The selective AMPA receptor antagonist GYKI 53784 blocks action potential generation and excitotoxicity in the guinea pig cochlea
Introduction
In the cochlea, glutamate (Glu) is the neurotransmitter used by the sensory inner hair-cells (IHCs) to transduce the mechanical displacement of the basilar membrane into activity of the primary auditory afferent nerve fibers (reviewed in Puel, 1995). The ionotropic receptors used by Glu for fast excitatory synaptic transmission are classically divided into three types of receptors named after their sensitivity to agonists: N-methyl-d-aspartate (NMDA), α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) and kainate. Analysis of ionotropic Glu receptors by gene expression, immunocytochemistry and in situ hybridization indicates that primary auditory nerve cells express NMDA (NR1 and NR2A-D), AMPA (GluR2-4) and kainate (GluR5-7) receptor subunits and the high-affinity kainate-binding proteins (KA1 and KA2) (reviewed in Puel, 1995). This suggests that NMDA, AMPA and kainate receptors coexist on primary auditory nerve cells. However, the role of each of these receptors at the IHC-to-auditory nerve cell synapse is controversial and unsettled. For instance, although iontophoretic application of NMDA appears to induce excitation of the primary auditory nerve fibers (Felix and Ehrenberger, 1990), no effect of NMDA has been observed on isolated primary auditory nerve soma (Nakagawa et al., 1991; Ruel et al., 1999) or in intact preparations (reviewed in Bobbin et al., 1984). Another reason for this controversy is that, in addition to activating kainate receptors, kainate can also act on AMPA receptors (Boulter et al., 1990; Keinanen et al., 1990; Patneau and Mayer, 1991). Isolated primary auditory nerve soma have been shown to respond to Glu and AMPA by a fast onset inward current that was rapidly desensitized, while kainate induced only a non-desensitizing, steady-state current (Nakagawa et al., 1991; Ruel et al., 1999). To help in determining whether AMPA or kainate receptors are active at this synapse, Ruel et al. (1999) used the receptor-specific desensitization blockers, cyclothiazide and concanavalin A. While no effect was observed with concanavalin A, cyclothiazide greatly enhanced the Glu-, AMPA- and kainate-induced steady-state currents and potentiated Glu-induced membrane depolarization. Consistent with the in vitro data, cyclothiazide reversibly increased spontaneous activity of single auditory nerve fibers, while concanavalin A had no effect, suggesting that AMPA receptors, rather than kainate receptors, are involved at this synapse (Ruel et al., 1999).
The role of AMPA, kainate and NMDA receptors in excitotoxicity, a form of neuronal degeneration in the cochlea (reviewed in Puel, 1995) is also unclear. The molecular mechanism appears to involve overactivation of ionotropic Glu receptors that are permeable to cations. Prolonged opening of these ligand-gated ions channels leads to excessive ion permeation, osmotic swelling, free radical generation, and eventual neuronal death. In the cochlea, noise trauma and ischemia induce excitotoxicity (Pujol et al., 1992; Puel et al., 1994; Puel, 1995). Local application of Glu (Puel et al., 1994), AMPA (Puel et al., 1991, Puel et al., 1994) and kainate (Pujol et al., 1985; Zheng et al., 1997), but not NMDA (Puel et al., 1994), mimics these pathologies by disrupting the IHC-auditory nerve synapses. Thus, AMPA and kainate receptors, but not NMDA receptors, may have a role in cochlear excitotoxicity.
To date, the lack of a specific antagonist that differentiates between AMPA receptor-evoked responses and kainate receptor-evoked responses has limited our ability to determine the involvement of AMPA versus kainate receptors in neurotransmission between the IHCs and the primary auditory nerve fibers and excitotoxicity to these fibers. Recently, GYKI 53784 (LY303070) has been demonstrated to be one of the most selective antagonists for AMPA receptors (Bleakman et al., 1996). Taking advantage of this new pharmacological tool, the role of AMPA receptors in fast synaptic transmission and excitotoxicity was addressed by comparing GYKI 53784 (LY303070) with AMPA/kainate antagonists, GYKI 52466 and DNQX, and the NMDA antagonist, D-AP5, in several electrophysiological, neurotoxicological and histochemical tests.
Section snippets
Materials and methods
The care and use of animals was carried out according to the animal welfare guidelines of the “Institut National de la Santé et de la Recherche Médicale” (INSERM), and under the approval of the “Ministère Français de l'Agriculture et de la Forêt”. Experiments were performed on adult pigmented guinea pigs weighing 250–300 g (Charles River, France). The animals were anaesthetized with urethane (1.4 g kg−1, i.p.) and artificially respired. Supplemental doses (0.35 g kg−1, i.p.) were administered
Comparative effects of DNQX, GYKI 52466 and GYKI 53784 on gross cochlear potentials
The compound action potential of the auditory nerve (CAP: N1–P1), N1 latency, cochlear microphonic (CM) and summating potential (SP) were recorded after cumulative 10-min perfusions of increasing doses of DNQX (n=5), GYKI 52466 (n=5) and GYKI 53784 (n=5). Neither the initial perfusion of artificial perilymph nor the second perfusion with or without 0.4% methanol induced significant changes in CAP, N1 latency, CM or SP (Fig. 1, Fig. 2). The drug effects were therefore compared with the cochlear
Discussion
Whereas the NMDA antagonist D-AP5 had no effect, GYKI 53784 had the same potency as DNQX in reducing CAP. Application of Glu, AMPA and kainate induced a fast transient increase in spontaneous firing only for the fibers that had the lower pre-perfusion SRs. This transient increase was followed by a reduction in the spontaneous discharge rate. At the electron microscopic level, the cochleas perfused with Glu, AMPA and kainate, but not NMDA, showed a massive destruction of all dendrites of primary
Acknowledgements
We would like to thank Margaret H. Niedenthal for the generous gift of GYKI 53784 (LY303070), G. Humbert, S. Ladrech and N. Renard for their technical assistance and J.L. Pasquier for artwork. This work was supported in part by research grant numbers R01 DC 00722 and PO1 DC00379 from the National Institute on Deafness and Other Communication Disorders, National Institutes of Health, and DAMD 17-93-V-3013, Kam's Fund for Hearing Research, and the Louisiana Lions Eye Foundation.
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