Motor learning promotes remyelination via new and surviving oligodendrocytes

Abstract

Oligodendrocyte loss in neurological disease leaves axons vulnerable to damage and degeneration, and activity-dependent myelination may represent an endogenous mechanism to improve remyelination following injury. Here we report that, while learning a forelimb reach task transiently suppresses oligodendrogenesis, it subsequently increases oligodendrocyte precursor cell differentiation, oligodendrocyte generation and myelin sheath remodeling in the forelimb motor cortex. Immediately following demyelination, neurons exhibit hyperexcitability, learning is impaired and behavioral intervention provides no benefit to remyelination. However, partial remyelination restores neuronal and behavioral function, allowing learning to enhance oligodendrogenesis, remyelination of denuded axons and the ability of surviving oligodendrocytes to generate new myelin sheaths. Previously considered controversial, we show that sheath generation by mature oligodendrocytes is not only possible but also increases myelin pattern preservation following demyelination, thus presenting a new target for therapeutic interventions. Together, our findings demonstrate that precisely timed motor learning improves recovery from demyelinating injury via enhanced remyelination from new and surviving oligodendrocytes.

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Fig. 1: Forelimb reach training modulates oligodendrogenesis and remodeling of preexisting myelin sheaths.
Fig. 2: Forelimb reach learning increases OPC differentiation.
Fig. 3: Demyelination results in incomplete oligodendrocyte replacement and functional deficits in motor cortex.
Fig. 4: Myelin sheath numbers on new oligodendrocytes are regulated during remyelination.
Fig. 5: Motor learning modulates oligodendrogenesis after demyelination in a timing-dependent manner.
Fig. 6: Delayed motor learning promotes remyelination via new oligodendrocytes.
Fig. 7: Delayed motor learning stimulates surviving mature oligodendrocytes to contribute to remyelination.

Data availability

All data that support the findings, and the tools and reagents will be shared on an unrestricted basis; requests should be directed to the corresponding author.

Code availability

All published code will be shared on an unrestricted basis; requests should be directed to the corresponding authors.

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Acknowledgements

We thank A. Chavez for technical assistance; A. Scallon and the Optogenetics and Neural Engineering Core (P30NS048154); D. Stitch and the University of Colorado Anschutz Medical Campus Advance Light Microscopy Core (P30NS048154); M. Hall for machining expertise; S. Bromley-Coolidge for assistance with reach videos; S. Rock Levinson for discussions on 3P logistic equation modeling; B. Cudmore (UC Davis College of Biological Sciences) for image processing expertise; M. Rasband (Baylor College of Medicine) for providing the βIV spectrin (C9581) antibody; M. Bhat (UT Health San Antonio) for providing the Caspr antibody; and members of the Hughes and Welle labs for discussions. M.A.T. is supported by the NIH Institutional Neuroscience Graduate Training Grant (5T32NS099042-17). Funding was provided by NIH 1R21EY029458-01 and the Boettcher Foundation Webb–Waring Biomedical Research Award to C.G.W. and the Boettcher Foundation Webb–Waring Biomedical Research Award, the Whitehall Foundation, the Conrad N. Hilton Foundation (17324), and the National Multiple Sclerosis Society (RG-1701–26733) and NINDS (NS106432, NS115975) to E.G.H.

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Contributions

E.G.H. and C.R.M. conceived the project. C.M.B. designed, analyzed and generated Figs. 1, 4, 6 and 7, Extended Data Figs. 2, 4, 8–10 and Supplementary Videos 2–17. H.J.B. designed, analyzed and generated Figs. 1, 3, 5 and 6, Extended Data Figs. 1, 3, 5–7, Supplementary Fig. 1 and Supplementary Video 1. C.R.M. contributed to the data analysis in Figs. 1, 3 and 5 and Extended Data Figs. 1 and 4–6. M.A.T. designed, analyzed and generated Fig. 2 and Extended Data Figs. 2–4. D.N. performed all electrophysiological experiments and analyses. C.G.W. supervised the electrophysiological experiments. E.G.H. supervised the project. C.M.B., H.J.B. and EGH wrote the manuscript with input from other authors.

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Correspondence to Ethan G. Hughes.

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The authors declare no competing interests.

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Extended data

Extended Data Fig. 1 Learning and rehearsal of a forelimb reach task induce skill refinement.

a, A large majority (93%) of mice successfully learn to perform the forelimb reach task. ‘Learners’ (black) gradually improve their reaching performance over the seven days of training, whereas ‘non-learners’ (grey) show a progressive decrease in success rate and eventually stop making reach attempts around day 4. Note, the lone point in the ‘non-learner’ group at day 7 is due to only one mouse making attempts on the last day of training. The other two mice had stopped trying. b, Successful reaches (%) significantly increase between learning days 1 and 7 (paired samples t-test; t(6) = 4.80, p = 0.003) for mice placed in ‘learning’ group. c, Peak performance during rehearsal (successful reaches; %) is significantly higher than peak performance during learning (paired samples t-test; t(6) = 5.47, p = 0.0016) for mice placed in ‘rehearsal’ group. Individual colors and traces reflect performance by individual mice. *p < 0.05, **p < 0.01, ***p < 0.001. Bars and errors represent Mean ± SEM, for statistics see Supplementary Table 2.1.

Extended Data Fig. 2 In vivo imaging of MOBP-EGFP faithfully reflects myelin sheath presence, length, and connection to oligodendrocyte cell body.

a, b, Maximum projections of cortical oligodendrocytes showing 98.24 ± 0.92% colocalization of in vivo MOBP-EGFP and SCoRe signal in myelin sheaths, confirming MOBP-EGFP faithfully reflects presence of myelin (ANOVA, nmice = 3, F2,6 = 5596.220, p < 0.0001). c, Maximum projection of 4% paraformaldehyde fixed tissue, stained for myelin (blue, MBP), paranodes (Caspr, red), and sodium channels (NavPan, green). d, No significant difference between sheath lengths measured using Simple Neurite Tracer in in vivo two-photon images of control and cuprizone-treated MOBP-EGFP mice, and in confocal images of sheaths immunostained for MBP in fixed tissue (nsheaths = 306, 297 and 233, respectively; points represent individual sheaths; ANOVA; F2,833 = 2.53, p > 0.08; red points and error bars represent group means ± SEM). e,f Semi-automated tracing with Simple Neurite Tracer faithfully reconstructs oligodendrocyte myelin sheaths and their connecting processes to the cell soma in layer I (e) and layer II/II (f). Top left: Maximum projection of an oligodendrocyte (OL) imaged using in vivo two-photon microscopy, spanning a depth of 3–33 μm (e) and 138–186 μm (f) in motor cortex. Bottom left: maximum projection of an isolated single sheath and process attached to the oligodendrocyte cell body. Center: maximum projection and pseudo-colored sheath and process (sheath and process pseudo-colored). Right: Three-dimensional (3D) reconstruction of the same oligodendrocyte generated from the raw in vivo imaging data using the Simple Neurite Tracer plugin in FIJI. View of 3D volume in xy plane from below (top) and view of 3D volume through z (bottom). *p < 0.05, **p < 0.01, ***p < 0.001. Bars and errors represent Mean ± SEM, for statistics see Supplementary Table 2.1.

