警觉信息素唤醒作用及其神经环路

Alarm pheromones (APs) are very important communicating substances in species survival, which conducts hazards, e.g. injury, distress, presence of predators, e.t.c., to the companions. Receptors of APs are located in the Grueneberg ganglion (GG) of nasal vestibule, and the axons of GG project to the posterior part of the main olfactory bulb through the dorsum of the nose. Previous studies suggested that olfaction is not sensitive to general odor, however recently our research group found that APs were able to awaken mice in non-rapid eye movement (NREM) sleep stage, and we proposed a hypothesis that "APs activates GG neurons, and again the signals were relayed by olfactory bulb, and amygdaloid body and other nuclei, and finally were transmitted to arousal related nuclei to awaken the sleeping mice and to maintain the arousal condition in awake mice". This study plans to use APs directional/quantitative delivery stimulation, EEG/EMG recording, resolution of sleep-wake states and EEG power analysis, optogenetics, transgenic mice, combined with anterograde or retrograde trans-synaptic tracing by neurotropic virus, clarity and immunofluorescence techniques to reveal the EEG/EMG characteristics of the arousal induced by APs in mice and the neural circuits involved. The expected results will enrich the theory of the connection between olfactory transmission and the regulation of sleep and wake, and enrich the manner of information exchange among individuals.

警觉信息素(APs)是种系生存中的重要通讯物质，供体释放的APs可向同伴传递受伤、痛苦或捕食动物来袭等危险因素信息。APs的感受器是位于鼻前庭的Grueneberg神经节(GG)， GG的纤维经鼻背投向主嗅球后部。前期研究认为睡眠期嗅觉对一般气味的刺激是不敏感的，但近期我们发现APs可唤醒非快动眼睡眠期小鼠并提出“APs激活GG神经元，经嗅球、杏仁体等中继到达觉醒相关核团引发睡眠期的唤醒作用和觉醒期的维持作用”假说。本项目拟利用APs定向、定量投递刺激，EEG/EMG记录，睡眠觉醒时相解析及脑波能谱分析，光遗传学，转基因小鼠，结合嗜神经病毒顺/逆跨突触追踪，透明脑与免疫荧光等技术手段，揭示APs对各睡眠时相的唤醒作用和觉醒维持作用的脑波特征及神经传导环路。预期结果将丰富嗅觉传递与睡眠觉醒调控理论及个体间信息交流方式。

警觉信息素（alarm pheromones, APs）是由个体释放并向同种其它个体传递危险和伤害信息的物质，对生活在食物链下游动物（如小鼠）的存活具有重要的意义。睡眠依据脑电、肌电等特征划分为非快动眼（non-rapid eye movement, NREM）和快动眼（rapid eye movement, REM）睡眠。小鼠生命1/2时间在睡眠中度过，已有的嗅觉“sensory gating”理论认为嗅觉在睡眠状态下对一般气味的感知减低，但需要躲避危险因素和天敌的捕食，APs能否唤醒睡眠中的小鼠尚且不知。本研究利用精准鼻腔气味投递睡眠记录、行为学评估、嗅觉亚感受器官损毁、嗅觉行为学评估、嗜神经病毒标记结合免疫荧光双标等技术阐明APs唤醒作用的环路基础，c-Fos功能标记验证APs唤醒作用的神经环路。重要成果与关键数据：（1）APs唤醒NREM睡眠期的小鼠，其主要成分SBT唤醒NREM睡眠期的作用强于APs。APs、SBT及小鼠天敌狐狸释放的TMT（trimethylthiazoline）均具有引发焦虑样行为的作用。SBT引起REM睡眠期小鼠的觉醒。（2）切断Grüeneberg神经节轴突（anotomy of axons of the Grüeneberg ganglion, GGAxo）不改变睡眠结构和主嗅觉功能。SBT可唤醒处于NREM和REM睡眠时期的GGAxo小鼠，但唤醒作用减弱，主要表现在投递结束后觉醒时间减少。犁鼻器切除（excision of the vomeronasal organ, VNOX）使睡眠结构片段化，觉醒时间增加。VNOX 不改变主嗅觉功能。SBT可唤醒处于NREM和REM睡眠时期的VNOX小鼠，但唤醒作用减弱，主要表现在投递期觉醒时间减少。（3）嗅球注射单纯疱疹病毒（herpes simplex virus, HSV）后双侧蓝斑（locus coeruleus, LC）可见HSV标记神经元大部分与络氨酸羟化酶（tyrosine hydroxylase, TH）共标记。鼻腔投递SBT与Air相比较，LC中活动神经元标记物c-Fos蛋白表达显著增多。提示APs可能经GG和VNO感知并激活LC去甲肾上腺素能神经元发挥唤醒作用。本项目成果丰富了嗅觉对睡眠调控的理论，为个体信息交流提供理论依据。亦可为农业生产中鼠害防控提供新策略。