GluA3 subunits are required for appropriate assembly of AMPAR GluA2 and GluA4 subunits on cochlear afferent synapses...

Cochlear sound encoding depends on α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptors (AMPARs), but reliance on specific pore-forming subunits is unknown. With 5-week-old male C57BL/6J Gria3-knockout mice (i.e., subunit GluA3^KO) we determined cochlear function, synapse ultrastructure, and AMPAR molecular anatomy at ribbon synapses between inner hair cells (IHCs) and spiral ganglion neurons. GluA3^KO and wild-type (GluA3^WT) mice reared in ambient sound pressure level (SPL) of 55-75 dB had similar auditory brainstem response (ABR) thresholds, wave-1 amplitudes, and latencies. Postsynaptic densities (PSDs), presynaptic ribbons, and synaptic vesicle sizes were all larger on the modiolar side of the IHCs from GluA3^WT, but not GluA3^KO, demonstrating GluA3 is required for modiolar-pillar synapse differentiation. Presynaptic ribbons juxtaposed with postsynaptic GluA2/4 subunits were similar in quantity, however, lone ribbons were more frequent in GluA3^KO and GluA2-lacking synapses were observed only in GluA3^KO. GluA2 and GluA4 immunofluorescence volumes were smaller on the pillar side than the modiolar side in GluA3^KO, despite increased pillar-side PSD size. Overall, the fluorescent puncta volumes of GluA2 and GluA4 were smaller in GluA3^KO than GluA3^WT. However, GluA3^KO contained less GluA2 and greater GluA4 immunofluorescence intensity relative to GluA3^WT (threefold greater mean GluA4:GluA2 ratio). Thus, GluA3 is essential in development, as germline disruption of Gria3 caused anatomical synapse pathology before cochlear output became symptomatic by ABR. We propose the hearing loss in older male GluA3^KO mice results from progressive synaptopathy evident in 5-week-old mice as decreased abundance of GluA2 subunits and an increase in GluA2-lacking, GluA4-monomeric Ca²⁺-permeable AMPARs.