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Pharmacology and Neuroscience

The objective of this exploratory/developmental grant is to investigate the use of differential display of mRNA (DDRT-PCR) to isolate, identify, and develop molecular probes for genes with ethanol-induced altered expression for use in future studies on alcoholism and associated disorders. This is part of a long-term goal to understand the consequences of chronic alcohol-induced alterations in brain gene products, knowledge of which can further our understanding of the etiology of alcoholism and the pathogenesis of alcohol-related brain disorders. The specific aim is to isolate and sequence gene transcripts differentially expressed following activation of chronic ethanol-exposed rat C6 glioma cells with lipopolysaccharide (LPS) + phorbol ester (PMA). Identification of differentially expressed genes under these conditions can elucidate intracellular mechanisms contributing to maladaptive processes underlying alcohol-induced brain damage, and provide insights into other aspects of alcoholism in as yet unknown ways. The experimental design is to perform side-by-side comparisons of mRNA expression in four groups: 1) unstimulated control cells; 2) unstimulated chronic ethanol cells; 3) activated control cells; 4) activated chronic ethanol cells. Upon isolation and verification of mRNAs differentially expressed by activated chronic ethanol cells, an additional comparison will be made between activated chronic ethanol cells and activated chronic ethanol-withdrawn cells. Conditions for activation and chronic treatment will be those used to demonstrate ethanol-suppression of induced nitric oxide synthase-2 (Nos2) expression in C6 glioma and normal rat glial cells. C6 cells are grown in 50 mM ethanol for 9 days prior to 24 h exposure to LPS+PMA. Media nitrite is assayed to confirm chronic ethanol suppressed Nos2 expression, and total RNA is extracted from the cells. DNA-free RNA is reversed transcribed into cDNA and amplified by PCR using primers and conditions optimized for the differential display technique. Following separation of PCR products by electrophoresis and visualization by autoradiography, cDNA bands exhibiting differential expression are excised from the gel, re-amplified by PCR and used for Northern blotting, cloning and sequencing. Searches of gene and protein sequence databases may identify homology to known genes and proteins; lack of homology could denote a novel gene.