GluA3 subunits are required for appropriate assembly of AMPAR GluA2 and GluA4 subunits on cochlear afferent synapses and for presynaptic ribbon modiolar-pillar morphology.

Abstract

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 <i>Gria3</i>-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 <i>Gria3</i> 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.