Hearing requires the hair cell synapse to maintain notable temporal fidelity (1 ms or less) while sustaining neurotransmitter release for prolonged periods of time (minutes). Details of the mechanisms that allow glutaminergic synapses to meet these remarkable demands of audition are poorly understood, but remain a critical step in elucidating cochlear function. Anatomical constraints have hampered or precluded conventional characterization of hair cell afferent fiber synaptic properties, and thus novel experimental strategies are needed to elucidate mechanisms underlying the neural code ultimately perceived as sound. Here, we propose to exploit the power of mouse genetics to develop an animal model specifically for the study of hair cell synaptic vesicle trafficking. This project aims to: 1) Generate a transgenic mouse pedigrees that can be used to visualize and study synaptic vesicle trafficking in the cochlear hair cell. SynaptopHlourins are a class of AFP-derived proteins targeted to synaptic vesicles that change their fluorescence upon changes in pH associated with different stages of synaptic vesicle cycle. 2) Validate these transgenic animal models by: a - confirming the synaptic targeting of the synpto-pHluorins with immuno- staining; b- verifying that the transgenic mice retain a normal synaptic phenotype in terms of normal auditory function and synaptic vesicle cycling as assessed with membrane capacitance measurements; c - employing two-photon excitation fluorescent confocal microscopy to study synaptic vesicle trafficking in the living cochlear hair cell. In addition to monitoring the kinetics of endocytosis, Fluorescence Recovery After Photobleaching is employed to measure the mobility of synaptic vesicles. The proposed pedigrees can be crossed into other transgenic or mutant mice strains to provide an endogenous reporter of synaptic activity in animals with synapse-related gene dysfunction. The new tools proposed here are of broad utility to hearing science, and promise new insight into synaptic mechanisms mediating function of the auditory system. Relevance to Public Health The proposed research provides new tools to addresses how the hair cell-afferent fiber synapse transforms the details of acoustic waves into a neural code that we perceive as sound. A better understanding of this important step in the auditory cascade provides the basis for improved design of cochlear protheses and the amelioration of hearing loss and deafness. ? ? ?