Neurosteroid regulation of cholinergic neurotransmission

以黄体酮为基础的神经类固醇，包括黄体酮及其代谢物5a-二氢黄体酮和3a，5a-四氢黄体酮（ALLO），在大脑内产生，并对大脑内的特定靶点产生快速的非基因组效应。这些黄体酮神经类固醇对高阶认知功能的影响似乎遵循倒u型曲线，例如，相对低水平的ALLO是有益的，而相对高水平的ALLO是有害的。ALLO损害认知功能的机制尚不清楚。我们已经确定，ALLO能迅速抑制前额叶皮层内的烟碱乙酰胆碱受体（nAChR），这是很重要的，因为在这个大脑区域，这种受体的胆碱能神经传递对正常的认知功能至关重要。机制实验表明，这种抑制作用是由以下两种因素共同介导的：(i) ALLO刺激膜孕酮受体（mPR）复合物的间接作用，从而启动抑制nAChR的细胞内信号通路；（ii） ALLO在nAChR处的直接变构作用。ALLO调节nAChR功能的间接和直接作用的具体机制，以及ALLO对nAChR依赖的认知功能的影响，将在拟议的研究中进行研究。我们将测试孕激素神经类固醇通过与nAChR间接和直接相互作用的组合调节胆碱能神经传递的总体假设。我们将完成三个主要目标：(1)确定连接ALLO激活mPR复合物和ALLO抑制nAChR的细胞内信号通路。我们将使用全细胞电生理学和蛋白质组学技术来测试mPR G蛋白偶联信号通路修改nAChR的磷酸化以调节其功能的假设。(2)确定ALLO是否是nAChR的直接变构调制器。我们将在脑突触体中使用放射性配体结合试验来验证ALLO是nAChR的正变构调节剂的假设。(3)确定ALLO对nachr依赖性认知功能的影响。我们将测试ALLO抑制nachr促进前额叶皮层依赖性注意力和认知灵活性的作用。拟议的研究将有助于对脑内神经类固醇作用的基本理解。这项工作很重要，因为大脑在生物体的整个生命过程中都暴露在黄体酮神经类固醇中，但这种暴露的神经生物学后果尚不完全清楚。本研究项目的发现将解决这一悬而未决的问题，并为研究不同孕激素神经类固醇水平状态下认知功能的神经生物学奠定基础，如发育、月经/发情周期、怀孕和暴露于压力环境中。

Progesterone-based neurosteroids, including progesterone and its metabolites 5a-dihydroprogesterone and 3a,5a-tetrahydroprogesterone (allopregnanolone; ALLO), are produced within the brain and induce rapid non-genomic effects at specific targets within the brain. The influence of these progesterone neurosteroids on higher-order cognitive functions appears to follow an inverted U-shaped curve, for example, as relative low levels of ALLO are beneficial whereas relative high levels of ALLO are detrimental. The mechanisms by which ALLO may impair cognitive functions are not known. We have determined that ALLO rapidly inhibits the nicotinic acetylcholine receptor (nAChR) within the prefrontal cortex, which is important because cholinergic neurotransmission at this receptor in this brain region is critical for normal cognitive functioning. Mechanistic experiments suggest that this inhibition is mediated by a combination of (i) indirect action of ALLO stimulating the membrane progesterone receptor (mPR) complex, which initiates an intracellular signalling pathway that inhibits the nAChR, and (ii) direct allosteric action of ALLO at the nAChR. The specific mechanisms of these indirect and direct actions of ALLO to regulate nAChR function, and the effects of ALLO on nAChR-dependent cognitive functions, will be investigated in the proposed research. We will test the overarching hypothesis that progesterone neurosteroids regulate cholinergic neurotransmission via a combination of indirect and direct interactions with the nAChR. We will complete three primary objectives: (1) To determine the intracellular signalling pathway linking ALLO activation of the mPR complex with ALLO inhibition of the nAChR. We will use whole-cell electrophysiology and proteomic techniques to test the postulate that the mPR G protein-coupled signalling pathway modifies the phosphorylation of the nAChR to regulate its function. (2) To determine whether ALLO is a direct allosteric modulator of the nAChR. We will use radioligand binding assays in brain synaptosomes to test the postulate that ALLO is a positive allosteric modulator of the nAChR. (3) To determine the influence of ALLO on nAChR-dependent cognitive functions. We will test the postulate that ALLO inhibits the role of nAChRs to facilitate prefrontal cortex-dependent attention and cognitive flexibility. The proposed research will contribute to the fundamental understanding of neurosteroid action within the brain. This work is important because the brain is exposed to progesterone neurosteroids throughout an organism's life, but the neurobiological consequences of this exposure are not fully known. Discoveries made within this research program will address this outstanding question and provide a foundation from which to study the neurobiology of cognitive functions during states of varying progesterone neurosteroid levels, such as development, the menstrual/estrous cycle, pregnancy, and exposure to stressful environments.