Micu Ileana, Ridsdale Andrew, Zhang Lingqing, Woulfe John, McClintock Jeff, Brantner Christine A, Andrews S Brian, Stys Peter K
Division of Neuroscience, Ottawa Health Research Institute, University of Ottawa, Ottawa K1Y 4E9, Canada.
Nat Med. 2007 Jul;13(7):874-9. doi: 10.1038/nm1568. Epub 2007 Jul 1.
Here we describe a technique for measuring changes in Ca2+ in the cytosolic domain of mature compact myelin of live axons in the central nervous system (CNS). We label the myelin sheath of optic nerve and dorsal column axons by using the Ca2+ indicator X-rhod-1 coupled with DiOC6(3) to produce bright myelin counterstaining, thereby providing unambiguous identification of the myelin sheath for analysis of two-photon excited fluorescence. We present evidence for localization of the Ca2+ reporter to the cytosolic domain of myelin, obtained by using fluorescence lifetime, spectral measurements and Mn2+ quenching. Chemical ischemia increased myelinic X-rhod-1 fluorescence (approximately 50% after 30 min) in a manner dependent on extracellular Ca2+. Inhibiting Na+-dependent glutamate transporters (with TBOA) or glycine transporters (with sarcosine and ALX-1393) reduced the ischemia-induced increase in Ca2+. We show that myelinic N-methyl-D-aspartate (NMDA) receptors are activated by the two conventional coagonists glutamate and glycine, which are released by specific transporters under conditions of cellular Na+ loading and depolarization in injured white matter. This new technique facilitates detailed studies of living myelin, a vital component of the mammalian CNS.
在此，我们描述了一种用于测量中枢神经系统（CNS）中活轴突成熟紧密髓鞘胞质域内Ca2+变化的技术。我们通过使用Ca2+指示剂X-rhod-1与DiOC6(3)偶联来标记视神经和背柱轴突的髓鞘，以产生明亮的髓鞘复染，从而为双光子激发荧光分析提供明确的髓鞘识别。我们通过使用荧光寿命、光谱测量和Mn2+淬灭，提供了Ca2+报告分子定位于髓鞘胞质域的证据。化学性缺血以依赖细胞外Ca2+的方式增加了髓鞘X-rhod-1荧光（30分钟后约增加50%）。抑制Na+依赖性谷氨酸转运体（使用TBOA）或甘氨酸转运体（使用肌氨酸和ALX-1393）可减少缺血诱导的Ca2+增加。我们表明，髓鞘N-甲基-D-天冬氨酸（NMDA）受体被两种传统共激动剂谷氨酸和甘氨酸激活，这两种物质在损伤白质中细胞Na+负载和去极化条件下由特定转运体释放。这项新技术有助于对活髓鞘进行详细研究，活髓鞘是哺乳动物中枢神经系统的重要组成部分。
