Project Summary - Abstract The cochlear synapses of auditory nerve fibers (ANFs) degenerate before the sensory cells in both noise- induced and age-related hearing loss, leaving may inner hair cells (IHCs) partially disconnected from the brain. Although this synaptopathy has minimal effects on threshold sensitivity, it compromises hearing performance on complex auditory tasks and may be a major risk factor for tinnitus and hyperacusis. We have shown, in mouse, that augmenting cochlear levels of neurotrophin-3 (NT3) by local delivery, or by transgenic or virally driven overexpression, can rescue IHC synapses after noise and prevent their loss in the aging ear. We have also shown that post-noise IHC synapse regeneration occurs spontaneously in guinea pig, but not the mouse. Here, we combine the resources of the Liberman and Corfas laboratories to apply a range of ultrastructural, neurophysiological and molecular techniques to further probe the mechanisms and dynamics of synaptopathy, characterize its functional effects, validate the minimally invasive tests used to assess it in humans, probe the role of NT3 signaling as the key determinant of synaptopathy, and further refine translational approaches to reverse and/or prevent synaptopathy after noise or in aging. Specifically, in Aim 1, we use FIB-SEM and machine learning to automate serial-section ultrastructural analysis of the post-exposure damage and recovery of the IHC afferent innervation in mature mice vs. guinea pigs, to shed light on the reasons for the profound differences in regenerative capacity in the two species. In Aim 2, we combine population studies of single ANFs with intracellular labeling in synaptopathic guinea pigs at various stages of post-exposure recovery to a) characterize the extent to which the recovered synaptic counts reflect recovered function and b) to understand the contributions of different fiber populations to the non- invasive metrics of ANF function used in human studies of synaptopathy. In Aim 3, we test the hypothesized pivotal role of NT3 in the development and rescue of age- and noise-induced synaptopathy by a) determining if NT3 expression levels are upregulated by noise in guinea pigs but not in mice, and b) if drug-mediated blockade of cochlear NT3 receptors prevents the spontaneous post-exposure recovery of synapses in guinea pig. In Aim 4, we continue our refinement of gene-therapy approaches to the treatment of synaptopathy using an IHC-specific promoter in an AAV viral capsid to drive prolonged NT3 overexpression a) after noise and b) in an aging mouse model. Together these Aims will deepen our understanding of, and treatments for, this major contributor to the impairments of sensorineural hearing loss.