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

Nature Medicine volume 13, pages 874–879 (2007)Cite this article

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 (∼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.

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Figure 1: X-rhod-1 and DiOC6(3) labeling of compact myelin and oligodendrocytes.
Figure 2: In-place immunohistochemistry localizes X-rhod-1 to myelin.
Figure 3: Fluorescence lifetime measurements by TCSPC.
Figure 4: Ca2+-dependent X-rhod-1 fluorescence measured from myelin and oligodendrocyte cytoplasm in optic nerve and dorsal columns.
Figure 5: Chemical ischemia induced a myelinic increase in Ca2+, dependent on glutamate and glycine release by Na+-coupled transporters.

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References

  1. Ranvier, L. & Weber, E. Leçons sur l'Histologie du Système Nerveux (Librairie F. Savy, Paris, 1878).

    Google Scholar 

  2. Virchow, R.L.K., Vogel, J. & Stiebel, S.F. Allgemeine Störungen der Ernährung und des Blutes (Erlangen, 1854).

    Google Scholar 

  3. Lazzarini, R.A. et al. (eds.) Myelin Biology and Disorders (Elsevier Academic, Amsterdam, 2004).

    Google Scholar 

  4. Taylor, C.M. et al. Proteomic mapping provides powerful insights into functional myelin biology. Proc. Natl. Acad. Sci. USA 101, 4643–4648 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Michailov, G.V. et al. Axonal neuregulin-1 regulates myelin sheath thickness. Science 304, 700–703 (2004).

    Article  CAS  PubMed  Google Scholar 

  6. Schlaepfer, W.W. Vesicular disruption of myelin simulated by exposure of nerve to calcium ionophore. Nature 265, 734–736 (1977).

    Article  CAS  PubMed  Google Scholar 

  7. Ellisman, M.H., Friedman, P.L. & Hamilton, W.J. Cytochemical localization of cations in myelinated nerve using TEM, HVEM, SEM and electron probe microanalysis. Scan. Electron Microsc. 1979, 793–800 (1979).

    Google Scholar 

  8. Micu, I. et al. NMDA receptors mediate calcium accumulation in central nervous system myelin during chemical ischaemia. Nature 439, 988–992 (2006).

    Article  CAS  PubMed  Google Scholar 

  9. Ness, J.K., Valentino, M., McIver, S.R. & Goldberg, M.P. Identification of oligodendrocytes in experimental disease models. Glia 50, 321–328 (2005).

    Article  PubMed  Google Scholar 

  10. Minta, A., Kao, J.P. & Tsien, R.Y. Fluorescent indicators for cytosolic calcium based on rhodamine and fluorescein chromophores. J. Biol. Chem. 264, 8171–8178 (1989).

    CAS  PubMed  Google Scholar 

  11. Lakowicz, J.R. Principles of Fluorescence Spectroscopy (Plenum, New York, 1999).

    Book  Google Scholar 

  12. Berridge, M.J., Lipp, P. & Bootman, M.D. The versatility and universality of calcium signalling. Nat. Rev. Mol. Cell Biol. 1, 11–21 (2000).

    Article  CAS  PubMed  Google Scholar 

  13. Dingledine, R., Borges, K., Bowie, D. & Traynelis, S.F. The glutamate receptor ion channels. Pharmacol. Rev. 51, 7–61 (1999).

    CAS  PubMed  Google Scholar 

  14. Danbolt, N.C. Glutamate uptake. Prog. Neurobiol. 65, 1–105 (2001).

    Article  CAS  PubMed  Google Scholar 

  15. Aragon, C. & Lopez-Corcuera, B. Structure, function and regulation of glycine neurotransporters. Eur. J. Pharmacol. 479, 249–262 (2003).

    Article  CAS  PubMed  Google Scholar 

  16. Stys, P.K. White matter injury mechanisms. Curr. Mol. Med. 4, 113–130 (2004).

    Article  CAS  PubMed  Google Scholar 

  17. Alexander, S.P.H., Mathie, A. & Peters, J.A. Cell-surface transmitter transporters: glycine. Br. J. Pharmacol. 147, S148–S149 (2006).

    Article  Google Scholar 

  18. Arundine, M. & Tymianski, M. Molecular mechanisms of calcium-dependent neurodegeneration in excitotoxicity. Cell Calcium 34, 325–337 (2003).

    Article  CAS  PubMed  Google Scholar 

  19. Mrsulja, B.J., Zalewski, A.A. & Coping, G. Ultracytochemical localization of ouabain-sensitive K+-dependent, p-nitrophenyl phosphatase in myelin. Brain Res. 343, 154–158 (1985).

    Article  CAS  PubMed  Google Scholar 

  20. Martenson, R.E. Myelin—Biology and Chemistry (CRC Press, Boca Raton, Florida, USA, 1992).

    Google Scholar 

  21. Cherksey, B., Durrie, R., Braun, P.E. & Sapirstein, V.S. In vitro analysis of ion channels in periaxolemmal-myelin and white matter clathrin coated vesicles: modulation by calcium and GTP gamma S. Neurochem. Res. 19, 1101–1106 (1994).

    Article  CAS  PubMed  Google Scholar 

  22. Ropte, S., Scheidt, P. & Friede, R.L. The intermediate dense line of the myelin sheath is preferentially accessible to cations and is stabilized by cations. J. Neurocytol. 19, 242–252 (1990).

