Journal of zyxwvutsrqponm Neurorhemis!ry Raven Press, Ltd., New York zyxwvutsrqpo 0 1988 International Society for Neurochernistry Rapid Communication Regional Distribution of the zyx GABAA /Benzodiazepine Receptor zyxw (a Subunit) mRNA in Rat Brain Pascale Montpied, Brian M. Martin, Sandra L. Cottingham, Barbara K. Stubblefield, Edward I. Ginns, and Steven M. Paul Sections on Molecular Pharmacology and Neurogenetics, Clinical Neuroscience Branch, NIMH, Bethesda, Maryland, U.S.A. Abstract: A human cDNA clone containing the 5’ coding region of the GABAA /benzodiazepine receptor a subunit was used to quantify and visualize receptor mRNA in various regions of the rat brain. Using a [32P]CTP-labelled antisense RNA probe (860 bases) prepared from the 01 subunit cDNA, multiple mRNA species were detected in Northern blots using total and poly A rat brain RNA. In all brain regions, mRNAs of 4.4 and 4.8 kb were observed, and an additional mRNA of 3.0 kb was detected in the cerebellum and hippocampus. The level of GABAA /benzodiazepine receptor mRNA was highest in the cerebellum followed by the thalamus zyxwvutsrq = frontal cortex = hip pocampus = parietal cortex = hypothalamus B pons = striatum = medulla. zyxwvutsrq In situ hybridization revealed high levels of a subunit mRNA in cerebellar gray matter, olfactory bulb, thalamus, hippo- campus/dentate gym, and the arcuate nucleus of the hypothalamus. These data suggest the presence of multiple GABAA/benzodiazepine receptor a subunit mRNAs in rat brain and demonstrate the feasibility of studying the expression of genes encoding the GABA,/benzodi- azepine receptor after pharmacological and/or environmental ma- nipulation. Key Words: GABAA/Benzodiazepine receptor zyxwvutsr a zyxwvu sub- unit-mRNA-cDNA probe-Northem blot analysis. Montpied P. et al. Regional distribution of the GABA,/benzodiazepine receptor (a subunit) mRNA in rat brain. J. Neurochern. 51,1651-1654 (1988). Benzodiazepines produce their major pharmacological ac- tions by augmenting the actions of the primary inhibitory neurotransmitter in brain, y-aminobutyric acid (GABA; 01- sen, 198 1; Skolnick and Paul, 1982). Extensive evidence demonstrates that benzodiazepines augment GABAergic neurotransmission by allosterically enhancing GABA recep- tor-mediated C1- conductance (Choi et al., 1977; Study and Barker, 1981). The latter is mediated by specific benzodiaze- pine recognition sites structurally associated with a subclass of GABA receptors: the GABAA /benzodiazepine receptor complex. The GABAA /benzodiazepine receptor also contains binding sites for a number of chemically dissimilar sedative/ hypnotic (Olsen, 198 1) and anxiogenic/convulsant drugs (Braestrup et a]., 1982), which, respectively, enhance or inhibit GABA-receptor-mediated Cl- conductance. Studies of the purification and structural characterization of the GABAA f benzodiazepine receptor suggest that the receptor exists as an oligomeric protein complex of at least four subunits (a2, z p2; Mamalaki et al., 1987),which form a central C1- channel. Photoaffinity labelling experiments further suggest that the benzodiazepine and GABA recognition sites reside on the a and @ subunits, respectively (Casalotti et al., 1986). Recently, Schofield and colleagues (1987) purified and partially sequenced the GABA,/benzodiazepine receptor from bovine brain. Based on the amino acid sequence of GABAA-receptor-denved polypeptides, synthetic oligode- oxynucleotide probes were synthesized and used to isolate cDNA clones for both the a and B subunits from a cDNA library of bovine cerebral cortex. The deduced amino acid sequences of these clones and their corresponding hydropathy profiles suggest that the GABAA fbenzodiazepine receptor is a member of a super-family of receptor-gated ion channels including the nicotinic-cholinergic and glycine receptors. Si- multaneous expression of the LY and p subunit mRNAs in frog oocytes produces a functional receptor with the electro- physiological and pharmacological properties of the native receptor (Schofield et al., 1987). Recently, we used oligodeoxynucleotidesderived from the reported cDNA sequences of the bovine brain GABAA /ben- zodiazepine receptor subunits to isolate human cDNA clones for this receptor (Martin et al., in preparation). Using a human cDNA clone homologous to the 5’ region of the bovine a subunit cDNA, a [32P]CTP-labelled antisense RNA probe was generated and used to characterize the regional distri- bution of the GABAA lbenzodiazepine receptor a subunit mRNA in rat brain by Northern analysis and in situ hybrid- ization. MATERIALS AND METHODS Isolation of GABAA /benzodiazepine receptor (a subunit) cDNA clones Oligodeoxynucleotides derived from the nucleotide se- quence of the bovine GABAA/benzodiazepine receptor a subunit (Schofield et al., 1987) were synthesized and used to Received July 15, 1988; accepted August 1, 1988. Address correspondence and reprint requests to Dr. Steven M. Paul at Section on Molecular Pharmacology, Clinical Neuroscience Branch, Building 10, Room 4N212, National institute of Mental Health, 9000 Rockville Pike, Bethesda, Maryland 20892, USA. dodecyl sulfate; SSC, standard saline citrate. Abbreviations used: GABA, y-aminobutyric acid; SDS, sodium 1651