Extended Data Fig. 3 Oligodendrocyte lineage cell dynamics throughout motor learning.

a, Genetic lines for in vivo imaging of oligodendrocyte precursor cells (OPCs; NG2-mEGFP) and oligodendrocytes (OLs; MOBP-EGFP). b, Motor cortex oligodendrogenesis from age 10–20 weeks across six mice, showing a plateau ~17 weeks. Dashed box represents age during standard experimental timeline. c, Rate of oligodendrogenesis is altered in learning vs. untrained mice during learning (Wilcoxon Rank-Sum, p = 0.014), days 8–18 post-learning (p = 0.038), and days 14–24 post-learning (p = 0.024). No differences are observed by days 25–35 post-learning (p > 0.9). Points represent mice. d, Main effect of diet restriction on oligodendrogenesis rate (%; ANOVA; F2,8 = 18.13, p = 0.001). Diet-restricted and non-diet-restricted controls have higher rates of oligodendrogenesis than diet-restricted learning mice (Tukey’s HSD, p = 0.001 and p = 0.005, respectively). e, Mean success rate is related to fold change in oligodendrogenesis rate post-learning (R-square = 0.98, p = 0.01). Line and shaded area represent fit and 95% confidence of fit. f, Trained mice (learning and rehearsal) have increased maximum rates of oligodendrogenesis relative to controls (t(10.61) = −2.49, p = 0.03). g, h, Proliferation rates from Fig. 2d; colors represent individual mice. All mice show reduced proliferation rate during learning relative to baseline (t(4) = −3.89, p = 0.018; paired student’s t-test), but no main effect of time on proliferation rate across the five weeks of experiment - possibly due to high variability post-learning (F4,15 = 2.341). i, Only a minority of proliferation and differentiation events occurred in OPCs that had migrated into the field of view throughout the course of the experiment. j, No effect of learning on rate of migration into or out of the field of view. *p < 0.05, **p < 0.01, ***p < 0.001. Bars and errors represent Mean ± SEM, for statistics see Supplementary Table 2.1.

Extended Data Fig. 4 Cuprizone treatment results in loss of myelin and oligodendrocytes.

a, b, Mean density of EGFP+ and PDGFRα + cells in control and cuprizone-treated MOBP-EGFP mice; individual points represent individual mice. Interaction effect between drug (control vs. cuprizone) and cell type (EGFP+ vs PDGFRα + ; (F1,5 = 22.39, p = 0.0052) to predict cell density. While EGFP+ cell density is decreased in cuprizone-treated mice relative to controls (Tukey’s HSD; p = 0.0086), there is no difference in PDGFRα+ cells between groups (p > 0.5). c, Maximum projection of a EGFP+/ASPA+ oligodendrocyte (top) and a EGFP+/ASPA oligodendrocyte (bottom). Note the large size of the ASPA/EGFP+ cell soma suggesting it is a recently born oligodendrocyte in the early stages of the maturation process. d, After three weeks of cuprizone treatment, 70.73 ± 12.78% of oligodendrocytes are EGFP+/ASPA+, 0.97 ± 0.84% of cells are ASPA+/EGFP, while the remainder are EGFP± only (nmice=3, ncells = 185). e, Maximum projection of an MBP+ myelin sheath with (top) and without EGFP (bottom) after three weeks of cuprizone. f, After three weeks of cuprizone, 76.21 ± 7.11% of sheaths are MBP+/EGFP+, and 20.6 ± 5.79% of sheaths are MBP+/EGFP (nmice=3, nsheaths = 351). g, h Maximum projections of oligodendrocytes showing colocalization of in vivo MOBP-EGFP and SCoRe imaging for myelin both before cuprizone administration (−21 days) and immediately following its removal (0 days). Note the surviving sheath (white arrow). i, Following 3 weeks of cuprizone diet, most myelin sheaths are MOBP-EGFP+/SCoRe+ (95.71 ± 1.16; ANOVA, F2,6 = 2012.94, p < 0.0001). j, Cuprizone administration modulates sheath density (F2,10 = 14.43, p = 0.001). Cuprizone-fed mice have a reduced density of MOBP-EGFP+/SCoRe+ positive sheaths relative to controls (p = 0.0001), but no difference in GFP-only or SCoRe-only sheaths. *p < 0.05, **p < 0.01, ***p < 0.001. Bars and errors represent Mean ± SEM, for statistics see Supplementary Table 2.3.

Extended Data Fig. 5 Dynamics of oligodendrocyte generation and loss during cuprizone treatment.

a, Oligodendrocyte loss occurred evenly across cortical depths. Shaded area represents cuprizone diet. b, Oligodendrogenesis is suppressed during cuprizone diet (n = 5 mice per group; t(6.54) = 4.10, p = 0.005; Student’s t-test). c, 85% of oligodendrocytes generated during cuprizone diet die within three weeks. d, Oligodendrocyte loss predicts gain (Spearman’s ρ = 0.89). e, Oligodendrocytes generated during remyelination are distributed across cortex similarly to developmental oligodendrogenesis (Wilcoxon Rank-sum, p > 0.1). f, Remyelination alters oligodendrogenesis rates (F29,2 = 27.67, p < 0.0001; ANOVA). Rates are higher during remyelination than in healthy trained and untrained mice (p < 0.0001 and p < 0.0001, respectively; Tukey’s HSD). g–i, Inter-individual variation in oligodendrocyte gain and loss is controlled for by normalizing gain to loss (‘oligodendrocyte replacement’). j, k, Representative diagram and images of peri-electrode immunohistochemistry. Myelinated neurons within 150 microns of the electrode (indicated with white arrowhead; layer II/III XZ maximum projection) were co-labelled with MBP (myelin; cyan), beta-IV spectrin (axon initial segment; purple) and NeuN/NFH (neuron cell soma / distal axon; green; top), whereas unmyelinated axons did not co-localize with MBP (bottom). l, Cuprizone administration alters peri-probe axonal myelination (Two-way ANOVA; F3,8 = 110.51, p < 0.0001). Control mice have more myelinated versus unmyelinated axons (Tukey’s HSD, p < 0.0001). At the cessation of cuprizone, cuprizone-fed mice have fewer myelinated (p < 0.0001) and more unmyelinated axons than healthy controls (p < 0.0001), and more unmyelinated than myelinated axons (p = 0.004). Note: myelin may be present elsewhere on the axon. m, The proportion of unmyelinated neurons observed via IHC does not differ from the proportion of myelin loss predicted by sigmoidal demyelination characterized in Fig. 3e-h (one-sample t-test, t(2) = 1.10, p > 0.3). *p < 0.05, **p < 0.01, ***p < 0.001. Bars and errors represent Mean ± SEM, points represent individual mice, for statistics see Supplementary Table 2.3.

