. Irreversible loss of hair cells is a main cause of sensorineural hearing loss, and currently, the only relatively effective clinical treatments are limited to digital hearing equipment like cochlear implants and hearing aids, but these are of limited benefit in patients. It is therefore urgent to understand the mechanisms of damage repair in order to develop new neuroprotective strategies. At present, how to promote the regeneration of functional hair cells is a key scientific question in the field of hearing research. Multiple signaling pathways and transcriptional factors trigger the activation of hair cell progenitors and ensure the maturation of newborn hair cells, and in this article, we first review the principal mechanisms underlying hair cell reproduction. We then further discuss therapeutic strategies involving the co-regulation of multiple signaling pathways in order to induce effective functional hair cell regeneration after degeneration, and we summarize current achievements in hair cell regeneration. Lastly, we discuss potential future approaches, such as small molecule drugs and gene therapy, which might be applied for regenerating functional hair cells in the clinic.

Optogenetics Reveals Roles for Supporting Cells in Force Transmission to and From Outer Hair Cells in the Mouse Cochlea.

Outer hair cells (OHCs) of the organ of Corti (OoC), acting as bidirectional cellular mechanoelectrical transducers, generate, receive, and exchange forces with other major elements of the cochlear partition, including the sensory inner hair cells (IHCs). Force exchange is mediated via a supporting cell scaffold, including Deiters' (DC) and outer pillar cells (OPC), to enable the sensitivity and exquisite frequency selectivity of the mammalian cochlea and to transmit its responses to the auditory nerve. To selectively activate DCs and OPCs in male and female mice, we conditionally expressed in them a hyperpolarizing halorhodopsin (HOP), a light-gated inward chloride ion pump, and measured extracellular receptor potentials (ERPs) and their DC component (ERPDCs) from the cortilymph, which fills the OoC fluid spaces, and compared the responses with similar potentials from HOP-/- littermates. The compound action potentials (CAP) of the auditory nerve were measured as an indication of IHC activity and transmission of cochlear responses to the CNS. HOP light-activated hyperpolarization of DCs and OPCs suppressed cochlear amplification through changing the timing of its feedback, altered basilar membrane (BM) responses to tones at all measured levels and frequencies, and reduced IHC excitation. HOP activation findings reported here complement recent studies that revealed channelrhodopsin activation depolarized DCs and OPCs and effectively bypassed, rather than blocked, the control of OHC mechanical and electrical responses to sound and their contribution to timed and directed electromechanical feedback to the mammalian cochlea. Moreover, our findings identify DCs and OPCs as potential targets for the treatment of noise-induced hearing loss.

Proteomic Analysis Reveals the Composition of Glutamatergic Organelles of Auditory Inner Hair Cells.

In the ear, inner hair cells (IHCs) employ sophisticated glutamatergic ribbon synapses with afferent neurons to transmit auditory information to the brain. The presynaptic machinery responsible for neurotransmitter release in IHC synapses includes proteins such as the multi-C2-domain protein otoferlin and the vesicular glutamate transporter 3 (VGluT3). Yet, much of this likely unique molecular machinery remains to be deciphered. The scarcity of material has so far hampered biochemical studies which require large amounts of purified samples. We developed a subcellular fractionation workflow combined with immunoisolation of VGluT3-containing membrane vesicles, allowing for the enrichment of glutamatergic organelles that are likely dominated by synaptic vesicles (SVs) of IHCs. We have characterized their protein composition in mice before and after hearing onset using mass spectrometry and confocal imaging and provide a fully annotated proteome with hitherto unidentified proteins. Despite the prevalence of IHC marker proteins across IHC maturation, the profiles of trafficking proteins differed markedly before and after hearing onset. Among the proteins enriched after hearing onset were VAMP-7, syntaxin-7, syntaxin-8, syntaxin-12/13, SCAMP1, V-ATPase, SV2, and PKCα. Our study provides an inventory of the machinery associated with synaptic vesicle-mediated trafficking and presynaptic activity at IHC ribbon synapses and serves as a foundation for future functional studies.

Stem Cell-Based Hair Cell Regeneration and Therapy in the Inner Ear / 神经科学通报·英文版

Hearing loss has become increasingly prevalent and causes considerable disability, thus gravely burdening the global economy. Irreversible loss of hair cells is a main cause of sensorineural hearing loss, and currently, the only relatively effective clinical treatments are limited to digital hearing equipment like cochlear implants and hearing aids, but these are of limited benefit in patients. It is therefore urgent to understand the mechanisms of damage repair in order to develop new neuroprotective strategies. At present, how to promote the regeneration of functional hair cells is a key scientific question in the field of hearing research. Multiple signaling pathways and transcriptional factors trigger the activation of hair cell progenitors and ensure the maturation of newborn hair cells, and in this article, we first review the principal mechanisms underlying hair cell reproduction. We then further discuss therapeutic strategies involving the co-regulation of multiple signaling pathways in order to induce effective functional hair cell regeneration after degeneration, and we summarize current achievements in hair cell regeneration. Lastly, we discuss potential future approaches, such as small molecule drugs and gene therapy, which might be applied for regenerating functional hair cells in the clinic.

