Peptidergic modulation of male sexual behavior in Lymnaea stagnalis: structural and functional characterization of -FVamide neuropeptides.

In the simultaneous hermaphrodite snail Lymnaea stagnalis, copulation as a male is controlled by neurons that send axons to the male copulatory organs via a single penis nerve. Using direct mass spectrometry of a penis nerve sample, we show that one of the molecular ions has a mass corresponding to GAPRFVamide, previously identified from the buccal ganglia, and named Lymnaea inhibitory peptide (LIP). The identity of this peptide is confirmed by partial peptide purification from the penis nerve, followed by post source decay mass spectrometry. We cloned the LIP-encoding cDNA, which predicts a prohormone that gives rise to five copies of LIP (now re-named LIP A), two other -FVamide peptides (LIPs B and C), and five structurally unrelated peptides. The LIP gene is expressed in neurons of the right cerebral ventral lobe that send their axons into the penis nerve. We show that the LIP A peptide is present in these neurons and in the penis nerve, and confirmed the presence of LIP B and C in the penis nerve by post source decay mass spectrometry. Finally, we demonstrate that LIP A, B and C inhibit the contractions of the penis retractor muscle, thereby implicating their role in male copulation behavior.

Diversity in cell specific co-expression of four neuropeptide genes involved in control of male copulation behaviour in Lymnaea stagnalis.

We report here the neuron-specific co-expression of four genes coding for neuropeptides involved in the control of male behaviour. These neurons are located in the anterior lobe of the right cerebral ganglion in the central nervous system of Lymnaea stagnalis and project via the penis nerve to the penial complex. In order to accomplish optimal assurance we applied in situ hybridization, immunocytochemistry and matrix-assisted laser desorption ionization mass spectrometry. The anterior lobe neurons express the gene encoding the amidated tetrapeptide APGWamide. Subsets of these cells are now shown to co-express the APGWamide gene exclusively with one of three other neuropeptide genes, encoding Lymnaea neuropeptide Y, conopressin or pedal peptide, respectively. All four genes are also expressed in other neurons in other centres projecting to the penial complex, but in these cells co-expression was not observed. The neuropeptides encoded by the genes could be identified in the anterior lobe cell bodies on the basis of immunocytochemistry and mass spectrometrical analysis. The neuropeptides APGWamide and Lymnaea neuropeptide Y, which are co-localized in the anterior lobe cells as well as in axons innervating the penis retractor muscle, do not induce muscle contraction but have a modulatory action by affecting the relaxation rate and amplitude of the contraction. APGWamide and conopressin had earlier been suggested to modulate peristalsis of the vas deferens. Thus, it seems that the neurons co-expressing the various combinations of neuropeptide genes in the anterior lobe represent functional units, each acting in the fine tuning of different muscles involved in specific aspects of male copulation behaviour.

Various isoforms of myomodulin identified from the male copulatory organ of Lymnaea show overlapping yet distinct modulatory effects on the penis muscle.

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.

Co-localized neuropeptides conopressin and ALA-PRO-GLY-TRP-NH2 have antagonistic effects on the vas deferens of Lymnaea.

We examined functional aspects of co-localization of neuropeptides involved in the regulation of male copulation behaviour in the simultaneous hermaphrodite snail Lymnaea stagnalis. The copulation behaviour is controlled by several types of peptidergic neurons that include a cluster of neurons in the anterior lobe of the right cerebral ganglion. All anterior lobe neurons express the gene encoding Ala-Pro-Gly-Trp-NH2 (APGWamide), and a subset of neurons also express the vasopressin-related conopressin gene. Immunocytochemical and peptide chemical experiments show that both APGWamide and conopressin are transported to the penis complex and the vas deferens via the penis nerve. Co-localization of the two peptides was also observed in some, but not all, axon bundles that run along the vas deferens. APGWamide and conopressin were structurally identified from the penis complex with vas deferens. Conopressin excites the vas deferens in vitro, whereas APGWamide inhibits the excitatory effects of conopressin, both in a dose-dependent fashion. We propose that the antagonistic effects of these peptides on the vas deferens underlie its peristalsis. Thus, these peptides play an important role in the control of ejaculation of semen during copulation.

Mutually exclusive neuronal expression of peptides encoded by the FMRFa gene underlies a differential control of copulation in Lymnaea.

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.

Structural and functional evolution of the vasopressin/oxytocin superfamily: vasopressin-related conopressin is the only member present in Lymnaea, and is involved in the control of sexual behavior.

It has been suggested that the gene duplication that led to the formation of the vasopressin/oxytocin two-gene family occurred early during vertebrate evolution. However, the existence of both vasopressin- and oxytocin-related peptides in invertebrates suggests that this duplication may have occurred much earlier, although there is no evidence for the co-occurrence of vasopressin- and oxytocin-related peptides in the same invertebrate species. We report here that in Lymnaea only the vasopressin-related peptide Lys-conopressin, but not an oxytocin-related peptide, is present. Moreover, it is very likely that an oxytocin-like cDNA or gene is absent. The conopressin gene is expressed in neurons that control male sexual behavior, and its gene products are present in the penis nerve and the vas deferens. Conopressin induces muscular contractions of the vas deferens and inhibits central neurons that control female reproductive behavior. Thus, although structurally related to vasopressin, conopressin has functional and behavioral characteristics typical for oxytocin. Physiological and receptor binding data suggest that conopressin and [Ile8]-conopressin, a synthetic oxytocin-like analog of conopressin, are functionally equivalent in Lymnaea, and that the chemical nature of the amino acid residue at position 8 does not result in a functional difference. Therefore, we suggest that invertebrates contain only a single member of the vasopressin/oxytocin gene family and that the amino acid change that distinguishes vasopressin from oxytocin is functionally neutral in invertebrates.

Identification of a novel amide peptide, GLTPNMNSLFF-NH2, involved in the control of vas deferens motility in lymnaea stagnalis.

Peptide messengers are crucial for the execution of male copulatory processes in the snail Lymnaea stagnalis. In this study, we report the purification of a novel peptide from the penis complex of L. stagnalis. This peptide enhances the contraction frequency and contraction amplitude of the vas deferens. Amino acid sequencing and mass spectrometry indicate that the primary structure is GLTPNMNSLFF-NH2. We propose that this amide peptide modulates the transfer rate of semen in the vas deferens.

Structural identification, neuronal synthesis, and role in male copulation of myomodulin-A of Lymnaea: a study involving direct peptide profiling of nervous tissue by mass spectrometry.

We used a strategy combining immunodetection, peptide chemistry, and a novel method, direct peptide fingerprinting of neurons and small pieces of nerve by using matrix-assisted laser desorption ionization mass spectrometry, to structurally identify and localize the neuropeptide myomodulin-A in the mollusc, Lymnaea stagnalis. Lymnaea myomodulin appeared to be identical to Aplysia myomodulin-A and is produced by many central neurons, including neurons located in the ventral lobe of the right cerebral ganglion that innervate the penis complex via the penis nerve. Myomodulin-A could also be characterized from the penis complex, and physiological concentrations of the peptide enhanced the relaxation rate of electrically induced contractions of the penis retractor muscle in vitro in a dose-dependent fashion.