黑质-纹状体DA系统对运动疲劳的调控及D2DR的干预作用研究

Exercise-induced fatigue is the main physiological limiting factor to improve sport performance. The central regulatory mechanism is closely related to the function of nigrostriatal DA system. At present, it has been suggested that the expression and distribution of D2DR not only regulating the transduction of postsynaptic signals, but also influencing on the regulation of presynaptic DA metabolism. However, it is not yet clear about the regulation of nigrostriatal DA systemin on exercise-induced fatigue. The model of exercise-induced fatigue was established by incremental repeated exhaustive exercise in this study. The nigrostriatal DA metabolism and regulated proteins will be determinated using microdialysis in vivo combined with HPLC, immunohistochemistry and immunoblotting techniques. The expression and distribution of striatal D2DR after exercise-induced fatigue were detected by laser scanning confocal microscopy, immunofluorescence and RT-PCR technology. In addition, the effects of antagonist and agonist on the changes in ethology and synchronized oscillatory activity of nigrostriatal neurons will be checked by Multi channel neural electrophysiological and behavioral investigation. The purpose of this study is to provide the theoretical support to the fatigue mechanism and its theory, and to provide new drug targets for the recovery of exercise-induced fatigue.

运动疲劳是限制运动成绩提高的主要生理因素，其中枢调控机制与黑质-纹状体DA系统功能改变密切相关。目前已知D2DR不仅调节突触后信号转导，而且对突触前DA代谢也有重要调节作用。但运动疲劳状态下，黑质-纹状体DA系统对运动疲劳的调控机制尚未明确。本研究拟通过递增负荷重复力竭运动建立疲劳模型，采用微透析HPLC联用、免疫组化/印迹技术，测定黑质-纹状体DA代谢及调节蛋白变化；采用LSCM、免疫荧光及RT-PCR技术，检测运动疲劳后纹状体D2DR表达量及空间分布；采用多通道神经电生理结合行为学方法，观察D2DR拮抗剂与激动剂介导的行为学改变及黑质-纹状体神经元同步振荡活动对皮层信息输出的影响。从分子-细胞-行为角度，阐述黑质-纹状体DA系统在运动疲劳中枢调控中的作用，为完善运动疲劳机制提供理论支持，并为运动疲劳恢复中枢干预提供新的药物靶点。

本项目利用在体微透析-HPLC、免疫组化/印迹、电刺激及微量注射药物干预、透射电子显微镜、在体多通道电生理记录等技术研究了递增负荷重复力竭运动疲劳对黑质-纹状体DA代谢（DA、DAT、VMAT2、DADR表达）、黑质-纹状体突触可塑性（DLS突触超微结构、PSD-95等蛋白）、D2DR拮抗剂Spiperone/激动剂 Pramipexole介导的行为学改变及皮层-纹状体神经核团间同步低频振荡的影响等内容。结果显示纹状体神经元参与运动疲劳中枢调控呈现区域特征，且在疲劳发生和积累的不同时段作用有所不同，这与纹状体背内侧(DMS)和背外侧(DLS)区分别接受不同类型投射神经元有关。运动疲劳后DLS突触间隙增宽，突触后致密物(PSD)厚度随着运动疲劳程度的加深而逐渐减小，影响突触传递效能，但支架蛋白PSD-95在运动疲劳后的改变与PSD的变化没有相关性，D2DR在七天重复力竭运动后表达显著下调，提示D2DR的调节作用可能与黑质-纹状体DA能通路的突触传递效能改变有关。运动疲劳后皮层M1区α(7-13Hz)及β频段(15-30Hz)放电升高，运动皮层处于过度激活状态；纹状体α及β频段变化趋势与皮层M1区一致，且皮层M1-纹状体之间α及β频段的相干系数增加，同步化程度增强；运动疲劳后皮层M1区、纹状体spike-LFP之间均出现明显的相位锁定关系，产生同步化，提示皮层-纹状体通路同步性增强是运动疲劳产生的可能机制之一。而D2DR药物干预会对大鼠运动行为能力产生显著影响：D2DR激动剂可以明显改善大鼠的运动能力，使力竭时间延长，且D2DR拮抗剂则降低了大鼠运动能力，力竭时间减少，并且D2DR激动剂可以使皮层M1区、纹状体α、β频段功率谱密度值降低，推测D2DR功能变化可能是造成低频振荡同步化影响疲劳后大鼠的运动行为能力的原因之一，提示D2DR可作为运动疲劳干预的药物靶点之一。