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，我们检测了缺氧缺糖（离体缺血）海马脑片细胞内游离Ca^<2 +>浓度（[Ca^<2+>] _i）和电活动的变化。用Fura-2负载Wistar大鼠的海马脑片，并在36-37 ℃下用Krebs溶液灌流，通过荧光测量来估计[Ca^<2+>] _i的变化。记录CA 1锥体细胞的场电位和膜电位。在正常pH（pH7.4）溶液中，离体心肌缺血6-7 min时，细胞内[Ca^<2+>] i迅速升高，膜电位迅速去极化。在体外缺血10分钟后，场电位没有恢复。在酸性溶液（pH6.8）中，[Ca^<2+>] _i的快速增加和快速去极化均被显著延迟和减慢。9个脑片中有7个脑片在离体缺血后出现了场电位。结果提示，酸中毒对缺血性损伤的CA 1神经元功能具有保护作用。(2)采用大鼠海马脑片研究了NMDA对神经元活动和细胞内Ca^<2+>的作用以及氨对NMDA引起的神经元活动变化的影响。应用NMDA（50 μ M，30秒）可引起海马CA 1区[Ca^<2+>] _i增加。5 mM NH_4Cl使NMDA引起的[Ca^<2+>] _i增加的持续时间延长。NMDA（20 μ M，10秒）可使CA_1神经元的膜电位去极化，NH_4Cl可使NMDA引起的膜电位去极化增强并延长。另一方面，NH_4Cl抑制刺激Schaffer侧支诱发的ep. s. p.和i. p. s. p.。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.