Time-lapse imaging of the dynamics of CNS glial-axonal interactions in vitro and ex vivo.
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.


RESULTS
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.


CONCLUSIONS
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.
Institute of Infection, Immunity and Inflammation, University of Glasgow, Glasgow, United Kingdom.
Kalliopi
K
Ioannidou
Kurt I
KI
Anderson
David
D
Strachan
Julia M
JM
Edgar
Susan C
SC
Barnett
D000818:Animals; D001369:Axons / Q000378:metabolism*; D015415:Biomarkers / Q000378:metabolism; D002449:Cell Aggregation; D002450:Cell Communication; D002454:Cell Differentiation; D002478:Cells, Cultured; D002490:Central Nervous System / Q000166:cytology*; D005456:Fluorescent Dyes / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000378:metabolism; D007150:Immunohistochemistry; D051379:Mice; D008810:Mice, Inbred C57BL; D036641:Microscopy, Fluorescence, Multiphoton; D009186:Myelin Sheath / Q000378:metabolism; D009457:Neuroglia / Q000166:cytology* / Q000378:metabolism; D009474:Neurons / Q000166:cytology / Q000378:metabolism / Q000637:transplantation; D009836:Oligodendroglia / Q000166:cytology / Q000378:metabolism; D015203:Reproducibility of Results; D013116:Spinal Cord / Q000166:cytology; D013234:Stem Cells / Q000166:cytology / Q000378:metabolism; D018800:Thy-1 Antigens / Q000378:metabolism; D059008:Time-Lapse Imaging / Q000379:methods*
D015415:Biomarkers; D005456:Fluorescent Dyes; D018800:Thy-1 Antigens; D049452:Green Fluorescent Proteins