Pergamon Neuroscience Vol. 85, No. 4, pp. 1235 1251, 1998 Copyright ~C 1998 IBRO. Published by ElsevierScienceLtd Printed in Great Britain. All rights reserved PII: S0306-4522(97)00684-2 O3O6 4522/98 $19.00+0.00 REDUCTION OF GABA AND GLUTAMATE TRANSPORTER MESSENGER RNAS IN THE SEVERE-SEIZURE GENETICALLY EPILEPSY-PRONE RAT M. T. AKBAR,* M. RATTRAY, + R. J. WILLIAMS, + N. W. S. CHONGt and B. S. MELDRUM*§ *Department of Clinical Neurosciences and tDepartment of Neuroscience, Institute of Psychiatry. London SE5 8AF, U.K. ~Biochemical Neuropharmacology Group, UMDS, Division of Biochemistry and Molecular Biology, Guy's Hospital, London SE1 9RT, U.K. Abstract The genetically epilepsy-prone rat is an animal model of inherited generalised tonic-clonic epilepsy that shows abnormal susceptibility to audiogenic seizures and a lowered threshold to a variety of seizure-inducing stimuli. Recent studies suggest a crucial role for glutamate and GABA transporters in epileptogenesis and seizure propagation. The present study examines the levels of expression of the messenger RNAs encoding the glial and neuronal glutamate transporters, GLT-I and EAAC-I, and the neuronal GABA transporter, GAT-1, in paired male genetically epileptic-prone rats and Sprague-Dawley control rats using the technique of in situ hybridization. In a parallel study, semiquantitative immuno- blotting was used to assess GLT-1 and EAAC-1 protein levels in similarly paired animals. Animals were assessed for susceptibility to audiogenic seizures on six occasions, and killed seven days following the last audiogenic stimulus exposure. Rat brains were processed for in situ hybridization with radioactive 35S-labelled oligonucleotide probes (EAAC-1 and GAT-1), 35S-labelled riboprobes (GLT-1), and Fluorescein-labelled riboprobes (GLT-1 and GAT-1) or processed for immunoblotting using subtype- specific antibodies for GLT-1 and EAAC-1. Semiquantitative analyses were carried out on X-ray film autoradiograms in several brain regions for both in situ hybridization and immunoblotting studies. Reductions in GAT-1 messenger RNA were found in genetically epileptic-prone rats in all brain regions examined (- 8 to 24% compared to control). Similar reductions in GLT-1 messenger RNA expression levels were seen in cortex, striatum, and CA1 ( - 8 to - 12%) of genetically epileptic-prone rats; the largest reduction observed was in the inferior colliculus (- 20%). There was a tendency for a reduced expression of EAAC-1 messenger RNA in most regions of the genetically epileptic-prone rat brain although this reached statistical significance only in the striatum (-12%). In contrast, no significant differences in GLT-1 and EAAC-1 protein between genetically epileptic-prone rats and control animals were observed in any region examined, although there was a tendency to follow the changes seen with the corresponding messenger RNAs. These results show differences in the messenger RNA expression levels of three crucial amino acid transporters. For the two glutamate transporters, GLT-1 and EAAC-I, differences in messenger RNA levels are not reflected or are only partially reflected in the expression of the corresponding proteins. ~2) 1998 IBRO. Published by Elsevier Science Ltd. Key words: GLT-1, GAT-1, EAAC-1, GEP rat, in situ hybridization, seizure. The genetically epilepsy-prone (GEP) rat is a nat- urally occurring model of generalized epilepsy which was originally derived from the Sprague-Dawley strain of rats by selective in-breeding. 57 Seizures are readily induced in GEP rats by high-intensity mixed frequency acoustic stimulation, usually by a loud §To whom correspondence should be addressed. Abbreviations: AS, audiogenic seizure; BSA, bovine serum albumin; EDTA, ethylenediaminetetra-acetate; GAD, glutamate decarboxylase; GEP rat, genetically epilepsy- prone rat; GFAP, glial fibrillary acidic protein; HRP, horseradish peroxidase; 1C, inferior colliculus; NMDA, N-methyl-D-aspartate; PBS, phosphate-buffered saline; PCR, polymerase chain reaction; SDS, sodium dodecyl sulphate; SSC, standard saline citrate; TBS, Tris-buffered saline. bell. At present two separate sub-strains exist (GEP rat-3 and GEP rat-9) that differ in their seizure severity: GEP-9 rats exhibit a severe audiogenic tonic-clonic (maximal) seizure, whereas GEP-3 rats exhibit (sub-maximal) clonus only, in response to the same stimulus. 34'57 Electrophysiological, pharmaco- logical and lesioning data have identified the inferior colliculus (IC) as the primary site for the initiation of audiogenic seizures (AS) in GEP rats. I°'L2'19 Pharmacological studies have demonstrated the involvement of the N-methyl-D-aspartate (NMDA) glutamate receptor and GABA A receptor as key components subserving initiation of AS in the IC in GEP rats. at ,24 In addition to susceptibility to sound-induced seizures, GEP rats are inherently predisposed to a 1235