Volumetric and Histological Brain Analysis

Animals in both groups were euthanized and intracardially perfused when behavioral testing completed. Brains including cerebellum were removed, weighed, and fixed for coronal sectioning and cresyl violet staining. Criteria for brain abnormality was chosen based on the brain weight after extraction^{33 (link)}, and no animal was excluded from analysis for abnormal brain weight. The stained sections were imaged using a Nanozoomer 2.0RS slide scanner (Hamamatsu, Japan) for histological analysis and presentation. From the coronal sections, measurements included cortical and dorsal hippocampal (dHPC) volumetrics, neuronal density, and cortical thickness in both hemispheres. Furthermore, in order to enhance the accuracy of the histology, six animals from B6 group and one animal from the BTBR group were excluded from histological assessments and also from all other corresponding volumetric analyses because of damages to one or both hemispheres, and technical issues such as missing and/or damaged sections.