Project Summary Auditory stimuli travel from the cochlea to the brainstem through type I and type II cochlear afferents. While type I afferents convey information about the frequency, intensity, and timing of sounds, the role of type II afferents remains unresolved. Limited recordings of type II afferents from the cochlear apex in pre-hearing rats reveal that they can be activated by widespread outer hair cell (OHC) stimulation and by ATP released by acute tissue damage. Secondly, synaptic activity in type II fibers has been shown to be glutamatergic but input from multiple OHCs is required to initiate action potential firing. A recent study suggests that type II afferents respond to non-damaging sound, via their glutamatergic pathway. Additionally, type II fibers exhibit properties similar to pain fibers, for example the expression of modulatory peptidergic and cannabinoid signaling pathways. Finally, type II fibers express receptor genes to inflammatory modulators, some of which are upregulated after noise exposure. These combined data suggest that type II afferents may sense non- damaging and damaging sound levels, and that multiple parallel pathways may contribute to the type II fiber response to different degrees, depending on history of sound exposure. To explore this hypothesis, we have developed Ca2+-imaging techniques that reveal type II fiber activity throughout the cochlear coil, from apex to base, of adult mice. Secondly, we propose to use loose patch recordings of type II afferent fibers to monitor firing rates in response to optogenetic stimulation of OHCs. Both approaches are utilized to determine the impact of non- damaging and damaging sound exposure on the activity of type II cochlear afferents. The goal of this proposal is to understand the contribution of multiple pathways (purinergic, glutamatergic, neuromodulatory and inflammatory) to these conditions. Our recent experiments show sensitization in the type II afferent response following noise exposure. Such sensitization might be reflective of a `gain-of-function pathology' like hyperacusis/noxacusis. The proposed comprehensive approach here aims to identify pharmacotherapeutic targets for such gain of function pathologies that can follow noise exposure and noise- induced hearing loss.