Dopaminergic axons directly make synaptic contacts with gabaergic and cholinergic neurons in the rat neostriatum

S8 PRODUCTION, CHARACTERIZATIONAND IMMUNOHISTOCHEMICALAPPLICATION OF MONOCLONALANTIBODIES TO RAT BRAIN GLUTAMINASE TAKESHI KANEKO*, YOSHIHIROURADE*, YASUYOSHIWATANABE* and NOBORUMIZUNO Department of Anatomy (Ist Division), Faculty of M~dicine, Kyoto University, Kyoto 606, Japan, and Hayaishi Bioinformation Transfer Project, Research Development Corporation of Japan, Kyoto 601, Japan Glutamatergic neurons are generally considered to contain phosphate-activated glutaminase (L-glutamine amidohydrolase, EC 3.5.1.2) which catalyzes the hydrolytic cleavage of glutamine to glutamate and ammonium ions. Thus, we attempted to produce monoclonal antibodies to rat brain glutaminase, and to identify immunohistochemically glutamatergic neurons. Phosphate-activated glutaminase (PAG) was obtained from rat brain mitochondria; i t was solubilized with Triton X-IO0 and purified 3000-fold. In polyacrylamide gel electrophoresis, the purified PAG showed a single band up to 1.7 ug after the s i l v e r stain at the position of molecular weight 62,000. Subsequently, 9 lines of monoclonal antibodies (IgMs) were produced against the purified PAG; these were named MAb-19, -21, -45, -48, -51, -55, -59, -97, and -120. Immunotitration tests revealed that 6 of the 9 antibodies (MAb-19, -21, -45, -48, -97, and -120) absorbed dose-dependently over 75% of glutaminase a c t i v i t y in the rat brain. In immunoelectroblotting after gel electrophoresis of the homogenate, a l l antibodies showed a positive band at the same position as that of the purified PAG. MAb-55, -59, and -97, however, cross-reacted with other proteins; the cross-reactive molecules binding to MAb-55 were found in Triton X-1OO-solubilized brain, and those binding to MAb-59 or -97 were detected in proteins solubilized with sodium dodecyl sulfate. On the basis of these results, of the 9 lines of antibodies produced in the present study, MAb-19, -21, -45, -48, and -120 were considered to be specific for PAG. The immunohistochemical a p p l i c a b i l i t y of these 5 lines of antibodies was then tested by using the rat brain, and the results indicated that they could immunohistochemically serve as good markers for glutamatergic neurons in the rat brain. Immunohistohcemical features of "false positive" labeling with non-specific antibodies were also examined by using MAb-51, -55, -59, and -97.

DOPAMINERGIC AXONS DIRECTLY MAKE SYNAPTIC CONTACTS WITH GABAERGIC AND CHOLINERGIC NEURONS IN RAT NEOSTRIATUM

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YOSHIYUKI KUBOTA, SHOZO KITC, SHOICHI SHIMADA and SHINOBU INAGAKI, Third Department of Internal Medicine, Hiroshima University School of Medicine, I-2-3 Kasumi, Minami-ku, Hiroshima 734 The synaptic relationships between the neuronal structures reacting with antibodies to tyrosine hydroxylase (TH) and glutamate decarboxylase (GAD) or choline acetyltransferase (CHAT) were studied by the electron microscopic "mirror technique" in adjacent sections of the rat neostriatum. GAD-IR neurons were divided into four groups. The first type cell ~ms medium in size (~2.7 ± 1.88 Nm, n=12) with an unindented nucleus, and thus it was suggested to belong to the medium spiny category that posseses a long axon. The second type of GAD-IR neuron was medium in size (13.9 ± 1.25 ~m, n=11) with nuclear indentations, suggesting this type was a medium aspiny neuron with a short axon. The others were large in size showing oval, triangular or multipolar shapes with nuclear indentations. The type I large neuron (20.4 ± 3.20 ~m, n=7) made a £ew synaptic contacts with their perikaryon and didn't have somatic spines, suggesting that this type belonged to the type I large neurons. The type 2 large neuron (20.0 ± 2.90 ~m, n=4) received many synaptic inputs on their cell soma (maximum 13 or 14 per one ultrathin section), suggesting that they belonged to the secondary projecting neuron. All types of GAD-IR neurons received synaptic inputs from TH-IR axons and also very frequently from GAD-IR axons. ChAT-IR neurons were large in size (22.7 + 2.93 >m, n=18) with indented nucleus and showed oval, triangular or multipolar shape, suggesting that they belonged to the type ] large neuron described by Chang and Kitai. Counterparts of all ChAT-IR neurons have synatic contancts with TH-IR axonal boutons. In our previous studies, leu- or met-enkephalin or substance P-IR neurons in the neostriatum received synaptic inputs from TH-IR axons, respectively. Thus, this and our previous studies provide ultrastructural evidence that TH-IR axons, presumably originated from dopaminergic nigrostriatal neurons, can influence a large variety of striatal neurons, such as ENK, SP, GABA and Ach neurons.