Real-time measurement of free Ca2+ changes in CNS myelin by two-photon microscopy

Here we describe a technique for measuring changes in Ca 2+ 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 Ca 2+ indicator X-rhod-1 coupled with DiOC 6 (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 Ca 2+ reporter to the cytosolic domain of myelin, obtained by using fluorescence lifetime, spectral measurements and Mn 2+ quenching. Chemical ischemia increased myelinic X-rhod-1 fluorescence (∼50% after 30 min) in a manner dependent on extracellular Ca 2+ . Inhibiting Na + -dependent glutamate transporters (with TBOA) or glycine transporters (with sarcosine and ALX-1393) reduced the ischemia-induced increase in Ca 2+ . 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.