Identification, molecular structure and expression of two cloned serotonin receptors from the pond snail, Helisoma trivolvis.

Helisoma trivolvis has served as a model system to study the functions of serotonin (5-HT) from cellular, developmental, physiological and behavioural perspectives. To further explore the serotonin system at the molecular level, and to provide experimental knockout tools for future studies, in this study we identified serotonin receptor genes from the H. trivolvis genome, and characterized the molecular structure and expression profile of the serotonin receptor gene products. Degenerate oligonucleotide primers, based on conserved regions of the Lymnaea stagnalis 5-HT(1Lym) receptor, were used to amplify G protein-coupled biogenic amine receptor sequences from H. trivolvis genomic cDNA, resulting in the cloning of two putative serotonin receptors. The deduced gene products both appear to be G protein-coupled serotonin receptors, with well-conserved structure in the functional domains and high variability in the vestibule entrance of the receptor protein. Phylogenetic analysis placed these receptors in the 5-HT(1) and 5-HT(7) families of serotonin receptors. They are thus named the 5-HT(1Hel) and 5-HT(7Hel) receptors, respectively. In situ hybridization and immunofluorescence studies revealed that these genes and gene products are expressed most heavily in the ciliated pedal and mantle epithelia of H. trivolvis embryos. In adults, widespread expression occurred in all ganglia and connectives of the central nervous system. Expression of both receptor proteins was localized exclusively to neurites when examined in situ. In contrast, when isolated neurons were grown in culture, 5-HT(1Hel) and 5-HT(7Hel) immunoreactivity were located primarily in the cell body. This is the first study to reveal a 5-HT(7) receptor in a molluscan species.

Regulation of early embryonic behavior by nitric oxide in the pond snail Helisoma trivolvis.

Helisoma trivolvis embryos display a cilia-driven rotational behavior that is regulated by a pair of serotonergic neurons named ENC1s. As these cilio-excitatory motor neurons contain an apical dendrite ending in a chemosensory dendritic knob at the embryonic surface, they probably function as sensorimotor neurons. Given that nitric oxide (NO) is often associated with sensory neurons in invertebrates, and has also been implicated in the control of ciliary activity, we examined the expression of NO synthase (NOS) activity and possible function of NO in regulating the rotational behavior in H. trivolvis embryos. NADPH diaphorase histochemistry on stage E25-E30 embryos revealed NOS expression in the protonephridia, buccal mass, dorsolateral ciliary cells and the sensory dendritic knobs of ENC1. At stages E35-40, the pedal ciliary cells and ENC1's soma, apical dendrite and proximal descending axon were also stained. In stage E25 embryos, optimal doses of the NO donors SNAP and SNP increased the rate of embryonic rotation by twofold, in contrast to the fourfold increase caused by 100 micro mol l(-1) serotonin. The NOS inhibitors L-NAME (10 mmol l(-1)) and 7-NI (100 micro mol l(-1)) decreased the rotation rate by approximately 50%, whereas co-addition of L-NAME and SNAP caused a twofold increase. In an analysis of the surge and inter-surge subcomponents of the rotational behavior, the NO donors increased the inter-surge rotation rate and the surge amplitude. In contrast, the NO inhibitors decreased the inter-surge rotation rate and the frequency of surges. These data suggest that the embryonic rotational behavior depends in part on the constitutive excitatory actions of NO on ENC1 and ciliary cells.