目的:观察电针对1-甲基-4-苯基-1,2,3,6-四氢吡啶(1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine,MPTP)诱导的帕金森病(Parkinson's disease,PD)模型小鼠纹状体抑制性突触前标志物囊泡γ-氨基丁酸转运蛋白(vesicular GABA transpor...目的:观察电针对1-甲基-4-苯基-1,2,3,6-四氢吡啶(1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine,MPTP)诱导的帕金森病(Parkinson's disease,PD)模型小鼠纹状体抑制性突触前标志物囊泡γ-氨基丁酸转运蛋白(vesicular GABA transporter,vGAT)、突触后标志物桥尾蛋白(Gephyrin)以及树突棘密度和无翅型MMTV整合位点家族成员5a(wingless-type MMTV integration site family member 5a,Wnt5a)表达的影响,探讨电针改善PD运动功能的部分作用机制。方法:将雄性C57BL/6小鼠随机分为空白组、模型组、电针组、左旋多巴组,每组10只;除空白组外,MPTP腹腔注射复刻PD小鼠模型。造模成功后第一天对电针组小鼠开始针刺“合谷”“太冲”穴,同日左旋多巴组小鼠腹腔注射左旋多巴注射液,各组小鼠干预2个疗程后采用爬杆及悬挂实验评估各组小鼠运动功能;苏木素伊红染色观察纹状体神经元细胞形态;免疫荧光标记纹状体抑制性突触前标志物vGAT和突触后标志物Gephyrin表达;Western Blot检测纹状体Wnt5a蛋白的表达。结果:与模型组相比,电针组及左旋多巴组小鼠爬杆耗时缩短(P<0.05),悬挂评分升高(P<0.05)。模型组小鼠神经元细胞稀疏,残留的多巴胺神经元萎缩,胞核皱缩偏移,水肿空泡变性增多;而电针组及左旋多巴组小鼠神经元细胞形态圆润,水肿变性细胞减少,形态清晰。各组小鼠纹状体抑制性突触前标志物vGAT表达无明显差异(P>0.05)。与模型组相比,纹状体神经元树突棘密度增加(P<0.05),电针组及左旋多巴组小鼠纹状体突触后标志物Gephyrin的阳性表达升高(P<0.05),Wnt5a蛋白表达水平升高(P<0.05)。结论:早期对帕金森病模型小鼠进行电针干预可改善其运动功能障碍,其治疗机制可能是电针激活Wnt5a信号进而调控抑制性突触,改善纹状体GABA能神经元功能。展开更多
OBJECTIVE Investigate the effects of diazepam(DZP) on γ2 subunit containing GABA type A receptor(GABA A R) trafficking.METHODS Immunofluorescence microscopy measured surface GABA A Rs and gephyrin in rat cortical neu...OBJECTIVE Investigate the effects of diazepam(DZP) on γ2 subunit containing GABA type A receptor(GABA A R) trafficking.METHODS Immunofluorescence microscopy measured surface GABA A Rs and gephyrin in rat cortical neurons after 24 h exposure of 1.0 μmol·L^(-1) DZP.Biochemical studies of mice injected with 10 mg·kg^(-1) DZP vs vehicle were assessed for γ2 subunit and total gephyrin cortical levels 12 h post-injection.Ubiquitination of the γ2 subunit was studied by immunoprecipitation after 12 h of 1.0 μmol·L^(-1) DZP exposure.A γ2 subunit encoding an N terminal fluorogen-activating peptide and pH-sensitive green fluorescent protein(γ2 pH FAP) measured lysosomal targeting of γ2 containing GABA A Rs.RFP-gephyrin and γ2 pH FAP synaptic diffusion rates were examined using fluorescence recovery after photobleaching(FRAP).RESULTS Extrasynaptic levels of γ2 GABA A Rs decreased by 12.2%,while synaptic gephyrin S270 phosphorylation increased by 18.3% in DZP-treated neurons after 24 h compared to control(P<0.05).Dendritic levels of gephyrin were also reduced to 74.1% of control,while S270 phosphorylation was elevated by 25.2%(P<0.05;P<0.01).Mice 12 h post-DZP injection demonstrated a 12.7% and 26.1% decrease in total γ2 and gephyrin levels,respectively(P<0.05;P<0.01).12 h DZP treatment