Rapid production of new oligodendrocytes is required in the earliest stages of motor-skill learning

Abstract

We identified mRNA encoding the ecto-enzyme Enpp6 as a marker of newly forming oligodendrocytes, and used Enpp6 in situ hybridization to track oligodendrocyte differentiation in adult mice as they learned a motor skill (running on a wheel with unevenly spaced rungs). Within just 2.5 h of exposure to the complex wheel, production of Enpp6-expressing immature oligodendrocytes was accelerated in subcortical white matter; within 4 h, it was accelerated in motor cortex. Conditional deletion of myelin regulatory factor (Myrf) in oligodendrocyte precursors blocked formation of new Enpp6+ oligodendrocytes and impaired learning within the same 2−3 h time frame. This very early requirement for oligodendrocytes suggests a direct and active role in learning, closely linked to synaptic strengthening. Running performance of normal mice continued to improve over the following week accompanied by secondary waves of oligodendrocyte precursor proliferation and differentiation. We concluded that new oligodendrocytes contribute to both early and late stages of motor skill learning.

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Figure 1: Time course of motor skill learning in mice and the requirement for Myrf.
Figure 2: Oligodendrocyte dynamics during motor-skill learning.
Figure 3: Enpp6 marks newly forming oligodendrocytes.
Figure 4: Enpp6highMbp+ newly formed oligodendrocytes express myelin structural proteins and synthesize myelin.
Figure 5: Visualization of Enpp6high cells in the developing mouse forebrain by ISH.
Figure 6: Rapid increase in Enpp6high newly forming oligodendrocytes in response to motor-skill learning.
Figure 7: Increased production of Enpp6+ newly formed oligodendrocytes was a response to motor learning, not physical exercise.

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Acknowledgements

We thank our colleagues at University College London, especially S. Jolly, U. Grazini and L. Magno, for advice and reagents, and M. Grist and U. Dennehy for technical help. We thank M. Wegner (University of Erlangen, Germany) for the Sox10 antibody. This work was supported by the European Research Council (grant agreement 293544 to W.D.R.), the Wellcome Trust (100269/Z/12/Z to W.D.R.) and the Biotechnology and Biological Sciences Research Council (BB/L003236/1 to H.L.). L.X. was supported by the National Natural Science Foundation of China (grant 31471013). Pdgfra-CreERT2 mice can be obtained through http://www.e-lucid.com/ with a material transfer agreement. Myrf loxP mice are available from Jackson Labs, strain 010607.

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Contributions

W.D.R. formed the hypotheses and obtained funding. I.A.M. adopted and developed the complex wheel test. B.E. provided MyrfloxP mice, advice and suggestions. W.D.R., I.A.M. and D.O. designed the experiments in Figure 1 and Supplementary Figure 1; D.O. and I.A.M. performed those experiments and DO analyzed the data. W.D.R., H.L. and L.X. designed all the other experiments and L.X. performed them, with assistance from A.S.-W., J.L.W. and A.D.F. H.L. identified Enpp6 and A.F. performed preliminary characterization. H.L. and W.D.R. supervised the work. W.D.R. wrote the paper with input from H.L., L.X. and B.E.

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Correspondence to William D Richardson.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Performance of P‑Myrf−/− versus P‑Myrf+/− mice on the complex wheel.

