吻内侧被盖核通过控制多巴胺系统调控睡眠觉醒的作用及机制

Sleep problems including insomnia are increasingly becoming serious medical and social puzzles. Severe sleep disorders were observed in nearly all patients with nervous and mental diseases involved in abnormal dopamine. The reason may be due to the unclear effects of DA in regulation of sleep and wakefulness because of the neglect of actions of GABAergic afferents on DA neurons. .Rostromedial Tegmental Nucleus (RMTg), a newly identified region in brainstem, is primarily comprised of gamma-aminobutyric acid (GABA) neurons. Its axons formed intensive synaptic contacts with midbrain DA cells. Compared with other GABAergic inputs, RMTg exerts a major inhibitory control on DA neurons. In our previous experiments, we found that nonselective lesions of RMTg neurons in rats produced a significant reduction in sleep, whereas, nonselective activation of RMTg neurons induced more sleep. Some references reported that inhibitory GABA neurons play important roles on brain functions elicited by DA. Taking into account the above considerations, we put forward the hypothesis: RMTg GABA neurons regulate sleep and wakefulness mainly through the control of DA system. .Here, we will take advantage of vesicular GABA transporter (VGAT)-cre mice and ontogenetic as well as DREADD manipulations to excite or inhibit activities of RMTg GABA neurons at upstream of midbrain DA system. Combined with neuroanatomical tracing, electrophysiology, immunohistochemistry and other approaches, we will investigate the roles, neural circuits, and neurobiological mechanisms of RMTg GABA neurons in sleep-wake regulation from molecular to behavioral levels. .The expected results will enrich and develop theories of sleep-wake regulation, have important significance for understanding the role of DA in sleep-wake regulation and DA-related sleep disorders, and lay the theoretical foundation for exploration of new targets for clinical therapy.

睡眠问题是医学及社会难题，特别是多巴胺（DA）相关神经精神疾病伴有严重睡眠紊乱。吻内侧被盖核（RMTg）的γ-氨基丁酸 (GABA)能神经轴突与DA神经元形成密集突触，对DA神经元活性有很强抑制作用，因此，RMTg可能与睡眠觉醒密切相关，但作用机制不明。前期实验发现非特异毁损RMTg的大鼠睡眠量显著减少，而非特异激活RMTg的大鼠睡眠量显著增加。我们假说：RMTg的GABA能神经元主要通过控制DA系统活性调控睡眠觉醒。研究将应用囊泡GABA转运体（VGAT）-cre小鼠、光遗传学和DREADD等方法，选择性激活或抑制DA系统上游RMTg的GABA能神经元，结合神经示踪、电生理、免疫组化等方法，从分子到行为，揭示RMTg调控睡眠觉醒的作用、神经通路及机制。预期结果将丰富和发展睡眠觉醒调节理论，对理解DA调控睡眠觉醒作用及DA相关性睡眠障碍具有重要意义，为探索临床治疗新靶点奠定理论基础。

抑郁、药物成瘾、帕金森病等神经精神疾患的发病率呈逐年上升趋势，严重危害人类健康。这些疾病均伴有严重的睡眠紊乱，究其原因与脑内多巴胺能神经元功能失调密切相关。吻内侧被盖核（RMTg）也被称为腹侧被盖区（VTA）的尾部，由γ-氨基丁酸（GABA）能神经元组成，主要投射到脑干结构腹侧被盖区、中缝背核、蓝斑等区域。鉴于RMTg的投射脑区均参与促觉醒作用，因此，我们推测RMTg可能与睡眠密切相关。. 在本研究中，首先应用囊泡GABA转运体（VGAT）-Cre小鼠、采用遗传药理学方法特异性激活小鼠RMTg 的GABA能神经元后，小鼠非快速眼动（NREM）睡眠明显增加，伴随快速眼动睡眠和觉醒减少。为了观察RMTg在睡眠觉醒不同状态的快速转化中的作用，我们在RMTg部位注射AAV-ChR2-mCherry病毒后，用蓝光刺激使RMTg GABA能神经元激活后，可诱导小鼠迅速由觉醒状态向NREM睡眠转变；在NREM睡眠期给予蓝光刺激，则发现NREM睡眠能继续维持。为了探究RMTg是否参与了生理性睡眠促进作用，我们将AAV-hM4Di-mCherry病毒注射到VGAT-Cre小鼠RMTg部位，当用外源性药物氧化氯氮平使RMTg GABA神经元抑制后发现，其NREM睡眠明显减少。为了进一步探索RMTg GABA能神经元发挥促睡眠作用的神经环路，我们采用光遗传学、神经示踪、电生理、免疫组化等技术明确了RMTg的下游核团在脑内的分布。当我们刺激RMTg的GABA神经元末梢时，可抑制中脑VTA或黑质致密部中多巴胺能神经元。当采用酪氨酸羟化酶（TH）-Cre小鼠，结合药理遗传学手段，抑制这些多巴胺能神经元也有促进睡眠的作用。我们的研究结果表明，RMTg是睡眠启动和维持不可或缺的核团，其机制主要与抑制中脑多巴胺神经元有关。.研究结果丰富和发展了睡眠觉醒调节理论，为延长NREM睡眠和治疗多巴胺相关精神疾病的睡眠障碍提供了潜在靶点。