IMMUNOHISTOCHEMICAL MAPPING OF ENKEPHALIN NEURONS

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The neuropeptides are a class of substances recently isolated from neural tissue and have been found to influence the activity of the nervous system at the level of the membrane, the neuron and the behaving organism. Immunohistochemistry of these substances has revealed discrete and rather extensive neuropeptide immunoreactivity in neuronal perikarya, axons and terminals. This proposal focuses on determining the details of the differential distribution of two of these peptides --methionine- and leucine-enkephalin. In addition to the extensiveness of enkephalinergic systems revealed in our preliminary studies, the enkephalins are of interist in light of their morphinomimetic properties at opiate receptors as well as on an organismic level. This proposal seeks to determine the differential distribution of enkephalins by purifying antisera to mono-specificity for each enkephalin by removing cross-reacting antibodies on a heterologous immunoabsorbent. The purified, monospecific antisera to each enkephalin will then be used in immunofluorescence histochemical procedures on sections of the rat brain to reveal the terminals, axons and perikarya containing either methionine- or leucine-enkephalin immunoreactivity. The results of these studies will be translated into detailed, schematic coronal maps of the rat brain with approximate stereotaxic coordinates. The connectivity of selected perikarya and terminals will be studied with immunofluorescence histochemistry on rat brains after stereotaxic lesions of enkephalinergic perikarya. Disappearance of normally-fluorescent terminals will indicate the perikarya of origin for these terminals. Selected regions will also be studied with antisera to other neuropeptides where convergence of enkephalinergic and other peptidergic systems has been reported. Finally, a small portion of this proposal (10%) aims to investigate possible changes in the differential distribution of the enkephalins in selected nuclei in the brains of mice made tolerant to and dependent upon opiates.