Immunocytochemical Localization and Biochemical Characterization of α‐Melanocyte‐Stimulating Hormon in the Brain of the Rainbow Trout, Salmo gairdneri

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Abstract
The distribution of alpha-melanocyte-stimulating hormone (alpha-MSH)-like immunoreactivity in the central nervous system of the rainbow trout Salmo gairdneri was investigated by indirect immunofluorescence and peroxidase-antiperoxidase techniques, using a highly specific antiserum generated in rabbits against synthetic alpha-MSH. Immunoreactive perikarya were exclusively observed in the basal hypothalamus within the pars anterioris of the nucleus lateralis tuberis. In this region, a moderate number of small stained cell bodies were observed surrounding the dorsal wall of the anterior infundibular recess. These immunoreactive cells were organized in rostro-caudal rows extending over the whole portion of the nucleus. Positive fibres originating from these perikarya were visualized in the dorsal posterior lobe and the ventral hypothalamus. A dense tract of immunoreactive fibres projected ventrally through the pituitary stalk and terminated in the neurohypophysis. The concentrations of alpha-MSH in different regions of the brain were measured by means of a sensitive and specific radioimmunoassay. The dilution curves obtained with synthetic alpha-MSH and serial dilutions of diencephalon, mesencephalon, medulla oblongata, telencephalon or pituitary extracts were strictly parallel. The highest concentration of alpha-MSH in brain was found in the diencephalon (1.31 +/- 0.07 ng/mg protein). In contrast alpha-MSH was not detectable in cerebellar extracts. Reverse-phase high-performance liquid chromatography and radioimmunoassay were used to characterize alpha-MSH-like peptides in the trout brain and pituitary. Two major forms of immunoreactive alpha-MSH were resolved by high performance liquid chromatography in hypothalamic extracts; these peptides exhibited the same retention times as des-Na-acetyl alpha-MSH and its sulfoxide derivative, respectively. Additional peaks of alpha-MSH immunoreactive material were detected in pituitary extract. These latter peptides coeluted with authentic alpha-MSH, diacetyl alpha-MSH and their sulfoxide forms. These results provide the first evidence for the presence of alpha-MSH in the brain of a teleostean fish. Our data indicate that, in the brain, the immunoreactivity corresponds to the non-acetylated form of alpha-MSH, while three different types of alpha-MSH-like molecules (namely deacetylated, monoacetylated, and diacetylated forms) coexist in the pituitary. It thus appears that, in salmonoid fish, mono- or diacetylation of the N-terminal serine residue of aL-MSH only occurs at the pituitary level.