Inner Hair Cell Ribbon Synapses Research Articles

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.

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.

Current Advances in Gene Therapies of Genetic Auditory Neuropathy Spectrum Disorder.

Auditory neuropathy spectrum disorder (ANSD) refers to a range of hearing impairments characterized by an impaired transmission of sound from the cochlea to the brain. This defect can be due to a lesion or defect in the inner hair cell (IHC), IHC ribbon synapse (e.g., pre-synaptic release of glutamate), postsynaptic terminals of the spiral ganglion neurons, or demyelination and axonal loss within the auditory nerve. To date, the only clinical treatment options for ANSD are hearing aids and cochlear implantation. However, despite the advances in hearing-aid and cochlear-implant technologies, the quality of perceived sound still cannot match that of the normal ear. Recent advanced genetic diagnostics and clinical audiology made it possible to identify the precise site of a lesion and to characterize the specific disease mechanisms of ANSD, thus bringing renewed hope to the treatment or prevention of auditory neurodegeneration. Moreover, genetic routes involving the replacement or corrective editing of mutant sequences or defected genes to repair damaged cells for the future restoration of hearing in deaf people are showing promise. In this review, we provide an update on recent discoveries in the molecular pathophysiology of genetic lesions, auditory synaptopathy and neuropathy, and gene-therapy research towards hearing restoration in rodent models and in clinical trials.

Optogenetics and electron tomography for structure-function analysis of cochlear ribbon synapses.

Ribbon synapses of cochlear inner hair cells (IHCs) are specialized to indefatigably transmit sound information at high rates. To understand the underlying mechanisms, structure-function analysis of the active zone (AZ) of these synapses is essential. Previous electron microscopy studies of synaptic vesicle (SV) dynamics at the IHC AZ used potassium stimulation, which limited the temporal resolution to minutes. Here, we established optogenetic IHC stimulation followed by quick freezing within milliseconds and electron tomography to study the ultrastructure of functional synapse states with good temporal resolution in mice. We characterized optogenetic IHC stimulation by patch-clamp recordings from IHCs and postsynaptic boutons revealing robust IHC depolarization and neurotransmitter release. Ultrastructurally, the number of docked SVs increased upon short (17-25 ms) and long (48-76 ms) light stimulation paradigms. We did not observe enlarged SVs or other morphological correlates of homotypic fusion events. Our results indicate a rapid recruitment of SVs to the docked state upon stimulation and suggest that univesicular release prevails as the quantal mechanism of exocytosis at IHC ribbon synapses.

MGluR7 is a presynaptic metabotropic glutamate receptor at ribbon synapses of inner hair cells.

Glutamate is the most pivotal excitatory neurotransmitter in the central nervous system. Metabotropic glutamate receptors (mGluRs) dimerize and can couple to inhibitory intracellular signal cascades, thereby protecting glutamatergic neurons from excessive excitation and cell death. MGluR7 is correlated with age-related hearing deficits and noise-induced hearing loss; however its exact localization in the cochlea is unknown. Here, we analyzed the expression and localization of mGluR7a and mGluR7b in mouse cochlear wholemounts in detail, using confocal microscopy and 3D reconstructions. We observed a presynaptic localization of mGluR7a at inner hair cells (IHCs), close to the synaptic ribbon. To detect mGluR7b, newly generated antibodies were characterized and showed co-localization with mGluR7a at IHC ribbon synapses. Compared to the number of synaptic ribbons, the numbers of mGluR7a and mGluR7b puncta were reduced at higher frequencies (48 to 64 kHz) and in older animals (6 and 12 months). Previously, we reported a presynaptic localization of mGluR4 and mGluR8b at this synapse type. This enables the possibility for the formation of homo- and/or heterodimeric receptors composed of mGluR4, mGluR7a, mGluR7b and mGluR8b at IHC ribbon synapses. These receptor complexes might represent new molecular targets suited for pharmacological concepts to protect the cochlea against noxious stimuli and excitotoxicity.

