Drug addiction is a complex state that results from compulsive drug intake, in which both neurotransmitter and hormonal systems are affected. The state is comprised of tolerance, sensitization, dependence and reward that lead to drug seeking and compulsive use. Many brain substances are altered with chronic drug use and are likely to be involved in the state of drug addiction. Therefore, the main goal of this proposal is to identify and characterize genes and gene products in specific brain regions in mice that are altered as the organism is exposed to acute and chronic opiates. We hypothesize that opiate use leads to altered neurohormonal levels mediated by changes in the processing enzymes that control the ratio of active hormone to prohormone. Two prohormone convertases, PC1 and PC2, are believed to be primarily responsible for the activation of many pro-neurohormones, e.g., PC1 generates ACTH and PC2 generates the endogenous opiate, b-endorphin, from the precursor, pro-opiomelanocortin (POMC). The regulation of PC1/PC2 is linked to the cAMP/cAMP response element binding protein (CREB)/cAMP response element (CRE) system. Agents such as opiates that alter CREB levels can be expected to change the activity of these prohormone processing enzymes and affect the biosynthesis of addiction-mediating hormones with major biochemical and behavioral consequences to the organism. By affecting CREmediated gene transcription, opiates also likely modulate other genes and gene products that have not been investigated due to the lack of technology and information available. In this grant application, we propose to conduct a series of interconnecting experiments to test the hypothesis that acute and chronic morphine exposure regulates CRE-mediated gene expression in discrete brain regions.
The specific aims are designed to test this hypothesis by: 1) determining the brain regions expressing CRE-responsive genes following morphine exposure; 2) monitoring the levels of the prohormone processing enzymes, PC1 and PC2, as well as bioactive peptide hormones in these regions; and 3) determining novel or uncharacterized genes/proteins regulated by various paradigms of morphine exposure in CRE-responsive mouse brain regions. The focus of this proposal will be to use two new technologies, ProteinChip Array (SELDI-TOF Mass Spectrophotometry) and Gene Chip Array, to characterize and identify genes and proteins involved in opiate use.
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