NEUROSTEROIDS & PROXIMAL INHIBITION IN THE HIPPOCAMPUS

项目总结 某些神经类固醇是g-氨基丁酸-A受体(GABAARs)的有效调节剂，在低浓度时增强GABA的作用，在较高浓度时直接激活GABAAR氯通道。目前的证据表明，GABAAR增强是神经类固醇抗焦虑、抗抑郁、镇静和抗惊厥作用的关键机制。我们开发了新的工具，帮助我们探索神经活性类固醇的影响，包括一系列类固醇对映体和荧光类固醇。这些药物表明，膜和细胞积累参与了神经类固醇对GABAARs的动力学和效力，并调节了一种特定形式的抑制，涉及神经元胞体附近的近端抑制输入。我们还开发了探索内源性神经类固醇作用的工具。尽管取得了这些进展，但关于各种类固醇池的生物学基础和生理作用的关键问题仍然存在。在目前的提案中，我们将解决以下目标：确定神经类固醇的膜和细胞内池对GABAAR调节的作用，重点是刺激GABA能输入到海马锥体神经元体体区所引起的一种形式的抑制。这些研究将使用生理学和细胞成像方法相结合的方法来检查类固醇对培养的大鼠海马神经元和海马片的影响，并将涉及基于我们对新型对映体荧光类固醇类似物和可用于细胞生理学和成像的光亲和标记的初步观察的机制研究。这些研究将提供关于中枢神经系统中神经类固醇作用的新信息，并更好地理解类固醇介导的GABAAR调节和神经精神效应。

PROJECT SUMMARY Certain neurosteroids are effective modulators of g-aminobutyric acid-A receptors (GABAARs), augmenting the actions of GABA at low concentrations and directly activating GABAAR chloride channels at higher concentrations. Present evidence indicates that GABAAR potentiation is a key mechanism in the antianxiety, antidepressant, sedative and anticonvulsant actions of neurosteroids. We have developed novel tools that have helped us to probe the effects of neuroactive steroids, including a series of steroid enantiomers and fluorescent steroids. These agents demonstrate that membrane and cellular accumulation participate in the kinetics and potency of neurosteroids on GABAARs and modulate a specific form of inhibition involving proximal inhibitory inputs near neuronal cell bodies. We have also developed tools for probing the roles of endogenous neurosteroids. Despite these advances, key questions remain with regard to the biological basis and the physiological roles of various steroid pools. In the present proposal, we will address the following aim: To determine the roles of membrane and intracellular pools of neurosteroids on GABAAR modulation, focusing on a form of inhibition resulting from stimulation of GABAergic inputs to the somatic regions of hippocampal pyramidal neurons. These studies will use a combination of physiological and cellular imaging methods to examine steroid effects in cultured rat hippocampal neurons and hippocampal slices, and will involve mechanistic studies based upon our initial observations with novel enantiomeric fluorescent steroid analogues and photoaffinity labels that can be used for cellular physiology and imaging. These studies will provide new information about neurosteroid effects in the CNS and a better understanding of steroid-mediated GABAAR modulation and neuropsychiatric effects.