Coexistence of substance P, neuropeptide Y, VIP, and CGRP in the nerve fibers of the carotid labyrinth of the bullfrog, Rana catesbeiana : a double-labelling immunofluorescence study in combination with alternate consecutive sections

Double immunohistochemical staining with rhodamine- and fluorescein isothiocyanate (FITC)-conjugated antisera revealed the coexistence of substance P (SP) and neuropeptide Y (NPY), and SP and calcitonin gene-related peptide (CGRP) in most nerve fibers in the intervascular stroma of the carotid labyrinth of the bullfrog, Rana catesbeiana, although there were a few fibers which showed only SP- or NPY-immunoreactivity. Approximately one third of SP-immunoreactive fibers also showed coexistence with vasoactive intestinal polypeptide (VIP)-immunoreactivity, and a few fibers contained VIP without SP. The combination of the double immunofluorescence technique and alternate consecutive sections further demonstrated the possible coexistence of SP, VIP, NPY, and CGRP. This coexistence of four different peptides in the same nerve fibers was proved by the following two evident facts: 1) some SP fibers which demonstrated coexistence with NPY-immunoreactivity were assumed to be continuous with those showing VIP-immunoreactivity, and 2) almost all of the SP fibers showed coexistence with CGRP-immunoreactivity. By this reasoning, nearly one third of SP fibers may demonstrate coexistence with NPY-, VIP-, and CGRP-immunoreactivities. These multiple peptides might be involved in vascular regulatory function, which is a possible function of the amphibian carotid labyrinth.