Three weeks after tamoxifen administration at P60-64 or P90-P94 (9), P‑Myrf−/− mice and their P‑Myrf+/− littermates [n=36 (20 males) and 32 (17 males) respectively] were housed singly in cages containing a complex wheel. Average wheel speeds were calculated for each 2-hour time window during the first seven nights (6pm-6am) and plotted as mean ± s.e.m (P‑Myrf−/−, red; P‑Myrf+/−, blue) were analyzed by two-way ANOVA with Bonferroni's post-hoc test. Each night was treated separately for multiple comparisons. *p < 0.05, **p < 0.01, ***p < 10−3, ****p < 10−4[Night 1: 2 h, p=0.43, t=1.81; 4 h, p=0.029, t=2.83; 6 h, p=0.0089, t=3.20; 8 h, p=0.0044, t=3.40; 10 h, p=0.0017, t=3.66; 12 h, p=0.0036, t=3.46. Night 2: 2 h, p=0.0067, t=3.28; 4 h, p=0.0033, t=3.48; 6 h, p=0.063, t=2.57; 8 h, p=0.016, t=3.03; 10 h, p=0.11, t=2.37; 12 h, p=0.034, t=2.79. Night 3: 2 h, p=0.0019, t=3.63; 4 h, p=0.0041, t=3.43; 6 h, p=0.020, t=2.95; 8 h, p=0.072, t=2.53; 10 h, p>0.99, t=1.05; 12 h, p=0.24, t=2.06. Night 4: 2 h, p=0.020, t=2.96; 4 h, p=0.0010, t=3.80; 6 h, p=0.0007, t=3.90; 8 h, p=0.020, t=2.96; 10 h, p=0.033, t=2.79; 12 h, p=0.30, t=1.96. Night 5: 2 h, p=0.0006, t=3.94; 4 h, p=0.0052, t=3.36; 6 h, p=0.015, t=3.04; 8 h, p=0.13, t=2.32; 10 h, p=0.59, t=1.66; 12 h, p=0.17, t=2.42. Night 6: p<0.0001, t=4.42; 4 h, p<0.0001, t=4.42; 6 h, p<0.0001, t=4.46; 8 h, p=0.0046, t=3.39; 10 h, p=0.020, t=2.96; 12 h, p=0.095, t=2.42. Night 7: 2 h, p<0.0001, t=4.65; 4 h, p<0.0001, t=4.39; 6 h, p=0.0001, t=4.32; 8 h, p=0.0004, t=4.05; 10 h, p=0.20, t=2.13; 12 h, p=0.097, t=2.42. Degrees of freedom=396 throughout.]

Supplementary Figure 2 Two populations of high- and low-expressing Enpp6+ cells in vivo.

(a,b) Coronal sections of P60 mouse forebrains were incubated with an Enpp6 ISH probe and the signal developed using the NBT/BCIP method (Methods). (a) At a relatively long development time (150 minutes) two populations of Enpp6-positive cells are detected in the subcortical white matter – a few strongly-labeled large cell bodies (arrows) against a background of more numerous, small, weakly-labeled cells (arrowheads), consistent with the RNA-seq data27 (Fig. 3A) and the likelihood that the strongly-labeled cells are newly-differentiating oligodendrocytes and the weakly-labeled cells more mature, myelinating oligodendrocytes. (b) Reducing the development time to 60 minutes allows the strongly-labeled cells to be visualized preferentially (arrows). Images are representative of >3 similar experiments. Scale bar, 50 μm.

Supplementary Figure 3 Enpp6 expression is oligodendrocyte-specific.

Forebrain sections of P90 mice (caged without wheels) were analyzed by ISH for Enpp6 mRNA followed by immuno-fluorescence labeling for stage-specific markers of the oligodendrocyte lineage (Sox10, Olig2, CC1). Alternatively, sections were analyzed by double fluorescence ISH for Enpp6 and Pdgfra. (a,b) All Enpp6+ cells were also Olig2+ and Sox10+. (c) No Enpp6+ cells were Pdgfra+ OPs. (d) Most Enpp6+ cells were CC1+ mature or maturing oligodendrocytes (Motor cortex, 98.2 ± 1.8%; Subcortical white matter, 86.9% ± 3.8%) (Supplementary Table 1). Images are representative of >3 similar experiments. Scale bar, 25 μm.

Supplementary Figure 4 The role of novel motor activity in stimulating OP differentiation.

OPs are continuously cycling in the young adult CNS in response to mitogenic growth factors such Pdgf. After division one or both daughter OPs can rest in the G1 phase of the cell cycle, sometimes for days or weeks, before either differentiating or entering another division cycle3,4,7,9,31. Our data suggest that electrical activity in axon(s) cans stimulate OPs that are paused in G1 to differentiate - losing Pdgfra expression and expressing Enpp6, Mbp and other myelin gene products instead. The newly-differentiating oligodendrocytes have a distinctive spidery morphology in vivo; they remain like this for several days in vivo in rodents31 before down-regulating Enpp6 (faint pink line) and assuming the typical morphology of mature myelinating oligodendrocytes. The Enpp6high early-differentiating oligodendrocytes and Enpp6low myelinating oligodendrocytes probably contribute to improving circuit performance in the early and late stages of motor learning, respectively.

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Xiao, L., Ohayon, D., McKenzie, I. et al. Rapid production of new oligodendrocytes is required in the earliest stages of motor-skill learning. Nat Neurosci 19, 1210–1217 (2016). https://doi.org/10.1038/nn.4351

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