Coronal brain sections (14 μM thickness) were cryostat cut and mounted 4 sections/slide. Cannabinoid receptor binding was performed using [3H]CP55,940 (specific activity 139.6 Ci/mmol; Research Triangle Institute, Research Triangle Park, NC, USA), as described previously (Herkenham et al., 1991, Hohmann and Herkenham, 1998, Hohmann et al., 1999). Nonspecific binding was determined in the presence of 10 μM CP55,940. Briefly, binding was performed in cytomailers (3 h at 37 °C) in 50 mM Tris-HCl (pH 7.4) containing 5% bovine serum albumin and either 4.6 or 3.3 nM [3H]CP55,940. Binding assays were performed by neuroanatomical level of section, so that brains from rats in all experimental groups were processed concurrently in the same assay. All slides were washed (4 h at 0 °C) in a buffer containing 1% bovine serum albumin, fixed in 0.5% formalin in 50 mM Tris-HCl (pH 7.4 at 25 °C) and blown dry. Sections were apposed to [3H]-sensitive film (Amersham Hyperfilm, GE Healthcare LifeSciences, Piscataway, NJ) with [3H] standards ([3H] microscales, Amersham, Arlington Heights, IL, USA) for 8 weeks for levels incubated in 4.6 nM [3H]CP55,940 and 9
