PROJECT SUMMARY The hair cell synapse is the first step in the ascending auditory pathway. Disruption of this ribbon-type synapse can lead to severe hearing disorders. The goal of this proposal is to investigate how hair cell synaptic activity provides rapid signaling for long periods of time without fatigue. To maintain a continuous release of neurotransmitter the hair cell requires a large pool of synaptic vesicles that are readily releasable. However, the mechanisms that regulate synaptic vesicle endocytosis and the replenishment of vesicle pools in hair cells are poorly understood. We propose here to study fundamental aspects of synaptic transmission at inner hair cells (IHCs) in mouse cochlea and at auditory hair cells in the adult bullfrog amphibian papilla (AP). The AP preparation allows us to routinely access single hair cells and their afferent fibers for high-time-resolution patch-clamp electrophysiology and structure/function studies. We propose to use paired recordings of the hair cell and its connected afferent fiber to study multivesicular release and simultaneously to measure membrane capacitance changes from the hair cell to assay the exocytosis and endocytosis of synaptic vesicles. We will pursue three Specific Aims: First, we hypothesize that the fast and phasic component of release from hair cells requires high levels of ATP synthesis and hydrolysis, whereas the sustained component of release persists at a reduced rate even when ATP levels are very low. Accordingly, hair cells contain numerous mitochondria that are located near synaptic ribbons suggesting a need for a large amount of local ATP. Second, we will test the role of internal pH in controlling the rate of endocytosis in hair cells. Finally, we will study a transient block of the Ca2+ current in post-hearing mouse IHCs that is caused by the release of protons during multivesicular exocytosis. This transient block of the Ca2+ current constitutes a new method to study multivesicular release at IHC synapses and a new mechanism to explain the rapid spike adaption that is observed in vivo at the mammalian auditory nerve. Together these experiments will determine how pH changes affect endocytosis at hair cells and they will clarify the contributions of metabolic mechanisms that influence the auditory hair cell's ability to continuously release neurotransmitter.