Many of the acute and chronic actions of cocaine and amphetamines are believed to be mediated by enhanced dopaminergic neurotransmission, particularly in the neostriatum and nucleus accumbens. At the molecular level, the effects of these psychomotor stimulants appear to involve activation of D1 and/or D2 receptor-mediated signal transduction pathways, which regulate the activity of cAMP-dependent protein kinase (PKA). One member of a family of basal ganglia-enriched PKA substrates, known as DARPP-32 (dopamine- and cyclic AMP-regulated phosphoproteins, M,=32,000) plays a central role in the regulation of downstream effects of this PKA- dependent pathway through its ability, upon phosphorylation, to inhibit the activity of protein phosphatase-1 (PP-1). The identification and characterization of specific targets of this D1/PKA/DARPP-32/PP-1 phosphorylaiton cascade, whose phosphorylaiton state is affected by administration of cocaine and amphetamines, will contribute to a greater understanding of the mode of action of these drugs of abuse, and may lead to novel therapeutic targets for the treatment of drug addiction. A multi- disciplinary approach will be undertaken by the Program Project to investigate the effects of cocaine and amphetamine on the phosphorylation state and functional regulation of key molecules in these dopaminergic signaling pathways. Studies will be performed at several levels of organizational complexity, encompassing in vitro biochemical studies with purified molecules, studies in cellular systems, and comparative studies in intact animals designed to identify and characterize differences in drug- induced biochemical and behavioral phenotypes between wild-type and knockout mice, harboring targeted deletions of key effector molecules in the D1/PKA phosphorylation cascade. The overall goal of the Scientific Core is to provide a range of support to all members of the Program Project. A research component of the Core will produce mouse lines which will enable tissue-specific inducible targeted deletion (""""""""knockout"""""""") of the genes for key effector molecules, including the PP-1 inhibitors, DARPP-32 and inhibitor-1, and the DARPP family members, DARPP-21 and DARPP-16 (Specific Aim 1). To ensure adequate supplies of mice, the Core will provide breeding, maintenance and genotyping for all colonies of transgenic mice, and the conventional and tissue-specific inducible knockout (""""""""TIKO"""""""") mouse lines to be produced and studied (Specific Aim 2). The Core will maintain stocks of key reagents, including purified PKA and protein phosphatases, DARPP-32 and antibodies, including phosphorylaiton state- specific antibodies, and will produce additional antibodies as required to support the other Projects (Specific Aim 3).
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