The auditory inner hair cell (IHC) ribbon synapse operates with an exceptional temporal precision and maintains a high level of neurotransmitter release. However, the molecular mechanisms underlying IHC synaptic exocytosis are largely unknown. We studied otoferlin, a predicted C2-domain transmembrane protein, which is defective in a recessive form of human deafness. We show that otoferlin expression in the hair cells correlates with afferent synaptogenesis and find that otoferlin localizes to ribbon-associated synaptic vesicles. Otoferlin binds Ca(2+) and displays Ca(2+)-dependent interactions with the SNARE proteins syntaxin1 and SNAP25. Otoferlin deficient mice (Otof(-/-)) are profoundly deaf. Exocytosis in Otof(-/-) IHCs is almost completely abolished, despite normal ribbon synapse morphogenesis and Ca(2+) current. Thus, otoferlin is essential for a late step of synaptic vesicle exocytosis and may act as the major Ca(2+) sensor triggering membrane fusion at the IHC ribbon synapse.

Hearing relies on faithful synaptic transmission at the ribbon synapse of cochlear inner hair cells (IHCs). At present, the function of presynaptic ribbons at these synapses is still largely unknown. Here we show that anchoring of IHC ribbons is impaired in mouse mutants for the presynaptic scaffolding protein Bassoon. The lack of active-zone-anchored synaptic ribbons reduced the presynaptic readily releasable vesicle pool, and impaired synchronous auditory signalling as revealed by recordings of exocytic IHC capacitance changes and sound-evoked activation of spiral ganglion neurons. Both exocytosis of the hair cell releasable vesicle pool and the number of synchronously activated spiral ganglion neurons co-varied with the number of anchored ribbons during development. Interestingly, ribbon-deficient IHCs were still capable of sustained exocytosis with normal Ca2+-dependence. Endocytic membrane retrieval was intact, but an accumulation of tubular and cisternal membrane profiles was observed in ribbon-deficient IHCs. We conclude that ribbon-dependent synchronous release of multiple vesicles at the hair cell afferent synapse is essential for normal hearing.

Hearing in mammals relies on the highly synchronous synaptic transfer between cochlear inner hair cells (IHCs) and the auditory nerve. We studied the presynaptic function of single mouse IHCs by monitoring membrane capacitance changes and voltage-gated Ca 2؉ currents. Exocytosis initially occurred at a high rate but then slowed down within a few milliseconds, despite nearly constant Ca 2؉ influx. We interpret the observed secretory depression as depletion of a readily releasable pool (RRP) of about 280 vesicles. These vesicles are probably docked close to Ca 2؉ channels at the ribbon-type active zones of the IHCs. Continued depolarization evoked slower exocytosis occurring at a nearly constant rate for at least 1 s and depending on ''long-distance'' Ca 2؉ signaling. Refilling of the RRP after depletion followed a biphasic time course and was faster than endocytosis. RRP depletion is discussed as a mechanism for fast auditory adaptation. influx at the ribbon-type active zones triggers exocytosis of synaptic vesicles, which probably release glutamate (2) onto glutamate receptors (3, 4) of the postsynaptic auditory nerve fibers. There is little information about the presynaptic function of IHCs, because the small diameter of auditory nerve fibers in mammals hinders postsynaptic recordings. Assumptions about transmitter release have, therefore, mainly been based on auditory nerve fiber spiking rate data (5) or on Furukawa's classical recordings of postsynaptic potentials from goldfish (6).To study the presynaptic function of mouse IHCs independently of postsynaptic recordings, we detected the exocytic fusion and endocytic retrieval of synaptic vesicle membrane as changes of the membrane capacitance (C m ; ref. 7). The specificity of C m measurements for reporting exocytosis of neurotransmitters has recently been confirmed in another ribbon-type presynapse, that of the goldfish retinal bipolar nerve terminal, by simultaneously recording transmitter release (8). In these neurons, as well as in neuroendocrine cells, several kinetic components of exocytosis have been observed and attributed to release of functionally different pools of vesicles (9-12). We compare the presynaptic properties of IHCs to the findings in other neurosecretory preparations and discuss them in the context of auditory adaptation and recovery from adaptation. ] e ; 10 mM CaCl 2 ) was used for all experiments except for those of Fig. 1b (2 mM CaCl 2 ). The extracellular solution further contained (in mM) 105 NaCl, 35 tetraethylammonium chloride (Pfaltz & Bauer), 2.8 KCl, 1 MgCl 2 , 10 NaOH-Hepes, and 10 D-glucose (pH 7.2). Solution changes were achieved by bath exchange. IHCs were stimulated electrically rather than mechanically, because C m measurements require voltage clamp. Unless stated otherwise, a resting period of Ϸ30 s was kept between depolarizations to allow recovery of exocytosis. EPC-9 amplifiers (HEKA Electronics, Lambrecht͞Pfalz, Germany) controlled by PULSE software (HEKA Electronics) were used for measurements. All voltages were co...