HENS, M.D.; FOWLER, K.A.; BURROWS, B.J.; DEAL, A.M.; LEISE, E.M.*; Univ. of North Carolina Greensboro; Univ. of North Carolina Greensboro; Univ. of North Carolina Greensboro; Univ. of North Carolina Greensboro; Univ. of North Carolina Greensboro: Neural Cell Birth and the Characterization of Larval Nitric Oxide Synthase Expression and Activity in the Caenogastropod Ilyanassa obsoleta

Exogenous serotonin induces metamorphosis in larvae of the caenogastropod Ilyanassa obsoleta, while nitric oxide (NO) blocks this response (reviewed in Leise et al., 2004). Pharmacological inhibition of nitric oxide synthase (NOS) triggers metamorphosis as well as programmed cell death in the apical ganglion of competent larvae. To determine other actions and targets for NO, we have amplified and cloned a portion of the Ilyanassa NOS gene and are using this cDNA as a template to generate probes for in situ hybridizations. The nucleotide sequence of this NOS cDNA was published in the GenBank database under the accession #AY763405. Preliminary results of in situ hybridization display NOS expression in the cerebral ganglia, mostly likely in the central neuropils. We are also investigating the possibility that neurogenesis in larval ganglia is under nitrergic control. Earlier studies with NADPH diaphorase histochemistry indicated that NOS production increases throughout larval development (Lin and Leise, 1996). To determine how neurogenesis relates to NOS production, we incubated larvae in 5-bromo-2-deoxyuridine (BrdU) for 3 hours to label cells undergoing replication, and presumably, mitosis, then localized BrdU by immunocytochemistry. Our results indicate that levels of mitosis are high early in the larval period, but that mitosis essentially ceases in metamorphically competent larvae and in metamorphosing individuals. We attempted to increase rates of neurogenesis in competent larvae by blocking NOS activity with 7-nitroindazole (7-NI), but to date results were negative. Longer incubation times or experimentation with younger larvae may elicit increased neuronal proliferation.