Extended Data Fig. 6 Demyelination induces deficits in early, but not delayed, motor learning.

a, Timeline for ‘early-learning’ intervention (3 days post-cuprizone). b, No difference in mean reach attempts per session during early-learning between control and cuprizone-treated mice (Student’s t-test, t(12.95) = 0.05, p > 0.9; coloured lines represent group means). c, Area plot of reach attempt outcome (success vs. failure) across forelimb reach learning days in both control and cuprizone-treated mice. d, Control mice have improved success rates day 7 of training relative to day 1 (Paired Student’s T-test; t(6) = 4.7, p = 0.003), but cuprizone-treated mice do not (t(7) = 1.96, p = 0.09). e, Maximum oligodendrocyte loss is related to peak performance during training (R2 = 0.95, p = 0.02; line and shaded area represent line of fit and 95% confidence). f, No relationship between mean learning success rate (%) and asymptote of oligodendrocyte replacement in early learners. g, Timeline for ‘delayed-learning’ intervention (10 days post-cuprizone) h, No difference in mean reach attempts per session during delayed-learning between control and cuprizone-treated mice (Student’s t-test, t(12.95) = 1.54, p > 0.1; coloured lines represent group means). i, Area plot of reach attempt outcome (success, rudimentary error, intermediate error, advanced error; see Supplementary Video 1) across delayed-learning days in both control and cuprizone-treated mice. j, Both control and cuprizone-treated mice improve their reaching success between days 1 and 7 of delayed-learning (Paired student’s t-test, p = 0.0005 and p = 0.004, respectively). k, No relationship between maximum oligodendrocyte loss and reaching performance during delayed learning. l, No relationship between delayed learning success rate and asymptote of oligodendrocyte replacement post-cuprizone. *p < 0.05, **p < 0.01, ***p < 0.001. Points represent individual mice, for statistics see Supplementary Table 2.5.

Extended Data Fig. 7 Motor skill rehearsal does not modulate remyelination.

a, Timeline of reach task rehearsal post cuprizone diet. b, Main effect of drug on reaching success during rehearsal (F(1,14) = 27.73, p < 0.0001). c,d, No effect of rehearsal on rate, inflection point, or asymptote of oligodendrocyte replacement. e, No effect of cuprizone on change in reaching behavior between learning and rehearsal. f, Area plot of reach attempt outcomes in control and cuprizone-demyelinated mice. g, Interaction effect between performance phase (learning vs. rehearsal) and drug (control vs. cuprizone) to predict success rate (F(1) = 4.62, p = 0.04). While control and cuprizone mice do not differ in success rate during pre-cuprizone learning, control mice perform significantly better during rehearsal relative to cuprizone-treated mice (Tukey’s HSD, p = 0.0004). Both cuprizone and cuprizone-treated mice have improved performance during rehearsal relative to learning (p = 0.0001 and p < 0.0001, respectively). h, No relationship between peak oligodendrocyte loss post-cuprizone and peak reaching success rate during rehearsal. i, No relationship between rehearsal success rate and asymptote of oligodendrocyte replacement. *p < 0.05, **p < 0.01, ***p < 0.001. Bars and errors represent Mean ± SEM, points represent individual mice, for statistics see Supplementary Table 2.5.

Extended Data Fig. 8 Identification of oligodendrocytes that survive demyelination.

a, Representative image outlining the methodology for following surviving oligodendrocytes over time. Single plane image of the same oligodendrocyte at baseline (−25d), one week after demyelination (7d), and six weeks after demyelination (44d). Red boxes highlight one example of the same oligodendrocyte processes lasting for the duration of the study. The maintenance of the spatial relationship between the oligodendrocyte of interest and other oligodendrocytes in the field of view (yellow arrowheads) provide further confirmation of oligodendrocyte identity. Note the new cell that appears at 7d. b, Change in centroid position of reference oligodendrocytes within the z-stack and surviving cell bodies from baseline to day of peak remodeling—that is the day where the largest number of sheaths were added by a given oligodendrocyte. c, Surviving oligodendrocytes at baseline are significantly smaller than new oligodendrocytes (t(21.91) = −5.81, p < 0.0001, Student’s t-test). d, Change in volume of surviving oligodendrocytes from baseline to peak remodeling is significantly smaller than the volume of new oligodendrocytes (t(23.88 = −7.59, p < 0.0001). e, Dynamics of sheath addition over time. Each line represents an individual oligodendrocyte.*p < 0.05, **p < 0.01, ***p < 0.001. Bars and errors represent Mean ± SEM, box plots represent Median and IQR, for statistics see Supplementary Table 2.7.

Extended Data Fig. 9 Dynamics of pre-existing and newly-generated myelin sheaths from surviving oligodendrocytes.

a, No oligodendrocytes are lost in healthy mice. b, No difference in percent of oligodendrocytes (OLs) surviving demyelination in untrained and delayed learning groups (Wilcoxon Rank-Sum, p > 0.5). c, d, No sheaths are lost (c) nor generated (d) on mature oligodendrocytes in healthy trained or untrained conditions. e, Behavior of pre-existing myelin sheaths that persist throughout study. Relevant sheaths are pseudo colored. f, Three weeks into remyelination, sheath retraction is significantly increased (F(3,22) = 18.65, p < 0.0001) when compared to age-matched controls (Tukey’s HSD, p = 0.0006) and when compared to the percent of sheaths growing in cuprizone-treated mice (p < 0.0001). g, No effect of delayed learning on sheath dynamics during remyelination. Sheaths retract more than they grow in both untrained (p = 0.016) and delayed learning mice (p = 0.0003). h, Maximum projection of new sheaths generated after cuprizone exhibiting growth (pseudo colored green, left) and retraction (pseudo colored red, right). i, New myelin sheaths change in length in the week following their generation, whether they are from new oligodendrocytes (control: F(3,302) = 47.94, p < 0.0001) or from surviving oligodendrocytes after cuprizone-demyelination (cuprizone diet: F(3,29) = 5.31, p = 0.0049). Sheaths in both control and cuprizone treatment stabilize their length within 3 days of sheath birth (d0 vs. d3, p < 0.0001 in control and p = 0.028 in cuprizone; Tukey’s HSD). Line and shading represent mean and SEM. j, Sheaths from pre-existing oligodendrocytes grow more often than they retract the first three days post-generation (Wilcoxon Rank-Sum, p = 0.0029). *p < 0.05, **p < 0.01, ***p < 0.001. Bars and errors represent Mean ± SEM, for statistics see Supplementary Table 2.7.