    Article  CAS  PubMed  Google Scholar 

  23. Lev-Ram, V. & Ellisman, M.H. Axonal activation-induced calcium transients in myelinating Schwann cells, sources, and mechanisms. J. Neurosci. 15, 2628–2637 (1995).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Hildebrand, C. & Hahn, R. Relation between myelin sheath thickness and axon size in spinal cord white matter of some vertebrate species. J. Neurol. Sci. 38, 421–434 (1978).

    Article  CAS  PubMed  Google Scholar 

  25. Simpson, P.B., Challiss, R.A. & Nahorski, S.R. Divalent cation entry in cultured rat cerebellar granule cells measured using Mn2+ quench of fura 2 fluorescence. Eur. J. Neurosci. 7, 831–840 (1995).

    Article  CAS  PubMed  Google Scholar 

  26. Li, S., Mealing, G.A., Morley, P. & Stys, P.K. Novel injury mechanism in anoxia and trauma of spinal cord white matter: glutamate release via reverse Na+-dependent glutamate transport. J. Neurosci. 19, RC16 (1999).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Spike, R.C., Watt, C., Zafra, F. & Todd, A.J. An ultrastructural study of the glycine transporter GLYT2 and its association with glycine in the superficial laminae of the rat spinal dorsal horn. Neuroscience 77, 543–551 (1997).

    Article  CAS  PubMed  Google Scholar 

  28. Ren, Y., Ridsdale, A., Coderre, E. & Stys, P.K. Calcium imaging in live rat optic nerve myelinated axons in vitro using confocal laser microscopy. J. Neurosci. Methods 102, 165–176 (2000).

    Article  CAS  PubMed  Google Scholar 

  29. Verbny, Y., Zhang, C.L. & Chiu, S.Y. Coupling of calcium homeostasis to axonal sodium in axons of mouse optic nerve. J. Neurophysiol. 88, 802–816 (2002).

    Article  CAS  PubMed  Google Scholar 

  30. Ouardouz, M. et al. Depolarization-induced Ca2+ release in ischemic spinal cord white matter involves L-type Ca2+ channel activation of ryanodine receptors. Neuron 40, 53–63 (2003).

    Article  CAS  PubMed  Google Scholar 

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Acknowledgements

We thank J. Wang and C. Morris for providing HEK293 cells for fluorescence lifetime and spectral measurements. This work was supported by grants from the National Institute of Neurological Disorders and Stroke (NINDS), the Canadian Institutes of Health Research (CIHR), the Canadian Institute for Photonic Innovations, and the Intramural Research Program of the National Institutes of Health, NINDS (operating); CIHR 90408 and the Heart and Stroke Foundation Center for Stroke Recovery (equipment); and the generosity of private donors. P.K.S. is supported by a Heart and Stroke Foundation of Ontario Career Investigator Award.

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Authors and Affiliations

  1. Division of Neuroscience, Ottawa Health Research Institute, University of Ottawa, Ottawa, K1Y 4E9, Canada

    Ileana Micu, Andrew Ridsdale, Lingqing Zhang & Peter K Stys

  2. Centre for Cancer Therapeutics, Ottawa Health Research Institute, University of Ottawa, Ottawa, K1Y 4E9, Canada

    John Woulfe

  3. Electron Microscopy, Children's Hospital of Eastern Ontario, University of Ottawa, Ottawa, K1H 8L1, Canada

    Jeff McClintock

  4. Laboratory of Neurobiology, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, 20892-4477, Maryland, USA

    Christine A Brantner & S Brian Andrews

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Contributions

I.M., experimental design, two-photon calcium imaging, fluorescence lifetime measurements, numerical data analysis, in-place immunohistochemistry, confocal microscopy, and a major role in the writing of the article. A.R. and L.Z., two-photon microscopy, fluorescence lifetime measurements and associated numerical data analysis, and editing and revision of the article. J.W. and J.M., electron microscopy and image interpretation. C.A.B. and S.B.A., electron probe microanalysis, analysis of data and scientific discussion. P.K.S., experimental concept and design, coordination of experiments, data analysis and interpretation, and writing of the article. All authors were involved with the analysis and interpretation of their respective data and contributed to the writing of the article.

Corresponding author

Correspondence to Peter K Stys.

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Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Fig. 1

In order to exclude the remote possibility that the failure of Mn2+ to quench X-rhod-1 fluorescence in resting, non-ischemic myelin was due to the inability of this cation to permeate uninjured myelin altogether, electron probe microanalysis was carried out on individual myelin sheaths from control optic nerves and those exposed to Mn2+-containing CSF (without ischemia, but both sets pre-treated with the same solutions normally used for dye loading). (PDF 326 kb)

Supplementary Fig. 2

X-rhod-1 emission spectrum in various micro-environments. (PDF 92 kb)

Supplementary Fig. 3

Effects of glutamate or glycine transport inhibition on myelinic and oligodendroglial Ca2+ levels in uninjured control optic nerves and dorsal columns. (PDF 110 kb)

Supplementary Fig. 4

Comparison of myelin integrity by electron microscopy in optic nerves maintained in normal Ca2+-CSF vs. nerves exposed to Ca2+-free loading solution indicated that the low Ca2+ exposure did not alter myelin structure to any appreciable degree. (PDF 782 kb)

Supplementary Methods (PDF 65 kb)

Supplementary Note (PDF 40 kb)

Supplementary Discussion (PDF 29 kb)

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Micu, I., Ridsdale, A., Zhang, L. et al. Real-time measurement of free Ca2+ changes in CNS myelin by two-photon microscopy. Nat Med 13, 874–879 (2007). https://doi.org/10.1038/nm1568

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