LY354740对青春期小鼠慢性氯胺酮暴露后焦虑样行为与海马区GABA及GAD67的影响

Effect of LY354740 on GABA and GAD67 in hippocampus and anxiety-like behavior of juvenile mice exposed to ketamine

目的:探讨II组代谢型谷氨酸受体激动剂LY354740对小鼠慢性氯胺酮暴露后焦虑样行为及海马区GABA及GAD67的影响.方法:SPF级健康雌性C57BL/6小鼠45只随机分成3组(n=15):对照组(C组),氯胺酮组(K组),LY354740组(L组).K组和L组腹腔注射质量分数为30 mg/kg的氯胺酮,间隔30 min注射1次,3次/d,连续14 d;L组在注射氯胺酮前30 min注射质量分数为10 mg/kg的LY354740;C组注射等量生理盐水.给药结束后行高架十字迷宫实验测定小鼠运动功能和情绪状态;免疫组化实验、Western Blot实验检测小鼠海马GAD67表达;酶联免疫吸附实验检测小鼠海马GABA浓度.结果:(1)酶联免疫吸附实验结果显示,与C组比较,K组小鼠海马GABA浓度降低(P<0.05);L组小鼠海马GABA浓度较K组升高(P<0.05).(2)高架十字迷宫结果显示,与C组比较,K组小鼠进入各臂端活动总频次减少、开放臂停留时间比降低(P<0.05);与K组比较,L组小鼠活动总频次增加、开放臂停留时间比升高(P<0.05).(3)免疫组化结果显示,与C组比较,K组小鼠海马CA3、DG区GAD67阳性染色平均光密度降低(P<0.05),CA1区平均光密度无明显统计学差异;L组小鼠海马CA3、DG区GAD67阳性染色平均光密度较K组升高(P<0.05).(4)Western Blot结果显示,与C组比较,K组小鼠海马GAD67表达降低(P<0.05);L组小鼠海马GAD67表达较K组增多(P<0.05).结论:II组代谢型谷氨酸受体激动剂LY354740改善小鼠慢性氯胺酮暴露后的焦虑样行为,可能与其增加海马GAD67表达、提高GABA浓度有关.

Objective:To investigate the effect of group II metabotropic glutamate receptor agonist LY354740 on GABA and GAD67 in hippocampus and anxiety-like behavior of juvenile C57BL/6 mice exposed to ketamine.Methods:Forty-five healthy female C57BL/6 mice were randomly divided into 3 groups(n=15),control group(group C),ketamine group(group K),and LY354740 group(group L).In group K and L,ketamine(30 mg/kg)was injected intraperitoneally three times a day at 30-minute interval for 14 consecutive days.In group L,mice were pretreated with LY354740(10 mg/kg)30 minutes before ketamine injection.In group C,same volume of normal saline was injected.After administration,the motor function and emotional state of mice were measured by elevated plus maze(EPM)test.The expression of GAD67 in hippocampus of mice was detected by immunohistochemistry and Western Blot assay.The concentration of GABA in hippocampus of mice was detected by ELISA.Results:The results of ELISA suggested significantly lower GABA content in hippocampus of mice of group K when compared with both group C and L(P<0.05).The results of EPM test indicated that the total number of entry in all of the arms and the proportion of staying on open arm was significantly reduced in group K when compared with the other two groups(P<0.05).The results of immunohistochemistry assay demonstrated that there was an apparent decrease of average optical density of positive staining of GAD67 in CA3 and DG region of hippocampus in mice of group K when compared with the other two groups(P<0.05).The results of Western Blot showed a down-regulated expression of GAD67 in hippocampus of mice of group K when compared with the other two groups(P<0.05).Conclusion:LY354740 improved the anxiety-like behavior of mice exposed to ketamine,which may be related to the increase of GAD67 and GABA in hippocampus of mice.