Fig. 3.

Ahr ablation modifies myelin structure and locomotor behavior in adult mice. (A) Immunohistochemistry was performed on WT sciatic nerve to localize AHR (green) and P0 (red). The slides were imaged using a confocal microscope. This experiment was repeated at least three times, and a typical experiment is presented here. (Scale bar, 100 μm.) (B) An immunocytochemistry experiment was performed on MSC80 cells. AHR localization is highlighted in red; nuclei were stained with Hoechst (blue). This experiment was repeated at least three times, and a typical experiment is presented here. (Scale bar, 10 μm.) (C) WB showing the expression of AHR in MSC80 cells and in sciatic nerve. (D) In the gait parameter, 8-wk-old WT and Ahr^−/− mice were placed on a treadmill at the speed of 10 cm/s and 16 cm/s. Stride time was measured, ***P < 0.001 by Student’s t test when Ahr^−/− were compared with control mice. (E) For the rotarod test, 8-wk-old WT and Ahr^−/− mice were subjected to the rotarod test for 5 d. The time that mice could stay on the rotarod was measured each day. At least five animals per group were tested. *P < 0.05, **P < 0.01 using Mann–Whitney test compared with control for each day of the experiment. (F) For the rod test, 8-wk-old WT and Ahr^−/− mice were placed on the rod to perform either static or dynamic rod tests. Representative pictures of posture of the mice on the rod are shown. The arrowhead indicates right hind limb where the Ahr^−/− mouse slipped (G) For the static rod test, measurement of the time that WT and Ahr^−/− mice could stay without dropping from the rod. The test was stopped when mice succeeded in staying 100 s. (H) For the dynamic rod test, the average speed to cross the rod was measured (cm/s). (I) The number of faults (slips) counted for either WT or Ahr^−/−. At least five animals per group were tested. *P < 0.05, **P < 0.01 by Student’s t test when Ahr^−/− were compared with WT mice. (J) Sciatic nerves were isolated from either WT or Ahr^−/− mice (8 wk old). Then, ultrathin (50–90 nm) cross-sections were prepared from Epon embedded nerves. (Scale bar, 2 µm.) (K) Myelin thickness was calculated by g-ratio determination. (L) Axonal perimeter was estimated using electronic microscopy pictures quantified by ImageJ software. At least five animals per genotype were used. (M) The g ratios were plotted against the axonal perimeter. (N) Adult male WT or Ahr^−/− mice were killed, and then their sciatic nerves were dissected (at least n = 6 per group). Total proteins were extracted, and WBs were performed using antibodies against P0, PMP22, SOX2, KROX20, β-catenin, active β-catenin, and α-tubulin (loading control). Figures represent a typical experiment. WBs were quantified using ImageJ software. Black horizontal line between the lanes in the gels signifies a crop was made. Error bars indicate SEM. *P < 0.05, **P < 0.01 by Student’s t test when Ahr^−/− were compared with control mice.