Meeting Abstract
Ganglia and innervated organs of the bivalve Crassostrea virginica contain serotonin and dopamine (DA)which mediate physiologic functions in gill and other organs. Gill lateral cells are controlled by serotonin and DA nerves from their ganglia that regulate cilia beating. DA slows down cilia beating and serotonin speeds them up. GABA is a neurotransmitter in nervous systems of vertebrates and invertebrates, but studies in bivalves have rarely been reported. Recently we used HPLC to show GABA is present in ganglia and tissues of C. virginica and GABA acts as a ganglionic neurotransmitter modulating gill lateral cell cilia activity. We used immunohistofluorescence to localize GABA in ganglia and gill, and identify DA receptors in gill lateral cells as D2-like (D2DR). We hypothesize Western Blot analysis would verify the presence of D2DR and GABA receptors in gill of C. virginica. For Western Blot analysis, gill cell lysate was prepared by polytron disruption in ice-cold NP-40 detergent buffer containing protease inhibitor, followed by centrifugation to obtain supernatant with solubilized membrane proteins. Up to 30 µg of solubilized protein was subjected to SDS-PAGE with 10% acrylamide gels and electroblotted onto nitrocellulose. D2DR and GABA receptor immunoreactivity was revealed after incubation with primary antibodies followed by incubation with HRP-conjugated secondary antibodies. The Western Blots showed strong bands between 70 – 75 kD corresponding to D2DR and GABA RA1-6 receptors in gill. The present project confirms our previous immunohistofluorescence studies showing the presence of DA GABA and furthers the understanding of their physiological roles in C. virginica.
