The Purinergic Receptor P2rx3 is Required for Spiral Ganglion Neuron Branch Refinement during Development

The mammalian cochlea undergoes a highly dynamic process of growth and innervation during development. This process includes spiral ganglion neuron (SGN) branch refinement, a process whereby Type I SGNs undergo a phase of "debranching" before forming unramified synaptic contacts with inner hair cells. Using Sox2^CreERT2 and R26R^tdTomato as a strategy to genetically label individual SGNs in mice of both sexes, we report on both a time course of SGN branch refinement and a role for P2rx3 in this process. P2rx3 is an ionotropic ATP receptor that was recently implicated in outer hair cell spontaneous activity and Type II SGN synapse development (Ceriani et al., 2019), but its function in Type I SGN development is unknown. Here, we demonstrate that P2rx3 is expressed by Type I SGNs and hair cells during developmental periods that coincide with SGN branching refinement. P2rx3 null mice show SGNs with more complex branching patterns on their peripheral synaptic terminals and near their cell bodies around the time of birth. Loss of P2rx3 does not appear to confer general changes in axon outgrowth or hair cell formation, and alterations in branching complexity appear to mostly recover by postnatal day (P)6. However, when we examined the distribution of Type I SGN subtypes using antibodies that bind Calb2, Calb1, and Pou4f1, we found that P2rx3 null mice showed an increased proportion of SGNs that express Calb2. These data suggest P2rx3 may be necessary for normal Type I SGN differentiation in addition to serving a role in branch refinement.