The addictive properties of opiate drugs are not well understood but have long been postulated to resulted from adaptations in endogenous opioid systems. Agonist induced feedback inhibition of endogenous opioid biosynthesis, as has been reported for monoamine transmitter systems, is a potential mechanism linking drug effects to the expression of endogenous ligands. It has been further suggested that such drug-induced changes in endogenous opioid gene expression may underly manly long-term consequences of opiate addiction including opiate dependence and drug craving which have been particularly difficult to understand at a mechanistic level. In agreement with the above hypothesis, opiate agonists have been reported to decrease proenkephalin mRNA levels in specific brain regions while opiate antagonists have been shown to increase mRNA levels. In addition, opiates are now known to regulate the expression of the immediate early genes, fos and jun, though to be critical regulators of proenkephalin and prodynorphin transcription. This research proposal will investigate transcriptional regulation of endogenous opioid peptides by opiate agonists and antagonists using transgenic animal models. Transgenic mice expressing proenkephalin/LacZ fusion genes have been produced, characterized, and will now be used to analyze the effects of opiate drugs on proenkephalin gene expression in the adult and developing mouse. These animal models will greatly facilitate analysis of proenkephalin expression across the entire nervous system and will detect changes in highly restricted populations of neurons in a fashion previously impossible. Transgenic animals will be used to screen the adult and developing mouse nervous system for cells in which endogenous opioid gene expression is regulated or altered by opiate agonists and antagonists. Most importantly, the transgenic animal system will allow us to analyze the effects of specific DNA elements and transcription factors on regulatory mechanisms coupling drug effects with proenkephalin transcription in an intact nervous system. Regulation will be investigated using knowledge of the promoter region, interacting transcription factors, and second messenger regulation developed over the past 10 years. Proenkephalin/LacZ fusion genes containing mutations within the well characterized cAMP, TPA, and Ca++ inducible enhancer which specifically block the binding of specific transcription factors will allow us to test specific models of trans-synaptic and opiate regulation in an intact nervous system.
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