运动通过上调mGluR2/3表达抑制帕金森病模型大鼠纹状体中等多棘神经元异常电活动

Exercise Inhibits the Abnormal Electrical Activity of Striatal Medium Spiny Neurons in Parkinson’s Disease Model Rats by Up-regulating mGluR2/3 Expression

目的:探讨运动通过上调代谢型谷氨酸受体2/3(mGluR2/3)表达对帕金森病(PD)模型大鼠纹状体中等多棘神经元(MSNs)异常电活动的影响。方法:清洁级SD大鼠随机分为假手术安静组(Control组,n=9)和6-羟基多巴胺(6-OHDA)造模组(6-OHDA组,n=40)。6-OHDA造模组采用神经毒素6-OHDA注射于大鼠右脑内侧前脑束(MFB),建立偏侧损毁PD模型大鼠,假手术组于相同部位给予同等剂量的生理盐水作为对照组。采用阿扑吗啡(APO)诱导旋转行为测试评价PD模型的可靠性。经鉴定符合PD模型的大鼠随机分为6-OHDA安静组(PD组,n=9)、6-OHDA+运动组(PD+Ex组,n=9)和6-OHDA+运动+mGluR2/3拮抗剂组(PD+Ex+APICA组,n=9)。运动组于手术后1周开始进行跑台训练干预(11 m/min,30 min/day,5 d/week,4 weeks)。运动+mGluR2/3拮抗剂组每次运动前,采用微量注射泵将mGluR2/3拮抗剂APICA注射到纹状体内,注射体积为1μL。采用免疫组织化学染色技术检测黑质酪氨酸羟化酶(TH)免疫阳性细胞数量和纹状体TH免疫阳性纤维终末含量。采用免疫印迹技术检测纹状体mGluR2/3的表达水平。采用多通道电生理记录系统对各组大鼠清醒静止状态下纹状体神经元电活动进行记录。结果:APO诱导的旋转行为测试和TH免疫组织化学检测结果表明,PD大鼠模型可靠,成模率为67.5%。免疫印迹技术检测结果显示,与Control组相比,PD组纹状体mGluR2/3表达水平显著下调(P<0.01);与PD组相比,PD+Ex组纹状体mGluR2/3表达水平显著上调(P<0.05)。电生理分析结果表明,与Control组相比,PD、PD+Ex和PD+Ex+APICA组纹状体MSNs平均放电频率均显著增加(P<0.01);与PD组相比,PD+Ex组纹状体MSNs平均放电频率显著降低(P<0.05);与PD+Ex组相比,PD+Ex+APICA组纹状体MSNs平均放电频率显著增加(P<0.01)。与Control组相比,PD组纹状体神经元局部场电位(LFPs)在β频段(10~30 Hz)节律性振荡功率出现异常增加(P<0.01),PD+Ex组纹状体神经元LFPs在β频段(10~30 Hz)节律性振荡功率较PD组显著降低(P<0.05),PD+Ex+APICA组纹状体神经元LFPs在β频段(10~30 Hz)节律性振荡功率较PD+Ex组异常增加(P<0.01)。与Control组相比,PD组动作电位(spike)在β频段(10~30 Hz)诱发的LFP波形平均(STWA)值显著升高(P<0.01);与PD组相比,PD+Ex组STWA值显著降低(P<0.05);与PD+Ex组相比,PD+Ex+APICA组STWA值显著升高(P<0.01)。结论:PD模型大鼠纹状体MSNs兴奋性显著增加,LFPβ频段节律性振荡的功率显著增加,spike与LFPβ频段节律性振荡的同步化程度显著增加;运动干预可使PD模型大鼠纹状体MSNs兴奋性、LFPβ频段节律性振荡的功率以及spike与LFPβ频段节律性振荡的同步化程度均显著降低;纹状体微量注射GluR2/3拮抗剂可使运动的积极效应消失,进一步证实mGluR2/3在PD模型大鼠纹状体MSNs运动依赖可塑性中发挥了重要作用,并将成为PD治疗药物研发的新的靶向分子。

