Cytotoxic Action of Lindane in Neocortical GABAergic Neurons Is Primarily Mediated by Interaction With Flunitrazepam-Sensitive GABA A Receptors Carmen Vale, 1 Inge Damgaard, 3 Cristina Sun ˜ ol, 1 Eduardo Rodrı ´guez-Farre ´, 2 and Arne Schousboe 3 * 1 Department of Neurochemistry, Institut d’Investigacions Biome `diques de Barcelona, CSIC, Barcelona, Spain 2 Department of Pharmacology and Toxicology, Institut d’Investigacions Biome `diques de Barcelona, CSIC, Barcelona, Spain 3 PharmaBiotec Research Center, Department of Pharmacology, The Royal Danish School of Pharmacy, Copenhagen, Denmark The cytotoxic action of the -isomer of hexachlorocy- clohexane (-HCH; lindane) was studied in cultured mouse neocortical neurons by measurements of the reduction in mitochondrial function using the MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) test. The cells were exposed to 30-300 μM lindane in the culture medium for different periods of time and lindane cytotoxicity was found to be time- and concentration-dependent. Lindane cytotoxicity could be ameliorated by addition of gamma aminobu- tyric acid (GABA) in a concentration-dependent man- ner but this effect of GABA was not blocked by bicuculline or picrotoxinin (PTX). Lindane induced cytotoxicity was also reduced by the GABA A receptor agonists muscimol and THIP (4,5,6,7-tetrahydroisoxa- zolo[5,4-c]pyridin-3-ol). This effect was enhanced by the simultaneous presence of flunitrazepam but only at the highest lindane concentrations studied (200 and 300 μM). Flunitrazepam by itself had no effect on lindane-induced cytotoxicity. The protective effect of GABA plus flunitrazepam was blocked by the benzodi- azepine receptor antagonist flumazenil and by the GABA A antagonist bicuculline, suggesting the involve- ment of central benzodiazepine receptors allosteri- cally coupled to the GABA recognition site at the GABA A receptor. When 100 μM PTX was used to suppress the protective effect of GABA and flunitraz- epam, a significant effect of PTX was observed only at 300 μM lindane. The GABA B receptor agonist, baclo- phen, only marginally reduced the cytotoxic effect induced by the highest lindane concentrations. It is concluded that the cytotoxic action of lindane in neocortical neurons in culture is mediated primarily through an interaction with allosterically coupled GABA-benzodiazepine recognition sites at the GABA A receptor. J. Neurosci. Res. 52:276–285, 1998.  1998 Wiley-Liss, Inc. Key words: neurons; cytotoxicity; lindane; -HCH; MTT-test; GABAreceptors; benzodiazepine receptors INTRODUCTION Hexachlorocyclohexane (HCH) comprises a series of isomers and their insecticide effects are highly depen- dent on the steric configuration of the molecules. The -isomer of HCH (lindane) is widely used as a pesticide in agriculture and against ectoparasites in human and veterinary medicine (Smith, 1991; Brown et al., 1995). Although lindane has some advantages as a pesticide because of its relatively low cost and wide spectrum of action, its use has been restricted in many countries due to its neurotoxicity. Its main neurotoxic effect in mammals involves different manifestations of hyperexcitability and induction of convulsions (Tusell et al., 1987). This effect seems to be related to inhibition of gamma aminobutyric acid (GABA) neurotransmission since lindane binds to the picrotoxinin (PTX) site of the GABA A receptor complex (Abalis et al., 1985; Cole and Casida, 1986; Llorens et al., 1990; Pome ´s et al., 1993; Huang and Contract grant sponsor: Danish State Biotechnology Programmes; Contract grant sponsor: Danish Medical Research Council; Contract grant number: 9700761; Contract grant sponsor: Lundbeck Founda- tion; Contract grant sponsor: Fondo de Investigacion Sanitaria; Con- tract grant number: 97/0656; Contract grant sponsor: CIRIT, Generali- tat de Catalunya; Contract grant numbers: SGR 95-00115, 95-00551. *Correspondence to: Prof. A. Schousboe, D.Sc., PharmaBiotec Res. Center, The Neurobiology Unit, Dept. of Pharmacology, The Royal Danish School of Pharmacy, 2 Universitetsparken, DK-2100 Copenha- gen, Denmark. Received 5 December 1997; Accepted 17 December 1997 Journal of Neuroscience Research 52:276–285 (1998)  1998 Wiley-Liss, Inc.