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2-28 ~;I~TUH~BRTDIZATION ANALYSIS FOR CELLS EXPRESSING NEUROTENSIN RECEPTOR IN

TAKESHI HOUTANI’, KAZUO SATO’, TEIZO UEYAMA’, MICHIKO IKEDA’, MASAYUKI MASU”, SHIGETADA NAKANISHI’ AND TETSUO SUGIMOTO’, ‘Department of Anatomy, Kansai Medical University, Moriguchi, Osaka 570 and ‘Institute for Immunology, Faculty of Medicine, Kyoto University, Kyoto 606, Japan

Previous studies provided evidence for regional distribution of neurotensin receptor (NTR)-expressing cells in the adult or developing rat brain. We now report results of in situ hybridization analysis using digoxigenin-labeled RNA probes generated from rat NTR cDNA. Consecutive brain sections from adult Sprague-Dawlcy rats were hybri- dized with the RNA probes of different size and different strand orientation. Hybridization with antisense probes yield- ed dtstmct accumulations of hybridization signal in neuronal perikarya of discrete brain sites, while hybridization with corresponding sense probes in adjacent sections gave no detectable amount of hybridization signal. In the forebrain, highly specific hybridization signal was observed in the olfactory bulb, the cerebral cortex, the pyramidal cell layer of the hippocampus, the polymorph layer of the dentate gyrus, the subiculum, the striatum, the amygdala and the diagonal band. In the thalamus, it was seen in the anterodorsal, ventral and reticular thalamic nuclei. In the hypothalamus, it was found in the paraventricular and supraoptic nucle.i, the lateral hypothalamic area and the anterior hypothalamic region. In the brainstem, areas such as the substantia mgra pars compacta, the ventral tegmental area, the motor nuclei for vagus and hypoglossal nerves and the spinal trtgeminal nucleus exhibited distinct distribution patterns of NTR- expressing neurons. The results provide detailed morphology for the NTR-expressing neuronal system on the basis of the superior resolution of hybrids.

2-29 BACTERIAL EXPRESSION OF METABOTROPIC GLUTAMATE RECEPTOR (mGluR1) FUSION PROTEIN AND GENERATION OF ANTISERUM.

TAROU OGURUSU, RYUZO SHINGAI, Department of Information Science, Facultv of Ensineerins, Iwate Universitv, 4-Ueda, Morioka 020, Japan.

The metabotropic glutamate receptor (mGluR1) is linked to inositol phosphate/calcium intracellular signaling pathway (Masu et al., Nature, Vol. 349, pp. 760-765, 1991). In the present study, we generated polyclonal antiserum against mGluR1.

The full-length cDNA clone for mGluR1 was kindly provided by S. Nakanishi. Recombinant bacterial expression plasmid was constructed that encodes the hydrophilic region at the amino terminus of mGluR1. The fusion protein was expressed in E. coli and used to produce rabbit antiserum. IgG from the serum was immobilized on agarose and used to immunoaffinity purify mGluR1 from rat brain. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the isolated proteins revealed a major band with an apparent Mr of -100 kDa and several minor bands.

2-30 MONOCLONAL ANTIBODY AGAINST GABAB RECEPTOR. HIROSHI NAKAYASU, ??HIROSHI KIMURA AND KINYA KURIYAMA, Department of Pharmacology, Kyoto Prefectural

University - of medicine, Kawaramachi-Hirokoji, Kamikyo 602. 'Institute of Molecular Neurobiology, Shiga University of medical Sciences, Seta, Otsu 52021 JAPAN -AA

We have prepared a ??onoclonal antibody against bovine cerebral GABA receptor using partially purified preparation as immunogen. This antibody recogn zed B only one protein band (80 kDa) among synaptic membrane proteins. This antibody could absorb GABAB binding activity from solubilized synaptic membrane fraction. On immunocytochemistry, this antibody stained synapses of neurons, but not glial cells. Using the antibody conjugated immunoaffinity beads, we could purify the 80 kDa protein which showed strong GABAB binding. To determine whether this protein is actually GABAB receptor or just only nonspecific GABA binding protein, the purified preparation was reconstituted with GTP binding protein and adenylate cyclase on phospholipid membrane. The cyclase activity on the membrane was inhibited by the addition of GABA or baclofen (specific GABAB receptor agonist), and this inhibition by GABA or baclofen was antagonized by hydroxysaclofen, a specific GABAB receptor-antagonist. These results: taken together, indicate that the 80 kDa protein recognized by this monoclonal antibody is actually the receptor molecule itself.

GABAB