We recently demonstrated that prolonged treatment with morphine increases the antinociceptive potency of the delta-opioid receptor (deltaOR) agonist deltorphin and promotes cell surface targeting of deltaORs in neurons of the dorsal horn of the rat spinal cord (Cahill et al., 2001b). In the present study we examined whether these effects were mediated selectively via muOR. Using the same intermittent treatment regimen as for morphine, we found that methadone and etorphine, but not fentanyl, enhanced [D-Ala2]-deltorphin-mediated antinociception. However, continuous delivery of fentanyl for 48 hr resulted in augmented deltaOR-mediated antinociception when compared with saline-infused animals. Time course studies confirmed that a 48 hr treatment with morphine was necessary for the establishment of enhanced deltaOR-mediated antinociception. The observed increases in deltaOR agonist potency and deltaOR plasma membrane density were reversed fully 48 hr after discontinuation of morphine injections. Wild-type C57BL/6 mice pretreated with morphine for 48 hr similarly displayed enhanced deltaOR-mediated antinociception in a tonic pain paradigm. Accordingly, the percentage of plasma membrane-associated deltaOR in the dorsal horn of the spinal cord, as assessed by immunogold electron microscopy, increased from 6.6% in naive to 12.4% in morphine-treated mice. In contrast, morphine treatment of muOR gene knock-out (KO) mice did not produce any change in deltaOR plasma membrane density. These results demonstrate that selective activation of muOR is critical for morphine-induced targeting of deltaOR to neuronal membranes, but not for basal targeting of this receptor to the cell surface.

An in vivo fluorescent deltorphin (Fluo-DLT) internalization assay was used to assess the distribution and regulation of pharmacologically available ␦ opioid receptors (␦ORs) in the rat lumbar (L4 -5) spinal cord. Under basal conditions, intrathecal injection of Fluo-DLT resulted in the labeling of numerous ␦OR-internalizing neurons throughout dorsal and ventral horns. The distribution and number of Fluo-DLT-labeled perikaryal profiles were consistent with that of ␦OR-expressing neurons, as revealed by in situ hybridization and immunohistochemistry, suggesting that a large proportion of these cells was responsive to intrathecally administered ␦OR agonists. Pretreatment of rats with morphine for 48 hr resulted in a selective increase in Fluo-DLT-labeled perikaryal profiles within the dorsal horn. These changes were not accompanied by corresponding augmentations in either ␦OR mRNA or 125 I-deltorphin-II binding levels, suggesting that they were attributable to higher densities of cell surface ␦OR available for internalization rather than to enhanced production of the receptor. Unilateral dorsal rhizotomy also resulted in increased Fluo-DLT internalization in the ipsilateral dorsal horn when compared with the side contralateral to the deafferentation or to non-deafferented controls, suggesting that ␦OR trafficking in dorsal horn neurons may be regulated by afferent inputs. Furthermore, morphine treatment no longer increased Fluo-DLT internalization on either side of the spinal cord after unilateral dorsal rhizotomy, indicating that OR-induced changes in the cell surface availability of ␦OR depend on the integrity of primary afferent inputs. Together, these results suggest that regulation of ␦OR responsiveness through OR activation in this region is linked to somatosensory information processing.

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