Opioid agonists are highly efficacious analgesics; however their clinical use is limited by the incidence of adverse effects, particularly the development of analgesic tolerance following repeated use. A growing body of evidence identifies an important role for the δOR in modulating morphine tolerance [5–10] and this phenomenon may involve the trafficking of δORs from internal stores toward the neuronal plasma membrane, thereby enhancing the effects of δOR-selective ligands [4, 14–18, 33]. The mechanism by which this contributes to morphine tolerance is unknown; however recent studies support an active role for spinal glia following chronic morphine treatment [31, 32]. In the current study, we investigated the relationship between δORs and glial activation and indeed demonstrate a functional role for spinal glia in morphine-induced changes in δOR agonist effects. Moreover, administration of a glial inhibitor effectively blocked these changes in δOR function.
The involvement of spinal glia in the modulation of morphine analgesia has been demonstrated in both preclinical [25, 27, 31, 32, 34–36] and clinical  studies. We hypothesized that the recruitment of glial cells is a gradual response to long-term morphine administration and may be detectable at time points earlier than those at which analgesic tolerance is established. We therefore assessed the spinal immune response using a 48 h morphine dosing schedule; one which has been shown to have substantive effects on δOR trafficking and function [14–16, 38]. This dosing regimen does not produce a state of tolerance ; however it may initiate mechanisms involved early in the cascade of events leading to opioid tolerance. In developing a means of assessing the three dimensional structures of GFAP- and OX42-immunoreactive cells, we observed significant increases in cell volume and surface area of fluorescent GFAP- and OX42-immunoreactive cells in the dorsal spinal cord following prolonged morphine treatment. These results are in accordance with previous studies [31, 32] illustrating the recruitment of glia in the events precipitating opioid tolerance. Morphine-induced glial hypertrophy was attenuated by co-administration with propentofylline. Interestingly, while propentofylline administration alone had no effect on astrocytes, it produced significant microglial hypertrophy in comparison with saline-treated rats. It is not clear why this occurs, since the combination of morphine and propentofylline did not show such an effect. The neuroprotective role of microglia in the CNS is well known and this cell population is very much attuned to its microenvironment, responding swiftly to even subtle physiological changes . It is possible that the localized administration of an exogenous compound into the spinal canal, in the absence of any 'pathological' events, was sufficient to produce a microglial response, although such an observation has not been reported previously . Nevertheless, additional functional studies are necessary to determine whether this propentofylline-induced increase in cell size was indeed accompanied by an inflammatory response. Despite microglial hypertrophy, however, neither baseline tail flick latencies nor deltorphin-mediated analgesia were altered following propentofylline administration alone, suggesting that this increase in microglial cell size was not functionally relevant in our study.
Activation of both glial cells and δORs appears to be important in the mechanisms of morphine tolerance, however it is unknown whether these two events are mutually exclusive or if, in fact, they represent important and related intermediary steps in the development of tolerance. Previous studies demonstrate that δORs are trafficked from internal stores toward the neuronal plasma membrane following morphine treatment, correlating with an increased functional competence of the receptor [14–16]; however it is not known if the spinal immune response observed following morphine is requisite for this δOR trafficking event. Therefore, our second series of experiments aimed to examine the functional role of spinal glia in morphine-induced changes in δOR function. Consistent with earlier reports [14, 15, 40, 41], we observed a significant augmentation in δOR-mediated effects in rats treated with morphine. This enhancement was effectively blocked by co-administration of morphine with propentofylline, demonstrating an integral role of spinal glial activation in the functional changes in δOR.
Taken together with previous reports that glial inhibition prevents the development of morphine tolerance [27, 31, 32], this study provides additional evidence for the role of δORs in opioid tolerance and suggests that glial activity may precipitate changes in the δOR, including receptor trafficking. Glial cell activity has been documented to modulate the trafficking of ionotropic channels such as AMPA receptors [42, 43]; however the current study is the first to our knowledge to suggest such a modulation of a G protein coupled receptor. The precise mechanism by which glial-modulated functional changes in δOR may occur is unclear; however two possible mechanisms include i) increased efficiency with which the receptor couples to intracellular signaling cascades, and/or ii) enhanced cell surface expression of the receptor. Future experiments will be required to investigate these possibilities.