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 relies on faithful sound coding at hair cell ribbon synapses, which use Ca 2ϩ -triggered glutamate release to signal with submillisecond precision. Here, we investigated stimulus-secretion coupling at mammalian inner hair cell (IHC) synapses to explore the mechanisms underlying this high temporal fidelity.

Summary At the presynaptic active zone, Ca2+ influx triggers fusion of synaptic vesicles. It is not well understood how Ca2+-channel clustering and synaptic vesicle docking are organized. Here we studied structure and function of hair cell ribbon synapses following genetic disruption of the presynaptic scaffold protein Bassoon. Mutant synapses - mostly lacking the ribbon - showed a reduction in membrane-proximal vesicles, with ribbonless synapses affected more than ribbon-occupied synapses. Ca2+-channels were also fewer at mutant synapses and appeared in abnormally shaped clusters. Ribbon absence reduced Ca2+-channel numbers at mutant and wild-type synapses. Fast and sustained exocytosis were reduced notwithstanding normal coupling of the remaining Ca2+-channels to exocytosis. In-vitro recordings revealed a slight impairment of vesicle replenishment. Mechanistic modeling of the in-vivo data independently supported morphological and functional in-vitro findings. We conclude that Bassoon and the ribbon (1) create a large number of release sites by organizing Ca2+-channels and vesicles, and (2) promote vesicle replenishment.

Cochlear inner hair cells (IHCs) transmit acoustic information to spiral ganglion neurons through ribbon synapses. Here we have used morphological and physiological techniques to ask whether synaptic mechanisms differ along the tonotopic axis and within IHCs in the mouse cochlea. We show that the number of ribbon synapses per IHC peaks where the cochlea is most sensitive to sound. Exocytosis, measured as membrane capacitance changes, scaled with synapse number when comparing apical and midcochlear IHCs. Synapses were distributed in the subnuclear portion of IHCs. High-resolution imaging of IHC synapses provided insights into presynaptic Ca(2+) channel clusters and Ca(2+) signals, synaptic ribbons and postsynaptic glutamate receptor clusters and revealed subtle differences in their average properties along the tonotopic axis. However, we observed substantial variability for presynaptic Ca(2+) signals, even within individual IHCs, providing a candidate presynaptic mechanism for the divergent dynamics of spiral ganglion neuron spiking.

KCNQ4 is an M-type K þ channel expressed in sensory hair cells of the inner ear and in the central auditory pathway. KCNQ4 mutations underlie human DFNA2 dominant progressive hearing loss. We now generated mice in which the KCNQ4 gene was disrupted or carried a dominant negative DFNA2 mutation. Although KCNQ4 is strongly expressed in vestibular hair cells, vestibular function appeared normal. Auditory function was only slightly impaired initially. It then declined over several weeks in Kcnq4 À/À mice and over several months in mice carrying the dominant negative allele. This progressive hearing loss was paralleled by a selective degeneration of outer hair cells (OHCs). KCNQ4 disruption abolished the I K,n current of OHCs. The ensuing depolarization of OHCs impaired sound amplification. Inner hair cells and their afferent synapses remained mostly intact. These cells were only slightly depolarized and showed near-normal presynaptic function. We conclude that the hearing loss in DFNA2 is predominantly caused by a slow degeneration of OHCs resulting from chronic depolarization.

channel open probability by the dihydropyridine agonist BayK8644. However, we observed substantial variability also for the fluorescence of immunolabeled CaV1.3 Ca 2؉ channel clusters. Moreover, the Ca 2؉ microdomain amplitude correlated positively with the size of the corresponding synaptic ribbon. Ribbon size, previously suggested to scale with the number of synaptic Ca 2؉ channels, was approximated by using fluorescent peptide labeling. We propose that IHCs adjust the number and the gating of CaV1.3 channels at their active zones to diversify their transmitter release rates.calcium microdomain ͉ coding ͉ imaging ͉ ribbon synapse ͉ modeling

The mechanisms underlying the large amplitudes and heterogeneity of excitatory postsynaptic currents (EPSCs) at inner hair cell (IHC) ribbon synapses are unknown. Based on electrophysiology, electron and superresolution light microscopy, and modeling, we propose that uniquantal exocytosis shaped by a dynamic fusion pore is a candidate neurotransmitter release mechanism in IHCs. Modeling indicated that the extended postsynaptic AMPA receptor clusters enable large uniquantal EPSCs. Recorded multiphasic EPSCs were triggered by similar glutamate amounts as monophasic ones and were consistent with progressive vesicle emptying during pore flickering. The fraction of multiphasic EPSCs decreased in absence of Ca(2+) influx and upon application of the Ca(2+) channel modulator BayK8644. Our experiments and modeling did not support the two most popular multiquantal release interpretations of EPSC heterogeneity: (1) Ca(2+)-synchronized exocytosis of multiple vesicles and (2) compound exocytosis fueled by vesicle-to-vesicle fusion. We propose that IHC synapses efficiently use uniquantal glutamate release for achieving high information transmission rates.

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