In pre-hearing mice, vesicle exocytosis at cochlear inner hair cell (IHC) ribbon synapses is triggered by spontaneous Ca2+ spikes. At the onset of hearing, IHC exocytosis is then exclusively driven by graded potentials, and is characterized by higher Ca2+ efficiency and improved synchronization of vesicular release. The molecular players involved in this transition are still unknown. Here we addressed the involvement of synaptotagmins and otoferlin as putative Ca2+ sensors in IHC exocytosis during postnatal maturation of the cochlea. Using cell capacitance measurements, we showed that Ca2+-evoked exocytosis in mouse IHCs switches from an otoferlin-independent to an otoferlin-dependent mechanism at postnatal day 4. During this early exocytotic period, several synaptotagmins (Syts), including Syt1, Syt2 and Syt7, were detected in IHCs. The exocytotic response as well as the release of the readily releasable vesicle pool (RRP) was, however, unchanged in newborn mutant mice lacking Syt1, Syt2 or Syt7 (Syt1−/−,Syt2−/− and Syt7−/− mice). We only found a defect in RRP recovery in Syt1−/− mice which was apparent as a strongly reduced response to repetitive stimulations. In post-hearing Syt2−/− and Syt7−/− mutant mice, IHC synaptic exocytosis was unaffected. The transient expression of Syt1 and Syt2, which were no longer detected in IHCs after the onset of hearing, indicates that these two most common Ca2+-sensors in CNS synapses are not involved in mature IHCs. We suggest that otoferlin underlies highly efficient Ca2+-dependent membrane-membrane fusion, a process likely essential to increase the probability and synchrony of vesicle fusion events at the mature IHC ribbon synapse.

Immature cochlear outer hair cells (OHCs) make transient synaptic contacts (ribbon synapses) with type I afferent nerve fibers, but direct evidence of synaptic vesicle exocytosis is still missing. We thus investigated calcium-dependent exocytosis in murine OHCs at postnatal day 2 (P2)-P3, a developmental stage when calcium current maximum amplitude was the highest. By using time-resolved patch-clamp capacitance measurements, we show that voltage step activation of L-type calcium channels triggers fast membrane capacitance increase. Capacitance increase displayed two kinetic components, which are likely to reflect two functionally distinct pools of synaptic vesicles, a readily releasable pool (RRP; ϭ 79 ms) and a slowly releasable pool ( ϭ 870 ms). The RRP size and maximal release rate were estimated at ϳ1200 vesicles and ϳ15,000 vesicles/s, respectively. In addition, we found a linear relationship between capacitance increase and calcium influx, like in mature inner hair cells (IHCs). These results give strong support to the existence of efficient calcium-dependent neurotransmitter release in immature OHCs. Moreover, we show that immature OHCs, just like immature IHCs, are able to produce regenerative calcium-dependent action potentials that could trigger synaptic exocytosis in vivo. Finally, the evoked membrane capacitance increases were abolished in P2-P3 OHCs from mutant Otof Ϫ/Ϫ mice defective for otoferlin, despite normal calcium currents. We conclude that otoferlin, the putative major calcium sensor at IHC ribbon synapses, is essential to synaptic exocytosis in immature OHCs too.

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The hair cell ribbon synapses of the mammalian auditory and vestibular systems differ greatly in their anatomical organization and firing properties. Notably, vestibular Type I hair cells (VHC-I) are surrounded by a single calyx-type afferent terminal that receives input from several ribbons, whereas cochlear inner hair cells (IHCs) are contacted by several individual afferent boutons, each facing a single ribbon. The specificity of the presynaptic molecular mechanisms regulating transmitter release at these different sensory ribbon synapses is not well understood. Here, we found that exocytosis during voltage activation of Ca 2ϩ channels displayed higher Ca 2ϩ sensitivity, 10 mV more negative half-maximum activation, and a smaller dynamic range in VHC-I than in IHCs. VHC-I had a larger number of Ca

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Transmitter release at auditory inner hair cell (IHC) ribbon synapses involves exocytosis of glutamatergic vesicles during voltage activation of L-type Ca v 1.3 calcium channels. At these synapses, the fast and indefatigable release of synaptic vesicles by IHCs is controlled by otoferlin, a six-C2-domain (C2-ABCDEF) protein that functions as a high-affinity Ca 2ϩ sensor. The molecular events by which each otoferlin C2 domain contributes to the regulation of the synaptic vesicle cycle in IHCs are still incompletely understood. Here, we investigate their role using a cochlear viral cDNA transfer approach in vivo, where IHCs of mouse lacking otoferlin (Otof Ϫ/Ϫ mice of both sexes) were virally transduced with cDNAs of various mini-otoferlins. Using patch-clamp recordings and membrane capacitance measurements, we show that the viral transfer of mini-otoferlin containing C2-ACEF, C2-EF, or C2-DEF partially restores the fast exocytotic component in Otof Ϫ/Ϫ mouse IHCs. The restoration was much less efficient with C2-ACDF, underlining the importance of the C2-EF domain. None of the mini-otoferlins tested restored the sustained component of vesicle release, explaining the absence of hearing recovery. The restoration of the fast exocytotic component in the transduced Otof Ϫ/Ϫ IHCs was also associated with a recovery of Ca 2ϩ currents with normal amplitude and fast time inactivation, confirming that the C-terminal C2 domains of otoferlin are essential for normal gating of Ca v 1.3 channels. Finally, the reintroduction of the mini-otoferlins C2-EF, C2-DEF, or C2-ACEF allowed us to uncover and characterize for the first time a dynamin-dependent ultrafast endocytosis in IHCs.

We show that a cage-shaped F-actin network is essential for maintaining a tight spatial organization of Cav1.3 Ca2+ channels at the synaptic ribbons of auditory inner hair cells. This F-actin network is also found to provide mechanosensitivity to the Cav1.3 channels when varying intracellular hydrostatic pressure. Furthermore, this F-actin mesh network attached to the synaptic ribbons directly influences the efficiency of otoferlin-dependent exocytosis and its sensitivity to intracellular hydrostatic pressure, independently of its action on the Cav1.3 channels. We propose a new mechanistic model for vesicle exocytosis in auditory hair cells where the rate of vesicle recruitment to the ribbons is directly controlled by a synaptic F-actin network and changes in intracellular hydrostatic pressure.DOI: http://dx.doi.org/10.7554/eLife.10988.001

SUMMARY A Ca2+ current transient block (ICaTB) by protons occurs at some ribbon-type synapses after exocytosis, but this has not been observed at mammalian hair cells. Here we show that a robust ICaTB occurs at post-hearing mouse and gerbil inner hair cell (IHC) synapses, but not in immature IHC synapses, which contain non-compact active zones, where Ca2+ channels are loosely coupled to the release sites. Unlike ICaTB at other ribbon synapses, ICaTB in mammalian IHCs displays a surprising multi-peak structure that mirrors the EPSCs seen in paired recordings. Desynchronizing vesicular release with intracellular BAPTA or by deleting otoferlin, the Ca2+ sensor for exocytosis, greatly reduces ICaTB, whereas enhancing release synchronization by raising Ca2+ influx or temperature increases ICaTB. This suggests that ICaTB is produced by fast multivesicular proton-release events. We propose that ICaTB may function as a submillisecond feedback mechanism contributing to the auditory nerve’s fast spike adaptation during sound stimulation.