Objective To explore the effect of exercise on the inhibition of abnormal electrical activi⁃ty of medium spiny neurons(MSNs)in the striatum of Parkinson’s disease(PD)model rats by up-regu⁃lating the expression of metabotropic glutamate receptor 2/3(mGluR2/3).Methods Clean Sprague-Daw⁃ley rats were randomly divided into a sham operation quiet group(Control,n=9)and a 6-hydroxydopa⁃mine(6-OHDA)model group(6-OHDA,n=40).In the 6-OHDA model group,neurotoxin 6-OHDA was injected into the medial forebrain bundle(MFB)of the right brain to establish PD model rats with hemiplegic lesions,and the sham group was given the same dose of saline at the same site as the control group.Apomorphine(APO)induced rotational behavior test was used to evaluate the reliability of the PD model.Then rats successfully modeled were randomly assigned to a 6-OHDA quiet(PD,n=9),a 6-OHDA+exercise(PD+Ex,n=9)and a 6-OHDA+exercise+mGluR2/3 antagonist group(PD+Ex+APICA,n=9).One week after surgery,treadmill training intervention(11 m/min,30 min/day,5 days/week for 4 weeks)was initiated in the exercise group.Before each exercise session in the exercise+mGluR2/3 antagonist group,1-μL mGluR2/3 antagonist APICA was injected into the stria⁃tum using a microinjection pump.Immunohistochemical staining was used to detect the number of tyro⁃sine hydroxylase(TH)immunopositive cells in the substantia nigra and the terminal content of TH im⁃munopositive fibers in the striatum.Western blotting was employed to detect the expression level of mGluR2/3 in the striatum.A multichannel electrophysiological recording system was used to record the electrical activity of striatal neurons in each group of rats in conscious and quiescent conditions.Re⁃sults The results of APO-induced rotational behavior test and TH immunohisto-chemistry showed that the PD rat model was reliable,with a successful modeling rate of 67.5%.Western blotting showed that striatal mGluR2/3 expression levels down-regulated significantly in the PD group compared with the control group(P<0.01),and striatal mGluR2/3 expression levels up-regulated significantly in the PD+Ex group compared with the PD group(P<0.05).Electrophysiological analysis showed that,com⁃pared with the control group,the mean firing frequency of striatal MSNs increased significantly in the PD,PD+Ex and PD+Ex+APICA groups(P<0.01).Moreover,compared with group PD,the mean firing frequency of striatal MSNs in group PD+Ex decreased significantly(P<0.05)and Compared with the PD+Ex group,the value increased significantly in the PD+Ex+APICA group(P<0.01).Compared with the control group,the local field potential(LFPs)in striatal neurons showed an abnormal increase in rhythmic oscillatory power in theβfrequency band(10 to 30 Hz)in the PD group(P<0.01),while that of the PD+Ex group had a significant decrease compared with the PD group(P<0.05),and the same case when that of the PD+Ex+APICA group compared with the PD+Ex group(P<0.01).Compared to the control group,the spike-triggered LFPs wave-forms average(STWA)in the beta band(10 to 30 Hz)increased significantly in the PD group(P<0.01),while that of the PD+Ex group decreased signifi⁃cantly compared with the PD group(P<0.05)and that of the PD+Ex+APICA group increased significant⁃ly compared with the PD+Ex group(P<0.01).Conclusion The significant increase in the excitability of Striatal MSNs,the power of rhythmic oscillations in the LFPβfrequency band and the synchroniza⁃tion degree of spike and LFPβband rhythmic oscillations of PD rats,can be significantly reduced by exercise intervention.However,microinjection of mGluR2/3 antagonist into striatum can eliminate the positive effect of exercise,which further confirms that mGluR2/3 plays an important role in the mo⁃tion-dependent plasticity of striatal MSNs in PD model rats,and will become a new target molecule for PD drug development.