Myosin VI Haploinsufficiency Reduced Hearing Ability in Mice

In human, myosin VI (MYO6) haploinsufficiency causes postlingual progressive hearing loss. Because the usefulness of mouse models remains unclear, we produced novel Myo6 null (−/−) mutant mice and analyzed the hearing phenotypes of Myo6+/− (+/−) heterozygous mutants. We first recorded and compared the auditory brainstem responses and distortion product otoacoustic emissions in control Myo6+/+ (+/+) wild-type and +/− mice. These hearing phenotypes of +/− mice were mild; however, we confirmed that +/− mice developed progressive hearing loss. In particular, the hearing loss of female +/− mice progressed faster than that of male +/− mice. The stereocilia bundles of +/− mice exhibited progressive taper loss in cochlear inner hair cells (IHCs) and outer hair cells (OHCs). The loss of OHCs in +/− heterozygotes occurred at an earlier age than in +/+ mice. In particular, the OHCs at the basal area of the cochlea were decreased in +/− mice. IHC ribbon synapses from the area at the base of the cochlea were significantly reduced in +/− mice. Thus, our study indicated that MYO6 haploinsufficiency affected the detection of sounds in mice, and we suggest that +/− mice with Myo6 null alleles are useful animal models for gene therapy and drug treatment in patients with progressive hearing loss due to MYO6 haploinsufficiency.

Excess extracellular K+ causes inner hair cell ribbon synapse degeneration

Inner hair cell (IHC) ribbon synapses are the first synapse in the auditory system and can be degenerated by noise and aging, thereby leading to hidden hearing loss (HHL) and other hearing disorders. However, the mechanism underlying this cochlear synaptopathy remains unclear. Here, we report that elevation of extracellular K+, which is a consequence of noise exposure, could cause IHC ribbon synapse degeneration and swelling. Like intensity dependence in noise-induced cochlear synaptopathy, the K+-induced degeneration was dose-dependent, and could be attenuated by BK channel blockers. However, application of glutamate receptor (GluR) agonists caused ribbon swelling but not degeneration. In addition, consistent with synaptopathy in HHL, both K+ and noise exposure only caused IHC but not outer hair cell ribbon synapse degeneration. These data reveal that K+ excitotoxicity can degenerate IHC ribbon synapses in HHL, and suggest that BK channel may be a potential target for prevention and treatment of HHL.

Localization of group II and III metabotropic glutamate receptors at pre- and postsynaptic sites of inner hair cell ribbon synapses.

Glutamate is the major excitatory neurotransmitter in the CNS binding to a variety of glutamate receptors. Metabotropic glutamate receptors (mGluR1 to mGluR8) can act excitatory or inhibitory, depending on associated signal cascades. Expression and localization of inhibitory acting mGluRs at inner hair cells (IHCs) in the cochlea are largely unknown. Here, we analyzed expression of mGluR2, mGluR3, mGluR4, mGluR6, mGluR7, and mGluR8 and investigated their localization with respect to the presynaptic ribbon of IHC synapses. We detected transcripts for mGluR2, mGluR3, and mGluR4 as well as for mGluR7a, mGluR7b, mGluR8a, and mGluR8b splice variants. Using receptor-specific antibodies in cochlear wholemounts, we found expression of mGluR2, mGluR4, and mGluR8b close to presynaptic ribbons. Super resolution and confocal microscopy in combination with 3-dimensional reconstructions indicated a postsynaptic localization of mGluR2 that overlaps with postsynaptic density protein 95 on dendrites of afferent type I spiral ganglion neurons. In contrast, mGluR4 and mGluR8b were expressed at the presynapse close to IHC ribbons. In summary, we localized in detail 3 mGluR types at IHC ribbon synapses, providing a fundament for new therapeutical strategies that could protect the cochlea against noxious stimuli and excitotoxicity.-Klotz, L., Wendler, O., Frischknecht, R., Shigemoto, R., Schulze, H., Enz, R. Localization of group II and III metabotropic glutamate receptors at pre- and postsynaptic sites of inner hair cell ribbon synapses.

