Hair cells in the cochlea connect to auditory afferent fibers via ribbon synapses, which convert presynaptic gracied potentials (analog signal) Into postsynaptic all-or-none spikes (digital signal). The long-term objective of this study is to investigate mechanisms of synaptic transmission and strategies for auditory coding at these synapses. Previously, by determining the quantal size of the hair cell ribbon synapse in bullfrog j amphibian papilla, we demonstrated unequivocally that hair cells can release more than one synaptic vesicle at a time (multivesicular release, MRV) from a single release site (i.e., ribbon). To study cellular mechanisms that control MRV, we will measure the calcium-dependence of MVR and determine its calcium threshold. To demonstrate the functional advantages of MRV, we will first stimulate hair cells with sinusoidal presynaptic depolarizations that mimic hair cell voltage responses in vivo, and quantify MVRs in the evoked EPSCs. Then we simulate two sets of EPSCs in which we either substitute MVRs with evenly distributed single vesicle releases within a time window (e.g., 0.1 ms), or fix the quantal content of MVRs (removing the variation in their quantal contents). We will feed both the original and simulated EPSCs to afferent fibers under current-clamp, and find out to what extent the phase-locking of spikes gets deteriorated. To investigate how synaptic vesicles are recycled, we will use a two-photon microscope to visualize FM1-43 dye loading to monitor vesicle recycling, and we will also make cell-attached capacitance measurement on hair cells to study vesicle recycling by monitoring capacitance changes with a submillisecond time resolution.