Extended Data Fig. 10 Surviving oligodendrocyte cell soma volume changes during remyelination.

a, Maximum projection of surviving oligodendrocyte cell bodies at baseline (left, magenta), peak remodeling (middle, cyan), and overlaid (right). Scale bar is 10 μm. b, Oligodendrocytes in normal untrained mice display little change in cell body volume throughout the study, from baseline (0d) to 43d. Surviving cells in delayed learning mice show dramatic increase in cell soma volume from baseline to day of peak remodeling when compared to oligodendrocytes in normal untrained mice (t(12.24) = 2.56, p = 0.025, Student’s t-test). c, Percent change in volume between baseline and day of sheath addition for surviving cells engaging in remodeling.*p < 0.05, **p < 0.01, ***p < 0.001. Bars and errors represent Mean ± SEM, for statistics see Supplementary Table 2.7.

Supplementary information

41593_2020_637_MOESM3_ESM.mov

Categorization of reach attempts during forelimb reach training. Categorization of four different possible outcomes of reach attempts during forelimb reach task. ‘Success’ involves correct targeting, grasping and retrieval of the pellet inside the training box. ‘Reach error’ (rudimentary) involves incorrect targeting of the pellet—no contact with the pellet is made. During a ‘grasp error’ (intermediate), the mouse correctly reaches for the pellet but does not successfully grasp its paw around the pellet. During a ‘retrieval error’ (advanced), the mouse is able to reach and grasp onto the pellet, but does not successfully retrieve it into the box.

41593_2020_637_MOESM4_ESM.mov

Developmental oligodendrogenesis in the motor cortex. Maximum intensity projection of 0–336 µm of the forelimb region of the motor cortex across 43 days of two-photon in vivo imaging in a MOBP–EGFP mouse implanted with a cranial window. New EGFP-positive oligodendrocytes (labeled in green) are generated across the scope of experiment, when the mouse is aged 10–17 weeks. Developmental oligodendrogenesis begins to slow after 16 weeks of age. (Frame rate: 2 frames per second).

41593_2020_637_MOESM5_ESM.mov

Using Simple Neurite Tracer to trace the majority of the oligodendrocyte arbor. In vivo two-photon imaging of sheaths from a mature oligodendrocyte. This field was acquired in the motor cortex of a MOBP–EGFP mouse implanted with a cranial window (P63; depth = 3–51 μm) and follows all traceable processes through z in both single plane images and with reconstructions. Note the yellow arrow pointing to the node of Ranvier and rotating 3D reconstruction of oligodendrocyte cell body (white), sheaths (magenta), and processes (green). (Frame rate: 15 frames per second).

41593_2020_637_MOESM6_ESM.mov

Categorization of existing sheath dynamics: retracting. In vivo two-photon imaging of the retraction of a myelin sheath from an existing oligodendrocyte in the motor cortex of a MOBP–EGFP mouse implanted with a cranial window (P69; depth = 3–9 μm). The sheath begins continuous retraction within the first week (7 d). Note the extended time period for which the sheath is retracting (7 d–39 d). (Frame rate: 2 frames per second).

41593_2020_637_MOESM7_ESM.mov

Categorization of existing sheath dynamics: growing. In vivo two-photon imaging of the growth of a myelin sheath from an existing oligodendrocyte in the motor cortex of an MOBPEGFP mouse implanted with a cranial window (P69; depth = 9–18 μm). This myelin sheath (green) is stable for 43 days, then extends over the next 19 days. (Frame rate: 2 frames per second).

41593_2020_637_MOESM8_ESM.mov

Dynamics of demyelination and remyelination. In vivo two-photon imaging of a field undergoing de- and remyelination in motor cortex of a MOBP–EGFP mouse implanted with a cranial window (P68; depth = 0–336 μm) and treated with 0.2% cuprizone diet for 3 weeks. Images were acquired every 2–3 days for 43 days. In this 3-week partial demyelination model, ~90% of EGFP-positive expressing oligodendrocytes are lost relative to baseline (−21 d) number and ~50% of oligodendrocytes are regained three weeks into remyelination (21 d). (Frame rate: 2 frames per second).

41593_2020_637_MOESM9_ESM.mov

Cuprizone-mediated myelin and oligodendrocyte loss. In vivo two-photon imaging of an individual oligodendrocyte undergoing demyelination and death in motor cortex of a MOBP–EGFP mouse implanted with a cranial window (P63; depth = 3–18 μm) and treated with 0.2% cuprizone diet for 3 weeks. Images were acquired every 2–3 days for 43 days. Note loss of EGFP-positive myelin sheaths at the end of cuprizone treatment (0 d) and loss of cell body 1.5 weeks later (11 d). (Frame rate: 2 frames per second).

41593_2020_637_MOESM10_ESM.mov

Myelin sheath generation by a new oligodendrocyte in the healthy brain. In vivo two-photon imaging of the myelin sheaths from a newly generated oligodendrocyte in a healthy mouse, as well as semiautomated traces that highlight the connection to the oligodendrocyte cell body for each sheath counted. This field was acquired in the motor cortex of a MOBP–EGFP mouse implanted with a cranial window (P67; depth = 3–57 μm). This video contains two time-points: 2 days pre-generation (−2 d) and 7 days post-generation (7 d). This newly generated oligodendrocyte forms a total of 40 new sheaths, 36 days after baseline imaging. This video is divided into three parts: first, maximum projections of each time point of the entire 54-μm z stack, which correspond to the figure images in Fig. 4a (top, control diet); second, single plane images of the z dimension; and third, the same images overlaid with semiautomated tracings. These semiautomated tracings were used to verify the connection between newborn sheaths and the newborn oligodendrocyte cell body. (Frame rate: 2 frames per second).

