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谢玉波　刘敬臣　徐林　曾邦雄

530021 　广西医科大学附属第一医院麻醉科(谢玉波、刘敬臣)

The effects of propofol on excitatory synaptic transmission in hippocampal CA1 neurons of rats

XieYubo , Liu Jingchen , Xu Lin , et al . Depa rtment of Anest hesiology , the First Hospital Affiliated to <?xml:namespace prefix = st1 ns = "urn:schemas-microsoft-com:office:smarttags" />Guangxi Medical University , Nanning 530021 CHINA

【Abstract】　Objective：To investigate the effect s of 500μmol/ L propofol on the whole-cell excitatory post synaptic current ( EPSC) in hippocampal CA1 pyramidal neurons of rat s and to analyze the mechanisms of propofol deeply. Methods：Wistar rat s (13-19 d) were killed by cervical dislocation and hippocampal slices (400μm) were prepared. EPSCs were recorded by wholecell patchclamp technique. The experiments were divided into two groups : the int ralipid group ( n = 6) and the propofol group ( n = 10) . Af ter hippocampal slices were preincubated with 50μmol/ L picrotoxin for 30 min ,the base value was recorded for 10 min and then 450μl int ralipid or propofol (500μmol/ L) was applied inperfusion for another 40 min. The Schaffer collateral/ commissural pathway was stimulated with pairpulses (interpulse interval was 50 ms) and the EPSC2/ EPSC1 ratio was analyzed. Moreover ,the holding potential was changed f rom - 80 to + 60mV and the current2voltage curve was drawn. Results：Intralipid didn’t affect EPSC ,but 500μmol/ L propofol reduced EPSC to (67.5 ±1.7) ％ of the basevalue after application of propofol. 500μmol/ L propofol decreased the EPSC2/ EPSC1 ratio and shifted the currentvoltage curve to the left . Conclusion：500μmol/ L propofol inhibits excitatory synaptict ransmission in hippocampal CA1 pyramidal neurons of rats. The activity of GABAA receptor in the presynaptic and post synaptic membrane may be increased with propofol applied.

Key words:Excitatory post synaptic current,GABA,Receptor,Propofol,Hippocampus

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丙泊酚是临床常用的静脉麻醉药,它易化或直接激活GABAA 受体,从而增强抑制性突触传递功能而抑制兴奋性突触传递^[1,2]。但丙泊酚的这种作用是突触前抑制还是突触后抑制,抑或二者兼而有之,到现在还不清楚。本研究采用全细胞膜片钳技术,分析丙泊酚对大鼠海马CA1 区电刺激诱发的兴奋性突触后电流(excitatory post synaptic current ,EPSC) 的影响,从而进一步揭示丙泊酚的作用机制。

材料与方法

脑片制备　将20 只13～19 d 雄性Wistar 大鼠(体重40～60 g ,成都军区昆明总医院动物中心提供) 快速断头,用刀片切取出海马半脑。利用振动式切片机(Vibroslice ,英国) 切出400μm 厚度的海马脑片。将脑片转至孵育槽孵育,孵育槽中为以95％O₂ / 5％CO₂饱和的人工脑脊液。

电生理学记录　所有实验均在室温(18～22 ℃)进行。脑片移至记录槽中,通过出入水管道以95％O₂ /5％CO₂饱和的人工脑脊液持续灌注,流速为2 ml/ min左右,循环液容积为50 ml.

利用拉制仪( Puller287 , 美国) 将硅硼玻璃管(Borosilicate , 内径1.5 mm ,美国) 拉制成实验用记录电极, 电极电阻为3.5 ～ 5 MΩ。刺激隔离器( PSIU6 ,美国) 连接双极刺激电极刺激Schaeffer 侧支/ 联合纤维(刺激冲动为0.1 ms) 。