Functional alteration of ribbon synapses in inner hair cells by noise exposure causing hidden hearing loss

For decades, studies on noise-induced hearing loss have been focusing on the loss of sensory hair cells and/or auditory afferent fibers following severe noise exposure. Recently, a condition of hidden hearing loss was characterized, in which moderate noise exposure that causes only temporary threshold elevation could induce persistent reduction in auditory brainstem response (ABR) amplitudes and loss of ribbon synapses in inner hair cells (IHCs). However, it is not clear whether and how moderate noise exposure alters the functionality of surviving and/or recovering ribbon synapses in IHCs. To address this issue, we applied moderate noise exposure to mice and combined auditory systems physiology, whole-mount immunofluorescence staining and patch-clamp electrophysiology to characterize changes of ribbon synapse functions in IHCs. After the noise exposure, the ABR threshold was elevated and then recovered, while the ABR Wave I amplitude was reduced but did not recover. Coincidently, whole-mount cochlea staining revealed the loss and recovery of ribbon synapses in IHCs. We then performed whole-cell patch-clamp recording in IHCs and we found that the Ca2+ current, the sustained exocytosis of synaptic vesicles, and the replenishment of synaptic vesicles were all significantly reduced one day after the noise exposure. Fourteen days after the noise exposure, however, only the sustained exocytosis failed to recover, and further examination revealed that this persistent reduction is due to a decrease in the Ca2+ efficiency of triggering exocytosis. In conclusion, our results suggest temporary and persistent alterations of ribbon synapse functions in IHCs contribute to the hidden hearing loss.

Disruption of Otoferlin Alters the Mode of Exocytosis at the Mouse Inner Hair Cell Ribbon Synapse.

Sound encoding relies on Ca2+-mediated exocytosis at the ribbon synapse between cochlear inner hair cells (IHCs) and type I spiral ganglion neurons (SGNs). Otoferlin, a multi-C2 domain protein, is proposed to regulate Ca2+-triggered exocytosis at this synapse, but the precise mechanisms of otoferlin function remain to be elucidated. Here, performing whole-cell voltage-clamp recordings of excitatory postsynaptic currents (EPSCs) from SGNs in otoferlin mutant mice, we investigated the impact of Otof disruption at individual synapses with single release event resolution. Otof deletion decreased the spontaneous release rate and abolished the stimulus-secretion coupling. This was evident from failure of potassium-induced IHC depolarization to stimulate release and supports the proposed role of otoferlin in Ca2+ sensing for fusion. A missense mutation in the Otof gene (pachanga), in which otoferlin level at the IHC plasma membrane was lowered without changing its Ca2+ binding, also reduced the spontaneous release rate but spared the stimulus-secretion coupling. The slowed stimulated release rate supports the hypothesis that a sufficient abundance of otoferlin at the plasma membrane is crucial for the vesicle supply. Large-sized monophasic EPSCs remained present upon Otof deletion despite the drastic reduction of the rate of exocytosis. However, EPSC amplitude, on average, was modestly decreased. Moreover, a reduced contribution of multiphasic EPSC was observed in both Otof mutants. We argue that the presence of large monophasic EPSCs despite the exocytic defect upon Otof deletion supports the uniquantal hypothesis of transmitter release at the IHC ribbon synapse. Based upon the reduced contribution of multiphasic EPSC, we propose a role of otoferlin in regulating the mode of exocytosis in IHCs.

Long‑term treatment with salicylate enables NMDA receptors and impairs AMPA receptors in C57BL/6J mice inner hair cell ribbon synapse.

Salicylate is widely used to produce animal models of tinnitus in mice and/or rats. The side effects on auditory function, including hearing loss and tinnitus, are considered the results of the auditory nerve dysfunction. A recent study indicated that chronic treatment with salicylate for several weeks reduces compressed action potential amplitude, which is contradictory to the studies reporting excessive activation of N-methyl-D-aspartate receptors (NMDAR) in tinnitus-induced animals. The specific aims of the experiment were to detect the effect of salicylate on the inner hair cells (IHCs), ribbon synapse, as well as the association between the hearing threshold and the number of mismatched ribbon synapses. In the present study, mice were injected intraperitoneally with a low dose of salicylate (200 mg/kg) for 14 days. The auditory brainstem response and otoacoustic emission were measured to assess auditory function of the mice. The postsynaptic regions of IHC were identified with two types of immunostaining targets: Postsynaptic density protein 95 and Glu2/3. The number of spheres was counted and the synapses were reconstructed in 3-dimensional images. Increases in distortion product otoacoustic emissions amplitudes of the salicylate group were detected, however, an elevation in the hearing threshold was also observed. A mismatch between pre-and post-ribbon synapses was observed. In addition, the cochlear components, including the numbers of outer hair cells and IHCs, were unlikely to be affected by salicylate. IHC ribbon synapses were more susceptible to salicylate stimuli. Furthermore, mismatch of pre- and post-ribbon synapses may indicate a competitive inhibition between NMDAR and α-amino-3-hydroxy-5-methyl-4-isoxa-zole-propionate receptors and dysfunction of ribbon synapses.

