Electron microscopy

Animals were sacrificed and transcardially perfused with PBS followed by a fixative containing 4% paraformaldehyde and 1.5% glutaraldehyde. After postfixation of the brains in the same fixative, sagittal sections were acquired using a vibrating microtome (Leica Microsystems, Wetzlar, Germany) with a thickness of 75 μm. The sections were stained with 0.5% osmium tetroxide in PBS for 30 min and subsequently rinsed in PBS followed by dehydration with 30, 50 and 70% ethanol. Afterward, the sections were treated with 1% uranyl acetate in 70% ethanol for 1 h, before the tissue was further dehydrated by using 80, 90, 96, 100% ethanol and finally propylene oxide. The sections were transferred in Durcupan (Sigma Aldrich, Steinheim, Germany) and embedded in between coated microscope slides and cover slips before the polymerization process at 56°C for 48 h. Regions of interest were located by light microscopy, marked, and transferred on blocks of resin for a second polymerization step. Finally, the embedded tissue was cut into semi‐thin sections and areas for ultrastructural analysis were cut into 55‐nm ultra‐thin sections using an ultra‐microtome (Leica Microsystems, Wetzlar, Germany) and transferred on Formvar‐coated grids and stained with lead citrate for 6 min. The analysis was performed using Zeiss SIGMA electron microscope (Zeiss NTS, Oberkochen, Germany).

Four different regions of the corpus callosum from each sample were analyzed. G‐ratios of transversally sectioned axons were calculated using ImageJ software and compared between groups. The analysis was performed by an investigator who was blinded to the experiment.