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In vivo gene delivery to tissues using adeno-associated vector (AAVs) has revolutionized the field of gene therapy. Yet, while sensorineural hearing loss is one of the most common sensory disorders worldwide, gene therapy applied to the human inner ear is still in its infancy. Recent advances in the development recombinant AAVs have significantly improved their cell tropism and transduction efficiency across diverse inner ear cell types to a level that renders this tool valuable for conditionally manipulating gene expression in the context of developmental biology studies of the mouse inner ear. Here, we describe a protocol for in utero micro-injection of AAVs into the embryonic inner ear, using the AAV-PHP.eB and AAV-DJ serotypes that respectively target the sensory hair cells and the supporting cells of the auditory sensory epithelium. We also aimed to standardize procedures for imaging acquisition and image analysis to foster research reproducibility and allow accurate comparisons between studies. We find that AAV-PHP.eB and AAV-DJ provide efficient and reliable tools for conditional gene expression targeting cochlear sensory and supporting cells in the mouse inner ear, from late embryonic stages on. [ABSTRACT FROM AUTHOR]

Clarin-1, a tetraspan-like membrane protein defective in Usher syndrome type IIIA (USH3A), is essential for hair bundle morphogenesis in auditory hair cells. We report a new synaptic role for clarin-1 in mouse auditory hair cells elucidated by characterization of Clrn1 total ([Clrn1.sup.ex4-/-]) and postnatal hair cell- specific conditional ([Clrn1.sup.ex4fl/fl] [Myo15-Cre.sup.+/-) knockout mice. [Clrn1.sup.ex4-/-] mice were profoundly deaf, whereas [Clrn1.sup.ex4fl/fl] [Myo15-Cre.sup.+/-] mice displayed progressive increases in hearing thresholds, with, initially, normal otoacoustic emissions and hair bundle morphology. Inner hair cell (IHC) patch-clamp recordings for the 2 mutant mice revealed defective exocytosis and a disorganization of synaptic F-actin and [Ca.sub.V]1.3 [Ca.sup.2+] channels, indicative of a synaptopathy. Postsynaptic defects were also observed, with an abnormally broad distribution of AMPA receptors associated with a loss of afferent dendrites and defective electrically evoked auditory brainstem responses. Protein-protein interaction assays revealed interactions between clarin-1 and the synaptic [Ca.sub.V]1.3 [Ca.sup.2+] channel complex via the [Ca.sub.V][[beta].sub.2] auxiliary subunit and the PDZ domain-containing protein harmonin (defective in Usher syndrome type IC). Cochlear gene therapy in vivo, through adeno-associated virus-mediated Clrn1 transfer into hair cells, prevented the synaptic defects and durably improved hearing in [Clrn1.sup.ex4fl/fl] [Myo15-Cre.sup.+/-] mice. Our results identify clarin-1 as a key organizer of IHC ribbon synapses, and suggest new treatment possibilities for USH3A patients., Introduction Usher syndrome type IIIA (USH3A), characterized by progressive hearing loss and retinitis pigmentosa, is caused by mutations in CLRN1 encoding clarin-1 (refs. 1, 2, and Figure 1A). CLRN1 transcripts [...]

Otoferlin, a predicted C2-domain transmembrane protein, which is defective in a recessive form of human deafness, is studied. It shows that otoferlin expression in the hair cells correlates with afferent synaptogenesis and otoferlin localizes to ribbon-associated synaptic vesicles.

Mutations in the potassium channel gene KCNQ4 underlie DFNA2, an autosomal dominant form of progressive hearing loss in humans. In the mouse cochlea, the transcript has been found exclusively in the outer hair cells. By using specific antibodies, we now show that KCNQ4 is situated at the basal membrane of these sensory cells. In the vestibular organs, KCNQ4 is restricted to the type I hair cells and the afferent calyx-like nerve endings ensheathing these sensory cells. Several lines of evidence suggest that KCNQ4 underlies the [I.sub.K,n] and [g.sub.K,L] currents that have been described in the outer and type I hair cells, respectively, and that are already open at resting potentials. KCNQ4 is also expressed in neurons of many, but not all, nuclei of the central auditory pathway, and is absent from most other brain regions. It is present, e.g., in the cochlear nuclei, the nuclei of the lateral lemniscus, and the inferior colliculus. This is the first ion channel shown to be specifically expressed in a sensory pathway. Moreover, the expression pattern of KCNQ4 in the mouse auditory system raises the possibility of a central component in the DFNA2 hearing loss.

