外侧隔核调节睡眠-觉醒的分子机制及神经环路

Insomnia and depression always coexist, and aggravate each other. However, there are no effective clinical ways to treat this insomnia because the mechanisms of sleep-wake regulation in depression are not clear. It has been reported that the lateral septum is involved in emotional responses, such as depression, and regulates delta wave activity in the cortex. Our preliminary study found that selective control of the lateral septal neuron activity significantly changed sleep-wake behavior. We hypothesized that the lateral septum is a key nuclei involved in insomnia with depression. In the present study, we are going to use Cre mice for vGAT and vGLUT2, AAV DIO Channelrhodopsin-2 (ChR2) or Halorhodopsin (NpHR)-mCherry, and designer receptors exclusively activated by a designer drug (DREADD) to specifically control neuron activity. This optogenetic technology allows targeted, fast control of precise defined events in freely moving animals by delivering optical control at the speed (millisecond scale) and with the precision (cell type–specific) required for biological processing. By optogenetic approach of transfecting ChR2 or NpHR in different neurons in the lateral septum, automatic sleep bioassay systems, neuron spike recording, neurochemistry, and histochemistry approaches, we are going to clarify the key neurons in the lateral septum to initiate/maintain sleep or wake, and neural circuits and molecular mechanisms. The outcome of the proposal will provide new insights into sleep-wake regulation by the key neurons in the lateral septum. This project will shed light on the neurobiological mechanisms for insomnia with depression, which in turn will aid in the development of novel treatment drugs.

失眠与抑郁共存，又相互加重。由于抑郁性失眠的机制不明，临床缺乏有效对策，因此，揭示抑郁性失眠的机制是重要的科学问题。有报道外侧隔核参与抑郁等情绪反应，并且影响大脑皮层delta波活性。我们预实验发现选择性激活或抑制外侧隔核神经元活性，显著改变睡眠觉醒行为。我们假说：外侧隔核可能是调节抑郁性失眠的重要核团。本课题拟运用Cre动物、Cre依赖AAV病毒、光遗传学、药理遗传学、神经元损毁、hrGFP神经纤维追踪、电生理膜片钳、Spike记录和免疫组化等方法，结合自动化睡眠解析平台，研究外侧隔核在生理及抑郁状态下对睡眠觉醒的调节作用，阐明其调控睡眠觉醒的主要神经元类型、神经环路和神经递质等神经生物学机制。预期结果将丰富和发展睡眠觉醒调节理论，为探索治疗抑郁性睡眠障碍有效靶点提供新思路。

外侧隔核调控大脑皮层活跃度，参与焦虑和抑郁等情绪反应，而焦虑或抑郁症患者出现睡眠障碍，提示外侧隔核可能参与睡眠觉醒的调控。因此，外侧隔核如何调节睡眠觉醒成为研究焦虑或抑郁性失眠的关键问题。我们利用光纤钙信号、化学遗传学、光遗传学、特异性神经元示踪和损毁技术，结合Vgat-Cre小鼠和高度自动化的睡眠记录解析系统，探究了外侧隔核调控睡眠觉醒和压力性失眠的机制。结果如下：（1）在体光纤钙信号和睡眠同步记录发现，外侧隔核GABA能神经元活性与睡眠觉醒时相高度相关，即在觉醒期神经元活性最高，非快动眼（non-rapid eye movement, NREM）睡眠期次之，REM睡眠期最低。（2）特异性损毁外侧隔核GABA能神经元，小鼠活动期的NREM睡眠总量降低16.4%，觉醒量相应增加13.9%，而对非活动期的睡眠觉醒量无明显影响。（3）在小鼠活跃期，化学遗传学激活外侧隔核GABA能神经元，两小时内NREM睡眠量增加130%；而抑制该类神经元，4小时内NREM睡眠总量降低了51%，表明外侧隔核GABA能神经元参与活动期生理性睡眠的调节。（4）利用神经元特异性示踪法，发现外侧隔核GABA能神经元主要投射到基底前脑和外侧下丘脑。离体脑片膜片钳显示，外侧隔核GABA能神经元的轴突末梢与外侧下丘脑中的神经元形成功能性联系。利用逆行Cre病毒结合hM3Dq受体，在小鼠活动期特异性激活外侧隔核向外侧下丘脑投射的神经元，激活后2 h内小鼠的NREM睡眠量比对照组增加了93%，提示外侧下丘脑是外侧隔核调控睡眠觉醒的重要下行通路。（5）特异性损毁外侧隔核GABA能神经元，小鼠换笼后的NREM睡眠潜伏期比对照组增加了44.7%；化学遗传学抑制外侧隔核GABA能神经元后，加重压力诱导的失眠症状。而特异性激活外侧隔核GABA能神经元，可显著改善换笼或腹腔注射生理盐水诱发的失眠。以上结果表明，外侧隔核是脑内调控压力性失眠的重要区域。该结果揭示了压力性失眠的机制，可能为开发新型促眠药物提供了新思路。