enhanced γ2 subunit ubiquitination 1.13-fold relative to control(P<0.05).Internalized γ2 pH FAP GABA A Rs associated with lysosomes was 8.0% higher in neurons treated with 12-16 h DZP compared to control.Pilot FRAP experiments suggest gephyrin and γ2 have increased mobility and turnover at synapses following DZP.CONCLUSION DZP treatment decreases γ2 GABA A R levels and gephyrin scaffolding function after one day of exposure,which may contribute to the formation of DZP tolerance.展开更多
目的探讨桥尾蛋白(gephyrin)丝氨酸S270位点磷酸化在水杨酸钠(sodium salicylate,SS)介导的GABA_(A)受体(GABA type A receptors,GABA_(A)Rs)内化的作用。方法将12只SD大鼠分成4组,分别为SS处理组、LiCl处理组、SS+LiCl处理组和对照组,...目的探讨桥尾蛋白(gephyrin)丝氨酸S270位点磷酸化在水杨酸钠(sodium salicylate,SS)介导的GABA_(A)受体(GABA type A receptors,GABA_(A)Rs)内化的作用。方法将12只SD大鼠分成4组,分别为SS处理组、LiCl处理组、SS+LiCl处理组和对照组,每组3只,急性分离各组耳蜗螺旋神经节神经元(spiral ganglion neurons,SGNs),SGNs原代培养48 h后分别用5 mM SS、1 mM LiCl、5 mM SS联合1 mM LiCl处理1 h,对照组不予处理;采用实时荧光定量PCR(RT-qPCR)检测GABA_(A)Rsα2亚基、桥尾蛋白基因表达的改变;采用Western blot技术检测各组GABA_(A)Rsα2膜蛋白和总蛋白表达的改变、桥尾蛋白及其S270位点磷酸化的改变。结果RT-qPCR结果显示,各组GABA_(A)Rsα2亚基mRNA、桥尾蛋白mRNA与对照组比较均无明显变化(P>0.05),Western blot结果显示,SS处理组GABA_(A)Rsα2亚基膜蛋白(0.08±0.02)较对照组(0.18±0.04)表达显著下调(P<0.01),LiCl处理组(0.14±0.03)有效逆转SS诱导的GABA_(A)Rsα2亚基膜蛋白下调(SS组为0.08±0.02)(P<0.05),LiCL组膜蛋白、各组总蛋白与对照组比较无明显变化(P>0.05);SS处理组桥尾蛋白S270磷酸化(0.84±0.02)较对照组(0.35±0.14)显著增强(P<0.001)。LiCl处理组(0.66±0.10)有效逆转SS诱导的桥尾蛋白S270磷酸化增强(SS组为0.84±0.02,P<0.05),LiCl处理组桥尾蛋白磷酸化、各处理组桥尾蛋白与对照组比较无明显变化。结论桥尾蛋白S270位点磷酸化可增强参与SS诱导的SGNs GABA_(A)Rs内化。展开更多
文摘目的:观察电针对1-甲基-4-苯基-1,2,3,6-四氢吡啶(1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine,MPTP)诱导的帕金森病(Parkinson's disease,PD)模型小鼠纹状体抑制性突触前标志物囊泡γ-氨基丁酸转运蛋白(vesicular GABA transporter,vGAT)、突触后标志物桥尾蛋白(Gephyrin)以及树突棘密度和无翅型MMTV整合位点家族成员5a(wingless-type MMTV integration site family member 5a,Wnt5a)表达的影响,探讨电针改善PD运动功能的部分作用机制。方法:将雄性C57BL/6小鼠随机分为空白组、模型组、电针组、左旋多巴组,每组10只;除空白组外,MPTP腹腔注射复刻PD小鼠模型。造模成功后第一天对电针组小鼠开始针刺“合谷”“太冲”穴,同日左旋多巴组小鼠腹腔注射左旋多巴注射液,各组小鼠干预2个疗程后采用爬杆及悬挂实验评估各组小鼠运动功能;苏木素伊红染色观察纹状体神经元细胞形态;免疫荧光标记纹状体抑制性突触前标志物vGAT和突触后标志物Gephyrin表达;Western Blot检测纹状体Wnt5a蛋白的表达。结果:与模型组相比,电针组及左旋多巴组小鼠爬杆耗时缩短(P<0.05),悬挂评分升高(P<0.05)。模型组小鼠神经元细胞稀疏,残留的多巴胺神经元萎缩,胞核皱缩偏移,水肿空泡变性增多;而电针组及左旋多巴组小鼠神经元细胞形态圆润,水肿变性细胞减少,形态清晰。各组小鼠纹状体抑制性突触前标志物vGAT表达无明显差异(P>0.05)。与模型组相比,纹状体神经元树突棘密度增加(P<0.05),电针组及左旋多巴组小鼠纹状体突触后标志物Gephyrin的阳性表达升高(P<0.05),Wnt5a蛋白表达水平升高(P<0.05)。结论:早期对帕金森病模型小鼠进行电针干预可改善其运动功能障碍,其治疗机制可能是电针激活Wnt5a信号进而调控抑制性突触,改善纹状体GABA能神经元功能。
基金supported by National Institute of Health(T32GM008424)Whitehall Foundation+1 种基金William C DEGROAT Neuropharmacology Departmental FellowshipPharmacology and Chemical Biology Startup Funds
