Cochlear inner hair cells (IHCs) develop from pre-sensory pacemaker to sound transducer. Here, we report that this involves changes in structure and function of the ribbon synapses between IHCs and spiral ganglion neurons (SGNs) around hearing onset in mice. As synapses matured they changed from holding several small presynaptic active zones (AZs) and apposed postsynaptic densities (PSDs) to one large AZ/PSD complex per SGN bouton. After the onset of hearing (i) IHCs had fewer and larger ribbons; (ii) Ca V 1.3 channels formed stripe-like clusters rather than the smaller and round clusters at immature AZs; (iii) extrasynaptic Ca V 1.3-channels were selectively reduced, (iv) the intrinsic Ca 2+ dependence of fast exocytosis probed by Ca 2+ uncaging remained unchanged but (v) the apparent Ca 2+ dependence of exocytosis linearized, when assessed by progressive dihydropyridine block of Ca 2+ influx. Biophysical modeling of exocytosis at mature and immature AZ topographies suggests that Ca 2+ influx through an individual channel dominates the [Ca 2+ ] driving exocytosis at each mature release site. We conclude that IHC synapses undergo major developmental refinements, resulting in tighter spatial coupling between Ca 2+ influx and exocytosis.

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 (RBEKO/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.

Ribbon synapses of cochlear inner hair cells (IHCs) undergo molecular assembly and extensive functional and structural maturation before hearing onset. Here, we characterized the nanostructure of IHC synapses from late prenatal mouse embryo stages (embryonic days 14-18) into adulthood [postnatal day (P)48] using electron microscopy and tomography as well as optical nanoscopy of apical turn organs of Corti. We find that synaptic ribbon precursors arrive at presynaptic active zones (AZs) after afferent contacts have been established. These ribbon precursors contain the proteins RIBEYE and piccolino, tether synaptic vesicles and their delivery likely involves active, microtubule-based transport pathways. Synaptic contacts undergo a maturational transformation from multiple small to one single, large AZ. This maturation is characterized by the fusion of ribbon precursors with membraneanchored ribbons that also appear to fuse with each other. Such fusion events are most frequently encountered around P12 and hence, coincide with hearing onset in mice. Thus, these events likely underlie the morphological and functional maturation of the AZ. Moreover, the postsynaptic densities appear to undergo a similar refinement alongside presynaptic maturation. Blockwise addition of ribbon material by fusion as found during AZ maturation might represent a general mechanism for modulating ribbon size. synaptogenesis | presynaptic development | ribbon synapse maturation | synaptic heterogeneity I n mammals, synaptic sound encoding occurs at the first auditory synapse between cochlear inner hair cells (IHCs) and postsynaptic neurites of afferent spiral ganglion neurons (SGNs). The highly specialized IHC presynaptic active zones (AZs) are characterized by the presence of proteinaceous electron-dense bodies, called "synaptic ribbons," which are primarily composed of the structural cytomatrix protein RIBEYE (1, 2). Ribbons provide presynaptic scaffolding, cluster and functionally regulate presynaptic Ca 2+ channels at the AZ membrane (3-5), and tether a halo of synaptic vesicles (SVs) (6). This latter feature is thought to enable rapid and indefatigable vesicular replenishment to the release site-even during periods of persistent stimulation (3,5,7,8).In mice, hearing onset occurs around postnatal day (P)12 (9) before which, IHC presynaptic AZs undergo a range of structural and functional refinements. For example, extrasynaptically localized Ca 2+ channels are progressively eliminated from the nonsynaptic basolateral plasma membrane and form-in concert with the corresponding postsynaptic glutamate receptor patchestightly confined clusters at mature presynaptic AZs (3, 10). Moreover, otoferlin-a large Ca 2+ -binding multi-C 2 domain protein (11, 12)-likely replaces synaptotagmins as the putative Ca 2+ sensor of IHC release during the first postnatal week (13). This finding reflects a key landmark of functional maturation of this unconventional high-throughput release machinery and is essentially required to faithfully orchestrate vesicular...