41593_2020_637_MOESM11_ESM.mov

Increased myelin sheath generation by a newly generated oligodendrocyte during remyelination. In vivo two-photon imaging of the myelin sheaths from a newly generated oligodendrocyte during the first week of remyelination, as well as semiautomated traces that highlight the connection to the oligodendrocyte cell body for each sheath counted. This field was acquired in the motor cortex of a MOBP–EGFP mouse implanted with a cranial window (P64; depth = 21–111 μm). This video contains two time-points: 2 days pre-generation (−2 d) and 7 days post-generation (7 d). This oligodendrocyte is generated during the first week of remyelination, 4 days after the cessation of cuprizone diet, and forms a total of 53 new sheaths. This video is divided into three parts: first, maximum projections of each time point of the entire 90-μm z stack, which correspond to the figure images in Fig. 4a (bottom, cuprizone diet); second, single plane images of the z dimension; and third, the same images overlaid with semiautomated tracings. These semiautomated tracings verify the connection between new sheaths and the oligodendrocyte cell body. (Frame rate: 2 frames per second).

41593_2020_637_MOESM12_ESM.mov

Generation of a remodeling myelin sheath by a newly generated oligodendrocyte during remyelination. In vivo two-photon imaging of the generation of a remodeling myelin sheath from a newly generated oligodendrocyte during the second week of remyelination, as well as semiautomated traces of the connection to the oligodendrocyte cell body for this myelin sheath. This field was acquired in the motor cortex of a MOBP–EGFP mouse implanted with a cranial window (P65; depth = 15–24 μm). This video contains three time-points: baseline (−25 d; note unmyelinated section), 4 days post-cuprizone (4 d) and 11 days post-cuprizone (11 d; note new oligodendrocyte and sheath). This oligodendrocyte was generated during the second week of remyelination and forms a new sheath on a never-before myelinated location. The yellow arrow corresponds to the eventual location of the junction between the new oligodendrocyte process and the new remodeling sheath. This video is divided into three parts: first, maximum projections of each time point of the entire 9-μm z stack, which corresponds to Fig. 4h (top, remodeling); second, single plane images of the z dimension, with relevant sheaths pseudocolored; and third, the same images overlaid with semiautomated tracings. These semiautomated tracings verify the connection between the new remodeling sheath and the new oligodendrocyte cell body. (Frame rate: 2 frames per second).

41593_2020_637_MOESM13_ESM.mov

Generation of a remyelinating sheath by a new oligodendrocyte during remyelination. In vivo two-photon imaging of the generation of a remyelinating myelin sheath from a new oligodendrocyte during the second week of remyelination, as well as semiautomated traces of the connection to the oligodendrocyte cell body for relevant sheaths. This field was acquired in the motor cortex of a MOBP–EGFP mouse implanted with a cranial window (P65; depth = 0–24 μm). This video contains three time-points: baseline (−25 d; note colored oligodendrocyte and sheath), 7 days post-cuprizone (7 d; note loss of baseline oligodendrocyte) and 11 days post-cuprizone (11 d; note new oligodendrocyte and sheath). The original myelin sheath and oligodendrocyte are lost in between 4 and 7 days after the cessation of cuprizone, and this location is then immediately remyelinated by a new oligodendrocyte, generated 7–9 days after the cessation of cuprizone diet. This video is divided into three parts: first, maximum projections of each time point of the entire 24-μm z stack, which correspond to the figure images in Fig. 4h (bottom, remyelinating); second, single plane images of the z dimension, with relevant sheaths pseudocolored; and third, the same images overlaid with semiautomated tracings through z. These semiautomated tracings verify the connection between the original oligodendrocyte cell body and original myelin sheath, as well as the connection between the new remyelinating sheath and the oligodendrocyte cell body. (Frame rate: 2 frames per second).

41593_2020_637_MOESM14_ESM.mov

Loss of a preexisting myelin sheath on a surviving oligodendrocyte. In vivo two-photon imaging of the loss of a preexisting myelin sheath from a surviving oligodendrocyte during and after the administration of cuprizone diet, as well as semiautomated traces of the connection to the oligodendrocyte cell body for this sheath. This field was acquired in the motor cortex of a MOBP–EGFP mouse implanted with a cranial window (P63; depth = 45–54 μm). This video contains three time-points: baseline (−25 d), 11 days pre-cuprizone cessation (−11 d) and 44 days post-cuprizone (44 d). Myelin sheath loss is a protracted process that, in this example, occurred over 29 days. This video is divided into three parts: first, maximum projections of each time point of the entire 9-μm z stack, which correspond to the figure images in Fig. 7b; second, single plane images of the z dimension, with relevant sheaths pseudocolored; and third, the same images overlaid with semiautomated tracings. These semiautomated tracings verify the connection between the surviving oligodendrocyte cell body and the lost myelin sheath. (Frame rate: 2 frames per second).

41593_2020_637_MOESM15_ESM.mov

Generation of a new myelin sheath by a surviving oligodendrocyte. In vivo two-photon imaging of the generation of a new myelin sheath from a surviving oligodendrocyte during remyelination, as well as semiautomated traces of the connection to the oligodendrocyte cell body for this sheath. This field was acquired in the motor cortex of a MOBP–EGFP mouse implanted with a cranial window (P63; depth = 51–66 μm). In contrast to sheath loss, sheath addition is a rapid process that results in a stable sheath within 3 days of initial myelin generation. This video contains four time-points: baseline (−25 d), 11 days post-cuprizone (11 d; note extensive sheath loss and presence of myelin debris), 18 days post-cuprizone (18 d; note brightening of oligodendrocyte cell body and cessation of sheath loss) and 44 days post-cuprizone (44 d; note addition of new sheaths by the surviving oligodendrocyte). This video is divided into three parts: first, maximum projections of each time point of the entire 15-μm z stack, which correspond to the figure images in Fig. 7c; second, single plane images of the z dimension, with relevant sheaths pseudocolored; and third, the same images overlaid with semiautomated tracings. These semiautomated tracings verify the connection between the surviving oligodendrocyte cell body and the new myelin sheath. (Frame rate: 2 frames per second).

41593_2020_637_MOESM16_ESM.mov

Extended generation of new sheaths by a surviving oligodendrocyte. In vivo two-photon imaging of the generation of myelin sheaths from a preexisting oligodendrocyte during remyelination. This field was acquired in the motor cortex of a MOBP–EGFP mouse implanted with a cranial window (P63; depth = 3–42 μm). This video contains two time-points: baseline (−25 d) and 44 days post-cuprizone (44 d). This oligodendrocyte began generating new myelin sheaths (green) on day 3 of learning (11 d) and continues over 33 additional days (44 d). Note that this surviving oligodendrocyte retains a number of sheaths (stable, cyan) for the duration of the imaging time period, in addition to losing sheaths (magenta, lost) and gaining new sheaths (yellow, new). This video is divided into two parts. The first part shows single plane images of the z dimension, overlaid with semiautomated tracings. These semiautomated tracings verify the connection between the surviving oligodendrocyte cell body, its processes and their associated myelin sheaths. The second part shows 3D reconstructions of these semiautomated traces over time. In this section, we show reconstructions of the processes and sheaths with verified connections to the surviving oligodendrocyte cell body over 69 days. (Frame rate: 2 frames per second). NB, a cell body from 44 days post-cuprizone was used in all time points for reconstruction consistency.

