Oligodendrocyte generation in the adult CNS provides a means to adapt the properties of circuits to changes in life experience. However, little is known about the dynamics of oligodendrocytes and the extent of myelin remodeling in the mature brain. Using longitudinal in vivo two-photon imaging of oligodendrocytes and their progenitors in the mouse cerebral cortex, we show that myelination is an inefficient and extended process, with half of the final complement of oligodendrocytes generated after 4 months of age. Oligodendrocytes that successfully integrated formed new sheaths on unmyelinated and sparsely myelinated axons, and they were extremely stable, gradually changing the pattern of myelination. Sensory enrichment robustly increased oligodendrocyte integration, but did not change the length of existing sheaths. This experience-dependent enhancement of myelination in the mature cortex may accelerate information transfer in these circuits and strengthen the ability of axons to sustain activity by providing additional metabolic support.
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We thank M. Pucak, N. Ye, H. Hsieh, and A. Doreswamy for technical assistance; T. Shelly for machining expertise; and members of the Bergles laboratory for discussions. We thank T. McCown (University of North Carolina) for the kind gift of AAV9-MBP-eGFP virus. E.G.H. was supported by a NRSA grant from the NIH (F32NS076098), the Boettcher Foundation, the Whitehall Foundation, the Conrad N. Hilton Foundation (17324), and the National Multiple Sclerosis Society (RG-1701–26733). J.O.M. was supported by a National Multiple Sclerosis Society Postdoctoral Fellowship (FG 2092-A-1) and the Conrad N. Hilton Foundation (17314). A.J.L. was supported by a National Multiple Sclerosis Society Postdoctoral Fellowship (FG 20114-A-1). Funding was provided by grants from the NIH (NS051509, NS050274), the Dr. Miriam and Sheldon G. Adelson Medical Research Foundation, a National MS Society Collaborative Center Award, the Brain Science Institute at Johns Hopkins University, and the Johns Hopkins Medicine Discovery Fund to D.E.B.
The authors declare no competing interests.
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Integrated supplementary information
a, Horizontal section from layer I cortex of a Mobp-EGFP mouse immunostained with CNP (left), EGFP (middle) and NG2 (right) (P30). b, Overlay of CNP and EGFP immunostaining shown in a. c, Overlay of CNP, EGFP, and NG2 immunostaining shown in a. Note lack of cells co-labeled with EGFP and NG2. All EGFP+ cells were labeled with CNP and are oligodendrocytes. d, Quantification of proportion of CNP+ colabeled with EGFP or NG2 (n = 3 mice; CNP+ cells = 356; mean ± SEM).
a, Depth of newly formed oligodendrocytes in young and middle-aged mice (young, 2–4 months, n = 8 mice; middle-aged, 11–14 months, n = 13 mice). b, Quantification of total oligodendrocyte number in layers I-IV of somatosensory cortex (2–4 months, n = 8 mice, P = 0.0233, one-way ANOVA with Tukey post hoc test, 300–350 μm vs. 0–50 μm, * = P = 0.0436, q(7) = 6.48; 300–350 μm vs. 100–150 μm, * = P = 0.0357, q(7) = 6.75 ; 11–14 months, n =13 mice, one-way ANOVA, P = 0.06, F(4, 51) = 2.40; mean ± SEM).
a-c, Immunostaining of coronal sections somatosensory cortex from young (P60; a), middle-aged (P365; b), and aged (P665; c) Mobp-EGFP mice. Antibodies against EGFP, NeuN, and NG2 were used to label mature oligodendrocytes, neurons, and oligodendrocyte precursor cells (OPCs). d, Quantification of number of EGFP+ oligodendrocytes in a 0.6 mm2 area over age (n = 4 mice per group; one-way ANOVA with Tukey's posthoc test, young vs. middle, P = 0.004, q(11) = 6.44; young vs. aged, P = 0.003, q(11) = 6.56; middle vs. aged, P = 0.90, q(11) = 0.12; n.s. = not significant; * = P < 0.005). e, Quantification of number of NeuN+ neurons in a 0.6 mm2 area over age (n = 4 mice each group; one-way ANOVA P = 0.44, F(2, 9) = 0.90; n.s. = not significant). f, Quantification of oligodendrocyte precursor cell number in a 0.6 mm2 area in young, middle-aged and aged mice (n = 4 mice each group; P = 0.95, F(2, 9) = 0.05, one-way ANOVA; n.s. = not significant). d-f, Data is presented as mean ± SEM.
