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Ca2+ activity signatures of myelin sheath formation and growth in vivo

Nature Neurosciencevolume 21pages1923 (2018) | Download Citation

Abstract

During myelination, individual oligodendrocytes initially over-produce short myelin sheaths, which are either retracted or stabilized. By live-imaging oligodendrocyte Ca2+ activity in vivo, we find that high-amplitude, long-duration Ca2+ transients in sheaths prefigure retractions, mediated by calpain. Following stabilization, myelin sheaths grow along axons, and we find that higher-frequency Ca2+ transient activity in sheaths precedes faster elongation. Our data implicate local Ca2+ signaling in regulating distinct stages of myelination.

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Acknowledgements

We to thank members of the Lyons lab, as well as T. Becker, P. Brophy, C. ffrench-Constant, M. Livesey, D. Meijer, W. Talbot and C. Wyart, for comments on the manuscript. We thank the University of Edinburgh CALM facility for imaging support, the zebrafish facility for excellent fish care, H. Baier for transgenic zebrafish and B. Vernay for support in image analysis. This work was supported by a Lister Institute Research Prize and a Wellcome Trust Senior Research Fellowship (102836/Z/13/Z) to D.A.L.

Author information

Affiliations

  1. Centre for Neuroregeneration, Centre for Discovery Brain Sciences, University of Edinburgh, Edinburgh, UK

    • Marion Baraban
    • , Sigrid Koudelka
    •  & David A. Lyons

Authors

  1. Search for Marion Baraban in:

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Contributions

M.B. designed and performed experiments and cowrote the manuscript. S.K. performed experiments using chemical inhibitors. D.A.L. designed experiments, managed the project and cowrote the manuscript.

Competing interests

The authors declare no competing financial interests.

Corresponding authors

Correspondence to Marion Baraban or David A. Lyons.

Integrated Supplementary Information

  1. Supplementary Figure 1 2D high-speed imaging of Ca2+ transients.

    A. Single plane confocal frames taken during high speed (4Hz) recording of GCaMP6s. Background ROI and sheath ROI are indicated. Scale bar= 10µm. See also Supplementary Movie 1. B. Equation used to calculate ∆F/F0 values and description of terms.C. Fluorescent intensity profiles over time taken from single plane high-speed imaging series, showing an individual Ca2+ transient. Raw intensity values for background ROI and sheath ROI are shown in the top and the corresponding ∆F/F0 trace bottom. D. Distribution of Ca2+ transient durations, imaged in high-speed mode (4-6.6Hz), indicates that essentially no Ca2+ transients have a duration<3.5s (34 of 35 Ca2+ transients are >3.5s). Therefore, we used this information to set the optimal acquisition delay (<3.5s) in our extended 3D time-lapse imaging protocol. Graph shows median and 1st and 3rd quartiles. 20 myelin sheaths were imaged in 12 different animals in 4 separate experiments.

  2. Supplementary Figure 2 Localised Ca2+ transients in myelin sheaths made by individual oligodendrocytes.

    Maximum intensity projections of individual time-points from 3D time-lapse movies of GCaMP6s expressing oligodendrocytes labelled by Tg (sox10:KalTA4; uas:GCaMP6s) that have average myelin sheath lengths per cell at the start of each movie of 2.8µm (a), see also Supplementary Movie 3, 8.1µm, (b) and 11.8µm (c). Scale bars= 10µm. Representative images of 40 cells imaged in 40 different animals in 25 separate experiments.

  3. Supplementary Figure 3 Directionality of Ca2+ transients over time in a sheath subsequently retracted.

    a. Frames from time-lapse movie of the GCaMP6s expressing cell described in Figure 2, which show the directionality of Ca2+ transient propagation from the sheath back along the process over time. See also Supplementary Movie 4. Arrowheads indicate Regions of Interest (ROI) where traces in corresponding plots in C and D were made. Scale bar= 5µm. Example from 12 cells imaged in 9 separate experimental sessions. b. Indicates Regions of Interest where traces in corresponding plots in C and D were made. c. ∆F/F over time from ROIs indicated in B with single y-axis scale. d. ∆F/F over time for individual ROIs indicated in B with y-axes that allow visualisation of how times of peaks relate to one another.

  4. Supplementary Figure 4 Directionality of myelin sheath retractions.

    Images from a time-lapse movie indicating that myelin sheaths are retracted in a manner wherein the sheath itself first shrinks along the length of the axon to the point of contact between the myelinating process and the axon. The oligodendrocyte process subsequently retracts back towards the cell body over time. Representative example of 46 cells imaged in 46 animals in 6 separate experimental sessions. See also Supplementary Videos 5+6. Scale bar= 5µm.

  5. Supplementary Figure 5 Calpastatin expression in oligodendrocytes lead in an increase of myelin sheath.

    a. Schematic representation of calpastatin protein show repetition of 4 domains (I, II, III, IV). Inset of one of the domains show the subdomains (A, B and C). Schematic modified from Ref 21. b. Protein alignment of the domain 1B, 2B, 3B and 4B (important for calpain inhibition) of Homo sapiens and Danio rerio Calpastatin orthologs show a consensus protein sequence. c. mbp:meGFP expressing oligodendrocytes (left) and mbp:meGFP and cast mRNA 300pg expressing animal (right) at 4 dpf. Scale bar= 10µm. See quantitation in panel D. d. Myelin sheath number per oligodendrocytes in mbp:meGFP expressing oligodendrocytes and mbp:meGFP and cast mRNA 300pg expressing animal at 4 dpf. (n=34 OLs in 28 mbp:meGFP expressing animals, and n=56 OLs in 37 mbp:meGFP and cast mRNA 300pg expressing animals. Graph shows mean and SD. Data from 4 separate experimental sessions. Two-tailed t-test, p=0.0173).

