Physiology and anatomy of glutamate receptors at the inner hair cell to auditory nerve synapse suggest GluA2-lacking, Ca2+-permeable AMPA receptors contribute to transmission in the mammalian cochlea

JUAN D. GOUTMAN; SHELBY PAYNE; BABAK V-GHAFFARI; SHASHANK CHEPURWAR; ADISH DANI; MARK RUTHERFORD

San Jose, California

Congreso; 43rd Annual MidWinter meeting of the Association for Research in Otolaryngology; 2020

Association for Research in Otolaryngology

Physiology and Anatomy of Glutamate Receptors at the Inner Hair Cell to Auditory Nerve Fiber Synapse Suggest GluA2-lacking, Ca2+-permeable AMPA Receptors Contribute to Transmission in the Mammalian CochleaJuan D. Goutman, Shelby Payne, Babak V-Ghaffari, Shashank Chepurwar, Adish Dani, Mark A. RutherfordEach cochlear inner hair cell provides the sole excitatory input to 10 or more auditory nerve fibers, via glutamatergic transmission dependent on post-synaptic AMPA receptors. AMPA receptors are glutamate-gated ion channels, and each post-synaptic density (PSD) contains thousands of receptors. AMPA receptors are hetero-tetrameric complexes, comprised of GluA2, GluA3, and GluA4 subunits. The absence of GluA2 from the tetrameric receptor-channel imparts several unique features which may be physiologically significant to sound encoding and excitotoxicity including inward current rectification, block by intracellular polyamines, and high permeability to Ca2+. All afferent synapses between cochlear inner hair cells and auditory nerve fibers appear to include GluA2. However, individual PSDs contains thousands of AMPARs, which may differ from each other in subunit stoichiometry. Here we show new anatomical analysis and, for the first time, physiological evidence for the presence of GluA2-lacking, Ca2+-permeable AMPA receptors at cochlear afferent synapses. We measured subunit localization within synapses and relative abundance among synapses by deconvolution confocal microscopy and by Stochastic Optical Reconstruction Microscopy (STORM) of subunit immunohistofluorescence. Among synapses, we observed a broad range of relative abundances of subunits. Within synapses, we observed nano-domains that appeared to lack GluA2 alongside regions of robust GluA2 immunofluorescence, suggesting the presence of Ca2+-permeable and Ca2+-impermeable AMPA receptors in the same PSD. Radial distributions of subunit fluorescence suggest that GluA2 tends to be more centrally located in the PSD, relative to the center of the presynaptic ribbon, than GluA3 or GluA4 subunits which tended to be more peripheral.With patch-clamp electrophysiology recordings on the post-synaptic terminals of auditory nerve fibers we used IEM-1460 to antagonize GluA2-lacking AMPA receptors, and found 50% block with a concentration of ~10 µM, consistent with the presence of GluA2-lacking AMPA receptors at cochlear inner hair cell afferent synapses. The addition of 100 µM spermine in the pipette produced a strong inward rectification of excitatory postsynaptic currents, consistent with the presence of GluA2-lacking AMPA receptors. Together, these results point to the presence of functional GluA2-lacking Ca2+-permeable AMPA receptors at cochlear inner hair cell afferent synapses, although the permeability to Ca2+ has not yet been demonstrated in cochlear nerve terminals. Understanding the significance of AMPA receptor heterogeneity may be key to elucidating the differences between auditory nerve fibers that innervate the same inner hair cell, in terms of their physiology and sensitivity to noise-induced damage.