Maximal projections from triple transgenic mice (Olig2-CreER; R26-lsl-tdTomato;NG2-mEGFP) at P150 (100 mg/kg tamoxifen injected for five days at P30, n = 3 mice) immunostained for EGFP, tdTomato and a marker of mature oligodendrocytes or OPCs. a, A subset of mature oligodendrocytes (ASPA+) express tdTomato. b, OPCs that express membrane-anchored EGFP and tdTomato were immunoreactive for PDGF receptor alpha (PDGFRa). Note mature oligodendrocyte that does not express EGFP and is not labeled with PDGF receptor alpha (yellow arrowhead). c, A small population of astrocytes are recombined and express tdTomato only. Astrocytes are not immunolabeled with PDGF receptor alpha and are distinguished by their distinct bushy morphology. Note the OPC labeled with PDGF receptor alpha that expresses both EGFP and tdTomato (yellow arrowhead).
a-b, Maximal intensity projection of dying OPCs from triple transgenic mice (Olig2-CreER; R26-lsl-tdTomato;NG2-mEGFP). a, P219; depth= 90–132 μm; b, P203; depth = 102–159 μm). Images were acquired every two days for six days. Inset panels show the cell soma at higher resolution and each individual fluorescent channel. c, Quantification of time-course of OPC death over 1.5 months in >P190 mice. (n = 3 mice; dying OPCs = 24).
a-c, Maximal intensity projection of newly generated oligodendrocytes (P96; Left- depth= 129–159 μm; Right- P426, depth = 78–108 μm). Images were acquired at baseline (0d) and after three weeks of sensory enrichment (21d). These new oligodendrocytes generate Isolated and Interrupted sheaths shown in b and c indicating they ensheath unmyelinated stretches of axons. (young adult, n = 8 mice; middle-aged, n = 13 mice) d-f, Maximal intensity projection of newly generated oligodendrocytes (P135; Left- depth = 15–33 μm; Right- P426, depth= 68–98 μm). Images were acquired at baseline (0d), after three weeks of sensory enrichment (21d), and following an additional three weeks in standard housing (42d). These newly generated sheaths are stable over the next three weeks shown in e (green) and f (pseudo-colored to cyan). (young adult, n = 8 mice; middle-aged, n = 13 mice).
a-b, Individual oligodendrocytes in a fixed horizontal section from layer I of somatosensory cortex immunostained with EGFP, CNPase (CNP), and MBP (P365; Mobp-EGFP mouse, n = 3 mice). Note the presence of myelin internodes with abnormal morphology near the cell soma (boxes). Areas from a, b are shown below at higher magnification. Note the internodes with abnormal morphologies are not MBP positive.
Supplementary Figure 8 Sensory enrichment results in the stable integration of new oligodendrocytes.
a, Maximal intensity projection of a newly generated oligodendrocytes (P365; top- depth = 105–114 μm; bottom- depth = 303–318 μm). Images were acquired at baseline (0d), after 3 weeks of sensory enrichment (21d), and following an additional 3 weeks in standard housing (42d). Pink arrowheads designate existing oligodendrocytes. Yellow arrows designate new oligodendrocytes. b, Quantification of oligodendrocyte addition after sensory enrichment (21d) and stability following 3 additional weeks in standard housing (42d; n = 4 mice, persistent new oligodendrocytes = 25/25).
