Research ArticleNEUROSCIENCE

Exercise training improves motor skill learning via selective activation of mTOR

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Science Advances  03 Jul 2019:
Vol. 5, no. 7, eaaw1888
DOI: 10.1126/sciadv.aaw1888
  • Fig. 1 Long-term exercise activates mTOR and facilitates PSD formation in mouse motor cortex.

    (A) Representative Western blotting bands using total protein extracts from the motor cortex. MW, molecular weight. (B to D) Quantification of protein expression levels between runner (n = 6) or nonrunner (n = 6) mice. (B) Exercise elevates matured BDNF (m-BDNF; two-sample student t test, t10 = 7.531, P = 0.0001), phosphorylated/total (p/t) Trkb (t10 = 2.362, P = 0.0398), and phosphorylated/total AKT (t10 = 3.391, P = 0.0069). (C) Phosphorylated/total mTOR protein (t10 = 2.281, P = 0.0181), phosphorylated/total ribosomal S6 protein (t10 = 2.395, P = 0.0376), and inhibited elongation factor 4E-BP2 (t10 = 6.543, P < 0.0001) in exercised mice. (D) Treadmill exercise also elevated postsynaptic protein PSD95 (t10 = 5.012, P = 0.0005) and vesicular protein SNAP25 (t10 = 2.401, P = 0.0373). (E) EM images in mouse motor cortex. Bo, axonal bouton; Sp, dendritic spine; white arrows, PSD complex. Scale bar, 500 nm. (F) Histogram for the PSD lengths between runners (n = 105 synapses from five mice) and nonrunners (n = 107 synapses from five mice). Inset: Mean PSD lengths were increased in runner mice (t210 = 5.495, P < 0.0001). (G) Histogram for PSD thickness. Inset: Mean PSD thickness were higher in runner group (t210 = 5.133, P = 0.0001). *P < 0.05, ***P < 0.001. Error bars, SEM.

  • Fig. 2 Exercise training potentiates synaptic transmissions via mTOR activation.

    (A) Illustration for whole-cell recordings of mEPSC of L5PRN in Thy1-YFP mice. (B) Schematic diagram for experimental protocols. Rapamycin administration was executed every 3 days during 21-day treadmill training. D0, day 0. (C) Bright-field (left) and epifluorescent (right) images of one YFP-labeled L5PRN for the whole-cell recording. (D to G) From left to right, representative traces of mEPSC extracted from nonrunner + saline (n = 8 neurons from four mice), runner + saline (n = 7 neurons from three mice), nonrunner + rapamycin (n = 8 neurons from four mice), and runner + rapamycin groups (n = 8 neurons from three mice). (H to K) Cumulative distributions of mEPSC amplitudes (H) and mEPSC frequencies (J) were plotted. Quantitative analysis (I and K) showed elevated mEPSC amplitudes in runner mice [one-way analysis of variance (ANOVA), F3,27 = 32.64, P < 0.0001; Tukey post hoc comparison, q27 = 4.966, P = 0.0081 (I)] that was subsequently abolished by rapamycin (q27 = 12.95, P < 0.0001). No significant change was found in mEPSC frequencies [q27 = 0.7616, P = 0.9488 for nonrunner + saline versus runner + saline and q27 = 3.112, P = 0.1487 for runner + saline versus runner + rapamycin (K)]. **P < 0.01, ***P < 0.001. Error bars, SEM.

  • Fig. 3 Exercise training enhances neuronal activity in vivo.