文摘OBJECTIVE Investigate the effects of diazepam(DZP) on γ2 subunit containing GABA type A receptor(GABA A R) trafficking.METHODS Immunofluorescence microscopy measured surface GABA A Rs and gephyrin in rat cortical neurons after 24 h exposure of 1.0 μmol·L^(-1) DZP.Biochemical studies of mice injected with 10 mg·kg^(-1) DZP vs vehicle were assessed for γ2 subunit and total gephyrin cortical levels 12 h post-injection.Ubiquitination of the γ2 subunit was studied by immunoprecipitation after 12 h of 1.0 μmol·L^(-1) DZP exposure.A γ2 subunit encoding an N terminal fluorogen-activating peptide and pH-sensitive green fluorescent protein(γ2 pH FAP) measured lysosomal targeting of γ2 containing GABA A Rs.RFP-gephyrin and γ2 pH FAP synaptic diffusion rates were examined using fluorescence recovery after photobleaching(FRAP).RESULTS Extrasynaptic levels of γ2 GABA A Rs decreased by 12.2%,while synaptic gephyrin S270 phosphorylation increased by 18.3% in DZP-treated neurons after 24 h compared to control(P<0.05).Dendritic levels of gephyrin were also reduced to 74.1% of control,while S270 phosphorylation was elevated by 25.2%(P<0.05;P<0.01).Mice 12 h post-DZP injection demonstrated a 12.7% and 26.1% decrease in total γ2 and gephyrin levels,respectively(P<0.05;P<0.01).12 h DZP treatment enhanced γ2 subunit ubiquitination 1.13-fold relative to control(P<0.05).Internalized γ2 pH FAP GABA A Rs associated with lysosomes was 8.0% higher in neurons treated with 12-16 h DZP compared to control.Pilot FRAP experiments suggest gephyrin and γ2 have increased mobility and turnover at synapses following DZP.CONCLUSION DZP treatment decreases γ2 GABA A R levels and gephyrin scaffolding function after one day of exposure,which may contribute to the formation of DZP tolerance.
文摘目的探讨桥尾蛋白(gephyrin)丝氨酸S270位点磷酸化在水杨酸钠(sodium salicylate,SS)介导的GABA_(A)受体(GABA type A receptors,GABA_(A)Rs)内化的作用。方法将12只SD大鼠分成4组,分别为SS处理组、LiCl处理组、SS+LiCl处理组和对照组,每组3只,急性分离各组耳蜗螺旋神经节神经元(spiral ganglion neurons,SGNs),SGNs原代培养48 h后分别用5 mM SS、1 mM LiCl、5 mM SS联合1 mM LiCl处理1 h,对照组不予处理;采用实时荧光定量PCR(RT-qPCR)检测GABA_(A)Rsα2亚基、桥尾蛋白基因表达的改变;采用Western blot技术检测各组GABA_(A)Rsα2膜蛋白和总蛋白表达的改变、桥尾蛋白及其S270位点磷酸化的改变。结果RT-qPCR结果显示,各组GABA_(A)Rsα2亚基mRNA、桥尾蛋白mRNA与对照组比较均无明显变化(P>0.05),Western blot结果显示,SS处理组GABA_(A)Rsα2亚基膜蛋白(0.08±0.02)较对照组(0.18±0.04)表达显著下调(P<0.01),LiCl处理组(0.14±0.03)有效逆转SS诱导的GABA_(A)Rsα2亚基膜蛋白下调(SS组为0.08±0.02)(P<0.05),LiCL组膜蛋白、各组总蛋白与对照组比较无明显变化(P>0.05);SS处理组桥尾蛋白S270磷酸化(0.84±0.02)较对照组(0.35±0.14)显著增强(P<0.001)。LiCl处理组(0.66±0.10)有效逆转SS诱导的桥尾蛋白S270磷酸化增强(SS组为0.84±0.02,P<0.05),LiCl处理组桥尾蛋白磷酸化、各处理组桥尾蛋白与对照组比较无明显变化。结论桥尾蛋白S270位点磷酸化可增强参与SS诱导的SGNs GABA_(A)Rs内化。