41593_2020_637_MOESM17_ESM.mov

Generation of a new myelin sheath by a surviving oligodendrocyte in layer II/III. In vivo two-photon imaging of the generation of a new myelin sheath from a surviving oligodendrocyte in layer II/III during remyelination, as well as semiautomated traces of the connection to the oligodendrocyte cell body for this sheath. This field was acquired in the motor cortex of an MOBP-EGFP mouse implanted with a cranial window (P63; depth = 183–198 μm). This video contains three time-points: baseline (−25d), 5 days post-cuprizone (5d), and 7 days post-cuprizone (7d; note new process and myelin sheath). Note that this sheath runs perpendicular to the imaging plane, in contrast to sheaths generated by layer I oligodendrocytes, which run parallel to the imaging plane. This video is divided into three parts: first, maximum projections of each time point of the entire 15 μm z-stack, which correspond to the figure images in Fig. 7k; second, single plane images of the z dimension, with relevant sheaths pseudo-colored; and third, the same images overlaid with semiautomated tracings. These semiautomated tracings verify the connection between the layer II/III surviving oligodendrocyte cell body and the newly generated myelin sheath. (Frame rate: 2 frames per second).

41593_2020_637_MOESM18_ESM.mov

Generation of a new remodeling sheath by a surviving oligodendrocyte. imaging of the generation of a new, remodeling myelin sheath from a surviving oligodendrocyte during remyelination, as well as semiautomated traces of the connection to the oligodendrocyte cell body for this sheath. This field was acquired in the motor cortex of a MOBP–EGFP mouse implanted with a cranial window (P63; depth = 6–18 μm). This video contains three time-points: baseline (−25 d), 28 days post-cuprizone (28 d; note sheath loss) and 44 days post-cuprizone (44 d; note sheath generation). This video is divided into three parts: first, maximum projections of each time point of the entire 12-μm z stack, which correspond to the figure images in Fig. 7m (top, remodeling); second, single plane images of the z dimension, with relevant sheaths pseudocolored; and third, the same images overlaid with semiautomated tracings. These semiautomated tracings verify the connection between the surviving oligodendrocyte cell body and the newly generated, remodeling myelin sheath. (Frame rate: 2 frames per second).

41593_2020_637_MOESM19_ESM.mov

Generation of a new remyelinating sheath by a surviving oligodendrocyte. In vivo two-photon imaging of sheath loss and subsequent remyelination by the same surviving oligodendrocyte, as well as semiautomated traces of the connection to the oligodendrocyte cell body for relevant sheaths. This video contains three time-points: baseline (−25 d), 28 days post-cuprizone (28 d; note sheath loss) and 44 days post-cuprizone (44 d; note sheath replacement with new connecting process). Sheath loss occurred over a period of 2 weeks following the cessation of the cuprizone diet and, once the sheath was lost by the surviving oligodendrocyte, this same surviving oligodendrocyte replaced the sheath within a week. This field was acquired in the motor cortex of a MOBP–EGFP mouse implanted with a cranial window (P63; depth = 12–18 μm). This video is divided into three parts: first, maximum projections of each time point of the entire 12-μm z stack, which correspond to the figure images in Fig. 7m (bottom, remyelinating); second, single plane images of the z dimension, with relevant sheaths pseudocolored; and third, the same images overlaid with semiautomated tracings. These semiautomated tracings verify the connection between the surviving oligodendrocyte cell body and the newly generated, remyelinating myelin sheath. (Frame rate: 2 frames per second).

Supplementary Information

Supplementary Fig. 1; Supplementary Video legends (for Supplementary Videos 1–17).

Reporting Summary

Supplementary Video 1

Categorization of reach attempts during forelimb reach training. Categorization of four different possible outcomes of reach attempts during forelimb reach task. ‘Success’ involves correct targeting, grasping and retrieval of the pellet inside the training box. ‘Reach error’ (rudimentary) involves incorrect targeting of the pellet—no contact with the pellet is made. During a ‘grasp error’ (intermediate), the mouse correctly reaches for the pellet but does not successfully grasp its paw around the pellet. During a ‘retrieval error’ (advanced), the mouse is able to reach and grasp onto the pellet, but does not successfully retrieve it into the box.

Supplementary Video 2

Developmental oligodendrogenesis in the motor cortex. Maximum intensity projection of 0–336 µm of the forelimb region of the motor cortex across 43 days of two-photon in vivo imaging in a MOBP–EGFP mouse implanted with a cranial window. New EGFP-positive oligodendrocytes (labeled in green) are generated across the scope of experiment, when the mouse is aged 10–17 weeks. Developmental oligodendrogenesis begins to slow after 16 weeks of age. (Frame rate: 2 frames per second).

Supplementary Video 3

Using Simple Neurite Tracer to trace the majority of the oligodendrocyte arbor. In vivo two-photon imaging of sheaths from a mature oligodendrocyte. This field was acquired in the motor cortex of a MOBP–EGFP mouse implanted with a cranial window (P63; depth = 3–51 μm) and follows all traceable processes through z in both single plane images and with reconstructions. Note the yellow arrow pointing to the node of Ranvier and rotating 3D reconstruction of oligodendrocyte cell body (white), sheaths (magenta), and processes (green). (Frame rate: 15 frames per second).

Supplementary Video 4

Categorization of existing sheath dynamics: retracting. In vivo two-photon imaging of the retraction of a myelin sheath from an existing oligodendrocyte in the motor cortex of a MOBP–EGFP mouse implanted with a cranial window (P69; depth = 3–9 μm). The sheath begins continuous retraction within the first week (7 d). Note the extended time period for which the sheath is retracting (7 d–39 d). (Frame rate: 2 frames per second).

Supplementary Video 5

Categorization of existing sheath dynamics: growing. In vivo two-photon imaging of the growth of a myelin sheath from an existing oligodendrocyte in the motor cortex of an MOBPEGFP mouse implanted with a cranial window (P69; depth = 9–18 μm). This myelin sheath (green) is stable for 43 days, then extends over the next 19 days. (Frame rate: 2 frames per second).

