title: Real-time measurement of free Ca2+ changes in CNS myelin by two-photon microscopy.
abstract: 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.
authors.affiliation: Division of Neuroscience, Ottawa Health Research Institute, University of Ottawa, Ottawa K1Y 4E9, Canada.
authors.forename: Ileana
authors.initials: I
authors.lastname: Micu
authors.forename: Andrew
authors.initials: A
authors.lastname: Ridsdale
authors.forename: Lingqing
authors.initials: L
authors.lastname: Zhang
authors.forename: John
authors.initials: J
authors.lastname: Woulfe
authors.forename: Jeff
authors.initials: J
authors.lastname: McClintock
authors.forename: Christine A
authors.initials: CA
authors.lastname: Brantner
authors.forename: S Brian
authors.initials: SB
authors.lastname: Andrews
authors.forename: Peter K
authors.initials: PK
authors.lastname: Stys
mesh_terms: D000818:Animals; D002118:Calcium / Q000378:metabolism*; D020013:Calcium Signaling; D002490:Central Nervous System / Q000166:cytology / Q000378:metabolism*; D005456:Fluorescent Dyes; D008853:Microscopy; D009186:Myelin Sheath / Q000378:metabolism*; D009474:Neurons / Q000166:cytology; D051381:Rats; D020318:Rats, Long-Evans; D013997:Time Factors
chemical_list: D005456:Fluorescent Dyes; D002118:Calcium