Neurosteroid regulation of cholinergic neurotransmission

孕酮类神经类固醇包括孕酮及其代谢物5a-二氢孕酮和3a，5a-四氢孕酮(allopregnanolone；allo)，在脑内产生，并在大脑内的特定靶点诱导快速的非基因组效应。例如，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形曲线，因为相对较低的Allo水平是有益的，而相对较高的Allo水平是有害的。ALLO可能损害认知功能的机制尚不清楚。我们已经确定，Allo迅速抑制前额叶皮质内的烟碱型乙酰胆碱受体(NAChR)，这一点很重要，因为该脑区该受体上的胆碱能神经传递对正常认知功能至关重要。机制实验表明，这种抑制是通过(I)Allo刺激膜孕酮受体(MPR)复合体的间接作用，启动抑制nAChR的细胞内信号通路，以及(Ii)Allo在nAChR上的直接变构作用共同介导的。这些间接和直接作用于nAChR功能的具体机制，以及Allo对nAChR依赖的认知功能的影响，将在拟议的研究中被探讨。我们将测试最重要的假设，即孕酮神经类固醇通过与nAChR的间接和直接相互作用组合来调节胆碱能神经传递。我们将完成三个主要目标：(1)确定细胞内连接Allo激活MPR复合体和Allo抑制nAChR的信号通路。我们将使用全细胞电生理学和蛋白质组学技术来验证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的直接变构调节剂。我们将使用脑突触体内的放射性配体结合分析来验证Allo是nAChR的正变构调节器这一假设。(3)探讨等位基因对nAChR依赖认知功能的影响。我们将测试这一假设，即Allo抑制nAChRs的作用，以促进前额叶皮质依赖的注意力和认知灵活性。这项拟议的研究将有助于从根本上理解神经类固醇在大脑中的作用。这项工作很重要，因为大脑在生物体的一生中都会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并不完全清楚。这项研究计划中的发现将解决这一悬而未决的问题，并为研究孕酮神经类固醇水平不同状态下认知功能的神经生物学提供基础，这些状态包括发育、月经/发情周期、怀孕和暴露在压力环境中。

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.