Supplementary Video 6

Dynamics of demyelination and remyelination. In vivo two-photon imaging of a field undergoing de- and remyelination in motor cortex of a MOBP–EGFP mouse implanted with a cranial window (P68; depth = 0–336 μm) and treated with 0.2% cuprizone diet for 3 weeks. Images were acquired every 2–3 days for 43 days. In this 3-week partial demyelination model, ~90% of EGFP-positive expressing oligodendrocytes are lost relative to baseline (−21 d) number and ~50% of oligodendrocytes are regained three weeks into remyelination (21 d). (Frame rate: 2 frames per second).

Supplementary Video 7

Cuprizone-mediated myelin and oligodendrocyte loss. In vivo two-photon imaging of an individual oligodendrocyte undergoing demyelination and death in motor cortex of a MOBP–EGFP mouse implanted with a cranial window (P63; depth = 3–18 μm) and treated with 0.2% cuprizone diet for 3 weeks. Images were acquired every 2–3 days for 43 days. Note loss of EGFP-positive myelin sheaths at the end of cuprizone treatment (0 d) and loss of cell body 1.5 weeks later (11 d). (Frame rate: 2 frames per second).

Supplementary Video 8

Myelin sheath generation by a new oligodendrocyte in the healthy brain. In vivo two-photon imaging of the myelin sheaths from a newly generated oligodendrocyte in a healthy mouse, as well as semiautomated traces that highlight the connection to the oligodendrocyte cell body for each sheath counted. This field was acquired in the motor cortex of a MOBP–EGFP mouse implanted with a cranial window (P67; depth = 3–57 μm). This video contains two time-points: 2 days pre-generation (−2 d) and 7 days post-generation (7 d). This newly generated oligodendrocyte forms a total of 40 new sheaths, 36 days after baseline imaging. This video is divided into three parts: first, maximum projections of each time point of the entire 54-μm z stack, which correspond to the figure images in Fig. 4a (top, control diet); second, single plane images of the z dimension; and third, the same images overlaid with semiautomated tracings. These semiautomated tracings were used to verify the connection between newborn sheaths and the newborn oligodendrocyte cell body. (Frame rate: 2 frames per second).

Supplementary Video 9

Increased myelin sheath generation by a newly generated oligodendrocyte during remyelination. In vivo two-photon imaging of the myelin sheaths from a newly generated oligodendrocyte during the first week of remyelination, as well as semiautomated traces that highlight the connection to the oligodendrocyte cell body for each sheath counted. This field was acquired in the motor cortex of a MOBP–EGFP mouse implanted with a cranial window (P64; depth = 21–111 μm). This video contains two time-points: 2 days pre-generation (−2 d) and 7 days post-generation (7 d). This oligodendrocyte is generated during the first week of remyelination, 4 days after the cessation of cuprizone diet, and forms a total of 53 new sheaths. This video is divided into three parts: first, maximum projections of each time point of the entire 90-μm z stack, which correspond to the figure images in Fig. 4a (bottom, cuprizone diet); second, single plane images of the z dimension; and third, the same images overlaid with semiautomated tracings. These semiautomated tracings verify the connection between new sheaths and the oligodendrocyte cell body. (Frame rate: 2 frames per second).

Supplementary Video 10

Generation of a remodeling myelin sheath by a newly generated oligodendrocyte during remyelination. In vivo two-photon imaging of the generation of a remodeling myelin sheath from a newly generated oligodendrocyte during the second week of remyelination, as well as semiautomated traces of the connection to the oligodendrocyte cell body for this myelin sheath. This field was acquired in the motor cortex of a MOBP–EGFP mouse implanted with a cranial window (P65; depth = 15–24 μm). This video contains three time-points: baseline (−25 d; note unmyelinated section), 4 days post-cuprizone (4 d) and 11 days post-cuprizone (11 d; note new oligodendrocyte and sheath). This oligodendrocyte was generated during the second week of remyelination and forms a new sheath on a never-before myelinated location. The yellow arrow corresponds to the eventual location of the junction between the new oligodendrocyte process and the new remodeling sheath. This video is divided into three parts: first, maximum projections of each time point of the entire 9-μm z stack, which corresponds to Fig. 4h (top, remodeling); second, single plane images of the z dimension, with relevant sheaths pseudocolored; and third, the same images overlaid with semiautomated tracings. These semiautomated tracings verify the connection between the new remodeling sheath and the new oligodendrocyte cell body. (Frame rate: 2 frames per second).

Supplementary Video 11

Generation of a remyelinating sheath by a new oligodendrocyte during remyelination. In vivo two-photon imaging of the generation of a remyelinating myelin sheath from a new oligodendrocyte during the second week of remyelination, as well as semiautomated traces of the connection to the oligodendrocyte cell body for relevant sheaths. This field was acquired in the motor cortex of a MOBP–EGFP mouse implanted with a cranial window (P65; depth = 0–24 μm). This video contains three time-points: baseline (−25 d; note colored oligodendrocyte and sheath), 7 days post-cuprizone (7 d; note loss of baseline oligodendrocyte) and 11 days post-cuprizone (11 d; note new oligodendrocyte and sheath). The original myelin sheath and oligodendrocyte are lost in between 4 and 7 days after the cessation of cuprizone, and this location is then immediately remyelinated by a new oligodendrocyte, generated 7–9 days after the cessation of cuprizone diet. This video is divided into three parts: first, maximum projections of each time point of the entire 24-μm z stack, which correspond to the figure images in Fig. 4h (bottom, remyelinating); second, single plane images of the z dimension, with relevant sheaths pseudocolored; and third, the same images overlaid with semiautomated tracings through z. These semiautomated tracings verify the connection between the original oligodendrocyte cell body and original myelin sheath, as well as the connection between the new remyelinating sheath and the oligodendrocyte cell body. (Frame rate: 2 frames per second).

Supplementary Video 12

Loss of a preexisting myelin sheath on a surviving oligodendrocyte. In vivo two-photon imaging of the loss of a preexisting myelin sheath from a surviving oligodendrocyte during and after the administration of cuprizone diet, as well as semiautomated traces of the connection to the oligodendrocyte cell body for this sheath. This field was acquired in the motor cortex of a MOBP–EGFP mouse implanted with a cranial window (P63; depth = 45–54 μm). This video contains three time-points: baseline (−25 d), 11 days pre-cuprizone cessation (−11 d) and 44 days post-cuprizone (44 d). Myelin sheath loss is a protracted process that, in this example, occurred over 29 days. This video is divided into three parts: first, maximum projections of each time point of the entire 9-μm z stack, which correspond to the figure images in Fig. 7b; second, single plane images of the z dimension, with relevant sheaths pseudocolored; and third, the same images overlaid with semiautomated tracings. These semiautomated tracings verify the connection between the surviving oligodendrocyte cell body and the lost myelin sheath. (Frame rate: 2 frames per second).

