Protection of cochlear synapses from noise-induced excitotoxic trauma by blockade of Ca2+-permeable AMPA receptors Ning Hua, Mark A. Rutherfordb, and Steven H. Greena,1 aDepartment of Biology, University of Iowa, Iowa City, IA 52242; and bDepartment of Otolaryngology, Washington University in St. Louis, St. Louis, MO 63110 Edited by David P. Corey, Harvard Medical School, Boston, MA, and accepted by Editorial Board Member David E. Clapham January 7, 2020 (received for review August 16, 2019) Exposure to loud sound damages the postsynaptic terminals the ribbon synapses between IHCs and SGNs (3, 4). This noise- of spiral ganglion neurons (SGNs) on cochlear inner hair cells induced cochlear synaptopathy (NICS) is detectable by counting (IHCs), resulting in loss of synapses, a process termed synaptopathy. synapses histologically or, noninvasively, by a persistent reduction Glutamatergic neurotransmission via α-amino-3-hydroxy-5- of auditory brainstem response (ABR) wave-I amplitude even methylisoxazole-4-propionic acid (AMPA)-type receptors is required after complete recovery of ABR thresholds. for synaptopathy, and here we identify a possible involvement of Available evidence strongly supports the presumption that NICS + GluA2-lacking Ca2 -permeable AMPA receptors (CP-AMPARs) using is excitotoxic damage caused by excessive release of glutamate IEM-1460, which has been shown to block GluA2-lacking AMPARs. from overstimulated IHCs. Direct application of glutamatergic In CBA/CaJ mice, a 2-h exposure to 100-dB sound pressure level agonists causes vacuolization of postsynaptic bouton terminals, octave band (8 to 16 kHz) noise results in no permanent threshold similar to the morphological changes seen after sound over- shift but does cause significant synaptopathy and a reduction in exposure (5, 6). Deficiency of the glutamate aspartate transporter auditory brainstem response (ABR) wave-I amplitude. Chronic intra- increases synaptic glutamate and exacerbates noise damage (7). cochlear perfusion of IEM-1460 in artificial perilymph (AP) into adult Conversely, blockade of glutamate receptors reduces noise dam- CBA/CaJ mice prevented the decrease in ABR wave-I amplitude and age to synapses (6), and genetic removal of vesicular glutamate NEUROSCIENCE the synaptopathy relative to intracochlear perfusion of AP alone. release from IHCs prevents synapse loss after noise exposure (8). Interestingly, IEM-1460 itself did not affect the ABR threshold, pre- Although NICS requires activation of glutamate receptors, the sumably because GluA2-containing AMPARs can sustain sufficient specific receptor types, the downstream mechanism(s) of damage, synaptic transmission to evoke low-threshold responses during and the reasons for selective vulnerability of a subset of synapses blockade of GluA2-lacking AMPARs. On individual postsynaptic den- are unknown. sities, we observed GluA2-lacking nanodomains alongside regions SGNs express a variety of glutamate receptor types, in- with robust GluA2 expression, consistent with the idea that individ- cluding N-methyl-D-aspartate (NMDA), α-amino-3-hydroxy-5- + ual synapses have both CP-AMPARs and Ca2 -impermeable AMPARs. methylisoxazole-4-propionic acid (AMPA), kainate, and metabo- SGNs innervating the same IHC differ in their relative vulnerability to tropic receptors (9, 10). The AMPA type is responsible for the noise. We found local heterogeneity among synapses in the relative abundance of GluA2 subunits that may underlie such differences in Significance vulnerability. We propose a role for GluA2-lacking CP-AMPARs in noise-induced cochlear synaptopathy whereby differences among Noise can cause excitotoxic trauma to cochlear synapses by synapses account for differences in excitotoxic susceptibility. These triggering excessive release of the neurotransmitter glutamate data suggest a means of maintaining normal hearing thresholds from the auditory sensory hair cells. We report that a specific while protecting against noise-induced synaptopathy, via selective + class of glutamate receptors, Ca2 -permeable α-amino-3-hydroxy- blockade of CP-AMPARs. 5-methylisoxazole-4-propionic acid (AMPA) receptors (CP-AMPARs), is largely responsible for this trauma. Because cochlear syn- excitotoxicity | cochlear synapse | spiral ganglion neuron | noise-induced + apses are heterogenous with respect to glutamate receptors, synaptopathy | Ca2 -permeable AMPA receptor the observation that a specific class is responsible might ex- plain the variability in susceptibility to noise among synap- piral ganglion neurons (SGNs) are bipolar neurons in the ses. Selective blockade of CP-AMPARs prevents excitotoxicity Scochlea that conduct auditory information from the sensory and noise-induced cochlear synaptopathy, while other glu- hair cells to the brain. Almost all (95%) SGNs are type I and tamate receptors continue to mediate neurotransmission and synapse on inner hair cells (IHCs); the remaining 5%, type II, allow hearing. synapse on outer hair cells. (Herein, “SGN” used without quali- fication refers to type I.) Each IHC in the rodent cochlea