Fig 1: Confirmation of the chemogenetic technique by immunohistochemistry 6 weeks after injection of rAAV8/hSyn-DIO-mCherry (Addgene #50459) into the median raphe region (MRR) of dopamine transporter Cre (DAT-Cre) mice. Subregions (MRR and PMR/paramedian raphe/) are defined based on the Mouse Brain Atlas [49]. Position of the coronal section is indicated in each image, relative to the Bregma. (a) Fluorescent micrographs show representative MRR sections with red fluorescent protein (RFP; mCherry, the fluorophore suggesting the presence of the designer receptor exclusively activated by designer drug (DREADD); (red) and tyrosine hydroxylase (TH, rate-limiting enzyme of catecholamine synthesis; green) labelling. (b–d) Confocal laser scanning images show high co-localization between the labelling of RFP and TH in representative MRR neurons (arrows). (e) Fluorescent micrographs show representative MRR sections with RFP (red) and dopamine beta-hydroxylase (DBH, the enzyme which catalyzes the conversion of dopamine to norepinephrine; green) labelling. (f–h) Confocal laser scanning images show no co-localization between the labelling of RFP and DBH. (i–m) Confocal laser scanning images of triple immunohistochemical staining of DREADD marker RPF-positive, TH-positive but DBH-negative neurons in the MRR, indicating dopaminergic phenotype. (n–q) Confocal laser scanning images show co-localization of RFP and DAT.