Postsynaptic GluA3 subunits are required for the appropriate assembly of AMPA receptor GluA2 and GluA4 subunits on mammalian cochlear afferent synapses and for presynaptic ribbon modiolar-pillar morphological distinctions

The encoding of acoustic signals in the cochlea depends on α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptors (AMPARs), but relatively little is known about their reliance on specific pore-forming subunits. With 5-week-old male GluA3 ^KO mice, we determined cochlear function, synapse ultrastructure, and AMPAR subunit molecular anatomy at ribbon synapses between inner hair cells (IHCs) and spiral ganglion neurons (SGNs). GluA3 ^KO and wild-type ( GluA3 ^WT ) mice reared in ambient sound pressure level (SPL) of 55-75 dB had similar ABR thresholds, wave-1 amplitudes, and latencies. Ultrastructurally, the IHC modiolar-pillar differences in presynaptic ribbon size and shape, and synaptic vesicle size seen in GluA3 ^WT were diminished or reversed in GluA3 ^KO . The quantity of paired synapses (presynaptic ribbons juxtaposed with postsynaptic GluA2 and GluA4) was similar, however, GluA2-lacking synapses (ribbons paired with GluA4 but not GluA2) were observed only in GluA3 ^KO . SGNs of GluA3 ^KO mice had AMPAR arrays of smaller overall volume, containing less GluA2 and greater GluA4 immunofluorescence intensity relative to GluA3 ^WT (3-fold difference in mean GluA4:GluA2 ratio). The expected modiolar-pillar gradient in ribbon volume was observed in IHCs of GluA3 ^WT but not GluA3 ^KO . Unexpected modiolar-pillar gradients in GluA2 and GluA4 volume were present in GluA3 ^KO . GluA3 is essential to the morphology and molecular composition of IHC-ribbon synapses. We propose the hearing loss seen in older male GluA3 ^KO mice results from progressive synaptopathy evident in 5-week-old mice as increased abundance of GluA2-lacking, GluA4 monomeric, Ca ²⁺ -permeable AMPARs.