Fig. 5: Cuprizone-induced demyelination is limited to gray matter regions of L5–POm axons.

Confocal immunofluorescence images of mCherry (magenta) and myelin (MBP, green) in cortex (a) and thalamus (b) were used for 3D colocalization analysis. White arrows point to mCherry⁺ MBP⁺ regions, yellow arrows point to mCherry⁺ demyelinated axons. Scale bars 20 µm (a), 15 µm (b). Data replicated in N = 4 mice (Ctrl and Cpz). c Population data of myelin coverage in Ctx and Thal revealing a significant myelin loss after Cpz treatment. Two-way ANOVA P < 0.0001 treatment; P = 0.0376 region; P = 0.0451 interaction. Šídák’s multiple comparisons Cpz vs. Ctrl for Ctx ***P = 0.0002 and Thal, *P = 0.0456, n = 58 internodes (Ctx Ctrl), n = 75 (Ctx Cpz), n = 70 (Thal Ctrl), and n = 112 (Thal Cpz) from N = 4 mice (Ctrl and Cpz). d Internode length decreased. Nested t-test **P = 0.0096, t = 3.74, n = 133 internodes (Ctrl, gray open circles), n = 110 (Cpz), N = 4 mice (filled circles). e Example confocal image of paranodes (Caspr⁺) in L5/6 of the cortex. Note the loss in Cpz. Scale bars, 50 µm. f Caspr density significantly reduced in Ctx but not WM or Thal. Kruskal–Wallis P < 0.0001, orange between treatments, multiple comparisons two-stage linear step-up procedure of Benjamini, Krieger, and Yekutieli *P = 0.0254. N = 9 mice (Ctrl Ctx and Thal), N = 6 (Ctrl and Cpz WM), N = 7 (Cpz Ctx), N = 8 (Cpz Thal). g In control axons, internodes were always flanked by two paranodes (white). In Cpz axons, internodes frequently lacked one (gray) or both (black) paranodes. Fischer’s exact test ***P < 0.0001. n = 121 internodes from N = 4 mice (Ctrl). n = 107 internodes from N = 4 mice (Cpz). h Example images of bare stretches of axons between nodal regions and internodes. White arrows, myelin ending and yellow arrows, Caspr⁺ paranodes. Scale bars, 5 µm. N = 4 mice (Ctrl and Cpz). i Immunofluorescence images of axonal spheroids after demyelination. Scale bars, 10 µm. j Percentage axons with spheroids in Ctx, WM and Thal significantly increased. Two-way ANOVA P = 0.0004 treatment; P = 0.0369 region; P = 0.1993 interaction. Šídák’s multiple comparisons, orange between treatment, *P = 0.0192 (WM Ctrl vs Cpz), *P = 0.0118 (Cpz Ctx vs Thal), **P = 0.0012, N = 6 mice (Ctrl), N = 5 mice (Cpz). k Size of swellings in the Ctx was significantly increased. Nested t-test **P = 0.0081, t = 3.65, n = 22 swellings (gray open circles), N = 3 mice (white filled circles, Ctrl), n = 106 swellings, N = 6 mice (black filled circles, Cpz). All data are plotted as mean ± SEM. Source data are provided as a Source data file.