Elsevier

Neuropharmacology

Volume 39, Issue 11, October 2000, Pages 1959-1973
Neuropharmacology

The selective AMPA receptor antagonist GYKI 53784 blocks action potential generation and excitotoxicity in the guinea pig cochlea

https://doi.org/10.1016/S0028-3908(00)00069-1Get rights and content

Abstract

The role of AMPA receptors in cochlear synaptic transmission and excitotoxicity was investigated by comparing the actions of a selective AMPA antagonist GYKI 53784 (LY303070) with additional AMPA/kainate antagonists, GYKI 52466 and DNQX, and the NMDA antagonist, D-AP5, in several electrophysiological, neurotoxicological and histochemical tests. GYKI 53784 had the same potency as DNQX and was 10 times more potent than GYKI 52466 in reducing auditory nerve activity. The NMDA antagonist D-AP5 had no effect on auditory nerve activity. When single-fiber activity was blocked with GYKI 53784, the effects of AMPA or kainate were also antagonized. GYKI 53784 completely blocked excitotoxicity (i.e. destruction of the afferent nerve endings) induced by AMPA and kainate. The histochemical detection of Co2+ uptake was used to study Ca2+ influx within the primary auditory nerve cells. Application of AMPA induced no significant Co2+ uptake into the cells, suggesting that these receptors normally have a very low permeability to Ca2+. Application of kainate induced significant Co2+ uptake that was blocked by the AMPA receptor antagonist GYKI 53784 suggesting that kainate stimulated Ca2+ entry through AMPA receptor channels. Results suggest that AMPA-preferring receptors are functionally located at the sensory cell-afferent synapse whereas NMDA and kainate receptors are not.

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.

References (38)

  • J.L Puel et al.

    α-Amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) electrophysiological and neurotoxic effects in the guinea pig cochlea

    Neuroscience

    (1991)
  • R Pujol et al.

    Kainic acid selectively alters auditory dendrites connected with cochlear inner hair cells

    Hearing Research

    (1985)
  • L.O Trussell et al.

    Glutamate receptor desensitization and its role in synaptic transmission

    Neuron

    (1989)
  • X.Y Zheng et al.

    Recovery of structure and function of inner ear afferent synapses following kainic acid excitotoxicity

    Hearing Research

    (1997)
  • C.F Zorumski et al.

    Blockade of desensitization augments quisqualate excitotoxicity in hippocampal neurons

    Neuron

    (1990)
  • M Baudry

    Functions and regulations of glutamate receptors

  • R.P Bobbin

    Glutamate and aspartate mimic the afferent transmitter in the cochlea

    Experimental Brain Research

    (1979)
  • R.P Bobbin et al.

    Neurotransmitters of the cochlea and the lateral line organ

  • J Boulter et al.

    Molecular cloning and functional expression of glutamate receptors subunit genes

    Science

    (1990)
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