PROENKEPHALIN GENE REGULATION IN HYPOTHALAMIC NEURONS

脑啡肽原基因参与许多生理过程 包括胚胎发育，压力，疼痛，阿片类药物成瘾和 阿片耐受性本研究提案概述了一项战略， 研究应激诱导下丘脑前脑啡肽原基因的调节 在转基因小鼠模型中， 启动子与报告基因融合，E.大肠杆菌β-半乳糖苷酶， 体外器官型转基因培养物。压力反应是一个主要的 导致急性和慢性情绪和焦虑症发病的因素 疼痛手术和停药虽然监管 压力引起的儿茶酚胺、ACTH和糖皮质激素已经被广泛地 研究，内源性阿片类药物只是最近才被牵连在 压力反应的神经化学机制。这项建议 重点是编码脑啡肽原的基因，脑啡肽原是内源性阿片样物质之一， 前体，因为它在下丘脑中受到压力的高度调节 并且因为其启动子被非常详细地理解。转基因 该模型提供了独特的能力，将联合收割机的分子和整合 因为β-半乳糖苷酶的组织化学检测是 简单并且容易与免疫组织化学（例如， 定义转录因子，如即刻早期基因c-fos） 或培养（器官型和原代）方法。这项建议旨在 进一步加深了我们对阿片调节脑啡肽原基因的理解， 使用压力模型。首先我们要确定高渗 应激和阿片调节（急性和慢性）应激诱导 表达和翻译后修饰阳性（例如CREB） 和负作用（例如CREM）转录因子， 转基因。我们还希望证明CREB是一个 转基因的显著正调节因子在急性 应激和应激急性阿片调节以及阿片诱导的 可以识别负调节剂。使用离体下丘脑 器官型培养我们将映射到脑啡肽原第二信使- 诱导增强子和特异性转录因子在 脑啡肽原基因在体内表达的调节可以通过 反义寡核苷酸的使用。使用转基因小鼠在 体内实验和作为器官型培养物的来源将允许 一个重要模式基因的调控机制分析 参与井中的高度受限的已识别神经元群体- 压力反应的生物学基础这些研究应 有助于了解外源性阿片类药物如何调节 脑啡肽原基因在大鼠下丘脑特定神经元中的表达 对急性应激的反应和长期可塑性的调节 脑啡肽原基因表达与慢性应激

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.