PARRIES, S.C.; TURNER, S.; GOLDBERG, J.I.; University of Alberta: Nitric oxide synthase localization in embryos of the pond snail Helisoma trivolvis.

Embryos of the pond snail Helisoma trivolvis have proven useful in elucidating the developmental roles of neurotransmitters, with the actions of serotonin (5-HT) being the most thoroughly studied. A pair of identified bilaterally symmetrical, serotonergic neurons (ENC1s) develops prior to the development of the CNS in H. trivolvis. The cilioexcitatory action of 5-HT released from these sensorimotor neurons regulates a simple rotational behavior that manifests itself at an early ontogenetic stage. More recently, neural messengers other than 5-HT, such as nitric oxide (NO), have been implicated in mediating the interaction between ENC1 and the embryonic ciliary bands. Here we describe the distribution of nitric oxide synthase (NOS), the enzyme that produces NO, over the course of development in H. trivolvis using NADPH-diaphorase (NADPHd) histochemistry and anti-NOS immunofluorescence. Positive NADPHd staining occurred in the paired protonephridia, the buccal mass, along the mantle edge, in the dorsolateral ciliary bands, and in the soma and sensory apparatus of ENC1. Immunocytochemical experiments revealed distinct staining patterns depending upon which mammalian isoforms were used to generate the antibodies. For example uNOS and iNOS antibodies showed positive staining in embryos, while nNOS antibodies were only effective in labeling regions of the adult CNS. This suggests that, as in mammals and other vertebrates, multiple isoforms of the NOS enzyme exist in this invertebrate. Furthermore, these data indicate that NO may be important in regulating development and behavior in H. trivolvis embryos.