Effects of extra and intracellular pH on the toxicity of excitatory amino acid to CNS neurons

(1)为了研究酸中毒对缺血性脑急性神经元损伤的影响，我们检测了缺氧（体外缺血）海马片细胞内游离Ca^<2+>浓度（[Ca^<2+>] _i）和电活动的变化。Wistar大鼠海马切片载fura-2，用Krebs溶液在36-37℃下进行复配。荧光测量估计了[Ca^<2+>] _i的变化。记录CA1锥体细胞的场电位和膜电位。在正常pH （pH 7.4）溶液中，[Ca^<2+>] _i在体外缺血后6-7 min呈显著性快速升高，膜电位快速去极化。体外缺血10分钟后，脑场电位未恢复。在酸性溶液（pH6.8）中，[Ca^<2+>] _i的快速增加和快速去极化都被明显地延缓和减慢。体外缺血后的场电位在9片中有7片重现。结果提示，酸中毒可保护CA1神经元的功能免受缺血性损伤。(2)采用大鼠海马切片，观察NMDA对神经元活性和细胞内Ca^<2+>的影响，以及铵对NMDA诱导的神经元活性变化的影响。应用NMDA （50muM,30sec）诱导CA1区[Ca^<2+>] _i升高。NMDA诱导的[Ca^<2+>] _i增加在5 mM NH_4Cl存在下延长的持续时间。NMDA （20muM,10sec）可使CA1神经元的膜电位发生去极化，且NMDA诱导的膜去极化在NH_4Cl的存在下被增强和延长。NH_4Cl对Schaffer侧枝刺激引起的e.p.s.p.p和i.p.s.p.p均有抑制作用。这些结果表明，铵通过增强nmda诱导的神经元兴奋而加重缺血性神经元损伤。

(1) To investigate the effect of acidosis on acute neuronal damage in ischemic brain, we examined changes in intracellular free Ca^<2+> concentration ([Ca^<2+>] _i) and electrical activities in hippocampal slices exposed to oxygen-glucose deprivations (ischemia in vitro) . Hippocampal slices from Wistar rats were loaded with fura-2 and superfused with Krebs solution at 36-37ﾟC.Changes in [Ca^<2+>] _i were estimated by fluorescence measurements. Field potentials and membrane potential were recorded from the CA1 pyramidal cells. In normal pH (pH 7.4) solution, a characteristic rapid increase in [Ca^<2+>] _i and a rapid depolarization of membrane potential were observed at 6-7 min in response to ischemia in vitro. Field potentials were not recovered following 10 min of ischemia in vitro. In acidic solution (pH6.8) , both of the characteristic rapid [Ca^<2+>] _i-increase and the rapid depolarization were significantly retarded and slown. Field potentials following ischemia in vitro were reappeared in 7 of 9 slices. The results suggest that acidosis protects functions of CA1 neurons against ischemic damage.(2) Actions of NMDA on neuronal activities and intracellular Ca^<2+> and effects of ammonium on the NMDA-induced changes in neuronal activities were investigated by using hippocampal slices of the rat. Application of NMDA (50muM,30sec) induced an increase in [Ca^<2+>] _i of the CA1 area. Duration of the [Ca^<2+>] _i-increase induced by NMDA elongated in the presence of 5 mM NH_4Cl. Membrane potential of the CA1 neuron was depolarized by NMDA (20muM,10sec) , and this NMDA-induced membrane depolarization was enhanced and elongated in the presence of NH_4Cl. On the other hand, NH_4Cl inhibited both e.p.s.p.and i.p.s.p.evoked by stimulation of the Schaffer collateral. These results suggest that ammonium aggravate ischemic neuronal damage by enhancing the NMDA-induced neuronal excitation.