Animals were deeply anesthetized and transcardially perfused with 1X phosphate-buffered saline (PBS) followed by 4% paraformaldehyde in 1X PBS. Brains were removed from the skull and postfixed in 4% paraformaldehyde. Brains were cryoprotected in 30% sucrose, embedded in optimal cutting temperature compound (VWR; Radnor, PA) and frozen in preparation for cryosectioning. Coronal 40-μm thick sections were made using a cryostat (Leica Biosystems; Wetzlar, Germany). Sections were Nissl-stained with 0.1% cresyl violet (Abcam; Cambridge, United Kingdom) to identify brain structures. Sections were dehydrated, cleared in histoclear, and coverslipped using cytoseal mounting media (VWR; Radnor, PA). They were imaged using a VS120 Virtual Slide Scanner (Olympus; Tokyo, Japan) using the 10X objective. ImageJ software was used for measurements (National Institutes of Health, Bethesda, MD, USA) by an observer blinded to genotype. For analysis of the width of several brain regions, the length of d1–d5 was measured at bregma, + 0.86 to +1.18 mm according previously published methods (Mizoguchi et al., 2017 (link)). The width of each brain region was defined as follows: d1=whole brain, d2-d3=striatum, d5=motor cortex, d1-d2=somatosensory cortex, d4=septum. The mouse brain in stereotaxic coordinates atlas (Franklin & Paxinos, 2008 ) was used to locate each brain region.