    (A) Illustrations of in vivo calcium recordings for apical dendrites and somas of L5PRN. (B) Schematic diagram of experimental protocols. Mice received AAV-GCaMP6s virus injection before 21-day treadmill training. Rapamycin was administrated every 3 days. At the end of exercise training, calcium imaging was performed. (C) Coronal section of mouse primary motor cortex showing GCaMP6s-transfected neurons after 3 weeks. (D) Representative z-stacked imaging planes (left) and calcium fluorescent traces (right) obtained from apical dendrites (top) and somas (bottom) of L5PRN. White arrowheads, active apical dendrite or somas. (E) Quantification of calcium peak ΔF/F0 values in apical dendrites (top) and somas (bottom). Runner mice showed significantly higher peak values (apical dendrite: one-way ANOVA, F3,195 = 16.89, P < 0.0001; Tukey post hoc comparison, q195 = 10.58, P < 0.0001 for nonrunner + saline versus runner + saline and q27 = 12.33, P < 0.0001 for runner + saline versus runner + rapamycin; soma: F3,456 = 14.78, P < 0.0001; q456 = 8.683 and P < 0.0001 for nonrunner + saline versus runner + saline and q456 = 12.03, P < 0.0001 for runner + saline versus runner + rapamycin). ***P < 0.001. n = 6 animals in each group. Error bars, SEM.

  • Fig. 4 The activation of mTOR is necessary for exercise-enhanced axonal myelination in the CC region.

    (A) Illustrations of L5PRN axonal myelination, which is formed by mature oligodendrocytes (OLs) in the CC region. (B) Schematic diagrams of experimental protocols. Mice received 21-day exercise and rapamycin or saline injection every 3 days. (C) Representative images for myelin basic protein (MBP) staining in the medial CC region. Scale bar, 250 μm. (D) Quantitative analysis of fluorescent intensities of MBP (n = 4 animals in each group) showed elevated myelination level in runner + saline group (one-way ANOVA, F3,11 = 3.353, P = 0.0591; Tukey post hoc test, q11 = 8.721, P = 0.0003) and subsequent decrease after mTOR inhibition (q11 = 8.852, P = 0.0003). A.U., arbitrary units. (E) EM images showing myelinated axonal fibers. Scale bars, 500 nm. (F) Distribution of g-ratios. Kruskal-Wallis test showed significantly lower g-ratios in runner + saline mice comparing to nonrunner + saline group (P < 0.0001). (G) Schematic illustrations for the experimental design of oligodendrogenesis. Daily 5-bromo-2-deoxyuridine (BrdU) injections were given during the last 5 days of exercise training. (H) Representative images of BrdU-incorporated (BrdU+) cells in the medial CC region. Scale bar, 200 μm. (I) Chronic exercise increased BrdU+ cells (F3,40 = 6.727, P < 0.001; q40 = 7.519, P < 0.0001), and rapamycin injection significantly decreased BrdU+ cells (q40 = 13.51, P < 0.0001). (J to M) Double immunostaining of BrdU+ cells with OL linage marker Olig2 (J), OL progenitor cells (OPCs) marker PDGFRα (K), mature OL marker CC1 (L), and cell proliferation marker Ki67 (M). Scale bar, 50 μm. (N to P) Quantification across groups showed that exercise increased the number of Olig2+BrdU+P (F3,11 = 20.89, P < 0.0001; q11 = 6.084, P = 0.0058) (N), BrdU+PDGFRα+-labeled OPCs (F3,11 = 47.05, P < 0.0001; q11 = 9.328, P = 0.0002) (O), and BrdU+CC1+-labeled, matured OL (F3,11 = 27.16, P < 0.0001; q11 = 7.906, P = 0.0008) (P) after exercise. The OLs proliferation and maturation were reduced by rapamycin treatment (runner + saline versus runner + rapamycin groups: BrdU+Olig2+, q11 = 10.77, P < 0.0001; BrdU+PDGFRα+, q11 = 15.27, P < 0.0001; BrdU+CC1+, q11 = 12.13, P < 0.0001). (Q) Exercise also facilitated OL differentiation as suggested by fewer BrdU+Ki67+ cells (F3,11 = 14.26, P = 0.0004; q11 = 6.655, P = 0.0031), which was not affected by mTOR (runner + saline versus runner + rapamycin groups, q11 = 1.838, P = 0.5819). *P < 0.05, **P < 0.01, ***P < 0.001. n = 4 animals in each group. Error bars, SEM.

  • Fig. 5 Treadmill exercise promotes cortical dendritic spine formations and motor learning via mTOR activation.

