Mammals and various invertebrates have excitatory glutamate neurons in their nervous systems. Human neurodegenerative diseases associated with glutamate dysfunctions include Parkinson’s, Alzheimer’s, Huntington’s, autism, depression and schizophrenia. Recently, the glutamate NMDA receptor was found in and reported to be involved in regulating bivalve metamorphosis in C. gigas , Mercenaria mercenaria and Mya arenaria. In addition, our la b found glutamate neurons present in visceral ganglia of Crassostrea virginica and showed they have a neurophysiological function as ganglionic neurons causing gill lateral cell cilia to increase their beating rates. Serotonin neurons from the visceral and cerebral ganglia of C. virginica innervate the gill lateral cells and increase the cilia beating rates. Based upon this and the presence of glutamate neurons in C. virginica , we hypothesize that glutamate neurons are present in the cerebral ganglia of C. virginica , and that they serve as ganglionic interneurons in the cerebral ganglia, as well as the visceral ganglia, innervating serotonin neurons. To test this, we first used immunohistofluorescence (IHF) microscopy to determine the presence of glutamate neurons in the cerebral ganglia. Briefly, the cerebral ganglia were excised, snap frozen, cryostat sectioned, fixed with EDAC (N-Ethyl-N’-(3-dimethyl-aminopropyl) carbodiimide hydrochloride), treated with blockers, and incubated with glutamate primary antibodies and FITC conjugated secondary antibodies. Sections were viewed on a Zeiss epilume fluorescence microscope with a ProgRes C3 Peltier cooled camera, 100 watt mercury lamps and FITC excitation/emission filters. Results showed the presence of glutamate neurons in the cortex of the cerebral ganglia. The next set of experiments were to determine if glutamate GluR-1 receptors in the cerebral and/or visceral ganglia innervated the serotonin neurons. Cerebral and visceral ganglia were excised and prepared for IHF as above. To view if GluR-1 receptors were co-localized on serotonin neurons in the cerebral and visceral ganglia, the same procedure was employed using glutamate GluR-1 receptors primary antibodies with Texas Red secondary antibodies and serotonin primary antibodies with FITC secondary antibodies. Sections were then viewed using FITC and Texas Red excitation/emission filters. The IHF results showed the presence of GluR-1 receptors co-localized on the serotonin neurons in the cortex of the cerebral and visceral ganglia. This study complements other work of our lab which showed a neurophysiology function of glutamate as an excitatory neurotransmitter in the cerebral and visceral ganglia of C. virginica . Together they demonstrate that C. virginica is a useful model to study neurophysiology as well as the pharmacology of drugs affecting nervous systems.
This work was supported in part by grants 2R25GM06003 of the Bridge Program of NIGMS , 0537231071 of the CSTEP Program of NYSED, P120A210054 of the MSEIP Program of the DoEd , and NIH grant K12GM093854-07A1 IRACDA Program of Rutgers University.