The experiments were conducted according to our published procedures, which were adapted from those of (Sim and Childers, 1997; Sim-Selley, Selley, Vogt et al., 2000). Coronal sections (20 µm) of regions of interest were cut on a cryostat (Leica CM3050 S) maintained at −20°C, thaw-mounted onto gelatin-subbed slides, and stored at −80°C until processed. Sections were incubated with 2 mM GDP in [35S]GTPγS binding buffer (50 mM Tris-HCl, 3 mM MgCl2, 0.2 mM EGTA, 100 mM NaCl, pH 7.4) for 15 min at 25°C, and then incubated for 2 hr at 25°C in [35S]GTPγS binding buffer with 0.04 nM [35S]GTPγS and 2 mM GDP with or without the µ agonist DAMGO (10 µM). After incubation, sections were rinsed twice (2 minutes each) in cold 50 mM Tris-HCl buffer, pH 7.4, once (30 seconds) in deionized H2O, and then dried thoroughly and exposed to phosphor screens for 48 hr in cassettes along with [14C]microscales for densitometric analysis.
