Enkephalin release promotes homeostatic increases in constitutively active mu opioid receptors during morphine withdrawal

We previously demonstrated that naloxone administration produces a robust conditioned place aversion (CPA) in opiate-naive rodents by blocking the action of enkephalins at μ opioid receptors (MORs). The aversive response to naloxone is potentiated by prior exposure to morphine. Morphine-induced MOR constitutive activity is hypothesized to underlie this enhanced effect of naloxone, an inverse agonist at the MOR. We sought additional evidence for the role of constitutively active MORs in this morphine-induced enhancement using the pro-enkephalin knockout (pENK−/−) mouse, which is devoid of naloxone CPA in the morphine-naive state. Naloxone, but not the neutral antagonist, 6-β-naloxol, produced CPA and physical withdrawal signs in pENK−/− mice when administered 2 h, but not 20 h, after morphine administration. Naloxone-precipitated physical withdrawal signs were attenuated in the pENK−/− mice relative to wild-type (WT) animals. In both WT and pENK−/− mice, naloxone-precipitated withdrawal jumping was greatest when naloxone was administered 2 h after morphine treatment and diminished at 3 h, in agreement with previous estimates of the time course for morphine-induced MOR constitutive activity in vitro. However, naloxone regained an ability to precipitate physical withdrawal in the WT, but not the pENK−/− mice when administered 4.5 h after morphine administration. Taken together, the data suggest that a compensatory increase in enkephalin release during spontaneous morphine withdrawal promotes a second period of MOR constitutive activity in WT mice that is responsible for the enhanced naloxone aversion observed in such animals even when naloxone is administered 20 h after morphine. The endogenous enkephalin system and MOR constitutive activity may therefore play vital roles in hedonic homeostatic dysregulation following chronic opiate administration.