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, Mr=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-l (PP-l). Identification and characterization of a number of specific targets of this DARPP-32/PP-l phosphorylation cascade, whose phosphorylation state is affected by administration of cocaine, amphetamines, opiates, and other drugs of abuse has been accomplished, and further studies will contribute to a greater understanding of the mode of action of these drugs, 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 drugs of abuse 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 heavy emphasis on comparative studies in vivo designed to identify and characterize differences in drug-induced biochemical and behavioral phenotypes between wild-type and knockout/mutant mice, harboring targeted deletion/mutation of key effector molecules in the DARPP-32/PP-l phosphorylation cascade. The goal of Core B is to provide technical support to all members of the Program Project. Core B will be responsible for the breeding, maintenance and genotyping for all mouse colonies to be utilized in Projects I-IV (Specific Aim 1). Core B will maintain stocks of key reagents, including purified enzymes, substrates and antibodies, including phosphorylation state-specific antibodies, and will produce additional antibodies as required to support the other Projects (Specific Aim II).
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