Profiling of neuropeptides released at the stomatogastric ganglion of the crab, Cancer borealis with mass spectrometry

Abstract

Studies of release under physiological conditions provide more direct data about the identity of neuromodulatory signaling molecules than studies of tissue localization that cannot distinguish between processing precursors and biologically active neuropeptides. We have identified neuropeptides released by electrical stimulation of nerves that contain the axons of the modulatory projection neurons to the stomatogastric ganglion of the crab, Cancer borealis. Preparations were bathed in saline containing a cocktail of peptidase inhibitors to minimize peptide degradation. Both electrical stimulation of projection nerves and depolarization with high K⁺ saline were used to evoke release. Releasates were desalted and then identified by mass using MALDI–TOF (matrix-assisted laser desorption/ionization–time-of-flight) mass spectrometry. Both previously known and novel peptides were detected. Subsequent to electrical stimulation proctolin, Cancer borealis tachykinin-related peptide (CabTRP), FVNSRYa, carcinustatin-8, allatostatin-3 (AST-3), red pigment concentrating hormone, NRNFLRFa, AST-5, SGFYANRYa, TNRNFLRFa, AST-9, orcomyotropin-related peptide, corazonin, Ala13-orcokinin, and Ser9-Val13-orcokinin were detected. Some of these were also detected after high K⁺ depolarization. Release was calcium dependent. In summary, we have shown release of the neuropeptides thought to play an important neuromodulatory role in the stomatogastric ganglion, as well as numerous other candidate neuromodulators that remain to be identified.