光遗传操控D2-MSNs揭示跑台训练改善帕金森病小鼠运动行为障碍的可能机制  

Optogenetic Manipulation for D-MSNs Reveals Possible Mechanisms of Improving Motor Behavior Disorder of PD Mice through Treadmill Training

作　　者：刘晓莉[1] 张丹昱 赵刚 乔德才[1] Liu Xiaoli;Zhang Danyu;Zhao Gang;Qiao Decai(College of Physical Education and Sports,Beijing Normal University,Beijing 100875,China)

机构地区：[1]北京师范大学体育与运动学院,北京100875

出　　处：《中国运动医学杂志》2020年第9期711-718,共8页Chinese Journal of Sports Medicine

基　　金：国家自然科学基金(31571221)。

摘　　要：目的:采用光遗传学技术揭示纹状体表达多巴胺Ⅱ型受体的中等多棘神经元(D2-MSNs)在改善帕金森病(PD)小鼠运动行为障碍中的作用。方法:雄性D2-Cre转基因小鼠,随机分成假手术安静组(Control组)、帕金森病组(PD组)、帕金森病运动组(PD+Ex组)和帕金森病光刺激组(PD+Laser组),最终每组样本量为6只。PD+Laser组小鼠纹状体注射包装抑制性光敏感蛋白病毒(rAAV-Ef1α-DIO-eNpHR3.0-EYFP-WPRE-pA),并采用全细胞膜片钳技术记录纹状体D2-MSNs对注入电流的反应,在激光共聚焦显微镜下鉴定病毒转染情况。采用右侧纹状体两点注射6羟基多巴胺(6-OHDA)药物建立偏侧PD小鼠模型,采用阿扑吗啡(APO)诱导旋转试验和纹状体酪氨酸羟化酶(TH)表达评价PD模型可靠性。PD+Ex组小鼠跑台运动干预方案为18 m/min,40 min/d,5 d/w,4 w。采用旷场实验通过运动总距离、平均运动速度和活动模式占比等参数评价各组小鼠自主活动能力,旷场实验中PD+Laser组光刺激方案为波长589 nm、10 mW,30 s光照、30 s关闭、30 s间隔的循环,重复7组,共10 min。结果:模型组小鼠APO旋转试验净旋转圈数>120 r/30 min,6-OHDA损毁侧纹状体TH阳性纤维大量丢失;光刺激前后D2-MSNs对注入电流反应不同,纹状体上光敏感蛋白eNpHR3.0在D2-MSNs表达,证实PD小鼠模型可靠及病毒转染成功。PD组和PD+Ex组总运动距离及平均运动速度较Control组显著降低(P<0.05),PD+Ex组和PD+Laser组总运动距离及平均运动速度较PD组显著增加(P<0.05),且PD+Ex组和PD+Laser组总运动距离、平均速度及活动模式占比等结果基本一致。结论:PD小鼠出现明显自主活动障碍与纹状体DA水平减少,引起D2-MSNs兴奋性改变有关。4周跑台运动干预有效改善了PD模型小鼠自主活动障碍,这一结果与光遗传操控D2-MSNs兴奋性对PD小鼠自主活动行为的影响基本相似。提示纹状体D2-MSNs可能是运动改善PD小鼠运动行为障碍的重�Objective To reveal the role of medium spiny neurons expressing dopamine type Ⅱ receptor(D2-MSNs) of the striatum in improving motor behavior disorder of Parkinson’s disease(PD)mice using optogenetics. Methods Male D2-Cre transgenic mice were randomly divided into a sham-operated quiet group(Control,n=6),a 6-OHDA quiet group(PD,n=6),a 6-OHDA exercise group(PD+Ex,n=6),and a 6-OHDA laser stimulation group(PD+Laser,n=6). PD+Laser group was injected the packaging inhibitory light-sensitive protein virus(rAAV-Ef1α-DIO-eNpHR3.0-EYFP-WPRE-pA),and the response of striatal D2-MSNs to the injection current was recorded using the whole-cell patch clamp technique, with the virus transfection identified under the laser scanning confocal microscope.The unilateral PD mouse model was established by injecting 6-OHDA into the right striatum and evaluated using the apomorphine(APO)-induced rotation test and striatum tyrosine hydroxylase(TH) expression. The PD+Ex group underwent 40-minute treadmill running at the speed of 18 m/min 5 days a week for 4 weeks. In the open field test,the total movement distance,average movement speed and activity mode ratio were used to evaluate the autonomous activity of the mice. The PD+Laser group was given laser stimulation at a wavelength of 589 nm and 10 mW 30 s on,30 s off,and 30 s interval as a group,repeated 7 times for 10 min. Results The net rotation circle of APO rotation test of 6-OHDA injection group was >120 r/30 min,and 6-OHDA damaged lateral striatum TH-positive fibers were largely lost.D2-MSNs reacted differently to the injection current before and after the laser stimulation. Light sensitive protein eNpHR3.0 in the striatum was expressed in D2-MSNs,which confirmed that viruses had transfected successfully. The average total movement distance and movement speed of PD and PD+Ex groups were significantly lower than the Control group(P<0.05),while those of PD+Ex and PD+Laser groups were significantly higher than the PD group(P<0.05). Moreover the total movement distance,average speed

关 键 词：运动 光遗传 D2-Cre转基因小鼠 纹状体中等多棘神经元Ⅱ亚型 帕金森病 自主活动能力 

分 类 号：R742.5[医药卫生—神经病学与精神病学]