UNLABELED ANTIBODY METHOD - CONTRASTING COLOR STAINING OF BETA-LIPOTROPIN AND ACTH-ASSOCIATED HYPOTHALAMIC PEPTIDES WITHOUT ANTIBODY REMOVAL

Staining patterns of three antisera to ACTH peptides and antiserum to ovine β-lipotropin were compared to reveal degrees of nonidentity of hypothalamic immunoreactivity with that found in the pituitary. Dual color immunocytochemistry was used to reveal association of these reactivities with those of other hypothalamic peptides. Cells of the pars intermedia were stained with each of the four antisera. Fibers in the dorsomedial nucleus and cell bodies and fibers in the arcuate nucleus and preoptic area were stained with anti-ACTH1-39 and 1-24, but remained unstained with anti-ACTH17-39 and anti-β-LPH. In the supraoptic and paraventricular nuclei and in the internal zone of the median eminence of all rats and in the pars nervosa of occasional rats, reactivity for anti-β-LHP and anti-ACTH1-39 were found in the same perikarya and fibers as vasopressin. Anti-ACTH1-24 and 17-39 failed to react in the magnocellular system. The hypothalamic reactivities of anti-ACTH1-24 and anti-β-LPH appeared to be specific for peptides resembling the immunizing antigens. Reactivity of anti-ACTH1-39 was due to ACTH-like material in the dorsomedial-arcuate area, but to neurophysin II in the magnocellular system. The data suggest that a) ACTH reactivity in the dorsomedial-arcuate area resembles that of pituitary ACTH; ACTH reactivity in the magnocellular system reflects an antigen unrelated to pituitary ACTH or at best possessing only partial homology with it. b) β-LHP and ACTH are derived from different precursors in the hypothalamus and pituitary, or alternatively, cleaved from a common precursor by different enzymes. The findings support the concept of regional diversity of neuropeptides and of neuronal idiotypy.