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PLOS ONE  2012 

Time-Lapse Imaging of the Dynamics of CNS Glial-Axonal Interactions In Vitro and Ex Vivo

DOI: 10.1371/journal.pone.0030775

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Background Myelination is an exquisite and dynamic example of heterologous cell-cell interaction, which consists of the concentric wrapping of multiple layers of oligodendrocyte membrane around neuronal axons. Understanding the mechanism by which oligodendrocytes ensheath axons may bring us closer to designing strategies to promote remyelination in demyelinating diseases. The main aim of this study was to follow glial-axonal interactions over time both in vitro and ex vivo to visualize the various stages of myelination. Methodology/Principal Findings We took two approaches to follow myelination over time: i) time-lapse imaging of mixed CNS myelinating cultures generated from mouse spinal cord to which exogenous GFP-labelled murine cells were added, and ii) ex vivo imaging of the spinal cord of shiverer (Mbp mutant) mice, transplanted with GFP-labelled murine neurospheres. We demonstrate that oligodendrocyte-axonal interactions are dynamic events with continuous retraction and extension of oligodendroglial processes. Using cytoplasmic and membrane-GFP labelled cells to examine different components of the myelin-like sheath, we provide evidence from time-lapse fluorescence microscopy and confocal microscopy that the oligodendrocytes' cytoplasm-filled processes initially spiral around the axon in a corkscrew-like manner. This is followed subsequently by focal expansion of the corkscrew process to form short cuffs, which then extend longitudinally along the axons. We predict from this model that these spiral cuffs must extend over each other first before extending to form internodes of myelin. Conclusion These experiments show the feasibility of visualizing the dynamics of glial-axonal interaction during myelination over time. Moreover, these approaches complement each other with the in vitro approach allowing visualization of an entire internodal length of myelin and the ex vivo approach validating the in vitro data.


