Dubinsky J M, Levi Y
Department of Physiology, University of Minnesota Medical School, Minneapolis 55455, USA.
J Neurosci Res. 1998 Sep 15;53(6):728-41. doi: 10.1002/(SICI)1097-4547(19980915)53:6<728::AID-JNR10>3.0.CO;2-U.
The mitochondrial permeability transition (mPT) has been implicated in both excitotoxic and apoptotic neuronal cell death, despite the fact that it has not been previously identified in neurons. To study the mPT in hippocampal neurons, cultures were loaded with the mitochondrial dye JC-1 and observed with confocal and conventional microscopy. After pretreatment with 4Br-A23187 and subsequent calcium addition, the initially rodlike mitochondria increased in diameter until mitochondria became rounded in appearance. Morphological changes reversed when calcium was removed by EGTA. When neurons were loaded with both fura-2-AM and rhodamine 123, calcium loading produced an increase in cytosolic calcium, mitochondrial depolarization, and similar alterations in mitochondrial morphology. Smaller calcium challenges produced calcium cycling, delaying morphological changes until after secondary depolarization and calcium release to the cytosol. In neurons exposed to glutamate, confocal observation of JC-1 fluorescence revealed comparable changes in mitochondrial morphology that were prevented when barium was substituted for calcium, or following pretreatment with the mPT inhibitor, cyclosporin A. These experiments establish conditions in which the mPT could be observed in situ in neurons in response to calcium loading. In addition, the timing of changes suggested that induction of the permeability transition in situ represents a sequence of multiple events that may reflect the multiple open conformations of the mPT pore.
线粒体通透性转换（mPT）已被认为与兴奋性毒性和凋亡性神经元细胞死亡有关，尽管此前尚未在神经元中发现这种现象。为了研究海马神经元中的mPT，将培养物用线粒体染料JC-1进行负载，并通过共聚焦显微镜和传统显微镜进行观察。在用4Br-A23187预处理并随后添加钙后，最初呈杆状的线粒体直径增大，直至线粒体外观变得圆润。当用EGTA去除钙时，形态变化逆转。当神经元同时负载fura-2-AM和罗丹明123时，钙负载导致胞质钙增加、线粒体去极化以及线粒体形态发生类似改变。较小的钙刺激会产生钙循环，将形态变化延迟至二次去极化和钙释放到胞质之后。在暴露于谷氨酸的神经元中，对JC-1荧光的共聚焦观察显示线粒体形态有类似变化，当用钡替代钙或用mPT抑制剂环孢素A预处理后，这种变化可被阻止。这些实验建立了在神经元中响应钙负载原位观察mPT的条件。此外，变化的时间表明原位通透性转换的诱导代表了一系列多个事件，这可能反映了mPT孔的多种开放构象。
