Glucocorticoid Negative Feedback: Intrinsic and Extrinsic Mechanisms

描述(申请人提供)：下丘脑-垂体-肾上腺(HPA)轴神经内分泌系统控制多能糖皮质激素的分泌。这些激素对生理系统产生动态调节影响，以响应昼夜节律和压力需求。HPA轴的活动受到糖皮质激素负反馈的严格调控。我的研究计划的重点是确定在心理压力背景下存在的糖皮质激素负反馈的分子、细胞和系统水平的机制。这些糖皮质激素的负反馈作用在解剖学和时间维度上各不相同，他们的研究需要综合的多组分方法。我建议在未来三年内开展两项职业发展活动，以最大限度地实现我对这项研究目标的追求：1)获得专业知识，在我的实验室内利用下丘脑器官型培养，研究糖皮质激素对CRH神经元功能的直接影响。这一能力将扩大我可以应用于糖皮质激素调节刺激分泌和刺激基因诱导偶联的分子和细胞生物学技术的范围，从而补充我对HPA轴调节的体内研究，并获得在我的实验室内实施病毒载体基因转移技术的专业知识。我将首先探索使用慢病毒在下丘脑器官型培养物中产生对靶基因表达的局部抑制，然后将有效的载体应用于选择大鼠大脑的区域。这项技术可能会有力地帮助确定体外和体内特定的分子和细胞是否是糖皮质激素负反馈效应所必需的。通过与Greti Aguera博士(器官培养使用培训)、Yosef Refaelli博士(表达短发夹RNA的慢病毒的生成和验证培训)和Robert Sapolsky博士(病毒载体在目标大鼠大脑区域微量注射和体内疗效验证培训)的访问和咨询，可以获得新的科学方法学专业知识。全面了解糖皮质激素负反馈机制对于确定HPA轴失调及其后果的临床基础至关重要。失控的心理应激和相关的HPA轴活动异常是导致许多社会普遍存在的病理生理状况的因素，如高血压、动脉粥样硬化、胰岛素抵抗、哮喘、免疫功能改变和精神情绪障碍。这项申请的拟议活动将极大地促进我的职业发展，并最大限度地促进我对HPA轴调节的理解。公共卫生相关性应激激素皮质醇的分泌增加，已被认为是导致慢性应激造成不利生理和心理后果的主要生物学因素。该项目将确定控制皮质醇分泌的新机制。

DESCRIPTION (provided by applicant): The hypothalamic-pituitary-adrenal (HPA) axis neuroendocrine system controls secretion of the multipotent glucocorticoid hormones. These hormones produce a dynamic regulatory influence on physiological systems in response to circadian and stress demands. Activity of the HPA axis is tightly regulated by glucocorticoid negative feedback. The focus of my research program is to determine the molecular, cellular and systems level mechanisms of glucocorticoid negative feedback present within the context of psychological stress. These glucocorticoid negative feedback actions vary across anatomical and temporal dimensions and their study requires a comprehensive multicomponent approach. I propose two career development activities over the next three years designed to maximize my pursuit of this research objective: 1] To acquire the expertise to implement within my laboratory the use of hypothalamic organotypic cultures for study of the direct glucocorticoid effects on CRH neuron function. This capability will extend the range of molecular and cell biology techniques that I can apply to mechanistic studies of glucocorticoid regulation of stimulus-secretion and stimulus-gene induction coupling, and will thereby complement my in vivo studies of HPA axis regulation, and 2] To acquire the expertise to implement within my laboratory the use of viral vector gene transfer technology. I will begin by exploring use of lentivirus to produce a localized suppression of targeted gene expression within hypothalamic organotypic cultures, and then apply effective vectors to select regions of rat brain. This technique may powerfully assist in determining whether specific molecules and cells in vitro and in vivo are necessary for glucocorticoid negative feedback effects. Acquiring new scientific methodological expertise will be made possible by visitation and consultation with Dr. Greti Aguilera (training in use of organotypic cultures), Dr. Yosef Refaelli (training in generation and validation of lentivirus expressing short hairpin RNAs) and Dr. Robert Sapolsky (training in viral vector microinfusion in target rat brain regions and validation of in vivo efficacy). A comprehensive understanding of glucocorticoid negative feedback mechanisms is essential to determining the clinical basis of HPA axis dysregulation and its ramifications. Uncontrolled psychological stress and associated abnormal HPA axis activity are contributing factors to a number of pathophysiological conditions prevalent in our society, such as hypertension, atherosclerosis, insulin resistance, asthma, altered immunity and psychiatric mood disorders. The proposed activities of this application will substantially advance my career development and maximize my research contributions to the understanding of HPA axis regulation. PUBLIC HEALTH RELEVANCE Increased secretion of the stress hormone, cortisol, has been implicated as a major biological factor that contributes to the adverse physical and psychological consequences of chronic stress. This project will identify new mechanisms by which cortisol secretion is controlled.