    (A) Illustration of Thy1-YFP mice and imaging planes. (B) Schematic diagram of experimental protocols. Mice received treadmill exercise from days 0 to 15 and were imaged on the same region of primary motor cortex on days 0, 3, and 7 or 15. (C) Z-axis stacked images showing the same dendritic branches of L5PRNs at day 0 (first imaging), day 3 (second imaging), and day 7 (third imaging) between runner or nonrunner mice. Arrowheads, eliminated spines; arrows, newly formed spines. Scale bars, 5 μm. (D) Overall spine turnover rates of runner mice were higher than nonrunner animals [two-way ANOVA(group effect), F1,36 = 127.1, P < 0.0001]. (E) Spine formation rates of L5PRN were persistently increased in runner mice compared to nonrunner controls [two-way ANOVA(group effect), F1,36 = 184.4, P < 0.0001]. (F) Spine elimination was not affected [two-way ANOVA(group effect), F1,36 = 0.5791, P = 0.4516]. (G) Newly formed spines on day 3 had higher survival rates in exercised mice on days 7 or 15 [two-way ANOVA(group effect), F1,24 = 79.33, P < 0.0001]. (H) Experimental protocols and (I) representative images of EM showing pre- and postsynaptic (in pink color) complexes in the motor cortex. Scale bars, 2 μm. (J) Histogram showed more synapses in runner mice (two-sample student t test, t58 = 5.791, P < 0.001). (K) Schematic illustration studying the effect of mTOR on spinogenesis. Rapamycin was applied daily for 3 days between repeated imaging. (L) Rapamycin treatment significantly decreased exercise-induced spine formation (one-way ANOVA, F3,18 = 15.75, P < 0.0001; Tukey’s post hoc test, q18 = 6.234, P = 0.0018). (M) Elimination of spines was not affected (F3,18 = 1.508, P = 0.2465). (N) Experimental designs of accelerating rotarod assay. (O) Similar motor performances on day 1 (nonrunner + saline, n = 8; runner + saline, n = 8; nonrunner + rapamycin, n = 9; runner + rapamycin, n = 8). (P and Q) Improvements of motor performances (in percentage) on days 2 and 3. Exercised significantly improved motor learning that was abolished by rapamycin injection (day 2: one-way ANOVA, F3,28 = 7.43, P = 0.0008; Tukey’s post hoc test, q28 = 4.70, P < 0.05; day 3: F3,28 = 10.64, P < 0.0001; q28 = 3.88, P < 0.05). *P < 0.05, **P < 0.01, ***P < 0.001. Error bars, SEM.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/5/7/eaaw1888/DC1

    Fig. S1. Exercise-activated ribosomal protein S6 in motor cortex.

    Fig. S2. Exercise-induced neuron activity of cortical pyramidal neurons.

    Fig. S3. Calcium spike dynamics of apical tuft and soma of L5PRN.

    Fig. S4. Calcium transients of L5PRN.

    Fig. S5. Subtyping of BrdU+ cells in the CC region.

    Fig. S6. Immunofluorescence labeling of BrdU and Olig2 in mouse motor cortex after exercise.

    Fig. S7. Energy requirement of cells under exercise.

    Table S1. Primary and secondary antibody used in Western blotting and immunofluorescence staining.

  • Supplementary Materials

    This PDF file includes:

    • Fig. S1. Exercise-activated ribosomal protein S6 in motor cortex.
    • Fig. S2. Exercise-induced neuron activity of cortical pyramidal neurons.
    • Fig. S3. Calcium spike dynamics of apical tuft and soma of L5PRN.
    • Fig. S4. Calcium transients of L5PRN.
    • Fig. S5. Subtyping of BrdU+ cells in the CC region.
    • Fig. S6. Immunofluorescence labeling of BrdU and Olig2 in mouse motor cortex after exercise.
    • Fig. S7. Energy requirement of cells under exercise.
    • Table S1. Primary and secondary antibody used in Western blotting and immunofluorescence staining.

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