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...

EF-hand Ca2+-binding proteins are thought to shape the spatiotemporal properties of cellular Ca2+ signaling and are prominently expressed in sensory hair cells in the ear. Here, we combined genetic disruption of parvalbumin-α, calbindin-D28k, and calretinin in mice with patch-clamp recording, in vivo physiology, and mathematical modeling to study their role in Ca2+ signaling, exocytosis, and sound encoding at the synapses of inner hair cells (IHCs). IHCs lacking all three proteins showed excessive exocytosis during prolonged depolarizations, despite enhanced Ca2+-dependent inactivation of their Ca2+ current. Exocytosis of readily releasable vesicles remained unchanged, in accordance with the estimated tight spatial coupling of Ca2+ channels and release sites (effective “coupling distance” of 17 nm). Substitution experiments with synthetic Ca2+ chelators indicated the presence of endogenous Ca2+ buffers equivalent to 1 mM synthetic Ca2+-binding sites, approximately half of them with kinetics as fast as 1,2-Bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA). Synaptic sound encoding was largely unaltered, suggesting that excess exocytosis occurs extrasynaptically. We conclude that EF-hand Ca2+ buffers regulate presynaptic IHC function for metabolically efficient sound coding.

The afferent inner hair cell synapse harbors the synaptic ribbon, which ensures a constant vesicle supply. Synaptic vesicles (SVs) are arranged in morphologically discernable pools, linked via filaments to the ribbon or the presynaptic membrane. We propose that filaments play a major role in SV resupply and exocytosis at the ribbon. Using advanced electron microscopy, we demonstrate that SVs are organized in sub-pools defined by the filament number per vesicle and its connections. Upon stimulation, SVs increasingly linked to other vesicles and to the ribbon, whereas single-tethered SVs dominated at the membrane. Mutant mice for the hair cell protein otoferlin (, ) are profoundly deaf with reduced sustained release, serving as a model to investigate the SV replenishment at IHCs. Upon stimulation, multiple-tethered and docked vesicles (rarely observed in wild-type) accumulated at active zones due to an impairment downstream of docking. Conclusively, vesicles are organized in sub-pools at ribbon-type active zones by filaments to support vesicle supply, transport, and finally release.

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 transmitter 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.

Inner hair cells (IHCs) are the primary receptors for hearing. They are housed in the cochlea and convey sound information to the brain via synapses with the auditory nerve. IHCs have been thought to be electrically and metabolically independent from each other. We report that, upon developmental maturation, in mice 30% of the IHCs are electrochemically coupled in 'mini-syncytia'. This coupling permits transfer of fluorescently-labeled metabolites and macromolecular tracers. The membrane capacitance, Ca 2+-current, and resting current increase with the number of dye-coupled IHCs. Dual voltage-clamp experiments substantiate low resistance electrical coupling. Pharmacology and tracer permeability rule out coupling by gap junctions and purinoceptors. 3D electron microscopy indicates instead that IHCs are coupled by membrane fusion sites. Consequently, depolarization of one IHC triggers presynaptic Ca 2+-influx at active zones in the entire mini-syncytium. Based on our findings and modeling, we propose that IHC-mini-syncytia enhance sensitivity and reliability of cochlear sound encoding.

Cochlear inner hair cells (IHCs) form specialized ribbon synapses with spiral ganglion neurons that tirelessly transmit sound information at high rates over long time periods with extreme temporal precision. This functional specialization is essential for sound encoding and is attributed to a distinct molecular machinery with unique players or splice variants compared to conventional neuronal synapses. Among these is the active zone (AZ) scaffold protein piccolo/aczonin, which is represented by its short splice variant piccolino at cochlear and retinal ribbon synapses. While the function of piccolo at synapses of the central nervous system has been intensively investigated, the role of piccolino at IHC synapses remains unclear. In this study, we characterize the structure and function of IHC synapses in piccolo gene‐trap mutant rats (Pclogt/gt). We find a mild hearing deficit with elevated thresholds and reduced amplitudes of auditory brainstem responses. Ca2+ channel distribution and ribbon morphology are altered in apical IHCs, while their presynaptic function seems to be unchanged. We conclude that piccolino contributes to the AZ organization in IHCs and is essential for normal hearing.