Male C57BL/6JRj mice (8 weeks; JanVier, Le Genest-Saint-Isle, France) received a single administration of ulotaront (3 mg/kg, ip), RO5263397 (0.3 mg/kg, ip) or the respective vehicle 105 min prior to the transcranial perfusion (heparinized PBS and 10% neutral buffered formalin (NBF)). Subsequently, the brains were dissected and postfixed overnight in 10% NBF at room temperature. The iDISCO+ (immunolabeling-enabled three-dimensional imaging of solvent-cleared organs) [31 (link),32 (link)] protocol was used for whole brain immunolabelling as previously described [33 (link),34 (link)]. For visualization of c-fos expression, rabbit anti-c-fos antibody (1:5000, Cell Signaling Technology) was used followed by incubation with the secondary donkey anti rabbit Cy-5 antibody (1:1000, Jackson ImmunoResearch). The optically transparent brain samples were imaged using LaVision ultramicroscope II (Miltenyi Biotec). Image processing, registration and cell detection was performed according to the method of Perens and colleagues [33 (link)]. In addition to voxel-level analysis, a total of 839 brain regions of interest (ROIs) were analyzed.
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