Figure 1

Regulation of neural MORs by icv administration of an analgesic dose of morphine. Insert: The mice were icv-injected with 10 nmol morphine and antinociception was determined by the warm water (52°C) tail-flick test at various time intervals post-injection. Antinociception was expressed as a percentage of the maximum possible effect after setting a cut-off time of 10 seconds. The values shown are the mean ± SEM from groups of 10–15 mice. Effect of morphine treatment on internalization and phosphorylation of the C terminal Ser375 of MOR. The PAG synaptosomes (P2) and supernatant (S3) were obtained at various intervals post-morphine administration. For each time point studied the PAG structures from 6 to 8 mice were pooled. To reduce the risk of interference with signals from proteins other than the MORs, the study of these receptors and their Ser375 phosphorylation was performed by immunoprecipitation after releasing the associated proteins by SDS solubilization (denaturing conditions, see Methods). In order to detect additional protein bands, the areas inside the rectangles were overexposed. The densitometric immunosignals associated with the 55–65 kDa band (average optical density of the pixels within the object area/mm2; Quantity One Software, BioRad) were normalized to those obtained probing the anti-MOR IgGs hc (heavy chain) with the appropriate secondary antibody. These IgGs were detached from the immunoprecipitated MORs and processed in parallel gels/blots (see Methods). Each bar is the mean ± SEM of three assays performed on PAG samples obtained from independent groups of mice. The data are expressed relative to the levels observed for the control group (attributed an arbitrary value of 1).