黑质orexin的电生理效应及其对帕金森病运动行为的调控作用

Substantia nigra occupies a very important position in the basal ganglia. Degeneration of dopaminergic neurons in substantia nigra leads to hypokinesia and other symptoms in Parkinson's disease. Orexins represent a recently characterized family of hypothalamic neuropeptides. Orexinergic system directly participates in central motor control. Morphological studies have demonstrated that the substantia nigra received a large number of intensive orexinergic fibers. Highest levels of orexin-1 and -2 receptor expression were observed in the substantia nigra. The patients with late-stage Parkinson's disease have a significant loss of the hypothalamic orexin-producing neurons, and the level of orexin was decreased in cerebrospinal fluid. Besides, orexin levels had a negative correlation with disease severity. Previous studies focused on the effects of orexin on sleep disturbance observed in parkinsonian patients. However, it is not clear its role in regulating motor activity under parkinsonian state. In the present study, in vivo extracellular recordings and in vitro patch clamp recordings will be employed to study the electrophysiological effects of orexin on gamma-aminobutyric acid (GABA)-ergic and dopaminergic neurons in the substantia nigra. Furthermore, immunohistochemistry will be used to observe the expression of orexin and orexin receptors in the substantia nigra under both normal and parkinsonian state. Finally, we will microinject orexin into the substantia nigra to observe its regulation on motor behavior in normal and parkinsonian rats. Our study will provide some theoretical and experimental basis for further investigations into the potential of orexinergic system of substantia nigra in the treatment of motor disorder related to basal ganglia.

黑质是基底神经节的重要核团之一，在机体运动功能调节中发挥重要作用。黑质致密带多巴胺能神经元退行性变可导致帕金森病运动减少等症状。Orexin是下丘脑神经肽家族成员，可直接参与调节中枢运动功能。形态学研究证实，黑质接受大量来自下丘脑的orexin能纤维支配，并表达高水平的orexin-1和orexin-2受体。帕金森病晚期患者下丘脑orexin能神经元缺失，脑脊液orexin水平降低，且orexin的水平与帕金森病严重程度呈负相关。以往人们主要关注orexin递质系统对帕金森病患者睡眠障碍的影响，而对其在帕金森病运动功能调节中的作用并不清楚。为此，本实验采用电生理记录、免疫组织化学、电化学、行为学等实验方法，系统探讨orexin对黑质GABA和多巴胺能神经元的电生理效应及其对帕金森病模型大鼠运动行为的调控作用。本研究将为进一步了解基底神经节运动障碍性疾病的发病及治疗提供一定的理论和实验依据。

黑质是基底神经节功能环路的重要核团之一，在机体运动功能调节中发挥重要作用。Orexin是下丘脑神经肽家族成员，可直接参与调节中枢运动功能。形态学研究证实，黑质接受大量来自下丘脑的orexin能纤维支配，并表达高水平的orexin 1和orexin 2（OX1和OX2）受体。帕金森病晚期患者下丘脑orexin能神经元缺失，脑脊液orexin水平降低，且orexin的水平与帕金森病严重程度呈负相关。本研究主要目的旨在探讨orexin-A和OX1受体阻断剂SB-334867对正常和6-羟基多巴胺（6-hydroxydopamine，6-OHDA）帕金森病模型大鼠黑质神经元电活动的调节，以及正常和帕金森病大鼠黑质OX1受体的表达。主要实验结果显示，1.正常大鼠黑质致密带微压力注射orexin-A可明显增加大多数多巴胺能神经元的自发放电频率。在正常大鼠黑质网状带，orexin-A也可明显增加大多数神经元的自发放电频率。2.为了观察内源性orexin-1受体是否参与调控神经元自发放电，黑质致密带微压力注射SB-334767可明显降低多数多巴胺能神经元的放电频率，且可降低部分黑质网状带的GABA能神经元的放电频率。该结果提示大鼠黑质内源性orexin系统参与神经元自发放电频露的调控。3.在6-OHDA帕金森病模型大鼠损毁侧，黑质致密带及网状带微压力注射orexin-A亦可显著增加多巴胺及GABA能神经元的自发放电频率。在正常和6-OHDA帕金森病模型大鼠，orexin-A对黑质致密带多巴胺能神经元的兴奋效应较黑质网状带明显增强。4.免疫组织化学染色显示，正常及帕金森病模型大鼠黑质均表达OX1受体。5. 进一步研究显示，orexin-A可明显增加大鼠苍白球神经元的自发放电频率，且其兴奋效应与神经元自发放电频率呈负相关。而内源性orexin系统对神经元自发放电频率发挥重要调节作用。orexin-A主要通过OX1受体发挥其对苍白球神经元的兴奋效应。6. 行为学实验观察到单侧苍白球微量注射orexin-A可使氟哌啶醇帕金森病僵直模型大鼠呈现明显的对侧偏转行为，在清醒大鼠证实orexin-A通过提高苍白球神经元兴奋性，从而发挥抗帕金森病效应。上述实验结果为基底神经节orexin在帕金森病发病和治疗中的作用提供了一定的理论和实验依据。