腺苷A2A受体启动和维持睡眠的分子机制

There are no effective ways to treat insomnia clinically because how physilogical sleep-wake cycle is regulated in the brain still remains unclear. The drive to sleep begins with the onset of wakefulness and dissipates slowly with the progression of sleep. Endogenous sleep factors acting on specific neurons in the brain are hypothesized to regulate the waxing and waning of the sleep drive. Two such sleep factors are adenosine, a naturally occurring purine nucleoside present in all cells, and prostaglandin (PG) D2, an eicosanoid acting as a tissue or local hormone, both of which are released as neuromodulators in the brain (Huang ZL et al. Curr Opin Pharmacol 2007; Curr Top Med Chem 2011). We have demonstrated that the endogenous somnogen PGD2 increases the level of extracellular adenosine. Adenosine diffuses into the brain parenchyma as the secondary somnogen, activates sleep-active ventrolateral preoptic area (VLPO) neurons in the hypothalamus via adenosine A2A receptor (R) to induce sleep potently. On the other hand, blockade of adenosine A2AR inhibits sleep (Huang ZL et al., Nat Neurosci 2005), indicating that A2AR is the most important receptor for sleep induction. However there is no any expression of A2AR in the VLPO, therefore, how sleep is induced and maintained remains to be elucidated. In the proposed project, we are going to use Cre mice for A2AR and so on, AAV DIO Channelrhodopsin-2 (ChR2) or Halorhodopsin (NpHR)-mCherry (adeno-associated viral vector double-floxed inverse open reading frame of ChR2-mCherry) and pulsed laser photostimulation of ChR2 or NpHR-transfected neurons to control neuron activity specifically. This optogenetic technology allows targeted, fast control of precisely defined events in freely moving mammals 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 A2AR positive neurons in the different regions of nucleus accumbens, automatic sleep bioassay systems, neuron spike recording, neurochemistry, and histochemistry approaches, we try to clarify the key regions in the brain to initiate/maintain sleep and molecular mechanisms involved. The outcome of the proposal will provide new insights into sleep-wake regulation under physiological conditions. This project will shed light on the neurochemical mechanisms underlying sleep disturbances, which in turn will aid in the development of novel treatment drugs.

由于生理性睡眠调节机制不明，失眠症治疗尚无有效对策。我们发现：内源性前列腺素D2和腺苷可能由腺苷A2A受体介导，活化下丘脑腹外侧视前区（VLPO），诱发最强大的生理性睡眠；咖啡因拮抗A2A受体，阻止睡眠，提示A2A是最重要的睡眠调节受体，但VLPO区无A2A受体表达，因此，睡眠启动和维持的机制亟待阐明。本课题将利用A2A受体-Cre小鼠，建立光遗传工学控制特定神经元活性法，在富含A2A受体的神经元如伏隔核和嗅结节等区，经不同频率光照射，瞬时、定点、高度选择性地调控神经元活性，运用自动化睡眠解析平台、神经元Spike记录、神经化学及组织学等手段，揭示睡眠启动和维持的关键神经元和神经递质及分子机制。研究结果将丰富和发展睡眠觉醒调节理论，为生理性促眠药物开发提供理论基础。

我们前期发现咖啡因拮抗腺苷A2A受体（A2AR），阻止睡眠，增加觉醒（Huang et al., Nat Neurosci 2005），提示：A2AR可能启动或维持睡眠，但作用和机制不明。本课题利用A2AR -Cre小鼠，通过光遗传或化学遗传学方法，在富含A2AR的伏隔核和嗅结节等区域的神经元上，人为表达光通道蛋白或化学遗传的DREADD受体，经不同频率光照射或CNO注射，时空特异性和高度选择性地调控A2AR阳性神经元活性，结合自动化睡眠解析平台、神经化学及组织学等手段，揭示睡眠启动和维持的关键神经元、神经环路和分子机制。主要科学发现如下：1）发现伏隔核(NAc)腺苷A2AR阳性神经元为重要的睡眠启动神经元，通过支配腹侧苍白球（VP），介导生理性睡眠效应；2）解析了伏隔核A2AR神经元的纤维投射。将携带人源绿色荧光蛋白基因的腺相关病毒（AAV）作为长轴突通路的示踪剂，对A2AR神经元的投射进行特异追踪，发现这些神经元投射至VP、斜角带核和无名质、下丘脑视前外侧区和下丘脑外侧区、中脑腹侧被盖核、中缝背核、背侧中缝核和导水管周围腹外侧区等；3）背侧纹状体A2AR神经元通过腹侧纹状体-外侧苍白球神经通路，促进慢波睡眠；4）揭示嗅结节中A2AR在睡眠调节中的作用及其机制；5）发现嗅球中表达的A2AR抑制快动眼睡眠，可能与嗅觉障碍性快动眼睡眠异常、抑郁有密切关系； 6) 腺苷A2AR介导内源性睡眠促进物质前列腺素D2的睡眠诱导作用；7）发现基底前脑胆碱能神经元通过向皮层的直接投射，抑制delta波能谱，提高皮层兴奋性，降低睡眠深度。本研究首次报道前脑中的A2AR调控生理性睡眠及神经生物学机制，丰富和发展睡眠觉醒调节理论，为将腺苷A2AR为靶点，开发生理性促眠药物提供理论基础。. 发表署名此基金号的SCI研究论文22篇；项目负责人的研究论文被广泛引用，被Elsevier评为中国论文被高引学者；培养研究生4人和博士后2人，参加出版科普书籍一本。在研期间，应邀到澳大利亚、日本、韩国及德国等作学术报告15次。