Opioid analgesics are powerful pain relievers; however, over time, pain control diminishes as analgesic tolerance develops. The molecular mechanisms initiating tolerance have remained unresolved to date. We have previously shown that desensitization of the μ-opioid receptor and interaction with β-arrestins is controlled by carboxyl-terminal phosphorylation. Here we created knockin mice with a series of serine- and threonine-to-alanine mutations that render the receptor increasingly unable to recruit β-arrestins. Desensitization is inhibited in locus coeruleus neurons of mutant mice. Opioid-induced analgesia is strongly enhanced and analgesic tolerance is greatly diminished. Surprisingly, respiratory depression, constipation, and opioid withdrawal signs are unchanged or exacerbated, indicating that β-arrestin recruitment does not contribute to the severity of opioid side effects and, hence, predicting that G-protein-biased µ-agonists are still likely to elicit severe adverse effects. In conclusion, our findings identify carboxyl-terminal multisite phosphorylation as key step that drives acute μ-opioid receptor desensitization and long-term tolerance.

Key pointsr The main cause of death from opioid overdose is respiratory depression due to the activation of µ-opioid receptors (MORs).r We conditionally deleted MORs from neurons in two key areas of the brainstem respiratory circuitry (the Kölliker-Fuse nucleus (KF) and pre-Bötzinger complex (preBötC)) to determine their role in opioid-induced respiratory disturbances in adult, awake mice.r Deletion of MORs from KF neurons attenuated respiratory rate depression at all doses of morphine.r Deletion of MORs from preBötC neurons attenuated rate depression at the low dose, but had no effect on rate following high doses of morphine. Instead, high doses of morphine increased the occurrence of apnoeas.r The results indicate that opioids affect distributed key areas of the respiratory network in a dose-dependent manner and countering the respiratory effects of high dose opioids via the KF may be an effective approach to combat overdose.Abstract The primary cause of death from opioid overdose is respiratory failure. High doses of opioids cause severe rate depression and increased risk of fatal apnoea, which correlate with increasing irregularities in breathing pattern. µ-Opioid receptors (MORs) are widely distributed throughout the brainstem respiratory network, but the mechanisms underlying respiratory depression are poorly understood. The medullary pre-Bötzinger complex (preBötC) and the pontine Kölliker-Fuse nucleus (KF) are considered critical for inducing opioid-related respiratory disturbances. We used a conditional knockout approach to investigate the roles and relative contribution of MORs in KF and preBötC neurons in opioid-induced respiratory depression in Adrienn Varga received her Ph.D. from Case Western Reserve University, where she studied the neural processes underlying navigation in an insect model. This work provided her with an appreciation for how the central nervous system integrates sensory information to shape motor commands. For her postdoctoral work at the University of Florida, she has continued to build on this previous training by moving into the rodent respiratory system, which provides a powerful model for relating sensory cues to the coordination of rhythmic behaviours. An important component of this research is seeking to define the neural mechanisms underlying opioid-induced respiratory depression.A. G. Varga and others J Physiol 598.1 awake adult mice. The results revealed dose-dependent and region-specific opioid effects on the control of both respiratory rate and pattern. Respiratory depression induced by an anti-nociceptive dose of morphine was significantly attenuated following deletion of MORs from either the KF or the preBötC, suggesting cumulative network effects on respiratory rate control at low opioid doses. Deletion of MORs from KF neurons also relieved rate depression at near-maximal respiratory depressant doses of morphine. Meanwhile, deletion of MORs from the preBötC had no effect on rate following administration of high doses of morphine. Instead, a severe ataxic breathing pa...

Key pointsr In addition to reductions in respiratory rate, opioids also cause aspiration and difficulty swallowing, indicating impairment of the upper airways. The Kölliker-Fuse (KF) maintains upper airway patency and a normal respiratory pattern.r In this study, activation of μ opioid receptors in the KF reduced respiratory frequency and tidal volume in anaesthetized rats.r Nerve recordings in an in situ preparation showed that activation of μ opioid receptors in the KF eliminated the post-inspiration phase of the respiratory cycle.r In brain slices, μ opioid agonists hyperpolarized a distinct population (61%) of KF neurons by activation of an inwardly rectifying potassium conductance.r These results suggest that KF neurons that are hyperpolarized by opioids could contribute to opioid-induced respiratory disturbances, particularly the impairment of upper airways.Abstract Opioid-induced respiratory effects include aspiration and difficulty swallowing, suggesting impairment of the upper airways. The pontine Kölliker-Fuse nucleus (KF) controls upper airway patency and regulates respiration, in particular the inspiratory/expiratory phase transition. Given the importance of the KF in coordinating respiratory pattern, the mechanisms of μ opioid receptor activation in this nucleus were investigated at the systems and cellular level. In anaesthetized, vagi-intact rats, injection of opioid agonists DAMGO or [Met 5 ]enkephalin (ME) into the KF reduced respiratory frequency and amplitude. The μ opioid agonist DAMGO applied directly into the KF of the in situ arterially perfused working heart-brainstem preparation of rat resulted in robust apneusis (lengthened low amplitude inspiration due to loss of post-inspiratory drive) that was rapidly reversed by the opioid antagonist naloxone. In brain slice preparations, activation of μ opioid receptors on KF neurons hyperpolarized a distinct population (61%) of neurons. As expected, the opioid-induced hyperpolarization reduced the excitability of the neuron in response to either current injection or local application of glutamate. In voltage-clamp recordings the outward current produced by the opioid agonist ME was concentration dependent, reversed at the potassium equilibrium potential and was blocked by BaCl 2 , characteristics of a G protein-coupled inwardly rectifying potassium (GIRK) conductance. The clinically used drug morphine produced an outward current in KF neurons with similar potency to morphine-mediated currents in locus coeruleus brain slice preparations. Thus, the population of KF neurons that are hyperpolarized by μ opioid agonists are likely mediators of the opioid-induced loss of post-inspiration and induction of apneusis.

ABSTRACT-Opioid receptor desensitization is considered an initial step in the development of tolerance. Curiously, the commonly used opioid morphine produces robust tolerance but minimal acute desensitization. This study was designed to test the hypothesis that desensitization is indeed present in morphine-treated animals and is distinguished from cellular tolerance by time course of recovery and mechanism. To induce tolerance, rats were treated with continuously released morphine for 1 week. Morphine-mediated activation of G protein-coupled inwardly rectifying potassium conductance was measured using voltage-clamp recordings from locus ceruleus neurons in brain slices from naive or morphine-treated rats. Cellular tolerance was observed as a decrease in morphine efficacy in slices from morphine-treated rats. This tolerance persisted for at least 6 h. An additional reduction in morphine-mediated current was observed when slices from morphine-treated rats were continuously maintained in morphine at approximately the circulating plasma concentration. This additional reduction recovered within 1 h after removal of morphine from the slice and represents desensitization that developed in the tolerant animal. Recovery from desensitization, but not long-lasting tolerance, was facilitated by protein phosphatase 1 (PP1) activity. Furthermore, desensitization, but not tolerance, was reversed by protein kinase C (PKC) inhibitor but not by an inhibitor of c-Jun N-terminal kinase. Therefore, morphine treatment leads to both long-lasting cellular tolerance and readily reversible desensitization, which are differentially dependent on PP1 and PKC activity and combine to result in a substantial decrease in morphine effectiveness. This PKC-mediated desensitization may contribute to the previously reported PKC-dependent reversal of behavioral tolerance.