A single injection of the psychostimulant, D-amphetamine (AMPH), increases opioid gene expression in the rat striatum (preprodynorphin and preproenkephalin in striatonigral and striatopallidal projection neurons, respectively). These inducible genomic responses are mediated by surface receptors (stimulus-transcription coupling) and considered as essential molecular steps for the development of psychoplasticity related to addictive properties of drugs of abuse. From studies conducted in the last period, metabotropic glutamate receptors (mGluRs) were found to play a profound role in mediating AMPH-stimulated opioid gene expression. Moreover, intracellular Ca2+ mobilization appears to be critical for the mGluRs' contribution, indicating the participation of Ca2+-Iinked group I mGluRs in this event. Based on these encouraging findings, a series of experiments was proposed in this continuation of project to explore and characterize the regulation of AMPH-stimulated opioid gene expression by the group 1 mGluRs and the Ca2+-sensitive signaling pathway. Our working hypothesis is that the group I mGluRs and associative signaling pathways participate in the mediation of AMPH-stimulated opioid gene expression in striatal neurons. With multidisciplinary approaches, this hypothesis will be tested in vivo in the following 4 aims designed to: (1) define the role of group I receptors in the mediation of AMPH action through examining effects of the selective group I agonists/antagonists on acute AMPH-stimulated striatal opioid gene expression in a well-characterized rat model, (2) differentiate the relative importance of the two group I subtypes, mGluR1 and mGluR5, in this event using the subtype-selective agonists/antagonists, antisense oligos and mutant mice (mGluR1 or 5 knockouts), (3) at the protein kinase level, examine the contribution of CaMK II to AMPH action by testing effects of AMPH on the group I-dependent phosphorylation of CaMK II in the striatum and effects of decreased CaMK II activity by antisense oligos on AMPH-stimulated opioid gene expression, and (4) at the transcription level, evaluate the importance of the CaMK II-responsive transcription factors, cAMP-response-element-binding protein (CREB) and Elk-1, in bridging group I mGluR stimulation on the membrane to opioid gene expression in the nucleus via monitoring the group I/CamK II-sensitive phosphorylation and DNA binding activity of these transcription factors in striatal neurons in response to AMPH stimulation. We will rely on quantitative in situ hybridization to analyze mRNA expression in vivo. Accomplishment of this project will elucidate how group I receptors and signaling pathways mediate opioid gene expression in matured striatal neurons in response to psychostimulant exposure. Since inducible gene expression is conceived to participate in the development of psychoplasticity, data from this project will provide valuable insight in cellular/molecular mechanisms underlying long-term actions of drugs of abuse.
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