Glucocorticoid Negative Feedback: Intrinsic and Extrinsic Mechanisms

性状（由申请方提供）：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轴的活性受到糖皮质激素负反馈的严格调节。我的研究项目的重点是确定在心理压力的背景下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，它们的研究需要一个全面的多组分方法。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的研究目标：1）获得专业知识，在我的实验室内使用下丘脑器官型培养来研究糖皮质激素对CRH神经元功能的直接影响。这种能力将扩大分子和细胞生物学技术的范围，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机制研究，从而补充我的HPA轴调节的体内研究，和2]获得在我的实验室内使用病毒载体基因转移技术的专业知识。我将开始探索使用慢病毒在下丘脑器官型培养物中产生靶基因表达的局部抑制，然后将有效的载体应用于大鼠脑的选择区域。该技术可以有力地帮助确定特定的分子和细胞在体外和体内是否是必要的糖皮质激素负反馈效应。通过与Greti Aguilera博士（器官型培养物使用培训）、Yosef Refaelli博士（表达短发夹RNA的慢病毒生成和验证培训）和Robert Sapolsky博士（靶大鼠脑区病毒载体微输注和体内疗效验证培训）的访问和咨询，将有可能获得新的科学方法学专业知识。对糖皮质激素负反馈机制的全面理解对于确定HPA轴失调及其后果的临床基础至关重要。不受控制的心理压力和相关的异常HPA轴活动是我们社会中流行的许多病理生理状况的促成因素，例如高血压、动脉粥样硬化、胰岛素抵抗、哮喘、免疫改变和精神情绪障碍。本申请的建议活动将大大促进我的职业发展，并最大限度地提高我对HPA轴调节的理解的研究贡献。应激激素皮质醇的分泌增加被认为是导致慢性应激的不良生理和心理后果的主要生物因素。本项目将确定皮质醇分泌控制的新机制。

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.