Fig 2: Validation of the OPC reporter line.(a) Confocal images of TC axons and inputs in V1 labeled with AAV-hSYN-eGFP (green) and immunostained for VGluT2 (magenta). Scale bar, 40 µm. (b) Representative image of tdTomato+ cells (magenta) in the NG2-CreERT2tdTomato mouse line. Cells of the oligodendrocyte lineage immunostained for Sox10 (cyan) and the OPC marker NG2 (yellow). White circles, OPCs confirmed by co-expression of tdTomato and NG2. Scale bar, 20 µm. Fluorescence scale bar, 40 µm. (c) Quantification of the percentages of tdTomato+ cells that co-stain for Sox10 and NG2. Individual data points and mean ± s.e.m. shown; n = 3 mice per group. (d) High resolution images of tdTomato+ cells (magenta) immunostained for NG2 (green). White arrow, confirmed OPC in which tdTomato and NG2 signal overlap. Compared to other tdTomato+ cells (magenta arrow), OPCs can be distinguished by their bean-shaped somata. Scale bar, 10 µm. (e) Confocal images of tdTomato+ cells (magenta) co-localizing with blood vessels (CD31, green). These are likely pericytes and are easy to distinguish from OPCs based upon morphology. Scale bar, 10 µm. (f) High magnification image of a tdTomato+ OPC (magenta) immunostained for NG2 (green). Bottom, volumetric reconstructions of the same cell based upon tdTomato versus NG2 signal, demonstrating a high level of overlap between the two channels. Scale bar, 10 µm.

Fig 3: The engulfment of synaptic inputs by OPCs is heightened by sensory experience and dampened by microglial depletion.a, Schematic of the LDR visual deprivation and stimulation paradigm. b, Volumetric reconstructions of microglia (Iba1, white) and engulfed VGluT2+ inputs (magenta) in normally reared mice at P27 (control), mice dark-reared between P20 and P27 (LDR) and mice re-exposed to light for 10 h following LDR (LDR + 10). Scale bar, 2 µm. c, Quantification of the volume of synaptic material within microglia from each condition. One-way ANOVA (P > 0.05; NS, not significant); n (P27/LDR/LDR + 10): 28/49/64, from three mice per group. d, Reconstructions of OPCs (NG2, green) and engulfed synaptic inputs (VGluT2, magenta) from control, LDR, and LDR + 10 mice. Scale bar, 2 µm. e, Quantification of the volume of synaptic material contained within each OPC from control, LDR, and LDR + 10 mice. One-way ANOVA (P = 0.0199) with Tukey’s posthoc test; n (P27/LDR/LDR + 10): 20/30/42, from three mice per group. f, Reconstructions of OPCs (NG2, green) and engulfed TC inputs (VGluT2, magenta) in P27 mice following depletion of microglia using PLX5622 for 1 week. Scale bar, 2 µm. g, Quantification of the volume of synaptic material contained within each OPC in the presence or absence of microglia. Two-tailed Mann–Whitney t-test, P = 0.0082; n (control/PLX): 26/42, from three mice per group. h, Reconstructions of OPCs (NG2, green) and synaptic inputs (VGluT2, magenta) from LDR and LDR + 10 mice fed PLX5622 or control chow between P20 and P27. Scale bar, 2 µm. i, Quantification of synaptic engulfment in LDR and LDR + 10 mice containing or lacking microglia. Two-way ANOVA (stimulation: P < 0.0001; treatment: P < 0.05; interaction between stimulation and treatment: P < 0.05); n (OPCs, control/PLX): LDR = 45/52, LDR + 10 = 51/54, from three mice per group. In c,e,g, and i, individual data points are shown with bars representing mean ± s.e.m. *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001.

Fig 4: Validation of microglial depletion using PLX5622.(a) Representative confocal images of microglia immunostained for Iba1 (green) in the visual cortex before and during PLX5622 administration. Scale bar, 20 µm. (b) Quantification of the number of microglia in V1 following three days (Bi) or ten days (Bii) of PLX5622 administration. Unpaired two-tailed T test, P < 0.0001 . (c) Confocal images of V1 stained for GFAP, a marker of astrocyte activation, after ten days of PLX5622 administration. Scale bar, 20 µm. (d) Quantification of the mean GFAP intensity normalized to the control group (Di) and the normalized area covered by GFAP (Dii). Unpaired two-tailed T test, P >0.05. (e) Confocal images of V1 with OPCs (NG2, green) and the oligodendroglial marker Sox10 (magenta) after ten days of PLX5622 administration. Scale bar, 20 µm. (f) Quantification of the number of OPCs (marked by co-expression of NG2+ and Sox10+) (Fi) and the percentage of Sox10+ cells that also expressed the OPC marker NG2 (Fii). Unpaired two-tailed T test, P > 0.05 (g) Representative images of myelin (MBP, green) in mice after ten days of PLX5622 administration. Scale bar, 20 µm. (h) Quantification of the mean MBP intensity normalized to the control group (Hi) and quantification of the area covered by MBP (Hii). Unpaired two-tailed T test, P > 0.05. All graphs: Individual data points with mean ± s.e.m.; n = 3 mice per condition. (f) n = 12 images from 3 mice per condition. Individual data points shown with bars representing mean ± SEM. ***P < 0.001, ****P < 0.0001.

Fig 5: Identification of the cell composition of peripheral nerve. (A) Representative confocal IF and EM images, showing the SC population in transverse sections of mouse sciatic nerve. SCs are labelled by staining with anti-S100 antibody (red) or endogenously in PLP-eGFP mice (green). In the EM image, a nmSC is coloured purple while a typical mSC is coloured green. (B) Quantification shows the proportions of SCs in mouse sciatic nerve (n=4 mice, mean±s.e.m.). (C) Pie chart showing the percentages of individual cell types within sciatic nerve. Cryosections of sciatic nerves were immunolabelled with cell type-specific markers as indicated (n=4). The NG2+/PDGFRß+/p75+/aSMA- population is classified as either associated or non-associated with CD31+ blood vessels (BV). Pericytes are defined as NG2+/PDGFRß+/aSMA+. (D) Representative images of transverse mouse sciatic nerve sections labelled as indicated. (i) Arrowhead indicates a p75+ cell (red) that is not associated with axons [labelled with antibody against neurofilament (NF), white]. (ii) Arrowhead indicates a p75+ cell (red) that is not associated with axons (labelled for NF, white) and does not express eGFP in nerves isolated from PLP-eGFP mice. (iii) Arrowhead indicates a PLP-eGFP-/p75+/NG2+ cell. (iv) Arrowhead indicates a PLP-eGFP-/NG2+/PDGFRß+ cell. (v) Arrow indicates an aSMA+/p75- pericyte in close contact with a CD31+ blood vessel (white). (vi) Arrow indicates a p75+ cell that co-expresses dsRed in nerves isolated from NG2-dsRed mice and is loosely associated with a CD31+ blood vessel. The asterisk indicates a p75+/dsRed+ cell that is not associated with a blood vessel. (v,vi) Dashed line boxes indicate the regions that are shown at higher magnification in insets. See also Fig. S1.