Immunohistofluorescence Study of the Actions of Manganese on the Phospholipase C Mechanism of Dopamine D2‐Like Post‐Synaptic Receptors in Crassostrea virginica

Abstract

Manganese (Mn) is a neurotoxin causing Manganism, a Parkinson's‐like disease. Mn neurotoxicity involves disruption of dopaminergic neurotransmission. The mechanism of how Mn produces its toxic effects is not fully resolved and reports postulate it to be more related to dopamine neuron dysfunction rather than degeneration. Lack of effective treatment for Mn toxicity is a major obstacle in clinical management of Manganism. Gill lateral cell (GLC) cilia of Crassostrea virginica are controlled by serotonergic‐dopaminergic innervations. Dopamine (DA) is the neurotransmitter causing cilio‐inhibition, serotonin cilio‐excitation. Previous work of our lab with C. virginica showed post‐synaptic DA receptors in GLC are D2 type (D2DR) G protein‐coupled metabotrophic receptors and that Mn disrupts the DA induced cilio‐inhibition of GLC cilia. These findings suggest the D2DR receptor is a potential site of action in Mn neurotoxicity. The D2DR signaling pathway involves inhibition of adenylyl cyclase and activation of phospholipase C (PLC). PLC has not been well studied in bivalves, nor has the effects of Mn on PLC been studied. PLC activation generates the second messengers inositol trisphosphate and diacylglycerol. In this study we hypothesize PLC can be visualized in oyster GLC by immunohistofluorescence, and if so we will determine if Mn effects its visualization. 1° antibodies to PLC and 2° FITC‐labeled antibodies were purchased from Abcam and Santa Cruz Biotechnology, respectively. Briefly, gills were dissected, snap frozen, cryostat sectioned at 10 microns, fixed with EDAC (N‐Ethyl‐N’‐(3‐dimethylaminopropyl) carbodiimide hydrochloride), treated with blockers, and incubated with 1°, then 2° antibodies. Sections were viewed on a Leica epilume fluorescence microscope with a Leica DFC400 camera, 50 watt mercury lamps and FITC excitation/emission filters. All sections were photographed with the same camera setting at 100, 200 and 400X. Phase contrast images also were photographed. We found gill sections showed bright green fluorescence in the cytoplasm and along cell membranes indicating the presence of PLC. In other experiments we treated gills for 1 or 48 hours with 500μM of Mn or zinc (Zn). Zn, which is known to cause allosteric inhibition of a number of enzymes, was used for comparisons. Fluorescence intensity of GLC was quantified using ImageJ from NIH. Results show fluorescence intensity in Mn treated GLC was 14 and 20% reduced compared to untreated cells for the 1 and 48 hour treatments, respectively. Gills treated with Zn did not show a difference. The study shows PLC is present in GLC of C. virginica and that Mn did cause a small, but significant reduction in PLC fluorescence intensity. This study provides new knowledge of the actions of Mn on the D2DR pathway in bivalve gill. Our future experiments will test if Mn negatively effects the physiological actions of PLC on GLC cilia activity.Support or Funding InformationSupported in part by grant 2R25GM06003 of the Bridge Program of NIGMS.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.