The goal of this research project is to elucidate the role of drugs of abuse in the regulation of dopamine-mediated protein phosphorylation in the rat brain. Specifically, these studies will evaluate the control of brain phosphoproteins as a possible mechanism of action for the acute and chronic effects of cocaine and amphetamine, two widely-abused psychostimulants which increase dopaminergic neurotransmission in the nucleus accumbens and neostriatum of rat brain. Recent work has identified several integral phosphoproteins as substrates for regulation by dopamine and/or by cyclic AMP-dependent protein kinase (cAMP-PK) in brain neurons. These include protein phosphatase inhibitors such as DARPP-32 (dopamine and cAMP- regulated phosphoprotein, Mr 32kD) and I-1 (protein phosphatase inhibitor- 1), other intermediary signal transduction proteins known as DARPP-16 and DARPP-21 (dopamine and cAMP-regulated phosphoprotein, Mr 16 kD and 2l kD, respectively), the ion pump Na, K-ATPase, voltage-dependent sodium channels, three classes of calcium channel (L-,N-, and P-type), and dopamine and glutamate receptor. Experiments will be performed to examine the acute and chronic effects of cocaine or amphetamine treatment on the in vivo phosphorylation state of these target protein. Brain tissue derived from drug-treated rats will be analyzed for phosphorylated phosphoproteins using selective protein immunoprecipitation techniques combined with the use of phosphorylation state-specific antibodies or radioactive labeling procedures. In some experiments, brain tissue from chronically drug- treated rats or cells prepared from drug-naive rats (i.e. primary culture or tissue slices) will be used to examine specific aspects of dopamine- regulated phosphorylation in vitro in nucleus accumbens or neostriatal cells in order to understand better how basic aspects of dopamine-regulated signal transduction are altered by extended drug use. Other major goals of the proposed studies are as follows: 1) To determine whether cocaine and amphetamine regulate the same or different cellular pathways and phosphoproteins targets in brain neurons; 2) To determine whether cocaine and amphetamine effects on phosphorylation are differentially expressed in two major brain loci for drug effects, nucleus accumbens and neostriatum; and 3) To evaluate possible contributions of other neurotransmitter systems (e.g. glutamate, GABA, CCK) in mediating the acute and chronic effects of cocaine and amphetamine. These studies are likely to provide novels and important information on the molecular actions of two major drugs of abuse which will be useful in the design and development of new interventions for the treatment and prevention of drug abuse.
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