Immunohistochemical distribution of a-neo-endorphin/dynorphin neuronal systems in rat brain: Evidence for colocalization (opioid peptide neurons/brain distribution/immunofluorescence)

a-Neo-endorphin anddynorphin immunoreactivities in rat brain were visualized by double antibody immunofluorescence of frozen sections. Antibodies were used that were specific for their respective antigens. The pattern of neuronal immunostaining produced by a-neo-endorphin and dynorphin antisera in adjacent serial sections was completely superimposible. No areas were found in which a-neo-endorphin or dynorphin immunoreactivities existed alone. The following brain regions contained a-neo-endorphin/dynorphin-immunoreactive fibers and terminals: the median forebrain bundle, the internal capsule, the substantia nigra, the hypothalamus, the nucleus accumbens, the hippocampus, and the medulla oblongata. A few fibers were seen in the cerebral cortex and in the corpus striatum. In many regions, this neuronal fiber system seems to overlap the neuronal system previously described to contain IMet]-/[Leu]enkephalin-immunoreactive material. In brains from colchicine-treated animals, numerous a-neo-endorphin/dynorphin-immunoreactive neuronal cell bodies were seen in the supraoptic, retrochiasmatic supraoptic, paraventricular, and magnocellular accessory nuclei of the hypothalamus. It is concluded that a-neo-endorphin-like and dynorphin-like immunoreactivities are part of the same neuronal system. Extracts from brain and posterior pituitary contain a number of opioid peptides (1-6) that are different in amino acid sequence from the classical opiate-like peptides [Met]and [Leu]enkephalin (7) and ,B-endorphin (8, 9). Two of these are a-neo-endorphin (3, 4) and dynorphin (1, 6, *), whose complete primary structure was elucidated recently (4, 6, *). Both peptides are related to the enkephalins inasmuch as they contain an NH2-terminal [Leu]enkephalin sequence attached to a series of more or less basic amino acid residues at the COOH terminus. The regional brain distributions of a-neo-endorphin and dynorphin were established by radioimmunoassay of extracts from dissected brain regions (10, 11). A preliminary immunohistochemical study of the localization -f dynorphin-immunoreactive material in rat brain-has been published (12). In that study, antibodies to an NH2-terminal fragment of dynorphin [dynorphin-(1-13)] (1) were used. Previously, we have shown that a-neo-endorphin and dynorphin immunoreactivity coexist in the neurons of the rat hypothalamus (13). Here, we report the distribution of a-neo-endorphinand dynorphin-immunoreactive nerve fibers, terminals, and cell bodies in brains from normal and colchicine-treated rats. The patterns ofneuronal immunostaining produced by the a-neo-endorphin and dynorphin -antisera were completely identical. No areas in the brain were detected in which a-neoendorphin or dynorphin immunoreactivity occurred alone. MATERIALS AND METHODS Antibodies. Antisera were raised against synthetic dynorphin-(1-17) (6, *) and against synthetic a-neo-endorphin (4). All peptides used in this study were synthesized by J.-K. Chang at Peninsula Laboratories (San Carlos, CA). Antibodies were generated by injecting a carbodiimide-treated peptide/thyroglobulin mixture into rabbits as described for the production of antibodies to NV-acetyl-,-endorphin (14). Two dynorphin antisera (R3-1 and R2-2) and two a-neo-endorphin antisera (R13 and R2-4) were used for the immunohistochemical studies. The specificity of all four antisera was tested extensively by radioimmunoassay (RIA), double antibody immunoprecipitation, and immunohistochemical blocking controls. In RIA using "WI-labeled dynorphin-(1-17) as tracer, dynorphin-(1-13) was 50% crossreactive with the dynorphin antibodies. The crossreactivity of dynorphin-(1-9) was <0.01%. Dynorphin-(6-13) and a.neo-endorphin did not crossreact. In the a-neo-endorphin RIA using "2I-labeled a-neo-endorphin as tracer, the two a-neo-endorphin antibodies had a crossreactivity of <0.01% with ,B-neo-endorphin [i.e., a-neo-endorphin(1-9)]. Dynorphin-(1-9), -(1-13), -(1-17), and -(6-13) were not crossreactive. [Leu]Enkephalin, [Met]enkephalin, a-neo-endorphin-(1-8), [Arg8]vasopressin, oxytocin, and substance P were not crossreactive with either antiserum. Because the antisera dilutions used in the immunohistochemical studies were much less than those used in the RIA, we also tested the a-neoendorphin and the dynorphin antibodies by double antibody immunoprecipitation at a dilution of 1:100 for binding of 'WIlabeled dynorphin and "WI-labeled a-neo-endorphin. Under these conditions, all four antisera immunoprecipitated only their own antigens: the dynorphin antisera did not immunoprecipitate "2I-labeled a-neo-endorphin and the a-neo-endorphin antisera did not precipitate 125I-labeled dynorphin. All four antisera were used throughout for visualizing a-neoendorphin/dynorphin-immunoreactive neurons in the brain. The immunostaining patterns produced by the four antisera were essentially identical; however; antisera R2-4 and R2-2 showed a somewhat brighter fluorescence of the a-neo-endorphin/dynorphin neuronal system. Immunohistochemistry. Serial sections (13 ,um thick) through 12 brains from normal male rats (Sprague-Dawley; 150-250 g) and through 6 brains from rats that had been injected intraventricularly with 50 ,g of colchicine in 25 ,ul of water 48 hr prior to death were immunofluorescently stained with a-neo-endorphin and dynorphin antisera. The fixation and double antibody Abbreviations: P1/NaCl, phosphate-buffered saline; RIA, radioimmunoassay. * Tachibana, S., Araki, K., Ohya, S. & Yoshida, S. (1981) in Proceedings of the International Narcotics Research Conference, Kyoto, Japan, p. 20 (abstr.). 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