Visualizing hM4Di DREADD Expression

hM4Di receptor expression in cell bodies and processes was visualized with immunohistochemistry for HA, GFP, or mCherry tags (described below). Containment of transduction sites within RVP or CVP, or VTA or SN dopamine cells, was confirmed by co-staining for substance P (SP; defining borders of VP), or TH (defining VTA and SN), and a brain atlas⁵⁰ . A synapsin promoter virus was employed in VP, so some expression was observed outside the borders of RVP or CVP in most animals. Animals with more than 30% of DREADD expression observed outside RVP or CVP were excluded from analyses (n=7). For animals with RVP virus injections, no more than 20% of DREADD-expressing tissue encroached into CVP (i.e., caudal of bregma), or vice versa. Animals injected with the Syn-GFP control virus in VP had comparable GFP expression patterns to hM4Di animals. For TH::Cre rats, hM4Di expression was robust in either VTA or SN, and was restricted almost exclusively to TH+ cell bodies and processes [% mCherry+ neurons coexpressing TH: m±SEM=97.5±0.7%; Fig. 6b–c, Supplemental Fig. 6]. mCherry expression was not observed in the contralateral hemisphere, nor did expression extend substantially into the SN of VTA-injected animals, or into VTA of SN-injected animals (Fig. 6b–c).