BF区GABA-Glutamate-Ach神经微环路在麻醉-觉醒调控中的作用机制研究

Although 230 million patients undergo anesthesia each year in the world, the mechanism of general anesthetics is still unknown. It is documented that the neural networks comprised of a number of neural circuits participate the anesthetic mechanism. Accumulated evidences from our group support that the basal forebrain (BF) is directly involved in the anesthesia-arousal regulation. Moreover, the orexinergic projection from Pef could activate the neurons in BF and then facilitate the emergence from general anesthesia. Our further study has demonstrated glutamate release is increased and GABA release is decreased in BF after anesthesia. Therefore, we hypothesized that general anesthetics have a dual-directional regulation on neurotransmitters for central nervous system. This hypothesis has been adopted by《Miller’s Anesthesia》. However, the mechanism of this phenomenon needs to be elucidated. According to the newest findings, it is considered that BF has a microcircuit of GABA-Glutamate-Ach which could shift the signaling between excitatory and inhibitory. This intrabasalis microcircuit initiate the difference of transmitters changes (excitatory transmitters increase while inhibitory ones decrease) and output signals (the inhibitory projections to the cortex), and then regulate the anesthesia–arousal cycle. The current project will use the advanced neuroscience techniques (optogenetics, DREADD and electrophysiological, et al) to explore how the microcircuit in BF regulate the ascending arousal signals. The results will expand the theory of neural network regulation of general anesthesia.

全球每年有2.3亿人接受麻醉，但全麻药物作用机制依然是未解之谜。现有证据显示，多个神经环路构成的神经网络参与了全麻机制。申请者前期研究发现基底前脑（BF）直接参与麻醉-觉醒调节，Pef区orexin神经投射可激活BF区促进麻醉觉醒。深入研究却发现麻醉后BF区glutamate释放增加而GABA释放减少，据此提出全麻药物对中枢神经递质的“双向调节”新假说，被写入《Miller’s Anesthesia》。这一有别于传统认识的发现其内在机制尚有待阐明。依据最新研究进展，我们认为BF核团内存在GABA-Glutamate-Ach神经微环路，从而实现抑制与兴奋相互转换，引起递质变化（兴奋增强而抑制减弱）与输出信号（向皮层的抑制投射）的差异表现，实现麻醉与觉醒调节。项目将利用光遗传学、DREADD及神经电生理等技术，研究BF区神经微环路调控对上行激活信号的整合机制，成果将进一步拓展网络调控学说内涵。

BF区是腹侧网状上行激活系统的重要组成部分，在意识状态调节中发挥重要作用。BF区包含GABA、Glu和Ach能在内的多种类型神经元。然而不同类型神经元在麻醉-觉醒意识转换中扮演何种角色仍未可知。本课题就上述问题进行深入研究。首先发现BF区Glu、Ach及GABA能神经元钙活动在麻醉后均显著下降，并于觉醒期逐渐恢复。其次，麻醉维持期用光/化学遗传方法兴奋BF区Glu能和Ach能神经元可显著降低小鼠脑电爆发抑制率，缩短觉醒时间。特异性敲除小鼠BF区上述神经元可显著提高麻醉敏感性。与此相反，兴奋BF区GABA能神经元显著增加麻醉期爆发抑制率，延长觉醒时间，提示BF区Glu、ACh能神经元在全麻中发挥促觉醒效应，而GABA能神经元发挥促麻醉效应。.为进一步明确BF区意识调节环路构成，环路示踪研究发现，BF区GABA能神经元主要投射至MS、CMT、MHb、LHb、LH、VTA及MRF；Glu能神经元主要投射至MS、LS、LHb及LH；ACh能神经元主要投射至MS、BLA、HIP及mRN。研究意外发现，麻醉后激活BF区向LH、LHb、CMT、VTA的GABA能神经投射，均可缩短麻醉后觉醒时间，而对麻醉诱导阶段没有影响。其中，向LH、LHb及CMT的投射功能主要由GABAA受体介导，而向VTA区的投射功能主要由GABAB受体介导。EEG监测结果同步证实，激活BF区GABA能神经元及其投射末梢可显著降低爆发抑制率（BSR），并降低浅麻醉状态下的脑电低频波段（δ/θ）能量密度，提高高频波段（β/γ）能量密度。.据此我们认为，BF区GABA能神经元与ACh、Glu能神经元构成局部环路，并抑制Glu、ACh神经元兴奋性以维持麻醉深度，而其长程投射在麻醉终止后促进觉醒，局部环路与长程投射间形成相反调节效应，在麻醉状态下脑内兴奋-抑制稳态维持，及觉醒期兴奋-抑制稳态重建中发挥重要作用。本研究深入阐明BF区神经元在麻醉中的环路调节机制，为未来描绘BF区相关投射及功能研究提供依据。