Rats were deeply anesthetized with sodium pentobarbital and transcardially perfused with ice cold 0.1M PBS followed by 4% paraformaldehyde (4°; pH=7.4). Brains were extracted and stored in 4% paraformaldehyde overnight at 4°C. Next, brains were moved to a 30% sucrose (w/v) in 0.1M PBS solution and stored at 4°C. 40 micron coronal sections were taken on a freezing sliding microtome. Immunohistochemistry staining and quantification procedures were similar to those we have previously described 18 (link), 33 (link), 34 (link). Free-floating coronal sections were incubated in rabbit anti-c-Fos antibody (1:10,000; Synaptic Systems; #226003; lot #: 3-37) or rabbit anti-Arc antibody (1:4,000; Synaptic Systems; #156003; lot#: 1-62) for 48 h at 4 °C with agitation. Images were acquired utilizing Olympus CX41 light microscope (Olympus America) and analyzed utilizing Image-Pro Premier image analysis software (Media Cybernetics, MD). Immunoreactivity (IR) data (c-Fos positive pixels/mm2 or Arc positive pixels/mm2) were acquired from a minimum of three sections/brain region/animal, and the data were averaged to obtain a single value per subject. The quantification was conducted by an experimenter blind to experimental conditions.