伏隔核表达腺苷A2A受体神经元调控睡眠—觉醒的神经解剖学通路

It is well documented that the adenosine A2A receptors in nucleus accumbens (NAc) may be involved in the regulation of sleep-wake cycle, but the projection pathways of the neurons expressing A2A receptor in the NAc are still remained unknown. We assume that activation of A2A receptors in the NAc may induce sleep by exciting the neurons of sleep centers in the ventrolateral preoptic area (VLPO) and inhibiting arousal histaminergic tuberomammillary nucleus（TMN）via specific neural pathways. Cre mice, overcoming the obstacles of lack of specificity in conventional neural tracing, can be used to selectively and specifically analyze the projection pathways of the neurons expressing A2A receptors in the NAc. The proposed project is designed to inject AAV-Flox-EGFP plasmid into different regions of NAc of adenosine A2A Cre mouse, slice the whole brain of the mouse, detect and map EGFP, analyze the neural pathways from A2A receptor-expressing neurons in the NAc to VLPO and TMN by stereological methods and examine the composition of synaptic connections and heteromeric composition of synapse receptors with immune electron microscopy. The previous work for the current proposal has been fund with a small amount of budget from NSFC, and we have obtained some expected results to move further. The final results will reveal the neural pathways between A2A receptor-expressing neurons in NAc and other sleep-wake systems at the cellular, subcellular and molecular levels and provide morphological basis for understanding the theory of physiologic sleep-wake regulation, which exerts an important role in establishing sleep-disorder animal models and in exploring the mechanism, etiology of insomnia.

伏隔核（NAc）神经元表达的腺苷A2A受体可能参与调控睡眠/觉醒，但神经投射通路不明。我们推测：NAc中A2A受体阳性神经元通过特定通路，兴奋腹外侧视前区（VLPO）睡眠中枢、抑制结节乳头核（TMN）觉醒系统，调控睡眠。Cre小鼠能突破传统追踪缺乏特异性的瓶颈，使选择性解析神经通路成为可能。本课题拟向腺苷A2A受体Cre小鼠NAc中注射AAV-Flox-EGFP质粒，切片后全脑Mappin检测EGFP，用体视学解析NAc A2A受体神经元与VLPO、TMN及相关神经元间的神经通路，用免疫电镜分析通路中的突触和突触受体异聚体的构成。本课题的前期工作获小额资助后，获得了部分结果，证明方法可行。预期最终研究结果将从细胞、亚细胞及分子水平，揭示NAc对睡眠/觉醒调控的神经通路，为完善和发展生理性睡眠调节的理论提供形态学依据；对建立睡眠障碍动物模型、探索失眠症的机制、病因和提出干预方法具有重要意义。

本课题围绕解决“伏隔核腺苷A2A受体神经元参与调控睡眠-觉醒投射通路”的科学问题，基于Cre/Loxp技术的特异性失踪方法、利用A2AR-Cre小鼠，在伏隔核处注射Cre依赖的AAV-Flox-hrGFP，以此特异性标记A2AR神经元及其轴突末梢，全面解析了伏隔核中A2AR神经元调控睡眠-觉醒的神经通路。另外，利用光遗传学技术操纵伏隔核中A2AR神经元活性，以确定伏隔核中A2AR神经元与睡眠-觉醒调控的关系。研究发现：1、伏隔核A2A受体表达神经元的传出投射至前脑、间脑和脑干，尤其在下丘脑外侧区分布密度最高，并与下丘脑中食欲肽神经元形成突触联系；2、伏隔核A2AR神经元投射至与睡眠-觉醒相关的脑区主要有：基底前脑的斜角带垂直臂核和无名质、下丘脑外侧区、中脑腹侧被盖、中缝核、腹侧导水管周围灰质。3、光遗传学特异性激活伏隔核中A2AR神经元后，实验动物可由觉醒状态快速进入非快速动眼睡眠状态，提示激活伏隔核A2AR神经元可促进非快速动眼睡眠。上述结果证实伏隔核腺苷A2A受体神经元参与非快速动眼睡眠的调控，并与多个参与睡眠-觉醒调控的核团有联系，提示伏隔核内腺苷A2A受体神经元参与睡眠调节是通过支配上述核团起作用的。我们的研究明确了伏隔核腺苷A2A受体神经元与睡眠-觉醒的确切关系，并揭示伏隔核腺苷A2A受体神经元参与睡眠-觉醒调控的神经通路，为完善和发展生理性睡眠调节的理论提供实验证据。