杏仁体-下丘脑食欲素/黑色素浓集激素神经元通路介导癫痫大鼠觉醒-睡眠调节紊乱

Epileptiform seizures could interfere with sleep structure disorder and cause low sleep quality, the related mechanism is still not be clarified. The hypothalamic orexin (ORX) neurons and melanin-concentrating hormone (MCH) neurons systems have already been confirmed playing important roles in regulation the wakefulness-sleep. Our previous study revealed that there are many neuronal axonal fibers projected from the amygdala to the hypothalamic region, where the ORX neurons and MCH neurons mainly distributed, and the synaptic connections were formed between amygdaloid axonal terminals and MCH neurons. In present study, we want to check the involvement of amygdala-hypothalamic ORX/MCH neurons pathway in the wakefulness-sleep disorder in rat epilepsy models. The epilepsy models in rat were established by electric stimulation in the rats' basolateral amygdaloid nuclus. With a combination of the neuronal axonal tracing technique, the immunohistochemical method and the in situ hybridization, the morphological and functional change of ORX neurons, MCH neurons and amygdaloid neurons in the different nuclei, will be evaluated after the epileptiform seizures. In the hypothalamus, the distribution pattern of neuronal axonal fibers arising from the different amygdaloid nuclei will be checked; the spatial distributions and synaptic connections between those axonal fibers and ORX/MCH neurons will be examined. Further, the type of neural transmitter of the amygdaloid neurons projecting to the hypothalamic ORX/MCH regions will be assayed. Finally, the amygdala-hypothalamic ORX/MCH neurons pathway regulating wakefulness-sleep in epilepsy models will be confirmed by evaluated the sleep character change after the amygdaloid nuclei lesions or the functional inhibition of ORX/MCH neurons. In our study, the role of the amygdala-hypothalamic ORX/MCH neurons pathway in the wakfulness-sleep disorder in epilepsy will be further clarified. These findings would provide a proof for the clinic treatment of the sleep disorder in epilepsy patients.

癫痫发作导致睡眠结构紊乱及睡眠质量下降，其机制尚不明确。下丘脑食欲素(ORX)神经元和黑色素浓集激素(MCH)神经元对觉醒-睡眠调节有重要作用。我们前期的实验证实杏仁体大量神经元投射到下丘脑ORX/MCH神经元调节其功能。本项目利用杏仁体电点燃癫痫大鼠模型，联合运用神经轴突示踪技术、免疫组织化学法以及原位杂交技术，首先研究癫痫发作导致的杏仁体内不同核团神经元以及下丘脑ORX和MCH神经元的形态和功能变化；进一步通过观察杏仁体不同核团神经纤维轴突在下丘脑的分布特点及其与ORX/MCH神经元的突触联系，以及投射到下丘脑的杏仁体神经元的神经递质类型，解析杏仁体-下丘脑ORX/MCH神经元通路的结构和功能属性在癫痫状态下的改变；最后结合损毁杏仁体核团或抑制ORX/MCH功能的实验，阐明杏仁体-下丘脑ORX/MCH神经元通路介导癫痫大鼠觉醒-睡眠调节紊乱的机制，为临床癫痫患者睡眠障碍的干预提供方向。

癫痫发作导致睡眠结构紊乱及睡眠质量下降，其机制尚不明确。下丘脑食欲素(ORX)神经元和黑色素浓集激素(MCH)神经元对觉醒-睡眠调节有重要作用。.本项目利用癫痫大鼠模型，联合运用神经轴突示踪技术、免疫组织化学法以及原位杂交技术，对癫痫发作导致的杏仁体内不同核团神经元以及下丘脑 ORX 和 MCH 神经元的形态和功能变化，杏仁体不同核团神经纤维轴突在下丘脑的分布特点及其与ORX/MCH神经元的突触联系，以及投射到下丘脑的杏仁体神经元的神经递质类型进行了研究，从而解析杏仁体-下丘脑 ORX/MCH神经元通路的结构和功能属性在癫痫状态下的改变。.实验结果显示，癫痫模型大鼠觉醒次数增加，觉醒时间增加，睡眠时间减少。大鼠癫痫发作2 h后，其杏仁体中心核（Ce）、内侧核（Me）、基底外侧核（BL）等亚核团以及下丘脑穹窿周区 Fos 表达量显著增加。其中，癫痫大鼠Orexin神经元表达FOS的数量增多，MCH神经元表达FOS的数量下降。CTb 注射到下丘脑PeF 、PLH等不同亚核团后，杏仁体内 Ce、Me、BMA、BL 及 ACo 内可见大量 CTb 逆行标记的神经元细胞。但在不同的核团，CTb标记神经元表现出不同的属性：在中心核，几乎所有CTb标记神经元都为GAD67 mRNA阳性；内侧核中的CTb神经元包含VGLUT2 mRNA和包含GAD67 mRNA的数量相当；在基底内侧核内， CTb标记的神经元胞体大都表达VGLUT2 mRNA。BDA注射到杏仁体BLA和Ce不同亚核团后，下丘脑内BDA标记的神经纤维在穹窿上区与 orexin 神经元的胞体均形成明显的相互重叠分布，表达 Fos 的 orexin 神经元的胞体或树突表面多见BDA标记的神经轴突终末贴附。BDA注射到杏仁体Me 后，下丘脑内BDA标记的神经纤维主要分布于VMH，与MCH神经元重叠分布但与orexin神经元重叠分布较少。杏仁体BMA神经元投射到下丘脑MCH神经元分布区域，并与MCH神经元之间形成突触联系，构成杏仁体联系并支配下丘脑功能的重要结构基础。综合以上结果，说明杏仁体-下丘脑ORX/MCH神经通路在癫痫引起觉醒-睡眠紊乱中发挥了重要作用，为临床癫痫患者睡眠障碍的干预提供了方向。