The mechanisms orchestrating transient and sustained exocytosis in auditory inner hair cells (IHCs) remain largely unknown. These exocytotic responses are believed to mobilize sequentially a readily releasable pool of vesicles (RRP) underneath the synaptic ribbons and a slowly releasable pool of vesicles (SRP) at farther distance from them. They are both governed by Cav1.3 channels and require otoferlin as Ca2+ sensor, but whether they use the same Cav1.3 isoforms is still unknown. Using whole-cell patch-clamp recordings in posthearing mice, we show that only a proportion (~25%) of the total Ca2+ current in IHCs displaying fast inactivation and resistance to 20 µM nifedipine, a L-type Ca2+ channel blocker, is sufficient to trigger RRP but not SRP exocytosis. This Ca2+ current is likely conducted by short C-terminal isoforms of Cav1.3 channels, notably Cav1.342A and Cav1.343S, because theirmRNAis highly expressed in wild-type IHCs but poorly expressed in Otof-/- IHCs, the latter having Ca2+ currents with considerably reduced inactivation. Nifedipine-resistant RRP exocytosis was poorly affected by 5 mM intracellular EGTA, suggesting that the Cav1.3 short isoforms are closely associated with the release site at the synaptic ribbons. Conversely, our results suggest that Cav1.3 long isoforms, which carry ~75% of the total IHC Ca2+ current with slow inactivation and confer high sensitivity to nifedipine and to internal EGTA, are essentially involved in recruiting SRP vesicles. Intracellular Ca2+ imaging showed that Cav1.3 long isoforms support a deep intracellular diffusion of Ca2+. [ABSTRACT FROM AUTHOR]

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 Ca² channels displayed higher Ca² 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 Ca² channels per ribbon (158 vs 110 in IHCs), but their Ca² current density was twofold smaller because of a smaller open probability and unitary conductance. Using confocal and stimulated emission depletion immunofluorescence microscopy, we showed that VHC-I had fewer synaptic ribbons (7 vs 17 in IHCs) to which Cavl.3 channels are more tightly organized than in IHCs. Gradual intracellular Ca² uncaging experiments revealed that exocytosis had a similar intrinsic Ca² sensitivity in both VHC-I and IHCs (KD of 3.3 ± 0.6 μM and 4.0 ± 0.7 μM, respectively). In otoferlin-deficient mice, exocytosis was largely reduced in VHC-I and IHCs. We conclude that VHC-I and IHCs use a similar micromolar-sensitive otoferlin Ca² sensor and that their sensory encoding specificity is essentially determined by a different functional organization of Ca² channels at their synaptic ribbons. [ABSTRACT FROM AUTHOR]

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 Syti, 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 Syti, Syt2 or Syt7 (Syti -/ -, Syt2 -/ - and Syt7-1- mice). We only found a defect in RRP recovery in Syti 7 -1- mice which was apparent as a strongly reduced response to repetitive stimulations. In post-hearing Syt2+ and Syt7 -1- mutant mice, IHC synaptic exocytosis was unaffected. The transient expression of Syti 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 Ca 2k-dependent membrane-membrane fusion, a process likely essential to increase the probability and synchrony of vesicle fusion events at the mature IHC ribbon synapse. [ABSTRACT FROM AUTHOR]

Otoferlin, a C2-domain-containing Ca2+ binding protein, is required for synaptic exocytosis in auditory hair cells. However, its exact role remains essentially unknown. Intriguingly enough, no balance defect has been observed in otoferlin-deficient (Otoƒ-/-) mice. Here, we show that the vestibular nerve compound action potentials evoked during transient linear acceleration ramps in Otoƒ-/- mice display higher threshold, lower amplitude, and increased latency compared with wild-type mice. Using patch-clamp capacitance measurement in intact utricles, we show that type I and type II hair cells display a remarkable linear transfer function between Ca2+ entry, flowing through voltage-activated Ca2+channels,andexocytosis. This linear Ca2+ dependencewasobservedwhenchanging the Ca2+ channel open probability or the Ca2+ flux per channel during various test potentials. In Otoƒ-/- hair cells, exocytosis displays slower kinetics, reduced Ca2+ sensitivity, and nonlinear Ca2+ dependence, despite morphologically normal synapses and normal Ca2+ currents. We conclude that otoferlin is essential for a high-affinity Ca2+ sensor function that allows efficient and linear encoding of low-intensity stimuli at the vestibular hair cell synapse. [ABSTRACT FROM AUTHOR]