Tracing PVHCrh Neuronal Projections

To track PVHCrh projections in the brain, we stereotaxically microinjected 40 nl of AAV8-FLEX-hsyn-ChR2-mCherry (UNC Vector Core) into the unilateral PVH (from bregma: A/P 0.26, M/L 0.76, D/V −4.8) of adult Crh-ires-Cre mice^{44 (link)} (a gift from Dr. Bradford Lowell) using a glass micropipette and an air pressure injection system. Mice were perfused 7 weeks later, the brains were removed and stored in 10% formalin overnight followed by transfer into 20% sucrose. Sectioned brain tissue (30um) was quenched in 0.3% H₂O₂ for 30 minutes, and then incubated overnight with rabbit polyclonal antiserum against mCherry (1:5000; Clontech #632496). Between this incubation and each subsequent incubation, tissue was washed with 1XPBS for 5 min × 5 times. On the second day, sections were incubated with biotinylated-conjugated donkey anti-rabbit antiserum (1:500, Jackson#712065152) for 2 h, followed by avidin-biotin complex (ABC) solution for 1h. Sections were incubated in DAB (3, 3′-Diaminobenzidine), TBS, H₂O₂ for less than 5 minutes. Sections were mounted and coverslipped (Cytoseal, Thermoscientific). Images were captured with a Zeiss AxioCam HRc microscope and AxioVision Rel software (v4.8).