  6. Supplementary Figure 6 No correlation between Ca2+ transient duration or amplitude and speed of sheath elongation

    Comparison of the rate of myelin sheath elongation shows no correlation with the average amplitude (A) or average duration (B) of 162 stabilised sheaths in 18 animals. Data from 9 separate experiments. Pearson’s Correlation Tests, p=0.11 and p=0.74.

  7. Supplementary Figure 7 Schematic model of relationship between Ca2+ activity and myelination

    Schematic overview of Ca2+ transients observed at early stages of myelination and proposed consequences indicated by arrows. The oligodendrocyte (OL) first forms myelin sheaths (left), which will either be retracted (top) or stabilised and elongate (bottom). Localised high-amplitude Ca2+ transient activity is observed in sheaths that become retracted, in a calpain-dependent manner, as indicated by arrows in top. Stabilised myelin sheaths with a high frequency Ca2+ activity elongate rapidly as indicated in the bottom panels.

Supplementary information

  1. Supplementary Text and Figures

    Supplementary Figures 1–7

  2. Life Sciences Reporting Summary

  3. Supplementary Table 1

    Confidence intervals for raw data distributions

  4. Supplementary Video 1 – 2D high-speed imaging of Ca2+ transients

    200 second sample from a time-lapse movie of a GCaMP6s expressing oligodendrocyte imaged in a single plane at 4Hz. Red arrowhead points to a sheath exhibiting a Ca2+ transient at the relevant time-point indicated in Supplementary Fig. 1A with the relevant traces in top graph in Supplementary Fig.1C. White arrowhead points to a sheath exhibiting another Ca2+ transient. 20 myelin sheaths were imaged in 12 different animals in 4 separate experiments.

  5. Supplementary Video 2 – Time-lapse movie of GCaMP6s expressing oligodendrocyte

    Time-lapse movie of GCaMP6s expressing cell shown in Figure 1. Image frames are Maximum Intensity Projections of individual 3D z-stacks. Frame interval 2.89s and imaged continuously for 21 minutes. Arrowheads points to sheaths exhibiting a Ca2+ transient at the relevant time-point. White arrowhead points to a Ca2+ transient occurring in another sheath, which is not sufficiently isolated in the z-axis to be analysed in Fig. 1E. Arrows point to sheaths without any evidence of Ca2+ activity. See corresponding traces in Fig. 1E. Representative movie of 13 cells imaged in 13 different animals in 9 separate experiments.

  6. Supplementary Video 3 – Time-lapse movie of GCaMP6s expressing oligodendrocyte at early stage of myelination

    Time-lapse movie of GCaMP6s expressing cell shown in Supplementary Figure 2 (top panels). Frames are Maximum Intensity Projections of individual 3D z-stacks. Frame interval 2.5s, imaged continuously for 20 mins. Arrowheads point to sheaths exhibiting Ca2+ transients at relevant time-point. Representative movie of 40 cells imaged in 40 different animals in 25 separate experiments.

  7. Supplementary Video 4 – Directionality of Ca2+ transients over time in a sheath subsequently retracted

    100 time-points from a time-lapse movie of the GCaMP6s expressing cell shown in Figure 2 and Supplementary Figure 3. Image frames are Maximum Intensity Projections of individual 3D z-stacks, frame interval 2.6s and imaged continuously for 20 minutes, as part of the extended time-lapse experiment. Arrowheads point to either the sheath or proximal process to indicate directionality of Ca2+ transient propagation. Representative movie of 12 cells imaged in 12 different animals in 9 separate experiments.

  8. Supplementary Video 5 – Time-lapse movie of myelin sheath formation by oligodendrocyte in control animal

    180 time-points from a time-lapse movie of meGFP expressing cell oligodendrocyte in a DMSO treated animal shown in Figure 2F. Image frames are Maximum Intensity Projections of individual 3D z-stacks, frame interval 5 minutes and imaged continuously from 81-96hpf. Arrowheads point to sheaths fully retracted over time. Representative movie of 14 cells imaged in 14 different animals in 2 separate experiments.

  9. Supplementary Video 6 – Time-lapse movie of myelin sheath formation by oligodendrocyte in PD150606-treated animal

    180 time-points from a time-lapse movie of meGFP expressing cell oligodendrocyte in a 75µM PD150606 treated animal shown in Figure 2F. Image frames are Maximum Intensity Projections of individual 3D z-stacks, frame interval 5 minutes and imaged continuously from 81-96hpf. Arrowheads point to sheaths fully retracted over time. Representative movie of 34 cells imaged in 34 different animals in 4 separate experiments.

  10. Supplementary Software

    Custom script to perform blinded analyses of imaging data.

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DOI

https://doi.org/10.1038/s41593-017-0040-x

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