N-Methyl-D-Aspartate Receptors Involvement in the Gentamicin-Induced Hearing Loss and Pathological Changes of Ribbon Synapse in the Mouse Cochlear Inner Hair Cells.

Cochlear inner hair cell (IHC) ribbon synapses play an important role in sound encoding and neurotransmitter release. Previous reports show that both noise and aminoglycoside exposures lead to reduced numbers and morphologic changes of synaptic ribbons. In this work, we determined the distribution of N-methyl-D-aspartate receptors (NMDARs) and their role in the gentamicin-induced pathological changes of cochlear IHC ribbon synaptic elements. In normal mature mouse cochleae, the majority of NMDARs were distributed on the modiolar side of IHCs and close to the IHC nuclei region, while most of synaptic ribbons and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) were located on neural terminals closer to the IHC basal poles. After gentamicin exposure, the NMDARs increased and moved towards the IHC basal poles. At the same time, synaptic ribbons and AMPARs moved toward the IHC bundle poles on the afferent dendrites. The number of ribbon synapse decreased, and this was accompanied by increased auditory brainstem response thresholds and reduced wave I amplitudes. NMDAR antagonist MK801 treatment reduced the gentamicin-induced hearing loss and the pathological changes of IHC ribbon synapse, suggesting that NMDARs were involved in gentamicin-induced ototoxicity by regulating the number and distribution of IHC ribbon synapses.

Otoferlin acts as a Ca2+ sensor for vesicle fusion and vesicle pool replenishment at auditory hair cell ribbon synapses.

Hearing relies on rapid, temporally precise, and sustained neurotransmitter release at the ribbon synapses of sensory cells, the inner hair cells (IHCs). This process requires otoferlin, a six C2-domain, Ca2+-binding transmembrane protein of synaptic vesicles. To decipher the role of otoferlin in the synaptic vesicle cycle, we produced knock-in mice (OtofAla515,Ala517/Ala515,Ala517) with lower Ca2+-binding affinity of the C2C domain. The IHC ribbon synapse structure, synaptic Ca2+ currents, and otoferlin distribution were unaffected in these mutant mice, but auditory brainstem response wave-I amplitude was reduced. Lower Ca2+ sensitivity and delay of the fast and sustained components of synaptic exocytosis were revealed by membrane capacitance measurement upon modulations of intracellular Ca2+ concentration, by varying Ca2+ influx through voltage-gated Ca2+-channels or Ca2+ uncaging. Otoferlin thus functions as a Ca2+ sensor, setting the rates of primed vesicle fusion with the presynaptic plasma membrane and synaptic vesicle pool replenishment in the IHC active zone.

Mechanisms of synaptic depression at the hair cell ribbon synapse that support auditory nerve function

Inner hair cells (IHCs) in the cochlea are the mammalian phono-receptors, transducing sound energy into graded changes in membrane potentials, the so called "receptor potentials." Ribbon synapses between IHCs and auditory nerve neurons are responsible for converting receptor potentials into spike rates. The characteristics of auditory nerve responses to sound have been described extensively. For instance, persistent acoustic stimulation produces sensory adaptation, which is revealed as a reduction in neuronal spike rate with time constants in the range of milliseconds to seconds. Since the amplitude of IHC receptor potentials is invariant during this period, the classic hypothesis pointed to vesicle depletion at the IHC as responsible for auditory adaptation. In this study, we observed that fast synaptic depression occurred in responses to stimuli of varying intensities. Nevertheless, release continued after this initial depression, via synaptic vesicles with slower exocytotic kinetics. Heterogeneity in kinetic elements, therefore, favored synaptic responses with an early peak and a sustained phase. The application of cyclothiazide (CTZ) revealed that desensitization of postsynaptic receptors contributed to synaptic depression, which was more pronounced during stronger stimulation. Thus, desensitization had a twofold effect: It abbreviated signaling between IHC and the auditory nerve and also balanced differences in decay kinetics between responses to different stimulation strengths. We therefore propose that both pre- and postsynaptic mechanisms at the IHC ribbon synapse contribute to synaptic depression at the IHC ribbon synapse and spike rate adaptation in the auditory nerve.

