Glutamate transporters in the guinea-pig cochlea: partial mRNA sequences, cellular expression and functional implications G. Rebillard, 1 J. Ruel, 1 R. Nouvian, 1 H. Saleh, 1 R. Pujol, 1 Y. Dehnes, 2 J. Raymond, 3 J. L. Puel 1 and G. Devau 3 1 Inserm U 254, Universite ´ Montpellier I, Neurobiologie de l’audition – Plasticite ´ synaptique, 71, rue de Navacelles 34090 Montpellier, France 2 University of Oslo, Anatomical Institute, PO Box 1105, Blindern, N-0317 Oslo, Norway 3 Inserm U 432, Universite ´ Montpellier II, Neurobiologie et de ´ veloppement du syste ` me vestibulaire, Place Euge ` ne Bataillon, 34095 Montpellier cedex 05, France Keywords: DHK, excitatory amino acid transporters, excitotoxicity, inner ear, PDC Abstract In the cochlea, glutamate plays a major role in synaptic transmission between the inner hair cell and the primary auditory neurons. Extracellular glutamate concentration must be regulated to prevent excitotoxicity. This regulation is mediated by excitatory amino acid transporters, membrane proteins that remove glutamate from the synaptic cleft. In this study, we investigated the distribution and activity of three excitatory amino acid transporters subtypes in the guinea-pig cochlea: glutamate aspartate transporter, glutamate transporter and excitatory amino acid carrier. A partial messenger ribonucleic acid sequence was determined for each of these transporters, by polymerase chain reaction with degenerate primers, using guinea-pig brain complementary deoxyribonucleic acid as the template. Primers specific for each transporter were then designed and used to screen a dissected organ of Corti complementary deoxyribonucleic acid library. The cellular distribution of each transporter was examined by immunocytochemistry. We investigated the functional consequences of inhibiting glutamate uptake by recording cochlear potentials during intracochlear perfusion with either L-trans-pyrrolidine-2,4-dicarboxylic acid or dihydrokainate. At the end of the electrophysiological session, cochleas were processed for electron microscopy. Only the glutamate aspartate transporter messenger ribonucleic acid was detected in the organ of Corti. Consistently, glutamate aspartate transporter protein was detected in the inner hair cell-supporting cells and in the ganglion of Corti satellite cells. Glutamate transporter and excitatory amino acid carrier were found in the afferent auditory neurons. Only intracochlear perfusions with L-trans-pyrrolidine-2,4-dicarboxylic acid resulted in a dose-dependent decrease in the amplitude of the cochlear compound action potential, leaving cochlear microphonic potential unaffected. After L-trans-pyrrolidine-2,4-dicarboxylic acid perfusion, cochleas displayed a swelling of the afferent endings typical of excitotoxicity. [(–)1-(4-aminophenyl)-4-methyl-7,8-methylenedioxy- 4,5-dihydro-3-methylcarbamyl-2,3-benzodiazepine], a selective a-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptor antago- nist protects the cochlea against L-trans-pyrrolidine-2,4-dicarboxylic acid effect. Introduction In the cochlea, glutamate is the main neurotransmitter acting between the inner hair cell (IHC) and the primary auditory afferent neurons (Puel, 1995; Ottersen et al., 1998). The activation of a-amino- 3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptors mediates fast sensory transmission (Ruel et al., 1999, 2000; Glowatzki & Fuchs, 2002). However, persistently high extracellular glutamate concentrations are excitotoxic and must be controlled to prevent damage to the afferent neurons endings (Olney, 1986; Choi, 1994). Glutamate transporters form a powerful uptake system that rapidly eliminates glutamate from the extracellular spaces (Seal & Amara, 1999; Sims & Robinson, 1999; Tanaka, 2000; Danbolt, 2001). Five subtypes of Na þ -dependent glutamate transporters have been cloned and identified to date, in various species, including humans: GLAST (excitatory amino acid transporter, EAAT1, glutamate aspartate trans- porter; Storck et al., 1992; Tanaka, 1993); GLT-1 (EAAT2, glutamate transporter; Pines et al., 1992); EAAC1 (EAAT3, excitatory amino acid carrier; Kanai & Hediger, 1992); EAAT4, which is present in the soma and dendrites of Purkinje cells (Fairman et al., 1995); and EAAT5, which is expressed in the retina (Arriza et al., 1997). GLAST messenger ribonucleic acid (mRNA) has been detected in the organ of Corti of the rat (Li et al., 1994). Immunocytochemical studies by light and electron microscopy have shown that GLAST is present in the supporting cells surrounding IHCs and in the satellite cells surrounding spiral ganglion neurons in rat and guinea-pig cochleas (Furness & Lehre, 1997). In GLAST-deficient mice, acoustic overstimulation exacerbates hearing loss (Hakuba et al., 2000), suggesting that glutamate trans- porters play a key role in protecting primary auditory neurons during sound stimulation. Damage to afferent neurons due to high glutamate concentrations are observed in various cochlear diseases induced by ischaemia or acoustic trauma, and causes severe hearing loss (for review, see Puel, 1995). A dysfunction of glutamate transporter activity may aggravate damage to the cochlea. In this study, we investigated the expression of GLAST, GLT-1 and EAAC1 mRNA and their protein distribution in the adult guinea-pig cochlea. We also European Journal of Neuroscience, Vol. 17, pp. 83–92, 2003 ß Federation of European Neuroscience Societies doi:10.1046/j.1460-9568.2003.02429.x Correspondence: Dr G. Devau, as above. E-mail: gdevau@iuniv-montp2.fr Received 18 July 2002, revised 14 October 2002, accepted 29 October 2002