Supplementary Figures 1–8
Supplementary Tables 1 and 2
Supplementary Video 1 - In vivo two-photon imaging of EGFP+ oligodendrocytes in the somatosensory cortex
In vivo two-photon imaging of a field of EGFP-positive expressing oligodendrocytes in the somatosensory cortex of a Mobp-EGFP mouse implanted with a cranial window (P90; depth = 0–501 μm). Images were acquired every 3 μm beginning in the pia, 21 μm above the brain surface. The increased density of oligodendrocytes with myelin sheaths oriented tangentially to the brain surface in the upper region of Layer I (0–50 μm) is the Plexus of Exner. The density of oligodendrocytes is low between 50 μm - ~200 μm below the cortical surface, then steadily increases with depth. Oligodendrocyte processes are oriented perpendicular to the imaging plane at these depths, corresponding to the orientation of axons in these cortical layers. EGFP intensity and the ability to resolve oligodendrocyte processes decreases with depth due to the limitations of two-photon microscopy. (n = 8 mice; Frame rate: 3 frames per second).
In vivo two-photon imaging of an individual EGFP+, tdTomato+ expressing oligodendrocyte precursor cell in the somatosensory cortex of a Olig2-CreER; R26-lsl-tdTomato; NG2-mEGFP mouse implanted with a cranial window (P240; depth = 111–120 μm). Images were acquired every 2–3 days for 28 days. Note the morphology change beginning at day 5, accompanied by a decrease in EGFP fluorescence indicating differentiation. The newly generated oligodendrocyte is successfully integrated and remains stable throughout the remaining imaging period (total = 35 days). (n = 3 mice; Frame rate: 3 frames per second).
In vivo two-photon imaging of an individual EGFP, tdTomato+ expressing oligodendrocyte precursor cell in the somatosensory cortex of a Olig2-CreER; R26-lsl-tdTomato; NG2-mEGFP mouse implanted with a cranial window (P240; depth = 234–252 μm). Images were acquired every 2–3 days for 28 days. Note the OPC divides on day 5 and the daughter cell on the right undergoes a morphology change beginning on day 19 which is accompanied by a decrease in EGFP fluorescence indicating differentiation. The differentiating cell (EGFP-negative, tdTomato+) loses its processes and displays a rounded cell body reminiscent of apoptotic cells. On day 28 the differentiating cell disappears most likely after having been cleared by microglia. (n = 3 mice; Frame rate: 3 frames per second).
In vivo two-photon imaging of two stable myelin sheaths (left) and one growing myelin sheath (right) in the somatosensory cortex of a young adult Mobp-EGFP mouse with a thinned-skull cranial window (P60; depth = 24–27 μm). The stable sheaths are from existing oligodendrocytes and the dynamic sheath extends from a recently generated oligodendrocyte. On day 4, the growing myelin sheath extends to form a node of Ranvier with one of the stable sheaths. (n = 8 mice; Frame rate: 1 frame per second).
In vivo two-photon imaging of the growth of a myelin sheath from an existing oligodendrocyte in the somatosensory cortex of a middle-aged adult Mobp-EGFP mouse implanted with a cranial window (P365; depth=63–67 μm). This myelin sheath (green) is stable for one week (0–7 days) then extends over the next 30 days. Note there were no newly generated or dying oligodendrocytes found in the surrounding 250 μm of this sheath. (n = 3 mice; Frame rate: 1 frame per second).
In vivo two photon imaging of the retraction of a myelin sheath from an existing oligodendrocyte in the somatosensory cortex of a middle-aged adult Mobp-EGFP mouse implanted with a cranial window (P365; depth = 63–67 μm). This sheath (red) continuously reduces its total length before coalescing at the process which connects the sheath to the cell body at day 48. Note there were no newly generated or dying oligodendrocytes found in the surrounding 250 μm of this sheath. (n = 20 mice; Frame rate: 1 frame per second).
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Hughes, E.G., Orthmann-Murphy, J.L., Langseth, A.J. et al. Myelin remodeling through experience-dependent oligodendrogenesis in the adult somatosensory cortex. Nat Neurosci 21, 696–706 (2018) doi:10.1038/s41593-018-0121-5
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