Agonist-induced GTP[γ -35S] activity was evaluated as described 35 on 20 µm coronal sections, by incubating GTP[γ-35S] (0.04 nM) with 3 µM DAMGO in assay buffer at 25°C for 2 hr. Agonist studies were also performed in the presence of the antagonist naloxone to verify the receptor specificity of G-protein activation. Basal activity was assessed in each experiment with GDP in the absence of agonist, and nonspecific binding was evaluated in the presence of 10 µM unlabeled GTP[γS] without GDP. Slides were rinsed, dried and exposed to tritium-sensitive screens (Fujifilm imaging plates).
