基底神经节多巴胺能神经通路的睡眠-觉醒调控作用及机制研究

Sleep disorder is an increasingly serious social and medical problem, while the mechanisms of physiological sleep-wake regulation are still unclear. Basal ganglia (BG) is important in regulating sleep and wakefulness, lesions of dopaminergic neurons located in substantia nigra pars compacta, result in dopamine (DA) de-innervation in dorsal striatum (CPu), and increase wakefulness in rats; while increase the release of DA in CPu results in sleep promotion. As DA is thought to be a wake-related neurotransmitter, we hypothesized that the different dopaminergic pathways may have opposite roles in sleep-wake regulation. To verify this hypothesis, we will apply a novel chemogenetic/optogenetic approach to selectively modulate the activity of nigrostriatal pathway and mesolimbic pathway in BG, to clarify the role of these two dopaminergic pathways in sleep-wake regulation; furthermore, By combining global or local gene knock down, and adopt a dopamine receptor D1 and D2 double gene reporter mice, to elucidate the mechanism of BG dopaminergic pathways in sleep-wake regulation. The results of the project will specific the influence of DA on sleep and wakefulness, bring contributions to the enrichment and development of sleep-wake regulation theory, and play an important role in exploring methods of treating sleep disorders.

睡眠障碍已成为日益严重的社会及医学问题，生理性睡眠-觉醒调节机制亟待阐明。我们发现基底神经节（BG）是参与睡眠-觉醒调控的重要结构，黑质致密部多巴胺（DA）能神经元毁损致背侧纹状体（CPu）去DA支配后，大鼠觉醒量显著增加；增强CPu DA的释放则促进大鼠睡眠。但DA被认为是促觉醒递质。我们推测不同DA能神经通路对睡眠-觉醒的调控作用相反，BG黑质纹状体通路的DA参与睡眠调控，而中脑边缘通路的DA促进觉醒。为验证这一假设，本题将采用化学遗传学、光遗传学技术，结合脑电记录、局部基因敲除、电生理及免疫组织化学等方法，通过特异调控BG DA黑质纹状体通路和中脑边缘通路观察其对睡眠-觉醒的影响，并进一步探讨BG DA能通路调控睡眠-觉醒的机制。研究结果将明确神经递质DA对睡眠-觉醒的影响，增添DA调控睡眠-觉醒的新机制，丰富和发展睡眠-觉醒调节理论，对探索临床治疗睡眠障碍具有重要意义。

睡眠障碍已成为严重的社会及医学问题，睡眠-觉醒调节机制亟待阐明。多巴胺（DA）是参与BG功能的重要神经递质，我们前期研究发现基底神经节（BG）是参与睡眠-觉醒调控的重要结构。本课题以BG的DA能神经通路为研究靶点，揭示黑质纹状体（SNc-CPu）和中脑边缘（VTA-NAc）DA通路的睡眠-觉醒调控作用，并探讨其可能机制。重要结果：1）借助逆行性腺相关病毒载体AAV-retro-Cre和cre依赖的M3受体腺相关病毒载体（AAV-DIO-hM3Dq），特异性激活SNc-CPu DA通路和VTA-NAc DA通路，发现：SNc-CPu DA通路激活增加睡眠，VTA-NAc DA通路激活则增加觉醒，表明CPu和NAc中的DA信号的睡眠-觉醒调控作用相反，分别促进睡眠和觉醒。2）特异性激活SNc-CPu DA通路后，通过c-Fos染色全脑观察发现：外侧下丘脑LH，结节乳头核TMN和蓝斑LC等觉醒核团c-Fos表达量显著降低，而睡眠相关核团外侧苍白球GP和黑质网状部SNr区域c-Fos表达显著增加，提示SNc-CPu DA通路激活后分别抑制和兴奋上述觉醒和睡眠相关核团而促进睡眠。3）借助DA受体基因报告小鼠D1R-tdTomato和D2R-EGFP，采用上述方法特异性激活SNc-CPu DA通路，发现：CPu DA D1R+神经元的活性有增加趋势但未达到显著性差异，D2R+细胞的活性则明显受到抑制。提示SNc-CPu DA通路可能通过作用于间接通路D2R促进睡眠。4）利用D2R基因敲除小鼠，发现：在D2R缺失的情况下，特异性激活SNc-CPu DA通路的促睡效应显著减弱，且小鼠的活动度显著增加，进一步提示SNc-CPu DA通路的促眠效应由D2R所介导。5）利用D2R-flox小鼠和Cre腺相关病毒表达载体，局部敲除CPu部位的D2R，发现：SNc-CPu DA通路激活后的促睡效应消失。进一步提示SNc-CPu DA通路通过D2R促进睡眠。以上研究结果，证实了BG中2条重要的DA能神经元通路对睡眠-觉醒的调控作用相反，SNc-CPu通路的 DA参与睡眠调控，而VTA-NAc通路中的DA促进觉醒。并支持了来自SNc的DA，通过作用于间接通路的D2R发挥促眠效应的假设。DA普遍被认为是促觉醒递质，DA调节睡眠新机制的阐明，对认识PD，HD等疾病的睡眠障碍机制及治疗具有重要意义。