(1:500 dilution; Abcam, Cambridge, MA) in 0.5% Triton-PBS overnight at 4°C. The following day, sections were incubated at room temperature for 2 hrs, rinsed in PBS, and then incubated in 1:300 dilution of goat anti-rabbit secondary IgG (Vector Labs) in 0.5% Triton X-100 in PBS for 2 hrs. Following rinsing, sections were incubated in ABC Elite (Vector Labs) for 90 min, rinsed in PBS, and immunoreactivity was revealed by using 3-diaminobenzidine (DAB) with nickel (Vector Labs). To reduce variability in the background and to standardize the staining, sections from subjects across groups were processed concurrently. Sections were mounted and coverslipped with Permount (Fisher Scientific). Cell numbers and region volumes for the interpeduncular nucleus, ventral tegmental area and ventromedial hypothalamus were quantified under 40× magnification using unbiased stereological methods and the optical fractionator probe with Stereo Investigator software (MicroBrightField, Inc., Williston, VT). This method of assessing total volume and cell number has been validated and employed in many prior studies. Total cell counts and area measurements were determined for each brain area, and cell density (number of cells per cubic millimeter) was calculated for each subject.
