Immunohistofluorescence Study of the Actions of Manganese on the DARPP‐32 Mechanisms of Dopamine D2 Signal Transduction Pathway in Crassostrea virginica

Abstract

Manganism is a human disease caused by inhalation of manganese (Mn) fumes. The neurotoxic mechanism is not fully understood. It involves disruption of dopamine (DA) neurotransmission rather than neuronal degeneration. There aren't effective treatments for manganism. Gill lateral cell (GLC) cilia of Crassostrea virginica are controlled by serotonergic‐dopaminergic innervation. DA causes cilio‐inhibition, serotonin cilio‐excitation. Our lab showed post‐synaptic DA receptors in GLC are D2 type (D2R) and Mn disrupts the DA induced cilio‐inhibition of GLC cilia. D2R signaling pathway involves inhibition of DARPP‐32, a protein phosphatase inhibitor that influences DA‐mediated behaviors in animal models of neuropsychiatric and neurodegenerative disorders. Activation of D1 receptors stimulates protein kinase A‐mediated phosphorylation of DARPP‐32, but activation of D2R reduces DARPP‐32 phosphorylation. DARPP‐32 is critically involved in regulating electrophysiological, transcriptional and behavioral responses to physiological and pharmacological stimuli, including antidepressants, neuroleptics and drugs of abuse. DARPP‐32 has not been well studied in bivalves, nor has the effects of Mn on it. In this study we hypothesize DARPP‐32 can be visualized in oyster GLC by immunohistofluorescence, and if so, we will determine if Mn effects its visualization. Briefly, gills were dissected, snap frozen, cryostat sectioned at 10μm, fixed with EDAC, treated with blockers, and incubated with 1° antibodies to DARPP‐32 and 2° FITC‐labeled 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. We found gill sections showed bright green fluorescence in the cytoplasm and along cell membranes indicating the presence of DARPP‐32. In other experiments gills were treated for 1 or 24 hours with 500μM of Mn or zinc (Zn). Fluorescence intensity of GLC was quantified using ImageJ from NIH. Results show fluorescence intensity in Mn treated GLC was not significantly different after 1 hour treatment, but significantly increased by 22% after 24 hour treatment. For Zn, the opposite was found, 18% increase after 1 hour, no change after 24 hour treatments. The study shows DARPP‐32 is present in GLC of C. virginica and Mn as well as Zn caused a significant changes in its fluorescence intensity. This study provides new knowledge of the actions of Mn on the D2R pathway in bivalve gill. Our future experiments will test if Mn negatively effects the physiological actions of DARPP‐32 on GLC cilia activity.Support or Funding InformationThis work was supported in part by grant 2R25GM06003 of the Bridge Program of NIGMS, NIH grant K12GM093854‐07A1 IRACDA Program of Rutgers University and 604060048 of PSC‐CUNY.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.