Distribution of gastrin releasing peptide-bombesin-like immunostaining in rat brain

Abstract

Several antisera were raised in rabbits against synthetic gastrin releasing peptide (1–27)(porcine)(GRP(1–27)) and GRP(1–16). In immunohistochemical staining experiments on frozen brain sections from rats, the antisera raised against GRP (1–27) also labeled substance P neurons. This substance P-like immunostaining was selectively blocked by incubation of the sections with GRP(1–27) antiserum in the presence of an excess concentration (50 μM) of synthetic substance P. A residual immunostaining was observed in sections stained with substance P absorbed GRP(1–27) antisera. This staining was blocked by addition of an excess concentration (10 μM) of synthetic GRP(1–27) or bombesin, but it was not blocked by a 10 μM concentration of GRP(1–16) or by a 100 μM concentration of vasoactive intestinal peptide, oxytocin, vasopressin, or by a further addition of 50 μM substance P. The antisera raised against GRP(1–16) did not label substance P neurons. The immunostaining produced by GRP(1–16) antisera was blocked by an excess (10 μM) concentration of GRP(1–16) or GRP(1–27). In the brainstem of normal rats both the substance P absorbed GRP(1–27) antisera and GRP(1–16) antisera labeled fibers in the ventral aspect of the medulla-pons region. In colchicine treated rats occasional cell bodies were seen in this ventral region. A group of cell bodies on the lateral border of the solitary nucleus was also visualized. Staining of 2.5 μm thick serial sections with substance P absorbed GRP(1–27) antisera and with GRP(1–16) antisera demonstrated that both antisera were labeling the same cells. In the forebrain of normal rats, the substance P absorbed GRP(1–27) antisera stained primarily axonal fibers and terminals in the suprachiasmatic nucleus. In colchicine treated rats the substance P absorbed GRP(1–27) antisera labeled cell bodies in the suprachiasmatic nucleus and in the parvocellular part of the paraventricular nucleus of hypothalamus. Unlike substance P absorbed GRP(1–27) antisera, antisera raised against GRP(1–16) produced only a faint immunostaining in these hypothalamic regions. These findings suggest that perhaps a differential processing of GRP-like peptides occurs in different brain regions or alternatively several forms of a GRP/bombesin-like peptide may exist.