Development of tolerance to sustained morphine treatment: changes in phosphorylation and surface presence of spinal MORs. Insert: Animals were subcutaneously implanted with an oily morphine suspension (time zero). Subsequently, the development of tolerance was monitored at various intervals post-opioid administration by measuring the analgesia produced by the release of the opioid. Groups of 10 mice were sacrificed at different intervals and the dorsal horns of the cervical-dorsal spinal cords were removed. To analyze the phosphorylation and presence of MORs in the plasma membrane, the immunoprecipitation was performed under denaturing conditions. For every post-opioid interval analyzed, densitometric signals associated with 55–65, 70–75, and 90–100 kDa were pooled and normalized to those obtained probing the anti-MOR IgGs (heavy chain). The assay was repeated twice and the results were comparable. Further details as in Fig. 1.