长期酒精摄入对小鼠小脑颗粒层场电位的影响及其机制

目的建立小鼠长期酒精注射中毒模型,探讨长期酒精摄入对小鼠小脑皮层苔藓纤维-颗粒细胞感觉信息突触传递的影响机制。方法20只6~8周龄的健康雄性ICR小鼠按照随机数字表法分为生理盐水组(对照组)和酒精摄入组(酒精组),每组10只。酒精摄入组每日腹腔注射浓度为20%的酒精,对照组则注射同等剂量的生理盐水,注射周期均为28 d。注射结束后,在小脑的CrusⅡ区清除头皮及肌肉组织,去除颅骨,剥离硬脑膜,利用气体喷射装置在同侧触须垫30 mm处给予吹风刺激,寻找最大反应部位后,在小鼠脑表面灌流NMDA受体阻断剂D-(-)-2-amino-5-phosphono-valerate,D-APV]、代谢性谷氨酸受体1阻断剂(JNJ16259685)及N-甲基-D-天冬氨酸(N-Methyl-D-aspartic acid,NMDA),每种药物灌流20 min,两种药物之间用人工脑脊液充分灌流直到波形恢复,期间通过膜片钳技术,记录小鼠小脑颗粒层电位波形的变化特点。采用Clampfit 10.3软件和SPSS 22.0软件对所有实验数据进行统计分析。结果给予吹风刺激后酒精组小鼠颗粒层场电位反应的潜伏期(11.8±0.7)ms较对照组(10.1±0.2)ms显著延长(t=-8.041,P<0.05),N1的振幅(1.2±0.1)mV明显小于对照组(0.6±0.1)mV(t=-12.728,P<0.05)。与对照组比较,酒精组小鼠颗粒层场电位反应抑制性成分P1波形上升时间(4.4±0.2)ms,(3.2±0.2)ms]、持续时间(12.1±0.5)ms,(10.3±0.2)ms]、消退时间(7.8±0.2)ms,(6.9±0.2)ms]、波形下面积(7.3±0.2)ms,(4.3±0.2)ms]均显著升高(t=16.100,-11.840,-11.673,-35.576,均P<0.05)。而两组小鼠刺激结束反应波形Roff波的振幅、波形半宽值、波形上升时间和衰减时间均差异无统计学意义(t=-1.909,-0.910,-0.789,1.462;均P>0.05)。当酒精组小鼠脑表面灌流JNJ16259685时,给予的5个吹风刺激诱发场电位的振幅较给药前均差异无统计学意义(P>0.05)。酒精组小鼠脑表面灌流D-APV后,吹风刺激诱发的场电位P1的振幅(42.3±1.5)mV]较给药前(101.1±0.9)mV]及洗脱后(100.1±2.2)mV]均显著降低(t=106.762,-69.605;均P<0.05),P1的波形下面积(42.6±1.3)%]较给药前(100.6±1.6)%]与洗脱后(97.6±2.2)%]也显著减小(t=88.862,-67.791;均P<0.05),且N2/N1比值(0.3±0.1)较给药前(0.4±0.1)与洗脱后(0.3±0.1)也明显降低(t=2.242,2.121;均P<0.05)。对照组小鼠脑表面灌流NMDA后,P1的振幅(110.7±3.2)mV]较给药前(100.1±0.9)mV]与洗脱后(102.0±1.7)mV]显著增加(t=-10.173,7.669,均P<0.05),P1的波形下面积(127.9±3.5)%]较给药前(100.0±3.1)%]与洗脱后(115.0±5.3)%]显著升高(t=-18.698,6.447,均P<0.05),而且N2/N1比值(0.5±0.1)较给药前(0.3±0.1)与洗脱后(0.3±0.1)也明显增高(t=-5.669,5.669,均P<0.05)。对照组小鼠脑表面灌流NMDA受体阻断剂D-APV后,面部吹风刺激诱发的场电位与给药前及洗脱后差异无统计学意义(P>0.05)。结论长期酒精摄入显著压制MF-GC兴奋性谷氨酸能突触传递,增强小脑皮层GABAA受体介导的抑制性反应,抑制性成分的增强依赖于NMDA受体,但不依赖于1型代谢性谷氨酸受体。

