Sakaguchi T, Okada M, Kuno M, Kawasaki K
Shionogi Research Laboratories, Toyonaka, Osaka, Japan.
Neuroscience. 1997 Jan;76(2):411-23. doi: 10.1016/s0306-4522(96)00403-4.
Hippocampal slices prepared from neonatal rats were cultured for several weeks, and excitotoxicity induced in CA1 pyramidal neurons by N-methyl-D-aspartate was evaluated at different culture stages. CA1 neurons cultured for one week exhibited cell death predominantly within 1-3 h after a 15-min N-methyl-D-aspartate insult (early death), whereas those cultured for three weeks showed cell death mainly a few hours to 24 h after the insult (delayed death). CA1 neurons cultured for two weeks were in a transitional state, expressing only weak early and delayed forms of cell death in response to N-methyl-D-aspartate. The N-methyl-D-aspartate-induced early cell death observed in the one-week group depended on external Cl- but did not require external Ca2+; rather, early cell death was enhanced in Ca2+-free solution. This early cell death was accompanied by cell swelling, but cell swelling alone produced by osmotic changes failed to induce cell death. There was no evidence that CA1 neurons in the one-week group were more responsive to N-methyl-D-aspartate than those in the two other groups. Delayed cell death examined in the three-week group depended on external Ca2+ but was independent of Cl-. The density of N-methyl-D-aspartate-induced whole-cell currents recorded from CA1 neurons in Mg2+-free solution remained unchanged during three weeks of culture. However, the N-methyl-D-aspartate receptor channel in the three-week group was more resistant to Mg2+ block than that in the one- or two-week group. The incidence of N-methyl-D-aspartate-induced delayed cell death was higher in the three-week group than in the two-week group in normal solution but not in Mg2+-free solution. Thus, Mg2+ block-resistant properties of the N-methyl-D-aspartate receptor channel acquired during prolonged culture may account for the high incidence of N-methyl-D-aspartate-induced delayed cell death in the three-week group. However, the N-methyl-D-aspartate receptor subunits expressed in the CA1 subfield did not show any feature specific to the three-week group. These results show that two mechanistically distinct modes of N-methyl-D-aspartate-induced neuronal death are manifested differentially at different culture stages, depending on the intrinsic neuron properties (i.e. early cell death) and on the properties or the responsiveness of the N-methyl-D-aspartate receptor (i.e. delayed cell death).
从新生大鼠制备的海马切片培养数周，在不同培养阶段评估N-甲基-D-天冬氨酸诱导的CA1锥体神经元兴奋性毒性。培养一周的CA1神经元在15分钟的N-甲基-D-天冬氨酸损伤后1-3小时内主要表现为细胞死亡（早期死亡），而培养三周的神经元在损伤后数小时至24小时主要表现为细胞死亡（延迟死亡）。培养两周的CA1神经元处于过渡状态，对N-甲基-D-天冬氨酸仅表现出微弱的早期和延迟形式的细胞死亡。在一周组中观察到的N-甲基-D-天冬氨酸诱导的早期细胞死亡依赖于细胞外Cl-，但不需要细胞外Ca2+；相反，在无Ca2+溶液中早期细胞死亡增强。这种早期细胞死亡伴随着细胞肿胀，但渗透压变化单独引起的细胞肿胀未能诱导细胞死亡。没有证据表明一周组的CA1神经元比其他两组对N-甲基-D-天冬氨酸更敏感。在三周组中检测到的延迟细胞死亡依赖于细胞外Ca2+，但与Cl-无关。在无Mg2+溶液中从CA1神经元记录的N-甲基-D-天冬氨酸诱导的全细胞电流密度在三周培养期间保持不变。然而，三周组的N-甲基-D-天冬氨酸受体通道比一周或两周组对Mg2+阻断更具抗性。在正常溶液中，三周组中N-甲基-D-天冬氨酸诱导的延迟细胞死亡发生率高于两周组，但在无Mg2+溶液中并非如此。因此，长时间培养过程中获得的N-甲基-D-天冬氨酸受体通道的抗Mg2+阻断特性可能是三周组中N-甲基-D-天冬氨酸诱导的延迟细胞死亡发生率高的原因。然而，在CA1亚区表达的N-甲基-D-天冬氨酸受体亚基未显示出三周组特有的任何特征。这些结果表明，N-甲基-D-天冬氨酸诱导的神经元死亡的两种机制不同的模式在不同培养阶段有不同表现，这取决于神经元的内在特性（即早期细胞死亡）以及N-甲基-D-天冬氨酸受体的特性或反应性（即延迟细胞死亡）。