Supplementary Video 13

Generation of a new myelin sheath by a surviving oligodendrocyte. In vivo two-photon imaging of the generation of a new myelin sheath from a surviving oligodendrocyte during remyelination, as well as semiautomated traces of the connection to the oligodendrocyte cell body for this sheath. This field was acquired in the motor cortex of a MOBP–EGFP mouse implanted with a cranial window (P63; depth = 51–66 μm). In contrast to sheath loss, sheath addition is a rapid process that results in a stable sheath within 3 days of initial myelin generation. This video contains four time-points: baseline (−25 d), 11 days post-cuprizone (11 d; note extensive sheath loss and presence of myelin debris), 18 days post-cuprizone (18 d; note brightening of oligodendrocyte cell body and cessation of sheath loss) and 44 days post-cuprizone (44 d; note addition of new sheaths by the surviving oligodendrocyte). This video is divided into three parts: first, maximum projections of each time point of the entire 15-μm z stack, which correspond to the figure images in Fig. 7c; second, single plane images of the z dimension, with relevant sheaths pseudocolored; and third, the same images overlaid with semiautomated tracings. These semiautomated tracings verify the connection between the surviving oligodendrocyte cell body and the new myelin sheath. (Frame rate: 2 frames per second).

Supplementary Video 14

Extended generation of new sheaths by a surviving oligodendrocyte. In vivo two-photon imaging of the generation of myelin sheaths from a preexisting oligodendrocyte during remyelination. This field was acquired in the motor cortex of a MOBP–EGFP mouse implanted with a cranial window (P63; depth = 3–42 μm). This video contains two time-points: baseline (−25 d) and 44 days post-cuprizone (44 d). This oligodendrocyte began generating new myelin sheaths (green) on day 3 of learning (11 d) and continues over 33 additional days (44 d). Note that this surviving oligodendrocyte retains a number of sheaths (stable, cyan) for the duration of the imaging time period, in addition to losing sheaths (magenta, lost) and gaining new sheaths (yellow, new). This video is divided into two parts. The first part shows single plane images of the z dimension, overlaid with semiautomated tracings. These semiautomated tracings verify the connection between the surviving oligodendrocyte cell body, its processes and their associated myelin sheaths. The second part shows 3D reconstructions of these semiautomated traces over time. In this section, we show reconstructions of the processes and sheaths with verified connections to the surviving oligodendrocyte cell body over 69 days. (Frame rate: 2 frames per second). NB, a cell body from 44 days post-cuprizone was used in all time points for reconstruction consistency.

Supplementary Video 15

Generation of a new myelin sheath by a surviving oligodendrocyte in layer II/III. In vivo two-photon imaging of the generation of a new myelin sheath from a surviving oligodendrocyte in layer II/III during remyelination, as well as semiautomated traces of the connection to the oligodendrocyte cell body for this sheath. This field was acquired in the motor cortex of an MOBP-EGFP mouse implanted with a cranial window (P63; depth = 183–198 μm). This video contains three time-points: baseline (−25d), 5 days post-cuprizone (5d), and 7 days post-cuprizone (7d; note new process and myelin sheath). Note that this sheath runs perpendicular to the imaging plane, in contrast to sheaths generated by layer I oligodendrocytes, which run parallel to the imaging plane. This video is divided into three parts: first, maximum projections of each time point of the entire 15 μm z-stack, which correspond to the figure images in Fig. 7k; second, single plane images of the z dimension, with relevant sheaths pseudo-colored; and third, the same images overlaid with semiautomated tracings. These semiautomated tracings verify the connection between the layer II/III surviving oligodendrocyte cell body and the newly generated myelin sheath. (Frame rate: 2 frames per second).

Supplementary Video 16

Generation of a new remodeling sheath by a surviving oligodendrocyte. imaging of the generation of a new, remodeling myelin sheath from a surviving oligodendrocyte during remyelination, as well as semiautomated traces of the connection to the oligodendrocyte cell body for this sheath. This field was acquired in the motor cortex of a MOBP–EGFP mouse implanted with a cranial window (P63; depth = 6–18 μm). This video contains three time-points: baseline (−25 d), 28 days post-cuprizone (28 d; note sheath loss) and 44 days post-cuprizone (44 d; note sheath generation). This video is divided into three parts: first, maximum projections of each time point of the entire 12-μm z stack, which correspond to the figure images in Fig. 7m (top, remodeling); second, single plane images of the z dimension, with relevant sheaths pseudocolored; and third, the same images overlaid with semiautomated tracings. These semiautomated tracings verify the connection between the surviving oligodendrocyte cell body and the newly generated, remodeling myelin sheath. (Frame rate: 2 frames per second).

Supplementary Video 17

Generation of a new remyelinating sheath by a surviving oligodendrocyte. In vivo two-photon imaging of sheath loss and subsequent remyelination by the same surviving oligodendrocyte, as well as semiautomated traces of the connection to the oligodendrocyte cell body for relevant sheaths. This video contains three time-points: baseline (−25 d), 28 days post-cuprizone (28 d; note sheath loss) and 44 days post-cuprizone (44 d; note sheath replacement with new connecting process). Sheath loss occurred over a period of 2 weeks following the cessation of the cuprizone diet and, once the sheath was lost by the surviving oligodendrocyte, this same surviving oligodendrocyte replaced the sheath within a week. This field was acquired in the motor cortex of a MOBP–EGFP mouse implanted with a cranial window (P63; depth = 12–18 μm). This video is divided into three parts: first, maximum projections of each time point of the entire 12-μm z stack, which correspond to the figure images in Fig. 7m (bottom, remyelinating); second, single plane images of the z dimension, with relevant sheaths pseudocolored; and third, the same images overlaid with semiautomated tracings. These semiautomated tracings verify the connection between the surviving oligodendrocyte cell body and the newly generated, remyelinating myelin sheath. (Frame rate: 2 frames per second).

Supplementary Tables

Supplementary Table 1: Antibody specification, which contains details about all antibodies used in this study; Supplementary Table 2: Statistics results table, which contains detailed descriptions of all statistics used for each figure panel.

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Bacmeister, C.M., Barr, H.J., McClain, C.R. et al. Motor learning promotes remyelination via new and surviving oligodendrocytes. Nat Neurosci (2020). https://doi.org/10.1038/s41593-020-0637-3

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