The repeated administration of DAMGO promotes incomplete recycling of internalized MORs. Insert: Groups of mice were icv-injected with three successive doses of 10 nmol morphine or 200 pmol DAMGO spaced 3 h apart, plus a fourth dose given 24 h after the first. Antinociception was determined by the warm water (52°C) tail-flick test at various time intervals post-injection. Details as in the Figs. 1 & 2. The mice were killed 3 h after the first, the second or the third dose of DAMGO and the MORs were immunoprecipitated under denaturing conditions from the P2 (membrane) and S3 (internalized) preparations. Mice that had received four doses of DAMGO were killed 30 min later and the PAG S3 fraction was subjected to subcellular fractionation and the presence of MOR was determined. Subcellular markers: EEA1 (early endosome antigen 1; BD 610456), Rab4 (BD 610888), Rab5 (BD 610281), Rab 11 (BD 610656), Lamp-1 (lysosomal-associated membrane protein 1; BD 611043).For further details see Methods.