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.