Mapping Neuronal Activation in Rat Brain

2 h prior to euthanasia, CTRL (CTRL + CNO: n = 3) and hM3d (hM3d + CNO: n = 3) rats received an intra-NAc microinfusion of 1uM CNO at a rate of 0.5ul/min for 4 min, followed by 2 min before removal of internal cannula. Rats were perfused with 1× PBS for 5 min followed by 4% paraformaldehyde for 15 min. Brains were removed and post-fixed for 24 h in 4% paraformaldehyde. Brains were stored in PBS glycerol sodium azide until slicing. Immunohistochemistry was conducted as previously described^{17 (link),18 (link),27 (link),31} . Phospho-cFos was immuno-enhanced using anti-rabbit phospho-cFos (Ser 32) (Cell Signaling, 5348), then visualized in both the PVN and the NAc. Tile scans were acquired at 10× using Leica True Confocal Scanner SPE in confocal mode and Leica Application Suite x software (Leica Microsystems). Images were analyzed for phospho-cFos expression using FIJI (Image J). A region of interest was created in FIJI and applied to all images. The following parameters were applied to all images for analysis: rolling ball radius: 20.0, threshold: 0–20, particle size: 50-infinity (pixel units), circularity: 0.00–1.00.