AGING-RELATED SUBUNIT EXPRESSION CHANGES OF THE GABA A RECEPTOR IN THE RAT HIPPOCAMPUS A. GUTIE u RREZ,* Z. U. KHAN,* D. RUANO,² C. P. MIRALLES,* J. VITORICA² and A. L. DE BLAS*‡ *Division of Molecular Biology and Biochemistry, School of Biological Sciences, University of Missouri—Kansas City, Kansas City, MO 64110-2499, U.S.A. ²Departamento de Bioquı ´mica, Bromatologı ´a y Toxicologı ´a, Facultad de Farmacia, Universidad de Sevilla, Seville, Spain Abstract––Aging-related changes in the subunit expression of some hippocampal GABA A receptors have been found. Quantitative in situ hybridization has revealed that  1 subunit messenger RNA expression was significantly increased in the hippocampus (34%) of old rats. The largest increases were observed in the dentate gyrus (76%) and in the CA1 field (30%). Quantitative immunocytochemistry also showed increased protein expression of the  1 subunit in the dentate gyrus (19%) and CA1 (14%) of old rats. The increased  1 messenger RNA and protein expression led to increased proportions of assembled GABA A receptors that contained  1 subunits, as revealed by quantitative immunoprecipitation of ( 3 H)flunitrazepam and ( 3 H)muscimol binding. In contrast, there were no significant changes in the expression of  2 ,  3 and total  2 ( 2S +  2L ) subunits, although a slightly increased expression of  2L peptide was detected in the hippocampus proper (7%), but not in the dentate gyrus. The results are consistent with the notion that in the rat hippocampus there is an aging-related change in the subunit composition of some GABA A receptors. Copyright  1996 IBRO. Published by Elsevier Science Ltd. Key words: GABA A receptor, benzodiazepine receptor, mRNA, aging, antibody, rat brain. The mammalian GABA A /benzodiazepine receptors are formed by heteropentameric combination of  1–6 ,  1–3 ,  1–3 and  subunits. 23,29 These subunits are encoded by different genes. In addition, two forms of the  2 subunit, short ( 2S ) and long ( 2L ), are generated by alternative RNA splicing. 21,39 Studies of the GABA A receptor by radioligand binding, 32,35 ion fluxes in membrane vesicles 3,6,26 and ionic currents in dissociated neurons 12 have revealed the existence of functional changes in GABA A receptor/benzodiazepine receptor during normal aging. In addition, aging-related decreases of GABA A receptor  1 subunit mRNA in the cortex and  2 mRNA in the cerebellum, as well as increases in  6 mRNA in the cerebellum, have been reported. 25,26 The GABA A receptor turnover rate is slower in older animals. 27 Impaired agonist-induced GABA A receptor down-regulation with aging has also been observed. 36 We have recently reported aging-related decreased expression of GABA A re- ceptor subunits (mRNA and protein) and ( 3 H)- muscimol binding in the rat inferior colliculus, 14 and aging-related changes of  2S and  2L mRNA and protein expression in the cerebral cortex and cerebellum. 15 In the present communication, we report the existence of aging-related increased expressions of  1 subunit mRNA and protein in the rat hippocampus, which leads to changes in the subunit composition of the hippocampal GABA A receptor. In these studies we have used quantitative immunoprecipitation, immunocytochemistry and in situ hybridization techniques. EXPERIMENTAL PROCEDURES Sprague–Dawley male rats were purchased from Sasco Inc. (Omaha, NE, U.S.A.) and Fischer 344 male rats from the International Center of Aging (Bethesda, MD, U.S.A.). M embrane preparation Membranes from Fischer 344 rat hippocampi were pre- pared as described elsewhere 38 in the presence of protease inhibitors: 1 mM EDTA, 1 mM dithiothreitol, 1 mM ben- zamidine, 50 g/ml trypsin inhibitor type II-S and 50 g/ml bacitracin. GABA A receptor solubilization GABA A receptors were solubilized at 4C for 60 min (4 mg of protein/ml) with 1% (w/v) Triton X-100, 1% (w/v) sodium deoxycholate, 0.1% (w/v) sodium dodecyl sulfate, 140 mM NaCl and 10 mM Tris–HCl, pH 7.5 (RIPA buffer), containing the aforementioned protease inhibitors. 34,38 Immunoprecipitation Three different antibodies were used for immunoprecipi- tations: the mouse monoclonal antibody (mAb) 62-3G1 4,37 ‡To whom correspondence should be addressed. A bbreviations: EDTA, ethylenediaminetetra-acetate; mAb, monoclonal antibody; PB, phosphate buffer; PBS, phosphate-buffered saline; RIPA buffer, Triton X-100, sodium deoxycholate, sodium dodecyl sulfate, NaCl, Tris–HCl; SSC, standard saline citrate. Pergamon N euroscience Vol. 74, No. 2, pp. 341–348, 1996 Copyright  1996 IBRO. Published by Elsevier Science Ltd Printed in Great Britain 0306–4522/96 $15.00+ 0.00 PII: S0306-4522(96)00137-6 341