Project Summary Neurons in the developing auditory system experience highly stereotyped bursts of activity prior to the onset of sensory experience. This activity is initiated within the cochlea when non-sensory inner supporting cells release ATP, triggering a cascade of events that ultimately induces trains of action potentials in spiral ganglion neurons (SGNs) that propagate throughout the auditory system. The spatially restricted release of ATP triggers correlated firing in groups of SGNs that will later encode similar frequencies of sound, providing a means to induce activity-dependent maturation and refinement of sound processing circuits in the brain prior to hearing onset. Despite the prominence of patterned activity during this critical developmental period, its role in maturation of the auditory system remains poorly understood, in part, due to an inability to selectively disrupt spontaneous activity while preserving sound transduction in the cochlea. Here, we propose to leverage newly developed mouse models that allow selective disruption of spontaneous activity within cochlear supporting cells yet preserve cochlear structure and the integrity of the auditory nerve. We will explicitly test the hypothesis that burst firing of auditory neurons is critical to initiate structural and functional maturation of nascent sound processing circuits. These studies will leverage genetic disruption of P2ry1 and Tmem16a, two components required to generate spontaneous activity in cochlear supporting cells, with in vivo widefield and two photon imaging of neuronal activity, RNA expression profiling and behavioral analyses of auditory function to rigorously test this hypothesis. We will extend our recent discovery that astrocytes in the inferior colliculus (IC) of pre-hearing mice are co-activated with surrounding neurons during spontaneous events, providing a means to coordinate spatial and temporal maturation of tripartite synapses (excitatory synapses ensheathed by astrocytes). Aim 1 will focus on the cochlea, determining how loss of P2RY1 and TMEM16A influence the properties and developmental trajectory of SGNs. Aim 2 will define the relationship between cochlear and extra-cochlear spontaneous activity in auditory cortex (AC), determine how disruption of cochlea-derived spontaneous activity alters spatial patterns of neuronal activation in the IC and AC and ultimately influence auditory discrimination. Aim 3 will define the patterns of neuronal activity required to induce calcium elevation in astrocytes and determine how selective genetic disruption of astrocyte mGluR5 expression, which is necessary to detect neuronal burst firing, influences astrocyte maturation and progressive refinement of tonotopic representation of sounds in vivo. These studies will provide greater insight into the fundamental mechanisms used to define circuits that process sound information and establish a framework to explore how genetic mutations, trauma and exposure to otot...