A high-resolution in vitro bioassay to identify neurons containing red pigment concentrating hormone

The release of red pigment concentrating hormone (RPCH) by single peptidergic neurons of the crayfish X organ/sinus gland system (XO-SG) was demonstrated using a novel in vitro bioassay in which XO neurons were co-cultured with tegumentary erythrophores. Local retraction of the pigmentary matrix within filipodia from erythrophores plated next to presumptive RPCH-containing neurons suggest spontaneous hormone release. Topical application of synthetic RPCH onto long filipodia also produced a local response. The time course of pigmentary matrix aggregation depended on the dose of synthetic RPCH. The effect of peptide on the cultured target cells was blocked by a polyclonal antiserum against RPCH. In co-culture conditions, the time course of pigmentary matrix aggregation was accelerated when presumptive RPCH-containing neurons were depolarized by intracellular current injection or by voltage-clamping to activate the Ca2+ current. The aggregation response evoked by these maneuvers was similar to that obtained with synthetic RPCH at a concentration of 1 fmol l-1. The immune serum was also used to identify a subset of 3-7 immunoreactive neurons localized in the external rim of the XO close to the medulla interna. Under culture conditions, this subset of neurons corresponded to the cells that induced the erythrophore response.