Zinc deficiency triggers hearing loss by reducing ribbon synapses of inner hair cells in CBA/N mice.

Zinc plays a vital role in our metabolism, encompassing antioxidant regulation, immune response, and auditory function. Several studies have reported that zinc levels correlate with hearing loss. We have previously demonstrated that the auditory brainstem response (ABR) threshold increased in mice fed a zinc-deficient diet. However, the effects of zinc deficiency on hearing were not fully elucidated. The present study investigated whether zinc deficiency affects hearing in association with neuronal components or cochlear structures. CBA/N mice were fed a normal or zinc-deficient diet for 8 weeks and assessed for ABR and distortion product otoacoustic emissions (DPOAE). The cochlear sections were stained with hematoxylin and eosin solution. Also, we observed the expression of synaptic ribbons, neurofilaments, and alpha-synuclein (α-Syn). The 8-week zinc-deficient diet mice had an elevated ABR threshold but no changed DPOAE threshold or cochlear structures. A reduced number of synaptic ribbons of inner hair cells (IHCs) and impaired efferent nerve fibers were observed in the zinc-deficient diet mice. The number of outer hair cells (OHCs) and expression of α-Syn remained unchanged. Our results suggest that zinc-mediated hearing loss is associated with the loss of neuronal components of IHCs.

Gentamicin administration leads to synaptic dysfunction in inner hair cells.

Ototoxicity is a major side effect of aminoglycosides, which can cause irreversible hearing loss. Previous studies on aminoglycoside-induced ototoxicity have primarily focused on the loss of sensory hair cells. Recent investigations have revealed that aminoglycosides can also lead to the loss of ribbon synapses in inner hair cells (IHCs). However, the functional implications of ribbon synapse loss and the underlying mechanisms remain unclear. In this study, we intraperitoneally injected C57BL/6 J mice with 300 mg/kg gentamicin once daily for 3, 10, and 20 days. Then, we performed immunofluorescence staining, patch-clamp recording, proteomics analysis and western blotting to characterize the changes in ribbon synapses in IHCs and the associated mechanisms. After gentamicin treatment, the auditory brainstem response (ABR) threshold was elevated, and the ABR wave I amplitude was decreased. We also observed loss of ribbon synapses in IHCs. Interestingly, ribbon synapse loss occurred on both the modiolar and pillar sides of IHCs. Whole-cell patch-clamp recordings in IHCs revealed a reduction in the calcium current amplitude, along with a shifted half-activation voltage and altered calcium voltage dependency. Moreover, exocytosis of IHCs was reduced, consistent with the reduction in the ABR wave I amplitude. Through proteomic analysis, western blotting, and immunofluorescence staining, we found that gentamicin treatment resulted in downregulation of myosin VI, a protein crucial for synaptic vesicle recycling and replenishment in IHCs. Furthermore, we evaluated the kinetics of endocytosis and found a significant reduction in IHC exocytosis, possibly reflecting the impact of myosin VI downregulation on synaptic vesicle recycling. In summary, our findings demonstrate that gentamicin treatment leads to synaptic dysfunction in IHCs, highlighting the important role of myosin VI downregulation in gentamicin-induced synaptic damage.

In situ regeneration of inner hair cells in the damaged cochlea by temporally regulated co-expression of Atoh1 and Tbx2.

Cochlear inner hair cells (IHCs) are primary sound receptors, and are therefore a target for developing treatments for hearing impairment. IHC regeneration in vivo has been widely attempted, although not yet in the IHC-damaged cochlea. Moreover, the extent to which new IHCs resemble wild-type IHCs remains unclear, as is the ability of new IHCs to improve hearing. Here, we have developed an in vivo mouse model wherein wild-type IHCs were pre-damaged and nonsensory supporting cells were transformed into IHCs by ectopically expressing Atoh1 transiently and Tbx2 permanently. Notably, the new IHCs expressed the functional marker vGlut3 and presented similar transcriptomic and electrophysiological properties to wild-type IHCs. Furthermore, the formation efficiency and maturity of new IHCs were higher than those previously reported, although marked hearing improvement was not achieved, at least partly due to defective mechanoelectrical transduction (MET) in new IHCs. Thus, we have successfully regenerated new IHCs resembling wild-type IHCs in many respects in the damaged cochlea. Our findings suggest that the defective MET is a critical barrier that prevents the restoration of hearing capacity and should thus facilitate future IHC regeneration studies.

Ultrastructure of noise-induced cochlear synaptopathy.

