We studied the role of the synaptic ribbon for sound encoding at the synapses between inner hair cells (IHCs) and spiral ganglion neurons (SGNs) in mice lacking RIBEYE (RBE^KO/KO). Electron and immunofluorescence microscopy revealed a lack of synaptic ribbons and an assembly of several small active zones (AZs) at each synaptic contact. Spontaneous and sound-evoked firing rates of SGNs and their compound action potential were reduced, indicating impaired transmission at ribbonless IHC-SGN synapses. The temporal precision of sound encoding was impaired and the recovery of SGN-firing from adaptation indicated slowed synaptic vesicle (SV) replenishment. Activation of Ca²⁺-channels was shifted to more depolarized potentials and exocytosis was reduced for weak depolarizations. Presynaptic Ca²⁺-signals showed a broader spread, compatible with the altered Ca²⁺-channel clustering observed by super-resolution immunofluorescence microscopy. We postulate that RIBEYE disruption is partially compensated by multi-AZ organization. The remaining synaptic deficit indicates ribbon function in SV-replenishment and Ca²⁺-channel regulation.

Significance: Hearing relies on temporally precise and indefatigable transmission of sensory information mediated by inner hair cell (IHC) ribbon synapses. Synaptic ribbons are electron-dense structures that tether numerous vesicles at the active zone (AZ) and drive neural encoding of acoustical signals by neurotransmitter release. The mechanisms of ribbon formation, AZ targeting and subsequent functional maturation in murine IHCs remain elusive. By combining various high-resolution imaging techniques, we analyzed multiple developmental stages and provide a new hypothesis for the assembly of auditory ribbon synapses from the apical cochlear turn. Our findings suggest that fusion of presynaptic ribbons contributes to the morphological AZ development that is essential for the functional maturation of afferent synaptic transmission within the cochlea.

Significance: Sound encoding relies on Ca ²⁺ -regulated transmitter release from inner hair cells (IHCs). Here we demonstrate a role of Ras-related in brain 3 (Rab3)–interacting molecule 2 (RIM2) in Ca ²⁺ channel-clustering and vesicle-tethering at the active zones of IHCs. Active zones of RIM2α-deficient IHCs cluster fewer synaptic voltage-gated Ca _V 1.3 Ca ²⁺ channels, resulting in reduced synaptic Ca ²⁺ influx. Exocytosis was diminished in RIM2α-deficient IHCs, likely contributing to the mild hearing impairment of RIM2α knockout mice. Hair cell-specific disruption of all RIM2 isoforms caused a stronger decrease of Ca ²⁺ current and exocytosis in IHCs and impaired the encoding of sound onset in spiral ganglion neurons. We conclude that RIM2α and RIM2β promote synaptic clustering of Ca ²⁺ channels at IHC active zones and are required for normal hearing.

Synaptic vesicle recycling sustains high rates of neurotransmission at the ribbon-type active zones (AZs) of mouse auditory inner hair cells (IHCs), but its modes and molecular regulation are poorly understood. Electron microscopy indicated the presence of clathrin-mediated endocytosis (CME) and bulk endocytosis. The endocytic proteins dynamin, clathrin, and amphiphysin are expressed and broadly distributed in IHCs. We used confocal vglut1–pHluorin imaging and membrane capacitance (C_m) measurements to study the spatial organization and dynamics of IHC exocytosis and endocytosis. Viral gene transfer expressed vglut1–pHluorin in IHCs and targeted it to synaptic vesicles. The intravesicular pH was ∼6.5, supporting only a modest increase of vglut1–pHluorin fluorescence during exocytosis and pH neutralization. Ca²⁺influx triggered an exocytic increase of vglut1–pHluorin fluorescence at the AZs, around which it remained for several seconds. The endocyticC_mdecline proceeded with constant rate (linear component) after exocytosis of the readily releasable pool (RRP). When exocytosis exceeded three to four RRP equivalents, IHCs additionally recruited a fasterC_mdecline (exponential component) that increased with the amount of preceding exocytosis and likely reflects bulk endocytosis. The dynamin inhibitor Dyngo-4a and the clathrin blocker pitstop 2 selectively impaired the linear component of endocyticC_mdecline. A missense mutation of dynamin 1 (fitful) inhibited endocytosis to a similar extent as Dyngo-4a. We propose that IHCs use dynamin-dependent endocytosis via CME to support vesicle cycling during mild stimulation but recruit bulk endocytosis to balance massive exocytosis.