甘丙肽及其受体对睡眠及情绪异常引起睡眠失调的调节作用及机理

The neurons in the ventrolateral preoptic nucleus (VLPO) are primarily active during sleep, and co-contain the inhibitory neurotransmitters, galanin (GAL) and γ-aminobutyric acid (GABA). The VLPO cluster more selectively projects to and heavily innervates the histaminergic neurons in the tuberomammillary nucleus (TMN). The extended VLPO neurons provide the main output from the VLPO to the noradrenergic (NA) locus coeruleus (LC) and serotoninergic dorsal raphe nucleus (DRN). A "flip-flop switch model" of sleep-wakefulness regulation between the VLPO and TMN has been presumed by several scientists according to the evidences above. However, the role and mechanism of galanin on the flip-flop circuit is unclear. A synthesized method including resolution of sleep-wake states, EEG power analysis, patch clamp electrophysiology, neuropharmacology, molecular biochemistry, clarity combined with immunofluorescence technigues will be employed in this proposed project for revealing that the role and mechanism of GAL and its receptors on sleep-wake regulation and ameliorative effects on emotional disturbances-induced sleep disorder, when GAL and its receptors agonists or antagonists act respectively on the histaminergic TMN, noradrenergic LC and serotoningic DRN. The expected results of the project will detail the role of GAL and its receptors in sleep-wake regulation, and enrich the theory of sleep control, and provide a new strategy for sleep research and sleep disorder treatment.

睡眠期主要活动的腹外侧视前核(VLPO)神经元共含抑制性神经递质甘丙肽(galanin, GAL)与γ氨基丁酸(GABA)，选择性的下行投射结节乳头体核(TMN)组胺(HA)能觉醒神经元并形成靶连接，同时也投射向蓝斑(LC)去甲肾上腺素(NA)能和中缝背核(DRN)五羟色胺(5-HT)能神经元，据此多名学者提出VLPO-TMN睡眠觉醒相互转化"跷跷板"调控模型。但GAL在此调控环路中的作用特征及机制尚不清。本项目利用睡眠觉醒时相解析，脑波能谱分析，膜片钳电生理技术，结合神经药理，透明脑结合免疫荧光等手段，从细胞到整体行为揭示GAL及其受体激动剂或拮抗剂分别作用于TMN HA能或LC NA与DRN 5-HT能神经元调节睡眠觉醒时相和改善情绪异常导致睡眠失调的机制。预期结果将为全面了解GAL及受体对睡眠调节作用，丰富睡眠中枢调节机制，为睡眠疾病治疗提供新策略。

睡眠期主要活动的腹外侧视前核(VLPO)神经元含神经递质甘丙肽(galanin, GAL)与γ氨基丁酸(GABA)，投射向结节乳头体核(TMN)组胺(HA)能觉醒神经元并形成靶连接，据此多名学者提出VLPO-TMN睡眠觉醒相互转化“跷跷板”调控模型。但GAL在此调控环路的作用特征和机制尚不清，缺少实验证据证明VLPO的GAL能神经元向TMN的HA能神经元投射神经解剖环路。本项目分别开展了GAL中枢作用对睡眠觉醒周期的影响及其作用靶点，应用病毒追踪技术证明是否存在VLPOGAL-TMNHA环路，GAL作用TMN或局部组胺释放条件下对睡眠觉醒的调节作用，以及GAL对抑郁样行为和睡眠障碍的作用研究。重要结果与关键数据：（1）GAL (0.1-0.5 nmol)中枢作用呈剂量依赖性的引起大鼠慢波睡眠(SWS)增加和EEG的delta (0.5-4 Hz)活动增强，同时觉醒减少；GAL 0.5 nmol促SWS可持续6小时，该作用被GAL受体拮抗剂所抑制。（2）c-Fos标记发现GAL促VLPO活动神经元增加，TMN活动神经元减少。（3）病毒顺行和逆行追踪结合免疫荧光标记证明下丘脑存在VLPOGAL-TMNHA环路，TMN的组胺能神经元表达GAL 1型受体(GalR1)。（4）GAL和GalR1激动剂作用TMN引起更为显著SWS增加和觉醒减少，GAL促SWS作用不能被GalR2拮抗剂抑制，提示GAL通过GalR1发挥促SWS作用。（5）GalR1激动剂TMN作用可抑制H3R拮抗剂引起组胺释放的促觉醒作用，提示GAL通过TMN表达GalR1的神经元抑制组胺释放发挥促SWS作用。（6）GAL中枢作用可改善幼鼠氯米帕明连续腹腔注射导致成年抑郁样行为和PS增加为特征的抑郁样睡眠。科学意义：本项目研究结果为深入了解GAL及受体对睡眠调节作用提供了新证据，丰富睡眠中枢调节机制，为睡眠疾病治疗提供新策略。