Effect of long-term alcohol intake on field potential of cerebellar granule layer in mice and its mechanism

Objective To investigate the effect of long-term alcohol intake on sensory information synaptic transmission of mossy fiber-granular cells in the cerebellar cortex of mice.Methods Twenty healthy male ICR mice aged 6 to 8 weeks were divided into normal saline group(control group)and alcohol intake group(alcohol group)according to random number table,with 10 mice in each group.The mice in alcohol group were injected intraperitoneally with 20%alcohol and the mice in control group were injected with the same amount of saline for 28 days.After the injection,the scalp,muscle tissue and skull were removed in turn,and the dura mater was removed to fully expose the crus II area of cerebellum.The mice were stimulated by air blowing at 30 mm of the ipsilateral tentacle pad with a gas jet device.When the the maximal response site was determined,the NMDA receptor antagonist(D-APV),metabolic glutamate receptor 1 antagonist(JNJ16259685)and N-methyl-D-aspartic acid(NMDA)were perfused on the brain surface of mice.Each drug was perfused for 20 minutes and ACSF was used between the two drugs until the waveform was recovered.Patch clamp amplifier was used to record the changes of potential waveform in mouse cerebellar granule layer.The data were analyzed by the softwares of Clampfit 10.3 and SPSS 22.0.Results After exposure to wind stimulation,the latency of field potential response in granular layer of mice in alcohol group(11.8±0.7)ms was significantly longer than that in the control group(10.1±0.2)ms(t=-8.041,P<0.05),and the amplitude of N1(1.2±0.1)MV was significantly lower than that in the control group(0.6±0.1)MV(t=-12.728,P<0.05).Compared with the control group,the rise time of P1 waveform((4.4±0.2)ms,(3.2±0.2)ms),duration((12.1±0.5)ms,(10.3±0.2)ms),extinction time((7.8±0.2)ms,(6.9±0.2)ms),volume under waveform((7.3±0.2)ms,(4.3±0.2)ms)were significantly increased in the alcohol group(t=16.100,-11.840,-11.673,-35.576,all P<0.05).There were no significant differences in the amplitude,half width,rise time and decay time of Roff wave between the two groups(t=-1.909,-0.910,-0.789,1.462,all P>0.05).When JNJ16259685 was perfused on the brain surface of mice in alcohol group,the amplitude of field potential evoked by five blowing stimuli had no significant difference compared with that before administration(all P>0.05).When D-APV was perfused into the brain surface of mice in the alcohol group,the amplitude of P1((42.3±1.5)Mv)was significantly lower than that before administration((101.1±0.9)mV)and after elution((100.1±2.2)mV)(t=106.762,-69.605,both P<0.05),and the area under waveform of P1((42.6±1.3)%)was also significantly lower than that before administration((100.6±1.6)%)and after elution((97.6±2.2)%)(t=88.862,-67.791,both P<0.05).The ratio of N2/N1(0.3±0.1)was significantly lower than that before administration(0.4±0.1)and after elution(0.3±0.1)(t=2.242,2.121,both P<0.05).When NMDA was perfused on the brain surface of mice in the control group,compared with before administration and after elution,the amplitude of P1((110.7±3.2)mV,(100.1±0.9)mV,(102.0±1.7)mV,t=-10.173,7.669,both P<0.05),the area under the waveform of P1((127.9±3.5)%,(100.0±3.1)%,(115.0±5.3)%,t=-18.698,6.447,both P<0.05),the ratio of N2/N1((0.5±0.1),(0.3±0.1),(0.3±0.1),t=-5.669,5.669,both P<0.05)were all significantly increased.When D-APV was perfused on the brain surface of mice in control group,the field potential evoked by blowing stimuli had no significant difference compared with that before administration and after elution(all P>0.05).Conclusion Long-term alcohol intake significantly suppresses the synaptic transmission of excitatory glutamate in MF-GC,and enhances the inhibitory response mediated by GABAA receptor in cerebellar cortex.The inhibitory component is enhanced by NMDA receptor,but not by type 1 metabolic glutamate receptor.