The proenkephalin gene is involved in a number of physiological processes including embryonic development, stress, pain, opioid addiction and opioid tolerance. The present research proposal outlines a strategy to study stress-induced regulation of the proenkephalin gene in hypothalamic neurons in vivo in a transgenic mouse model in which the proenkephalin promoter is fused to the reporter gene, E. coli beta-galactosidase and in vitro organotypic transgenic cultures. The stress response is a major contributor to morbidity in mood and anxiety disorders, acute and chronic pain, surgery, and drug withdrawal. While the regulation of catecholamines, ACTH, and glucocorticoids by stress have been extensively studied, endogenous opioids have-only recently been implicated in the neurochemical mechanisms underlying the stress response. This proposal focuses on the gene encoding proenkephalin, one of the endogenous opioid precursors, because it is highly regulated in the hypothalamus by stress and because its promoter is understood in great detail. The transgenic model provides unique capabilities to combine molecular and integrative studies because the histochemical detection of beta-galactosidase is simple and is easily combined with both immunohistochemistry (e.g. for defining transcription factors such as the immediate early gene c-fos) or culture (organotypic and primary) methods. This proposal aims to further our understanding of opioid regulation of the proenkephalin gene using the stress model. Initially, we will determine whether hypertonic stress and opioid modulation (acute and chronic) of stress induces expression and post-translational modification of positively (e.g. CREB) and negatively acting (e.g. CREM) transcription factors that interact with the transgene. We also wish to demonstrate that CREB is a significant positive regulator of the transgene in conditions of acute stress and acute opioid modulation of stress and that opioid-inducible negative regulators can be identified. Using-isolated hypothalamic organotypic cultures we will map to the proenkephalin second messenger- inducible enhancer and the role of specific transcription factors in regulation of proenkephalin gene expression in vivo can be analyzed by the use of antisense oligonucleotides. The use of transgenic mice for in vivo experiments and as a source for organotyPic cultures will allow a mechanistic analysis of the regulation of an Important model gene in a highly restricted population of identified neurons involved in the well- established biology of the stress response. These studies should contribute significantly to understanding how exogenous opioids regulate proenkephalin gene expression in specific hypothalamic neurons in response to acute stress and the regulation of long-term plasticity of proenkephalin gene expression with chronic stress.
