延髓面旁区调控慢波睡眠的作用及分子机制研究

Insomnia is the most common symptom of psychiatric disorders, affecting more than 27% of the worldwide population. However, the neural regulation of sleep is incompletely understood. A mutually inhibitory interaction between sleep-active neurons of the ventrolateral preoptic nucleus (VLPO) and monoaminergic and cholinergic arousal systems regulates sleep-wake behavior. However, lesions in these cell groups have caused relatively limited alterations of sleep and wakefulness. In the recent study, we uncovered the location and identity of medullary neurons that might contribute to the regulation of sleep. We started by retrogradely labeling inputs to the medial parabrachial nucleus (MPB) - a key brainstem arousal node - from the medullary brainstem. Our tracer results revealed a substantial input from sleep-active (Fos-positive) medullary neurons located lateral and dorsal to the facial nerve, a region we termed the parafacial zone (PZ). Ablation of PZ neurons - and therefore 'disinhibition' of the wake-promoting MPB - resulted in insomnia in rats. These studies suggest a critical role of PZ in sleep regulation. Our recent preliminary data revealed GABAergic PZ neurons to drive slow-wave-sleep (SWS) in vivo. Previous studies in rats have shown that PZ sleep-active neurons project to the wake-promoting parabrachial nucleus (PB) and that projections from the PB to the basal forebrain (BF) are indispensable for maintaining cortical EEG activation and wakefulness. We therefore hypothesized that GABAergic PZ neurons might promote SWS by inhibiting the wake-promoting MPB-BF-cortex circuit. We will down-regulate the vesicular GABA transporter of PZ in rats by AAV-shRNA approach to end GABAnergic transmission, in the meanwhile, record sleep electroencephalogram (EEG) by a state-of-the-art high-efficiency sleep bioassay system to clarify the essential role of PZ on SWS regulation. Next, we will apply the optogenetic and pharmacogenetic DREADD (designer receptors exclusively activated by a designer drug) approach to control the PZ neuron activity of rats or Vgat-Cre mice to study the roles of PZ on induction and maintenance of SWS. And we will apply techniques of cell electrophysiological, spike discharge recording, neuroanatomical, pharmacological and etc. to dissect the role of the PZpathway in the sleep-wake neural network mechanism. This study will enrich and develop the theory of sleep regulation and provide new ideas for the insomnia treatment.

睡眠机制未明，失眠无有效对策。Lu等发现：睡眠期延髓面旁区（PZ）神经元FOS增多；毁损PZ，觉醒量增加。预实验显示：激活PZ，慢波睡眠量及delta波成分显著增加。提示：PZ可能是调控慢波睡眠新脑区，亟待解明作用与机制。PZ区主要为GABA能神经元，投射纤维至觉醒相关的内侧臂旁核（MPB），后者投射纤维至基底前脑。由此假说：PZ兴奋，抑制MPB，协同基底前脑，降低皮层兴奋性，调控慢波睡眠。本题利用高度自动化睡眠解析平台，采用AAV-shRNA技术下调或终止PZ区GABA信号，光遗传学及遗传药理学DREADD技术人为调控大鼠及Vgat-Cre小鼠PZ区GABA能神经活性，考察PZ区GABA能神经元调控慢波睡眠的作用；结合细胞电生理、Spike放电和睡眠脑电同步记录、神经纤维示踪、药理学等手段，揭示PZ调控慢波睡眠的神经环路及分子机制。研究结果将丰富和发展睡眠调节理论，为治疗失眠提供新思路。

睡眠机制未明，失眠无有效对策。项目主要参与者陆军教授利用遗传药理学DREADD技术和光遗传技术，对促进慢波睡眠的PZ神经元的下游通路进行了检测，发现该通路特异性、高效启动了慢波睡眠并增强了慢波睡眠的脑电活动。 提示，PZ是调控慢波睡眠重要的脑区。但是，PZ中GABA能神经元受到哪些核团或者神经元的调控，仍不清楚。我们利用改造后的狂犬病毒标记核团中特异性神经元，并将其上游神经元整体标记，以此来观察PZ中GABA能神经元的上游神经支配核团或者神经元。纤维镜下全脑追踪发现，PZ内GABA神经元接受的传入投射脑区主要有岛叶皮质、运动皮质、前边缘皮质、扣带皮质、感觉皮质、伏隔核、内侧隔核、斜角带、终纹床核、腹侧苍白球、内侧视前区、外侧视前区、无名质、中央杏仁核、下丘脑室旁核、下丘脑、弓状核、深部中脑核、前顶盖前核、黑质致密部、红核，但尚未完成理想结果。后期调整了研究方向，围绕帕金森病人睡眠障碍，聚焦纹状体多巴胺受体，有重大发现。研究结果提示，多巴胺受体D1阳性神经元是重要的觉醒神经元，期望为防治帕金森病睡眠障碍提供新思路。