Conditional deletion of pejvakin in adult outer hair cells causes progressive hearing loss in mice

Mutations in the Pejvakin (Pjvk) gene cause autosomal recessive hearing loss DFNB59 with audiological features of auditory neuropathy spectrum disorder (ANSD) or cochlear dysfunction. The precise mechanisms underlying the variable clinical phenotypes of DFNB59 remain unclear. Here, we demonstrate that mice with conditional ablation of the Pjvk gene in all sensory hair cells or only in outer hair cells (OHCs) show similar auditory phenotypes with early-onset profound hearing loss. By contrast, loss of Pjvk in adult OHCs causes a slowly progressive hearing loss associated with OHC degeneration and delayed loss of inner hair cells (IHCs), indicating a primary role for pejvakin in regulating OHC function and survival. Consistent with this model, synaptic transmission at the IHC ribbon synapse is largely unaffected in sirtaki mice that carry a C-terminal deletion mutation in Pjvk. Using the C-terminal domain of pejvakin as bait, we identified in a cochlear cDNA library ROCK2, an effector for the small GTPase Rho, and the scaffold protein IQGAP1, involved in modulating actin dynamics. Both ROCK2 and IQGAP1 associate via their coiled-coil domains with pejvakin. We conclude that pejvakin is required to sustain OHC activity and survival in a cell-autonomous manner likely involving regulation of Rho signaling.

Auditory neuropathy--neural and synaptic mechanisms.

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.

Fine Tuning of CaV1.3 Ca2+ channel properties in adult inner hair cells positioned in the most sensitive region of the Gerbil Cochlea.

Hearing relies on faithful signal transmission by cochlear inner hair cells (IHCs) onto auditory fibres over a wide frequency and intensity range. Exocytosis at IHC ribbon synapses is triggered by Ca2+ inflow through CaV1.3 (L-type) Ca2+ channels. We investigated the macroscopic (whole-cell) and elementary (cell-attached) properties of Ca2+ currents in IHCs positioned at the middle turn (frequency ∼2 kHz) of the adult gerbil cochlea, which is their most sensitive hearing region. Using near physiological recordings conditions (body temperature and a Na+ based extracellular solution), we found that the macroscopic Ca2+ current activates and deactivates very rapidly (time constant below 1 ms) and inactivates slowly and only partially. Single-channel recordings showed an elementary conductance of 15 pS, a sub-ms latency to first opening, and a very low steady-state open probability (P o: 0.024 in response to 500-ms depolarizing steps at ∼−18 mV). The value of P o was significantly larger (0.06) in the first 40 ms of membrane depolarization, which corresponds to the time when most Ca2+ channel openings occurred clustered in bursts (mean burst duration: 19 ms). Both the P o and the mean burst duration were smaller than those previously reported in high-frequency basal IHCs. Finally, we found that middle turn IHCs are likely to express about 4 times more Ca2+ channels per ribbon than basal cells. We propose that middle-turn IHCs finely-tune CaV1.3 Ca2+ channel gating in order to provide reliable information upon timing and intensity of lower-frequency sounds.

