臂旁核谷氨酸能神经元在异氟醚麻醉-苏醒调控中的作用机制研究

Understanding the role and mechanism of general anesthetics is conducive to improve surgical effect and reduce postoperative side effects, but the mechanism of general anesthetics is still unclear. Although reports indicate general anesthetics play role through influencing some important wake or sleep neurons, degeneration of these neurons did not exert significant influences. Pre-experiments showed that the parabrachial nucleus (PB) was crucial for awakening behavior, but its role and underlying mechanism in the occurrence of general anesthesia remained unknown. We hypothesis that PB may be an important "switch" for controlling the action of general anesthetics, deactivating the switch enhances anesthesia, and activating it accelerates anesthesia recovery. The research project will utilize a highly automated EEG analytical platform, vGLUT2-Cre mice, AAV-shRNA, optogenetic and DREADD (designer receptors exclusively activated by a designer drug) approaches which specifically regulate the glutamatergic neuronal activity in the PB, pharmacological methods, etc., combine cellular electrophysiology, neuro-anatomy and neuronal tracing means, to investigate the roles of PB glutamatergic neurons in the inhalation anesthetics isoflurane-induced anesthesia, and molecular mechanism and neural circuit. The expected results will provide new insights for revealing the mechanism of general anesthetics, offer new ideas for the safe and rational usage of general anesthetics, and supply a new target for the development of new general anesthetics.

认识全麻药的作用和机制有利于外科手术效果和降低术后不良反应，但目前全麻药的作用机制尚不清楚。虽有报道显示全麻药可能通过影响某些重要的觉醒或睡眠神经元而发挥作用，但变性这些神经元并没有产生显著影响。预实验发现臂旁核（parabrachial nucleus, PB）对觉醒行为至关重要，但影响全麻药的作用和机制不明。我们假说：PB可能是全麻药作用的重要“开关”，失活该开关加深麻醉，激活则加快麻醉苏醒。本研究将利用高度自动化脑波解析平台、vGLUT2-Cre小鼠、AAV-shRNA、光遗传及DREADD技术特异性调控PB核谷氨酸能神经元活性、药理学方法等，结合电生理、神经解剖与神经纤维示踪等手段，研究PB核谷氨酸能神经元在吸入性全麻药异氟醚诱导的麻醉-苏醒中的作用、分子机制和神经环路。预期结果为揭示全麻药的作用机制提供新内容，为临床安全使用全麻醉药提供新思路，亦可能为研制新型全麻药提供新靶点。

项目围绕“具有促觉醒作用的臂旁核（parabrachial nucleus, PBN）谷氨酸能神经元是全身麻醉-觉醒调控神经环路中的一个重要‘开关’，失活该开关麻醉加深，激活则麻醉苏醒加快”这一假说，利用自动化脑波解析平台记录分析脑波，采用AAV-shRNA技术下调PB谷氨酸信号，借助光遗传及化学遗传技术特异性操控Vglut-cre小鼠PBN谷氨酸能神经元活性，研究PBN谷氨酸能神经元在吸入性全麻药诱导的全身麻醉-觉醒调控中的作用及机理。同时结合细胞电生理、神经解剖与神经纤维示踪等手段，探讨PBN谷氨酸能神经环路调控麻醉-觉醒行为的发生机制。结果显示，（1）七氟醚浓度依赖性地抑制PBN神经元的放电频率，抑制PBN神经元诱导麻醉，其机理为通过突触后GABAA受体和背景钾通道抑制PBN神经元活性。（2）用化学遗传技术激活PBN谷氨酸能神经元可以降低七氟醚敏感性，并加快动物复苏；抑制PBN谷氨酸能神经元则延长七氟醚诱导的动物麻醉复苏时间。（3）用光遗传技术激活PBN谷氨酸能神经元可以诱导七氟醚麻醉期动物大脑皮层觉醒。（4）激活PBN谷氨酸能神经元可以增加七氟醚麻醉期动物大脑皮层和皮层下觉醒核团（基底前脑、中缝背核、外侧下丘脑等）c-Fos表达。这些结果充分证明了PBN谷氨酸能神经元在全身麻醉-觉醒调控神经环路中的重要“开关”作用，为揭示七氟醚麻醉作用机制提供了新内容，为临床安全使用七氟醚这类全麻醉药提供了新思路，亦为新型全麻药的研制提供靶点。