At the first auditory synapse in mammals, one ribbon-type AZ of the IHC drives one postsynaptic spiral ganglion neuron (SGN) to spike at rates exceeding 100 Hz in silence and 1 kHz upon sound onset 1, 2 . Moreover, SGNs sustain firing rates of several hundred Hz during ongoing acoustic stimulation. In such a steady state, vesicle replenishment has to balance vesicle fusion at the IHC AZ. Accordingly, high rates of initial and sustained exocytosis have been found in hair cells [3][4][5][6][7][8] . Ribbon-type AZs of IHCs replenish readily releasable vesicles at hundreds of Hz over several seconds of stimulation, faster than ribbon synapses in the eye 9-15 and most non-ribbon-type AZs 16(but see ref. 17). This efficient vesicle re-supply maintains a large standing pool of fusion competent synaptic vesicles, which appears to be critical for reliable and temporally precise sound encoding [18][19][20] . and Otof +/+ mice ( Fig. 2a-b The observations of normal RRP size after resting the synapse for more than 30 seconds and of reduced vesicle re-supply during ongoing stimulation prompted us to explore RRP recovery from depletion in paired-pulse experiments (Fig. 3a). RRP recovery, assessed as the paired-pulse ratio for different inter-pulse intervals, was impaired in Otof Pga/Pga mice (Fig. 3b). This indicated a deficit in vesicle replenishment also in the rest period between stimuli. The ΔC m pattern elicited by trains of ten short (10 ms) depolarizations demonstrates the exocytosis phenotype found after a period of rest (30 s voltage-clamp at -84 mV): normal RRP exocytosis but subsequent failure (Fig. 3c). Studying the C m decline after exocytosis we observed normal endocytic membrane retrieval (Fig. 3d). Normal synaptic ultrastructure in Otof Pga/Pga IHCsIn order to explore whether a docking or a priming defect underlies the impairment of vesicle replenishment at Otof Pga/Pga IHC synapses, we studied their ultrastructure using electron microscopy (EM). Both, EM of single ultrathin sections (perpendicular to the 7 plasma membrane and the long axis of the ribbon; Supplementary Fig. 2) as well as EM tomography (Fig. 3e-f High-resolution EM tomography ( Fig. 3e-f) was used to measure the distance of membrane-proximal synaptic vesicles (labeled orange in Fig. 3f) from the plasma membrane under both conditions. The average membrane-membrane distance was approximately 6 nm regardless of condition and genotype (Supplementary Reduced rates but maintained size variability of EPSCsThe stark contrast between the absence of auditory neuron population responses in vivo (Fig. 1) (Fig. 4a-b). We pooled the data from recordings in 5. Fig. 4h-i; Kolmogorov-Smirnov test, p = 0.14). Moreover, we detected action potential generation by recording action currents in the loose-patch configuration (Fig. 4g). Together these results suggest that Otof Pga/Pga synapses should be capable of encoding sound into spiking activity in auditory nerve fibers, albeit at lower rates.In addition, we recorded from SGNs of Otof -/-mice of the same age a...

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.

Inner hair cells (IHCs) of the cochlea use ribbon synapses to transmit auditory information faithfully to spiral ganglion neurons (SGNs).In the present study, we used genetic disruption of the presynaptic scaffold protein bassoon in mice to manipulate the morphology and function of the IHC synapse. Although partial-deletion mutants lacking functional bassoon (Bsn mice. Both genotypes showed impaired sound onset coding and reduced evoked and spontaneous spike rates. In summary, reduced bassoon expression or complete lack of full-length bassoon impaired sound encoding to a similar extent, which is consistent with the comparable reduction of the readily releasable vesicle pool. This suggests that the remaining loosely anchored ribbons in Bsn gt IHCs were functionally inadequate or that ribbon independent mechanisms dominated the coding deficit.