On PD 90, animals were deeply anesthetized via sodium pentobarbital and perfused intracardially with phosphate-buffered saline followed by 4% paraformaldehyde in phosphate-buffered saline. Brains were removed, post-fixed overnight in the 4% paraformaldehyde solution and then incubated in a 15% followed by a 30% sucrose solution. Brains were frozen at −80°C until sectioned. Brains were sectioned coronally (40 μm) beginning anterior to the rostral NAC (Plate 9, Paxinos and Watson, 2005) and continuing through the hippocampus (Plate 57, Paxinos and Watson, 2005). First section was randomly selected and every 6th succeeding section throughout the caudal extent of the NAC was mounted on glass slides and cover slips were attached with Permount. Serial sets were stained using the classical Nissl stain. Glia and neurons in both core and shell were counted using the optical fractionator method using Stereo Investigator (MBF Bioscience). Glial cells were defined by dense heterogeneous staining in the nucleus only compared to neurons which had staining throughout the soma and dense nuclear staining (see Cotter et al., 2002). Separate contours were used for NAC core and shell based on matched sections from the atlas of Paxinos and Watson (2005).