provides Author contributions: N.H., M.A.R., and S.H.G. designed research; N.H., M.A.R., and S.H.G. the sole afferent presynaptic input to ∼10 to 20 SGNs, depending performed research; N.H., M.A.R., and S.H.G. analyzed data; and N.H., M.A.R., and S.H.G. on species and tonotopic location. Each SGN has a myelinated wrote the paper. axon with an unbranched dendritic terminal making one synapse Competing interest statement: A provisional patent application titled “Targeting Calcium-Permeable AMPA Receptors for Inner Ear Therapy with IEM-1460 and Related with one IHC. Each of these synapses consists of a single post- Compounds” was filed on 23 December 2019. Some data presented in this paper was synaptic density (PSD) apposed to a presynaptic ribbon-type active cited in the application. zone in the IHC. Each PSD includes the scaffold protein PSD95 This article is a PNAS Direct Submission. D.P.C. is a guest editor invited by the and is estimated to contain a few thousand glutamate receptors Editorial Board. receiving excitation from the IHC ribbon synapse (1, 2). Published under the PNAS license. Exposure to sounds at high levels can destroy cochlear hair 1To whom correspondence may be addressed. Email: [email protected]. cells, resulting in permanent elevation of the hearing threshold. This article contains supporting information online at https://www.pnas.org/lookup/suppl/ Even at a sound pressure level (SPL) too low to permanently doi:10.1073/pnas.1914247117/-/DCSupplemental. impair hair cell function, exposure may impair hearing by destroying www.pnas.org/cgi/doi/10.1073/pnas.1914247117 PNAS Latest Articles | 1of11 Downloaded by guest on September 25, 2021 large fast excitatory currents necessary for synaptic transmission nerve fibers spiking at sound onset. The ABR waveform is a series at cochlear afferent synapses (11–14) and for excitotoxicity (15). of peaks with latencies corresponding to electrical activity at AMPA-type glutamate receptors are tetrameric combinations of successive levels of auditory processing in the brain. The am- GluA1, GluA2, GluA3, and GluA4 subunits, of which primarily plitude of the first peak (i.e., wave-I amplitude) is proportional the latter three are expressed in the PSDs of SGNs (9, 16). Any to the number of synchronously spiking SGNs. Synapses lost as a combination of AMPA receptor (AMPAR) subunits makes a re- consequence of NICS reduce the number of SGNs responding, + + + ceptor permeable to Na ,K ,andCa2 ions, but combinations that resulting in a reduced wave-I amplitude but, if hair cells are + include GluA2 have greatly reduced Ca2 permeability (17, 18). intact, no change in auditory threshold (27). Because GluA2 appears to be expressed at every synapse, it might The experiment was designed to allow us to establish for each + be assumed that all synapses lack Ca2 -permeable AMPAR re- mouse in each experimental group the effects of noise and the ceptors (CP-AMPARs), but this does not appear to be the case. effects of IEM-1460 while correcting for any possible effects In synapses on frog ear and fish lateral line hair cells, CP- of surgery on the cochlea (Fig. 1A). ABR was measured five AMPARs in particular appear to play a major role in synaptic times in each mouse that underwent surgery and three times in transmission and excitotoxicity (16). This is potentially significant unoperated mice. The first measurement was done before + because studies of excitotoxicity in the brain have implicated Ca2 surgery and the second was done at 3 d postsurgery, to de- influx as a key mediator of excitotoxic trauma (19), and although termine any effects on the threshold or the wave-I growth curve + NMDA-type glutamate receptors play the major role in Ca2 in- (amplitude as a function of sound level). These two measure- flux, CP-AMPARs also have a role in glutamate toxicity in the ments provided a baseline for any effects of surgery or IEM- brain (20–22). Moreover, previous in vitro (23) and in vivo (24) 1460 on the ABR. The cannula connecting the minipump to the + studies have suggested that, unlike in the brain, Ca2 entry via round window was filled with artificial perilymph (AP) only, so NMDA-type glutamate receptors is not a major contributor to that the minipump contents reached the cochlea at 3.5 d after excitotoxicity in the cochlea. Rather, we suggest that cochlear the surgical implantation. Approximately 1.5 d after the entry excitotoxicity is mediated by GluA2-lacking CP-AMPARs. To of IEM-1460 into the cochlea, a third ABR measurement was directly test this hypothesis, we used IEM-1460, a selective performed to assessed the effect of IEM-1460 itself on hearing. blocker of GluA2-lacking AMPARs (25, 26), in noise-exposed This measurement also provided the prenoise baseline for mice, with the results implicating a role of GluA2-lacking/CP- comparison to and normalization of postnoise measurements AMPARs in NICS. for each mouse. At age 13 to 14 wk and with minipump contents already pre- Results sent in the cochlea for ∼2.5 d, two groups of mice were exposed Experimental Design and Timeline of ABR Measurements.