eCB介导的DSI效应在麻醉-觉醒调节中的作用及机制

Although 200 million patients are undergone anesthesia procedures each year in the world, the mechanism of general anesthetics is still a fundamental question puzzling the neuroscience world. It has been listed as one of the 125 leading unsolved science questions by Science in 2005. Up to date, it is well accepted that neural signals network may be attributed to the mechanism of general anesthesia.Accumulated evidences from our and other groups support that orexinergic and GABAergic neural pathways are crucial for regulating anesthesia-arousal cycle.However,how do the neural pathways regulate each other is unclear. A novel retrograde synaptic signals regulation, which called DSI (depolarization induced suppression of inhibitory), has been found recently. Orexinergic signal can induce DSI effect on GABAergic neural through endocannabinoids system. The aim of current project is to explore the role of DSI in regulative effect of orexin to GABA neuronal pathway in the transformation from anesthesia to wakefulness. This hypothesis will be tested through in vivo and in vitro studies by the neuropharmacological and electrophysological techniques as well as the transgenic animal models. The findings of current research project will facilitate the understanding of proactive role of orexin in awakening, and to lay the foundation for exploring the regulation of neuronal network among various neuronal pathways in anesthesia-arousal transformation.

全球每年近2亿人需要接受麻醉，而全麻机制研究已被Science杂志列为全球125个未解科学谜团之一。申请者及国外研究证实网络调控是全麻机制的核心，并且orexin及GABA等神经递质调控麻醉-觉醒，然而其通过何种调控途径实现相互抑制或激动，从而实现麻醉-觉醒这一转换过程尚缺乏线索。新近发现去极化诱导的去抑制（DSI）在不同种类的神经突触调控中发挥重要作用，且orexin可通过内源性大麻素2-AG及其受体产生DSI效应，减少GABA的释放。我们的假说是，麻醉觉醒过程中orexin含量急剧增高，激活突触后orexin受体，通过DSI逆行性抑制突触前膜GABA信号，从而产生麻醉向觉醒转换的主动调节过程。本研究拟构建神经元特异性基因敲除小鼠，利用free-moving动物模型结合微注射、在体微透析及电生理技术，阐明DSI突触信号在全麻觉醒主动调节中的作用机制，为揭示麻醉-觉醒神经网络调控奠定基础。

全球每年近2亿人需要接受麻醉，而全麻机制研究已被Science杂志列为全球125个未解科学谜团之一。申请者及国外研究证实网络调控是全麻机制的核心，并且orexin及GABA等神经递质调控麻醉-觉醒，然而其通过何种调控途径实现相互抑制或激动，从而实现麻醉-觉醒这一转换过程尚缺乏线索。通过文献回顾，我们认为内源性大麻素系统（eCBs）很可能通过DSI和DSE效应调控了上述全麻神经网络平衡。本研究中，我们应用模式动物、电生理、化学遗传学和免疫电镜等技术，发现在无论是全身给予，还是下丘脑背内侧核（DMH）核团微注射大麻素一型受体（CB1R）拮抗剂AM281，都可以加速小鼠全麻后觉醒；而在相邻的穹窿周围区（Pef）或腹外侧视前区（VLPO）微注射AM281则并不影响全麻后意识恢复。具体说来，全麻药物可以选择性地激活DMH核团中谷氨酸能突触上的内源性大麻素（eCB）信号，而对GABA能突触没有影响。DMH核团谷氨酸能突触上eCB信号的激活导致了对下游谷氨酸能DMH-Pef投射和GABA能DMH-VLPO投射的双重抑制。选择性敲除全脑或前额叶皮质（Prefrontal Cortex, PFC）内谷氨酸能神经元上的CB1R，就可以模拟AM281的促觉醒作用。相反，如果敲除脑内的GABA能神经元或者下丘脑的谷氨酸能神经元上的CB1R则不影响全麻后的苏醒时间。应用化学遗传学手段抑制PFC-DMH，DMH-VLPO或DMH-Pef投射都可以逆转AM281促进觉醒的作用，而激活这些神经通路则可以模拟AM281的促觉醒作用。至此，我们认为抑制PFC投射至DMH的谷氨酸能突触上的eCB信号可以加速全麻后觉醒，这种作用可能是由加强DMH-Pef的兴奋性投射，或DMH-VLPO的抑制性投射，或两者同时参与来介导的。上述发现不仅为揭示麻醉-觉醒神经网络调控奠定了重要理论基础，更为临床调控麻醉-觉醒提供了重要的干预靶点。