The coupling of plasma membrane MORs to G proteins reduces tolerance to opioids. The mice were icv-injected with one or two doses of 10 nmol morphine spaced 6 h apart, or they received a single icv dose of 200 pmol DAMGO. Groups of mice that had received the same opioid treatment were sacrificed at various intervals post-opioid administration. The control mice received icv saline instead of the opioid treatment. The PAG synaptosomes (P2) and supernatant (S3) were obtained and the variations in the surface and internalized MORs (representative data in Figs 1-3), and in the association of surface MORs with Gαi2 subunits, was analyzed (see data in Fig. 6). The densitometric signals corresponding to MORs and the associated Gαi2 subunits that were observed in PAG from control mice injected with saline alone were attributed an arbitrary value of 1. The MOR and Gαi2 values corresponding to mice killed at the post-opioid intervals studied were then normalized to the levels observed for the controls. After normalization of the data, the levels of surface MORs observed at the different post-opioid intervals were correlated with the co-precipitation with Gαi2 subunits, as well as with the levels of internalized MORs. Regression lines, regression coefficients and their confidence intervals of 95% are shown (Sigmaplot v10/Sigmastat v 3.5). The data were pooled from two independent assays.