DESCRIPTION (provided by applicant): Hair cells in the cochlea connect to auditory afferent fibers via ribbon synapses. Presynaptic graded potentials are converted here into postsynaptic spikes. The long-term objective of this study is to investigate the strategies for auditory signal encoding at this synapse. It has been suggested that hair cells tend to release more than one synaptic vesicle at a time (multivesicular release: MVR). However, the cellular mechanisms underlying MVR are poorly understood and its functional advantages are not known. Our first hypothesis is that MVR occurs when [Ca2+]j in hair cells crosses a threshold that triggers neighboring vesicles on a ribbon to pre-fuse with each other and release all their contents into the synaptic cleft simultaneously. We will determine the quantal response size (i.e., excitatory postsynaptic current (EPSC) amplitude evoked by a single vesicle fusion) and use this to quantify EPSC quantal content. We will find out if vesicles in MVR are from a single ribbon and investigate the Ca2+-dependence of MVR (i.e. determine its Ca2+ threshold). The second hypothesis concerns the function of MRV and has two parts. One part is that MVR can charge and discharge the membrane of afferent fibers more rapidly, helping them to fire spikes with higher temporal precision for phase-locking. The second part is that MRV provides a necessary varying factor on EPSC amplitudes evoked by repeated sinusoidal depolarizations of hair cells. This allows afferent fibers to avoid firing spikes at every sinusoidal cycle and the timing of spikes will not deteriorate due to spike refractory periods. We will measure EPSCs in response to a sinusoidal presynaptic depolarization and then simulate these EPSCs to either substitute MVR with evenly distributed single vesicle releases within a time window (e.g. 0.1 ms), or limit the variation of EPSC amplitudes within the variation of quantal responses (removing thus the variation in their quantal content). These simulated EPSCs will then be experimentally injected into afferent fibers under current-clamp to determine to what extent the phase-locking of spikes becomes deteriorated when MVR is absent. The third hypothesis to be tested is that fused synaptic vesicles can be recycled through fast endocytosis following MVR. We will use a 2-photon microscope to visualize FM1-43 dye loading to monitor vesicle recycling, and we will also make cell-attached capacitance measurements on hair cells to study vesicle recycling by monitoring capacitance changes with a time resolution of 50 ps or higher. RELEVANCE: In the United States, roughly 23,000 adults and 15,500 children have received cochlear implants, which restore part of their hearing by directly stimulating auditory nerve fibers with electrodes. However, the algorithms to stimulate the fibers according to the sound signal have been determined only empirically. The fundamental studies of afferent fiber spiking proposed here will provide guidance for significantly improving these algorithms, especially for adult patients whose auditory systems are fully developed and may thus have lost some of their plasticity and adaptability to different stimulus protocols.