Mass spectrometry (MS) was employed to detect and structurally characterize peptides in two functionally related neurons, named VD1 and RPD2, which form a network involved in the modulation of heartbeat in Lymnaea. Matrix-assisted laser desorption/ionization MS, directly applied to single neurons VD1 and RPD2, showed overlapping yet distinct mass profiles, with a subset of putative peptides specifically present in neuron VD1. Direct tandem MS of a single VD1 neuron revealed the primary structures of the VD1-specific peptides, which were identified as members of the family of small cardioactive peptides. Based on the tandem MS data, a degenerate oligonucleotide was made for use in a polymerase chain reaction strategy to isolate the cDNA encoding the precursor to the small cardioactive peptides from a brain-specific cDNA library. The calculated masses of the mature, posttranslationally modified peptides, as predicted from the corresponding cDNA, agreed with the measured masses of the actual peptides, as detected in single-cell MS analysis. In situ hybridization studies showed that the transcript encoding the precursor is present in VD1, but not in RPD2, thus corroborating the single-cell MS analysis. Finally, the small cardioactive peptides were shown to enhance the contractions of the auricle in vitro.

Direct matrix-assisted laser desorption ionization mass spectrometry of neurons and nerves was used to study the diversity, localization and transport of neuropeptides that are produced in the neuronal network that underlies male copulation behaviour in the mollusc Lymnaea stagnalis. These studies reveal a large peptide diversity and distinct peptide profiles in different parts of the network, suggesting a complex regulation of copulation behaviour. Two peptides, e.g., conopressin and APGWamide, which are colocalized in neurons of the network, are involved in the control of vas deferens activities. Conopressin, which is structurally related to vasopressin, induces muscular contractions of the vas deferens, whereas APGWamide inhibits the conopressin-induced contractions. Together, these peptides may be involved in the modulation of peristaltic movements of the vas deferens.KEY WORDS: direct peptide profiling by mass spectrometry, peptidergic neurons, male sexual behaviour, vas deferens, co-transmission of antagonistic peptides, APGWamide, conopressin, mollusc, Lymnaea stagnalis.