Morphine-Induced Changes in δ Opioid Receptor Trafficking Are Linked to Somatosensory Processing in the Rat Spinal Cord

Abstract

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.

Morphine priming in rats with chronic inflammation reveals a dichotomy between antihyperalgesic and antinociceptive properties of deltorphin

“…injection of the fluorescent deltorphin analog, Fluo-DLT. As previously described elsewhere (Morinville et al, 2004a), this assay is based on the principle that the amount of Fluo-DLT internalized over a 20 min period, as quantified by densitometry in confocal images of the spinal cord, reflects the density of cell surface receptors available to bind and internalize the fluorescent ligand during this period. In sections from the lumbar spinal cord of salinetreated, CFA-injected, and CFA-injected plus morphinetreated rats, numerous fluorescently-labeled neurons were detected by confocal microscopy throughout the gray matter of the spinal cord.…”

“…with 30 l of -Bodipy 576/589 deltorphin-I 5-aminopentylamide (Fluo-DLT) as described (Morinville et al, 2004a). Briefly, animals were anesthetized with sodium pentobarbital (Somnotol; MTC Pharmaceuticals, Cambridge, Ontario, Canada, 6.5 mg/100 g of body weight) and injected i.t.…”

“…By contrast, drugs acting on ␦OR produce more limited analgesia, but also give rise to considerably less undesirable side-effects and induce virtually no tolerance (Porreca et al, 1984;May et al, 1989; Sheldon et al, 1990;Szeto et al, 1999;Gallantine and Meert, 2005). For these reasons, ␦OR agonists have been proposed as possible alternatives to OR agonists for the treatment of chronic pain, including neuropathic (Mika et al, 2001;Petrillo et al, 2003;Morinville et al, 2004a) and chronic inflammatory pain (Desmeules et al, 1993;Stewart and Hammond, 1994;Fraser et al, 2000;Hurley and Hammond, 2000;Qiu et al, 2000;Cahill et al, 2003;Petrillo et al, 2003).One of the reasons for the relatively poor analgesic efficiency of ␦OR agonists may be that only a small proportion of ␦OR is actually present on neuronal plasma membranes under baseline conditions (Cheng et al, 1995(Cheng et al, , 1997Elde et al, 1995;Zhang et al, 1998;Cahill et al, 2001a). However, under conditions of chronic inflammation, such as produced in rodents by injection of complete Freund's adjuvant (CFA) in the hind paw, we observed a massive recruitment of ␦OR from intracellular stores to the plasma membrane in neurons of the dorsal horn of the spinal cord Morinville et al, 2004b).…”

“…By contrast, drugs acting on ␦OR produce more limited analgesia, but also give rise to considerably less undesirable side-effects and induce virtually no tolerance (Porreca et al, 1984;May et al, 1989;Sheldon et al, 1990;Szeto et al, 1999;Gallantine and Meert, 2005). For these reasons, ␦OR agonists have been proposed as possible alternatives to OR agonists for the treatment of chronic pain, including neuropathic (Mika et al, 2001;Petrillo et al, 2003;Morinville et al, 2004a) and chronic inflammatory pain (Desmeules et al, 1993;Stewart and Hammond, 1994;Fraser et al, 2000;Hurley and Hammond, 2000;Qiu et al, 2000;Cahill et al, 2003;Petrillo et al, 2003).…”

Gene transfer to the rhesus monkey brain using SV40-derived vectors is durable and safe

“…To identify neurons and stain neurons, we used Neurotrace (NT), a fluorescent stain that has been applied as a neuronal marker in numerous studies focusing on the characterization of neurons, [54][55][56] and NT staining was performed as reported previously. 21,22,24,57 After rehydration in 0.1 M PBS, pH 7.2, sections were treated with PBS plus 0.1% Triton X-100 10 min, washed twice for 5 min in PBS and then stained by NT (Molecular Probes Inc., Eugene, OR, USA) (1:100) for 20 min at room temperature.…”