针刺调整睡眠-觉醒周期紊乱的TRN胆碱能-GABA能神经调控机制研究

Sleep disorders are risk of health. Sleep-waking cycle disorder is a key induced sleep disorder. The thalamic reticular nucleus serves as a gate for information between the cerebral cortex and thalamus. The earlier studies suggested that cholinergic input fibers from basal forbrain and brain stem promoted the process of waking from sleep. However, some researches reversed this view last year that provided critical insight into cholinergic roles in mediating biological rhythms. Cholinergic neurons play an important role in waking-to-sleep cycles and in sleep stabilization by activating GABAergic neurons in the TRN through a7-nAChRs. Combined with our studies on the neural mechanism of acupuncture on sleep-waking cycle disorder, focused on cholinergic fibers-GABAergic neurons in TRN, we employed PCPA insomnia mice, Chat - ChR2 mice, electrophysiological technique, optogentics and two-photon fluorescence imaging techniques to study the relationship between acupuncture on sleep-wake cycle disorder and cholinergic fiber- GABAergic neural regulation in TRN .The research results will provide scientific support that helping for the clinical application of acupuncture treatment on insomnia and related diseases due to sleep-waking cycle disorder such as anxiety, depression and so on. in additon, The research results also are very important for promoting to solve the resulting psychological barriers, unemployment and life safety.

睡眠障碍是危及人类健康的潜在致命因素，睡眠－觉醒周期紊乱是导致睡眠障碍的关键。传统认为胆碱能神经是促进觉醒的重要因素，但去年睡眠机制研究颠覆了这一观点，发现选择性激活丘脑网状核（TRN）内胆碱能投射可促睡眠。结合前期研究发现针刺调整睡眠-觉醒周期紊乱与丘脑、基底前脑有关，围绕“针刺可能通过调节TRN中胆碱能-GABA能调控交通皮层与丘脑，实现对睡眠-觉醒周期紊乱的整复。”的假说，从TRN胆碱能-GABA能神经元调控入手，选择PCPA失眠小鼠、Chat-ChR2小鼠等为载体，采用光遗传学、双光子荧光成像等技术，在体研究针刺调整睡眠-觉醒周期紊乱与TRN胆碱能－GABA能调控的相关性，以期揭示针刺调整睡眠-觉醒周期紊乱的中枢神经机制，为临床应用针刺治疗失眠及睡眠-觉醒周期紊乱相关疾病如焦虑症、抑郁症等提供科学支撑。研究结果对促进解决由此引发的心理障碍、失业及生命安全问题，具有重要社会价值。

睡眠障碍是危及健康的潜在致命因素，睡眠-觉醒周期紊乱是睡眠障碍的关键。丘脑网状核（Thalamus Reticular Nucleus，TRN）是调节睡眠-觉醒周期性转换的重要中枢。TRN内胆碱能－GABA能调控是实现睡眠觉醒转换调节的核⼼环节。本课题选择PCPA失眠模型及VGAT-Cre鼠为载体，采用行为学、在体电生理、免疫组化、化学遗传学、药理学方法揭示针刺调整睡眠-觉醒周期紊乱的TRN中枢机制。研究结果提示：针刺内关、足三里能有效调整PCPA失眠大鼠睡眠-觉醒周期紊乱。其可能与针刺调节了TRN区PV阳性GABA 能神经元数量，即PCPA失眠大鼠TRN 区 PV 阳性神经元数量减少，针刺后TRN区PV阳性GABA能神经元数量增多。针刺后TRN内神经元以连续簇状放电为主，神经元兴奋性增高；光纤钙信号发现针刺激活了TRN内GABA能神经元。化学遗传方法特异性的抑制TRN区GABA能神经元，化学遗传抑制组的针刺治疗效果消失，即依然处于失眠状态。针刺可能通过上调PSD-95蛋白改善失眠大鼠TRN区PV神经元的突触可塑性，并且TRN区PV神经元的突触可塑性增强可能与PV神经元的α7nAChRs数量上调有关，即造模后，失眠大鼠TRN区PV神经元α7nAChRs数量下降，针刺干预可以增加失眠大鼠TRN区PV神经元α7nAChRs数量。使用α7nAChR特异性抑制剂MLA注射，针刺后TRN内神经元活性显著降低，放电频率显著下降。针刺治疗可以抑制小胶质细胞活性，降低TRN区IL-1β、TNF-α的浓度从而改善失眠后认知缺陷。P2X7受体可能与CT8时电针对小鼠自发性活动相位超前转移相关；电针调整小鼠自发活动节律具有明显的依时相性，CT8 针刺相位超前，可能是通过GABA能突触信号通路中的GABAB受体、Gi/o蛋白磷酸化修饰水平下调。CT16 针刺相位迟后，可能与胰岛素信号通路AC蛋白修饰水平上调，抑制胰岛素分泌有关。研究结果揭示了针刺调整睡眠觉醒周期紊乱的部分中枢神经机制，对进一步阐释针灸治疗失眠的神经环路具有重要的学术意义。亦可为临床治疗多种原因所致睡眠-觉醒周期紊乱提供最佳治疗时间选择及优化针灸处方提供支撑，具有重要临床意义。