Marks J D, Bindokas V P, Zhang X M
Department of Pediatrics, The University of Chicago, 5841 S. Maryland Avenue, Chicago, IL 60637, USA.
Brain Res Dev Brain Res. 2000 Nov 30;124(1-2):101-16. doi: 10.1016/s0165-3806(00)00096-1.
Neuronal vulnerability to excitotoxicity changes dramatically during postnatal maturation. To study the intracellular mechanisms by which maturation alters vulnerability in single neurons, we developed techniques to maintain hippocampal neurons from postnatal rats in vitro. After establishing their neuronal phenotype with immunohistochemistry and electrophysiology, we determined that these neurons exhibit developmentally regulated vulnerability to excitotoxicity. At 5 days in vitro, NMDA-induced cell death at 24 h increased from 3.6% in 3-day-old rats to >90% in rats older than 21 days. Time-lapse imaging of neuronal morphology following NMDA demonstrated increasingly prevalent and severe injury as a function of postnatal age. Neither high- nor low-affinity calcium dyes demonstrated differences in peak NMDA-induced Ca(2+) increases between neurons from younger and older animals. However, neurons from older animals were uniformly distinguished from those from younger animals by their subsequent loss of Ca(2+) homeostasis. Because of the role of mitochondrial Ca(2+) buffering in Ca(2+) homeostasis, we measured NMDA-induced changes in mitochondrial membrane potential (DeltaPsi) as a function of postnatal age. NMDA markedly dissipated DeltaPsi in neurons from mature rats, but minimally in those from younger rats. These data demonstrate that, in cultures of postnatal hippocampal neurons, (a) vulnerability to excitotoxicity increases as a function of the postnatal age of the animal from which they were harvested, and (b) developmental regulation of vulnerability to NMDA occurs at the level of the mitochondrion.
神经元对兴奋性毒性的易感性在出生后成熟过程中会发生显著变化。为了研究成熟过程改变单个神经元易感性的细胞内机制，我们开发了在体外维持新生大鼠海马神经元的技术。在用免疫组织化学和电生理学确定其神经元表型后，我们发现这些神经元对兴奋性毒性表现出受发育调节的易感性。在体外培养5天时，NMDA诱导的24小时细胞死亡从3日龄大鼠的3.6%增加到21日龄以上大鼠的>90%。NMDA处理后神经元形态的延时成像显示，随着出生后年龄的增加，损伤越来越普遍且严重。高亲和力和低亲和力钙染料均未显示年轻和年长动物来源的神经元在NMDA诱导的Ca(2+)峰值增加方面存在差异。然而，年长动物来源的神经元与年轻动物来源的神经元的区别在于，它们随后失去了Ca(2+)稳态。由于线粒体Ca(2+)缓冲在Ca(2+)稳态中的作用，我们测量了NMDA诱导的线粒体膜电位（DeltaPsi）变化作为出生后年龄的函数。NMDA使成熟大鼠神经元中的DeltaPsi显著消散，但对年轻大鼠神经元的影响最小。这些数据表明，在新生海马神经元培养物中，(a)对兴奋性毒性的易感性随着收获它们的动物的出生后年龄而增加，并且(b)对NMDA易感性的发育调节发生在线粒体水平。