[1]  Peters A (1960) The formation and structure of myelin sheaths in the central nervous system. J Biophys Bioch Cytol 8: 431–446.
[2]  Remahl S, Hildebrand C (1990) Relations between axons and oligodendroglial cells during initial myelination. II. The individual axon. J Neurocytol 19: 883–898.
[3]  Franklin RJ, Ffrench-Constant C (2008) Remyelination in the CNS: from biology to therapy. Nat Rev Neurosci 9: 839–855.
[4]  Bauer NG, Richter-Landsberg C, ffrenceh-Constant C (2009) Role of the oligodendroglial cytoskeleton in differentiation and myelination. Glia 57: 1691–1705.
[5]  Werner HB, Jahn O (2010) Myelin matters: proteomic insights into white matter disorders. Exp Rev Prot 7: 159–164.
[6]  Pfeiffer SE, Warrington AE, Bansal R (1993) The oligodendrocyte and its many cellular processes. Trends Cell Biol 3: 191–197.
[7]  Raff MC, Miller RH, Noble MD (1983) A glial progenitor cell that develops in vitro into an astrocyte or an oligodendrocyte depending on culture medium. Nature 303: 390–396.
[8]  Li H, Lu Y, Smith HK, Richardson WD (2007) Olig1 and Sox10 interact synergistically to drive myelin basic protein transcription in oligodendrocytes. J Neurosci 27: 14375–14382.
[9]  Kirby B, Takada BN, Latimer AJ, Shin J, Carney TJ (2006) In vivo time-lapse imaging shows dynamic oligodendrocyte progenitor behavior during zebrafish development. Nature Neurosci 9: 1506–1511.
[10]  Czopka T, Lyons DA (2011) Dissecting mechanisms of myelinated axon formation using zebrafish. Methods Cell Biol 10525–62.
[11]  Watkins TA, Emery B, Mulinyawe S, Barres BA (2008) Distinct stages of myelination regulated by gamma-secretase and astrocytes in a rapidly myelinating CNS coculture system. Neuron 60: 555–569.
[12]  Sobottka B, Ziegler U, Kaech A, Becher B, Goebes N (2011) CNS live imaging reveals a new mechanism of myelination: the liquid croissant model. Glia, 2011 Sep 1. doi: 10.1002/glia.21228. [Epub ahead of print].
[13]  Pedraza L, Huang JK, Colman D (2009) Disposition of axonal caspr with respect to glial cell membranes: Implications for the process of myelination. J Neurosci Res 87: 3480–3491.
[14]  Okabe M, Ikawa M, Kominami K, Nakanishi T, Nishimune Y (1997) Green mice' as a source of ubiquitous green cells. FEBS Letts 407: 313–319.
[15]  Chernoff GF (1981) Shiverer: an autosomal recessive mutant mouse with myelin deficiency. The J Hered 72: 128.
[16]  Feng G, Mellor RH, Bernstein M, Keller-Peck C, Nguyen QT, et al. (2000) Imaging neuronal subsets in transgenic mice expressing multiple spectral variants of GFP. Neuron 28: 41–51.
[17]  Sorensen A, Moffat K, Thomson C, Barnett SC (2008) Astrocytes, but not olfactory ensheathing cells or Schwann cells, promote myelination of CNS axons in vitro. Glia 56: 750–763.
[18]  Thomson CE, Hunter AM, Griffiths IR, Edgar JM, McCulloch MC (2006) Murine spinal cord explants: a model for evaluating axonal growth and myelination in vitro. J Neurosci Res 84: 1703–1715.
[19]  Bottenstein JE, Sato GH (1979) Growth of a rat neuroblastoma cell line in serum-free supplemented medium. Proc Natl Acad Sci U S A 76: 514–517.
[20]  Kimura M, Inoko H, Katsuki M, Ando A, Sato T, et al. (1985) Molecular genetic analysis of myelin-deficient mice: shiverer mutant mice show deletion in gene(s) coding for myelin basic protein. J Neurochem 44: 692–696.
[21]  Molineaux SM, Engh H, de Ferra F, Hudson L, Lazzarini RA (1986) Recombination within the myelin basic protein gene created the dysmyelinating shiverer mouse mutation. Proc Natl Acad Sci U S A 83: 7542–7546.
[22]  Dupouey P, Jacque C, Bourre JM, Cesselin F, Privat A, et al. (1979) Immunochemical studies of myelin basic protein in shiverer mouse devoid of major dense line of myelin. Neurosci Letts 12: 113–118.
[23]  Barbarese EML, Nielson ML, Carson JH (1983) The effect of the shiverer mutation on myelin basic protein expression in homozygous and heterozygous mouse brain. J Neurochem 40: 1680–1686.
[24]  Privat A, Jacque C, Bourre JM, Dupouey P, Baumann N (1979) Absence of the major dense line in myelin of the mutant mouse “shiverer”. Neurosci Letts 12: 107–112.
[25]  Thomson CE, McMulloch M, Sorensen A, Barnett SC, Seed BV, et al. (2008) Myelinated, synapsing cultures of murine spinal cord- valisdation as an in vitro moels of the central nmervous system. E J Neurosci 28: 1518–1535.
[26]  Edgar JM, McLaughlin M, Yool D, Zhang SC, Fowler JH, et al. (2004) Oligodendroglial modulation of fast axonal transport in a mouse model of hereditary spastic paraplegia. J Cell Biol 166: 121–131.
[27]  Yamada M, Ivanova A, Yamaguchi Y, Lees MB, Ikenaka K (1999) Proteolipid protein gene product can be secreted and exhibit biological activity during early development. J Nurosci 19: 2143–2151.
[28]  Sommer I, Schachner M (1981) Monoclonal antibodies (O1 to O4) to oligodendrocyte cell surfaces: an immunocytological study in the central nervous system. Dev Biol 83: 311–327.
[29]  Kerschensteiner M, Reuter MS, Lichtman JW, Misgeld T (2008) Ex vivo imaging of motor axon dynamics in murine triangularis sterni explants. Nature Prot 3: 1645–1653.
[30]  Colman DR, Kreibich G, Sabatini DD (1982) Synthesis and incorporation of myelin polypeptides into CNS myelin. J Cell Biol 95: 598–608.
[31]  Trajkovic K, Dhaunchak AS, Goncalves JT, Wenzel D, Schneider A, et al. (2006) Neuron to glia signaling triggers myelin membrane exocytosis from endosomal storage sites. J Cell Biol 172: 937–948.
[32]  Laursen LS, Chan CW, Ffrench-Constant C (2009) An integrin-contactin complex regulates CNS myelination by differential Fyn phosphorylation. J Neurosci 29: 9174–9185.
[33]  Matthews MA, Duncan ID (1971) A quantitative study of morphological changes accompanying the initiation and progress of myelin production in the dorsal funiculus of the rat spinal cord. J Comp Neurol 142: 1–22.
[34]  Bunge MB, Bunge RP, Pappas GD (1962) Electron microscopic demonstration of connections between glia and myelin sheaths in the developing mammalian central nervous system. J Cell Biol 12: 448–453.
[35]  Uzman BG (1964) The Spiral Configuration of Myelin Lamellae. J Ultrast Res 11: 208–212.
[36]  Hirano A, Dembitzer HM (1967) A structural analysis of the myelin sheath in the central nervous system. J Cell Biol 34: 555–567.
[37]  Knobler RL, Stempak JG, Laurencin M (1976) Nonuniformity of the oligodendroglial ensheathment of axons during myelination in the developing rat central nervous system. A serial section electron microscopical study. J Ultrastruct Res 55: 417–432.
[38]  Webster HD (1971) The geometry of peripheral myelin sheaths during their formation and growth in rat sciatic nerves. J Cell Biol 48: 348–367.
[39]  Luse SA (1956) Formation of myelin in the central nervous system of mice and rats, as studied with the electron microscope. J Biophys Biochem Cytol 2: 777–784.
[40]  Bunge MB, Bunge RP, Ris H (1961) Ultrastructure study of remyelination in an experimetnal lesion in adult spinla cord. J Biophys Biochem Cytol 10: 67–94.
[41]  Ridley AJ (2011) Life at the leading edge. Cell 145: 1012–1022.
[42]  Sherman DL, Brophy PJ (2005) Mechanisms of axon ensheathment and myelin growth. Nat Rev Neurosci 6: 683–690.


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