Acoustic overexposure can eliminate synapses between inner hair cells (IHCs) and auditory nerve fibers (ANFs), even if hair-cell function recovers. This synaptopathy has been extensively studied by confocal microscopy, however, understanding the nature and sequence of damage requires ultrastructural analysis. Here, we used focused ion-beam scanning electron microscopy to mill, image, segment and reconstruct ANF terminals in mice, 1 day and 1 week after synaptopathic exposure (8-16 kHz, 98 dB SPL). At both survivals, ANF terminals were normal in number, but 62% and 53%, respectively, lacked normal synaptic specializations. Most non-synapsing fibers (57% and 48% at 1 day and 1 week) remained in contact with an IHC and contained healthy-looking organelles. ANFs showed a transient increase in mitochondrial content (51%) and efferent innervation (34%) at 1 day. Fibers maintaining synaptic connections showed hypertrophy of pre-synaptic ribbons at both 1 day and 1 week. Non-synaptic fibers were lower in mitochondrial content and typically on the modiolar side of the IHC, where ANFs with high-thresholds and low spontaneous rates are normally found. Even 1 week post-exposure, many ANF terminals remained in IHC contact despite loss of synaptic specializations, thus, regeneration efforts at early post-exposure times should concentrate on synaptogenesis rather than neurite extension.

Ca2+ regulation of glutamate release from inner hair cells of hearing mice.

In our hearing organ, sound is encoded at ribbon synapses formed by inner hair cells (IHCs) and spiral ganglion neurons (SGNs). How the underlying synaptic vesicle (SV) release is controlled by Ca2+ in IHCs of hearing animals remained to be investigated. Here, we performed patch-clamp SGN recordings of the initial rate of release evoked by brief IHC Ca2+-influx in an ex vivo cochlear preparation from hearing mice. We aimed to closely mimic physiological conditions by perforated-patch recordings from IHCs kept at the physiological resting potential and at body temperature. We found release to relate supralinearly to Ca2+-influx (power, m: 4.3) when manipulating the [Ca2+] available for SV release by Zn2+-flicker-blocking of the single Ca2+-channel current. In contrast, a near linear Ca2+ dependence (m: 1.2 to 1.5) was observed when varying the number of open Ca2+-channels during deactivating Ca2+-currents and by dihydropyridine channel-inhibition. Concurrent changes of number and current of open Ca2+-channels over the range of physiological depolarizations revealed m: 1.8. These findings indicate that SV release requires ~4 Ca2+-ions to bind to their Ca2+-sensor of fusion. We interpret the near linear Ca2+-dependence of release during manipulations that change the number of open Ca2+-channels to reflect control of SV release by the high [Ca2+] in the Ca2+-nanodomain of one or few nearby Ca2+-channels. We propose that a combination of Ca2+ nanodomain control and supralinear intrinsic Ca2+-dependence of fusion optimally links SV release to the timing and amplitude of the IHC receptor potential and separates it from other IHC Ca2+-signals unrelated to afferent synaptic transmission.

Isolation and Culture of Primary Cochlear Hair Cells from Neonatal Mice.

Cochlear hair cells are the sensory receptors of the auditory system. These cells are located in the organ of Corti, the sensory organ responsible for hearing, within the osseous labyrinth of the inner ear. Cochlear hair cells consist of two anatomically and functionally distinct types: outer and inner hair cells. Damage to either of them results in hearing loss. Notably, as inner hair cells cannot regenerate, and damage to them is permanent. Hence, in vitro cultivation of primary hair cells is indispensable for investigating the protective or regenerative effects of cochlear hair cells. This study aimed to discover a method for isolating and cultivating mouse hair cells. After manual removal of the cochlear lateral wall, the auditory epithelium was meticulously dissected from the cochlear modiolus under a microscope, incubated in a mixture consisting of 0.25% trypsin-EDTA for 10 min at 37 °C, and gently suspended in culture medium using a 200 µL pipette tip. The cell suspension was passed through a cell filter, the filtrate was centrifuged, and cells were cultured in 24-well plates. Hair cells were identified based on their capacity to express a mechanotransduction complex, myosin-VIIa, which is involved in motor tensions, and via selective labeling of F-actin using phalloidin. Cells reached >90% confluence after 4 d in culture. This method can enhance our understanding of the biological characteristics of in vitro cultured hair cells and demonstrate the efficiency of cochlear hair cell cultures, establishing a solid methodological foundation for further auditory research.

Diversity matters - extending sound intensity coding by inner hair cells via heterogeneous synapses.

Our sense of hearing enables the processing of stimuli that differ in sound pressure by more than six orders of magnitude. How to process a wide range of stimulus intensities with temporal precision is an enigmatic phenomenon of the auditory system. Downstream of dynamic range compression by active cochlear micromechanics, the inner hair cells (IHCs) cover the full intensity range of sound input. Yet, the firing rate in each of their postsynaptic spiral ganglion neurons (SGNs) encodes only a fraction of it. As a population, spiral ganglion neurons with their respective individual coding fractions cover the entire audible range. How such "dynamic range fractionation" arises is a topic of current research and the focus of this review. Here, we discuss mechanisms for generating the diverse functional properties of SGNs and formulate testable hypotheses. We postulate that an interplay of synaptic heterogeneity, molecularly distinct subtypes of SGNs, and efferent modulation serves the neural decomposition of sound information and thus contributes to a population code for sound intensity.