An innovative method, direct peptide profiling of small samples of nervous tissue by matrix-assisted laser desorption ionization mass spectrometry, in combination with peptide characterization, immunocytochemistry in conjunction with specific neuronal labeling by backfilling of the penis nerve, and bioassay of peptides was used to study the intrinsic neuronal expression patterns of distinct sets of related FMRFa peptides and their significance for the organization of male copulation behavior in the mollusk, Lymnaea stagnalis. Previous studies indicate that the sets of FMRFa-related and GDPFLRFa-related peptides are encoded by two alternatively spliced transcripts of the single FMRFa gene. Direct mass spectrometry revealed that both FMRFa-related and GDPFLRFa-related peptides are present in the penis nerve, the sole nerve that innervates the penis complex. Accordingly, authentic FMRFa, GDPFLRFa, and related peptides were purified from the penis complex. The loci of synthesis of FMRFa and related peptides could be traced to the right cerebral ventral lobe, those of GDPFLRFa and related peptides to the B group neurons in the right parietal ganglion and to a few unidentified neurons in the right pleural ganglion. Notwithstanding their related structures, the two sets of peptides have distinctly different actions on the penis retractor muscle.

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Male copulatory behavior in the snail Lymnaea stagnalis is controlled by several types of peptidergic neurons, including a cluster of neurons in the ventral lobe of the right cerebral ganglion that show immunoreactivity to myomodulin‐A of Aplysia and innervate the penis complex. We identified structurally myomodulin‐A and three related peptides from Lymnaea and showed that they are present in a characteristic ratio in both the penis nerve and penis complex, suggesting that they are processed from a single precursor and transported from the ventral lobe to the penis complex. All four peptides decreased the relaxation time of electrically evoked contractions of the penis retractor muscle. However, their effects on the amplitude of contraction were different, ranging from no effect to an increase or a decrease in the amplitude. A mixture of the peptides in a ratio as determined by direct mass spectrometry of the penis nerve decreased the contraction time, the relaxation time, and the amplitude. These effects resemble those of one particular peptide in the mixture. The direct mass spectrometry determinations of the peptide profile in the penis nerve suggest that many more, as yet unidentified, neuropeptides are involved in modulation of muscle activities of the penis complex.