Purinergic Modulation of Acetylcholine Release at the Efferent-Inner Hair Cell Synapse in the Developing Inner Ear

Before the onset of hearing (postnatal day 12 in mice), inner hair cells (IHCs) are transiently innervated by medial olivocochlear (MOC) efferent fibers and fire spontaneous sensory-independent action potentials essential for normal auditory pathway development. Several studies suggest that this activity is driven and/or modulated by ATP released from cochlear supporting cells. We investigated the role of ATP in modulating MOC-IHC synapses using pharmacology and electrophysiology. IHCs from BALB/C mice at P8-11 were patch-clamped, MOC fibers extracellularly stimulated, and quantal content (m) estimated. ATP reversibly decreased m in a concentration-dependent manner. Suramin, a non-specific P2 antagonist, abolished this effect, while TNP-ATP, a specific P2X antagonist, did not. The P2X agonist α,β-MeATP also had no effect on m. Both ATPγS, a non-hydrolyzable ATP analog, and the P2Y agonist 2-MeSADP mimicked ATP. To identify the P2Y subunit involved, we used specific pharmacology based on RNAseq studies indicating P2Y1 expression in supporting cells and P2Y12 and P2Y14 in MOC neurons. MRS2500, a P2Y1 antagonist, did not prevent ATP-induced inhibition. MRS2690, a P2Y14 agonist, produced no inhibition of ACh release. However, PSB-0739, a P2Y12 antagonist, blocked ATP inhibition, indicating that P2Y12 receptors mediate ATP-dependent reduction in quantal content from MOC fibers.