C-Terminal Modulation of Cav1.3 L-Type Calcium Channels Modifies their Gating Properties in Cochlear Inner Hair Cells

The Ca2+ currents in inner hair cells (IHCs) are crucial for synaptic transmission and flow through voltage-gated calcium channels (VGCCs) formed by the α1 subunit Cav1.3. VGCCs exhibit a calmodulin (CaM) mediated calcium-dependent inactivation (CDI), via binding CAM to the channel's C-terminus. In IHCs, Cav1.3 exhibits unusually weak CDI, probably caused by calcium binding proteins (CaBP) competing with CaM. IHCs express long and short Cav1.3 splice variants either including (long variant, Cav1.3L) or excluding (short variants) a C-terminal modulatory domain. In expression systems - lacking CaBPs - the C-terminal modulatory mechanism (CTM) functions via intramolecular interaction of a proximal (PCRD) and a distal C-terminal regulatory domain (DCRD) by inhibiting CaM binding near the PCRD, thereby inhibiting CaM-mediated CDI (Bock et al., JBC 2011). Here, the role of the CTM for IHC VGCCs was investigated in Cav1.3L-DCRDHA/HA mice in which CTM was disrupted by partial replacement of the DCRD with an HA tag.Localization of HA-tagged Cav1.3 channels in IHCs was determined by immunohistochemistry. Channel properties were investigated by whole-cell patch-clamp recordings. Hearing was assessed using auditory brainstem responses (ABR) and distortion products of otoacoustic emissions (DPOAE).Anti-HA immunolabeling was present at all IHC ribbons. Patch-clamp recordings revealed significantly reduced CDI and increased amplitudes of Ca2+ and Ba2+ currents in Cav1.3L-DCRDHA/HA IHCs. Non-stationary fluctuation analysis showed unchanged numbers of Cav1.3 channels and single channel currents. Voltage dependence and activation kinetics of ICa and IBa, ABR thresholds and DPOAEs were unaffected.Our data demonstrate that the long Cav1.3 isoform is an intrinsic component of Cav1.3 clusters at all IHC ribbon synapses and that its DCRD is required for normal CDI and ICa amplitude.Funding:Austrian Science Fund (SFB-F4402), EC-project MRTN-CT-2006-35367 (“CavNet”), Innsbruck University, DFG (SFB-894)

Cholinergic efferent synaptic transmission regulates the maturation of auditory hair cell ribbon synapses

Spontaneous electrical activity generated by developing sensory cells and neurons is crucial for the maturation of neural circuits. The full maturation of mammalian auditory inner hair cells (IHCs) depends on patterns of spontaneous action potentials during a ‘critical period’ of development. The intrinsic spiking activity of IHCs can be modulated by inhibitory input from cholinergic efferent fibres descending from the brainstem, which transiently innervate immature IHCs. However, it remains unknown whether this transient efferent input to developing IHCs is required for their functional maturation. We used a mouse model that lacks the α9-nicotinic acetylcholine receptor subunit (α9nAChR) in IHCs and another lacking synaptotagmin-2 in the efferent terminals to remove or reduce efferent input to IHCs, respectively. We found that the efferent system is required for the developmental linearization of the Ca2+-sensitivity of vesicle fusion at IHC ribbon synapses, without affecting their general cell development. This provides the first direct evidence that the efferent system, by modulating IHC electrical activity, is required for the maturation of the IHC synaptic machinery. The central control of sensory cell development is unique among sensory systems.

Burst activity and ultrafast activation kinetics of CaV1.3 Ca2+ channels support presynaptic activity in adult gerbil hair cell ribbon synapses

Auditory information transfer to afferent neurons relies on precise triggering of neurotransmitter release at the inner hair cell (IHC) ribbon synapses by Ca2+ entry through CaV1.3 Ca2+ channels. Despite the crucial role of CaV1.3 Ca2+ channels in governing synaptic vesicle fusion, their elementary properties in adult mammals remain unknown. Using near-physiological recording conditions we investigated Ca2+ channel activity in adult gerbil IHCs. We found that Ca2+ channels are partially active at the IHC resting membrane potential (−60 mV). At −20 mV, the large majority (>70%) of Ca2+ channel first openings occurred with an estimated delay of about 50 μs in physiological conditions, with a mean open time of 0.5 ms. Similar to other ribbon synapses, Ca2+ channels in IHCs showed a low mean open probability (0.21 at −20 mV), but this increased significantly (up to 0.91) when Ca2+ channel activity switched to a bursting modality. We propose that IHC Ca2+ channels are sufficiently rapid to transmit fast signals of sound onset and support phase-locking. Short-latency Ca2+ channel opening coupled to multivesicular release would ensure precise and reliable signal transmission at the IHC ribbon synapse.