At the inner hair cell (IHC) ribbon synapse, the first synapse in the auditory pathway, 'analog' sound information is converted into a 'digital' pattern of action potentials at auditory nerve fibers and transmitted to the brain. The relative time of each spike within a train of action potentials carries important information that needs to be faithfully represented in the auditory pathway. A well characterized form of temporal coding is phase locking: auditory neurons are capable of firing at a particular time within each cycle of a low-frequency stimulus. This phenomenon is required for localizing a sound source by computing the small difference in time at which the wave arrives at the two ears. Interaural delays can be as small as 10 microseconds, emphasizing the precision of temporal coding by the auditory periphery. The goal of this proposal is to investigate the mechanisms that allow the IHC ribbon synapse to release neurotransmitter with high precision and over long periods of time. In acutely excised rat cochlear preparations, simultaneous patch-clamp recordings will be performed from IHCs and postsynaptic terminals of auditory nerve fibers. Recently, we have shown that short-term facilitation occurs at this synapse, producing not only an increase in release but also a reduction in latency. Our first aim is to investigate the mechanisms underlying this phenomenon. We will study the role of the residual intracellular Ca2+ concentration by controlling its spread with specific buffers, and by monitoring its decay time course with fluorescent dyes. Facilitation will also be studied by uncaging Ca2+ in the cytosolic space. Secondly, the underlying mechanisms of phase-locked synaptic responses will be investigated. Preliminary experiments show that synaptic responses at the IHC ribbon synapse phase-lock to periodic stimuli. This feature will be further explored by applying stimuli with variable amplitude and by testing whether the preferred phase is conserved. These experiments will be compared with responses to single step depolarizations. We will evaluate the role of short-term facilitation and depression in establishing phase-locking. Finally, the ability of the IHC ribbon synapse to signal continuously with high precision will be studied. It has been shown that in response to steady IHC depolarization, this synapse exhibits short-term depression. The time course of recovery of synaptic responsiveness following a depleting stimulus will be evaluated. Given that neural synchrony is required for complex tasks such as speech intelligibility, the outcome of this study will hopefully provide the basis for potential improvements in cochlear implant design and a better understanding of hearing deficits that originate at the IHC afferent synapse. This research will be done primarily in Argentina, at the Instituto de Investigaciones en Ingeniera Gentica y Biologa Molecular (INGEBI) in collaboration with Dr. Juan Goutman, with the companion grant being R01 DC006476, 01-01-2004 to 11-29-2013.