面旁核γ氨基丁酸能神经元对七氟烷诱导麻醉-苏醒行为调控作用及神经环路机制研究

General anesthesia is an important part of surgical treatment. Due to the unclear mechanism of anesthetics, there is no effective prevention and treatment strategy for adverse reactions such as delayed recovery of anesthesia, delirium and cognitive impairment. Therefore, it’s an important scientific problem to reveal the mechanism of general anesthetics. Previous studies have found that the parafacial nucleus is an important sleep center in the brain stem. Our results also suggest that the parabrachial nucleus, an important downstream nucleus of the parafacial nucleus, plays an important regulatory role in anesthesia recovery. We hypothesized that the parafacial nucleus is a “launcher” nuclei involved in general anesthesia. In the present study, we are going to use Cre mice for vGAT, calcium imaging in neurons, optogenetics and designer receptors exclusively activated by a designer drug (DREADD) which specifically control neuron activity, cellular electrophysiology, hrGFP neural tract tracing , histochemistry approache and automatic electroencephalogram bioassay systems to clarify the roles of parafacial nucleus GABAergic neurons in the inhalation anesthetics sevoflurane-induced anesthesia, and molecular mechanism and neural circuit. The outcome of the proposal will provide new insights for revealing the mechanism of general anesthetics by the key neurons in the parafacial nucleus, offer new ideas for the safe and rational usage of general anesthetics, and supply a new target for the development of new anesthetics.

全身麻醉是外科手术治疗过程中重要一环。但由于全麻药作用机制不明，目前临床上对术后麻醉苏醒延迟、谵妄和认知功能障碍等不良反应缺乏有效防治策略。因此，揭示全麻药作用机制是亟待解决的重要科学问题。近年研究证实脑干面旁核参与睡眠觉醒调控并与皮层兴奋性密切相关，我们前期研究利用特异性神经元活性操纵技术也证实了面旁核重要下游核团——臂旁核在麻醉苏醒过程中起到重要调控作用。我们提出假说：面旁核可能是启动麻醉苏醒的关键脑区。本课题拟运用转基因Cre小鼠、Cre依赖AAV病毒、神经元钙成像、光遗传学、化学遗传学、hrGFP神经纤维示踪和免疫组化等方法，结合生物脑电解析、麻醉行为学评价和神经电生理技术，从分子到整体水平研究面旁核对吸入性全麻药七氟烷诱导麻醉-苏醒行为的调控作用及功能性神经环路机制。预期结果为完善全麻药中枢作用机制提供新内容，为临床安全、高效使用全麻药提供新思路，亦能为研制新型全麻药提供新靶点。

全身麻醉机制一直是个未解之谜，深入了解其中枢机制有助于应对麻醉药物使用过程中出现的不良反应。面旁核区域GABA能神经元活性与睡眠行为的发生和维持呈正相关，且与皮层兴奋性密切相关，提示面旁核可能参与麻醉苏醒调控。因此，面旁核如何参与调节麻醉苏醒对理解全麻药物中枢作用机制至关重要。我们利用化学遗传学、光遗传学、逆行病毒标记技术，结合Vgat-Cre小鼠探究了面旁核GABA能神经元调控麻醉苏醒的机制。结果如下：（1）七氟烷麻醉状态下利用化学遗传学激活面旁核GABA能神经元，实验动物麻醉诱导时间缩短22.3%，麻醉苏醒时间延长47.3%，且麻醉敏感性上升，丙泊酚诱导的麻醉也观察到相似结果。（2）化学遗传学特异性抑制面旁核GABA能神经元后，七氟烷麻醉诱导时间延长28%，麻醉苏醒时间缩短13.8%；丙泊酚麻醉苏醒时间缩短25.7%。表明面旁核GABA能神经元参与全麻药物麻醉苏醒行为的调节。（3）利用光遗传学在七氟烷麻醉过程中瞬时激活面旁核GABA能神经元观察到小鼠出现皮层活动度抑制现象。而瞬时抑制该类神经元后，出现皮层觉醒现象。（4）臂旁核作为面旁核GABA能神经元调控重要下游脑区，利用逆行Cre病毒结合化学遗传学受体，在小鼠全麻状态下特异性激活面旁核-臂旁核通路后，七氟烷麻醉诱导时间缩短16.4%，苏醒时间延长18.9%。而抑制该通路后，七氟烷麻醉苏醒时间显著缩短18.4%。提示臂旁核是面旁核调控麻醉苏醒行为的重要下行通路。（5）利用免疫组织化学c-fos染色对特异性激活面旁核GABA能神经元后，皮层及皮层下觉醒系统神经元兴奋性变化情况进行探究。在七氟烷麻醉状态下特异性激活面旁核GABA能神经元后，小鼠皮层区域，基底前脑，外侧下丘脑和丘脑中央区c-fos表达量均出现显著降低。证实面旁核调控麻醉苏醒是通过抑制皮层下觉醒系统和抑制皮层兴奋性。以上结果表明，面旁核是脑内调控麻醉苏醒的重要区域。抑制该区域神经元有助于机体由麻醉状态向觉醒转换，而激活该区域显著提高麻醉药物的敏感性，这一发现为缩短临床手术后麻醉苏醒时间提供了新思路。