16 张脑片分为两组:10％脂肪乳剂组( n = 6) 和500μmol/ L 丙泊酚组( n = 10) 。两组均先以50μmol/ L 印防己毒素(picrotoxin) 预孵脑片30 min ,Schaeffer 侧支/ 联合纤维给予单刺激(刺激间隔为20 s) ,CA1 区锥体神经元记录基础EPSC 10 min ,然后加入450μl 10 %脂肪乳剂或丙泊酚(相当于500μmol/ L) ,继续记录EPSC 40 min ,并且进行如下实验: (1) 在Schaeffer 侧支/ 联合纤维连续给予配对刺激P1 、P2 共10 次, P1 、P2 间隔(interp ul se interval , IPI) 为50 ms ,记录配对刺激诱发的EPSC 的比率,即EPSC2/ EPSC1 ; (2) 给予单刺激,同时改变CA1 区锥体神经元细胞膜钳制电压( - 80～ + 60mV ,变化幅度为20 mV) ,制作电流2电压( I2V) 曲线,每个钳制电压下给予7 次刺激。除实验(2) 外,膜钳制电压均为- 70mV 。印防己毒素为GABAA受体非竞争性拮抗剂。

全细胞记录采用Axopatch 200B (美国) 膜片钳放大器,电刺激脉冲的给出及电信号的采集均通过计算机程序Clamp x 8.0 及digidata 1320A 接口来完成。

试剂与药品　实验液体配方(mmol/ L) : 人工脑脊液(NaCl 118 , KCl 4.75 , KH₂ PO₄ 1.19 ,NaH₂ CO₃ 25 ,MgSO₄ 1.19 ,Dglucose 11 ,CaCl₂ .2H₂O 2.94) , pH = 7.4 ; 电极内液( Kgluconate 170 ,HEPES 10 , NaCl 10 , MgCl₂ 2 , EGTA 0.2 ,Mg₂A TP 3.5 ,Na₂GTP 1.0) ,pH = 7.2 。

实验所用丙泊酚(10 mg/ ml) 购自Zeneca 公司(英国) ,10％脂肪乳剂购自乐山裕恒药业公司,其余所用药品及试剂均购自Sigma 公司(美国) 。

统计分析　采用Clamfit 8.0 、Origin 5.0 和Excel 2000 软件进行数据分析。EPSC 幅值做标准化处理,即数据以相对于每组基线均值的比值或百分率表示为均数或均数±标准差(_{<?xml:namespace prefix = v ns = "urn:schemas-microsoft-com:vml" />} ±s) 。组间及组内比较均采用ANOVA 单因素方差分析, P < 0.05为差异有显著意义。

丙泊酚对大鼠海马CA1 区EPSC 的影响如图1 所示,在膜钳制电压为- 70mV 条件下,脂肪乳剂对CA1 区锥体神经元细胞EPSC 值无影响,加入脂肪乳剂前为0.997 ±0.015 , 加入后为0.991 ±0.012 ,两者差异无显著意义( P > 0.05) 。500μmol/ L 丙泊酚降低大鼠海马CA1 区EPSC值,25～30 min 左右达最大抑制效果,加入丙泊酚前为1.000 ±0.021 , 加入丙泊酚后为0.675 ±0.017 ,两者差异有显著意义( P < 0.05) ;加药后与脂肪乳剂组比较,差异也有显著意义( P < 0.05) 。  

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与脂肪乳剂组比, *P < 0.05

图1 　丙泊酚对EPSC的影响

丙泊酚对大鼠海马CA1 区EPSC2/ EPSC1 比率的影响应用配对刺激代替单刺激( IPI 50 ms) ,则脂肪乳剂组EPSC2/ EPSC1 值为1.071 ±0.061 ,丙泊酚组为0.672 ±0.041 ,两组间差异有显著意义( P <0.05) (图2) 。

与脂肪乳剂组比, *P < 0105

图2 　丙泊酚对EPSC2/ EPSC1 比率的影响

丙泊酚对大鼠海马CA1 区I2V 曲线的影响改变膜钳制电压( - 80～ + 60 mV ) ,则发现脂肪乳剂组反转电位为+ 20 mV 左右,丙泊酚组为-35 mV 左右(图3) 。

以- 60 mV 的EPSC 值为- 1

图3 　丙泊酚对I-V曲线的影响

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