Mu-opioid Receptor Knockout Mice Research Articles

NCP, a dual kappa and mu opioid receptor agonist, is a potent analgesic against inflammatory pain without reinforcing or aversive properties.

While agonists of mu (MOR) and kappa (KOR) opioid receptors have analgesic effects, they produce euphoria and dysphoria, respectively. Other side effects include respiratory depression and addiction for MOR agonists and sedation for KOR agonists. We reported that 17-cyclopropylmethyl-3,14β-dihydroxy-4,5α-epoxy-6β-{[4'-(2'-cyanopyridyl)]carboxamido}cmorphinan (NCP) displayed potent KOR full agonist and MOR partial agonist activities (58%) with 6.5x KOR-over-MOR selectivity in vitro Herein, we characterized pharmacological effects of NCP in rodents. In mice, NCP exerted analgesic effects against inflammatory pain in both the formalin test and the acetic acid writhing test, with A50 values of 47.6 and 14.4 microg/kg (s.c.), respectively. The analgesic effects in the acetic acid writhing test were mediated by the KOR. NCP at doses much higher than those effective in reducing inflammatory pain did not produce antinociception in the hot plate and tail flick tests, inhibit compound 48/80-induced scratching, cause conditioned place aversion (CPA) or preference, impair rotarod performance, inhibit locomotor activity, cause respiratory depression, or precipitate morphine withdrawal. However, NCP (10~100 microg/kg) inhibited gastrointestinal transit with a maximum of ~40% inhibition. In MOR knockout mice, NCP caused CPA, demonstrating that its lack of CPA is due to combined actions on the MOR and KOR. Following s.c. injection, NCP penetrated into the mouse brain. In rats trained to self-administer heroin, NCP (1~320 microg/kg/infusion) did not function as a reinforcer. Thus, NCP produces potent analgesic effects via KOR without side effects except constipation. Therefore, dual full KOR/partial MOR agonists with moderate KOR-over-MOR selectivity may be promising as non-addictive analgesics for inflammatory pain. Significance Statement Developing non-addictive analgesics is crucial for reducing opioid overdose deaths, minimizing drug misuse, and promoting safer pain management practices. Herein, pharmacology of a potential non-addictive analgesic, NCP, is reported. NCP has full KOR agonist / partial MOR agonist activities with a 6.5 x selectivity for KOR over MOR. Unlike MOR agonists, analgesic doses of NCP do not lead to self-administration or respiratory depression. Furthermore, NCP does not produce aversion, hypolocomotion, or motor incoordination, side effects typically associated with KOR activation.

Review of the role of the endogenous opioid and melanocortin systems in the restless legs syndrome.

Restless legs syndrome (RLS) is responsive to opioid, dopaminergic and iron-based treatments. Receptor blocker studies in RLS patients suggest that the therapeutic efficacy of opioids is specific to the opioid receptor and mediated indirectly through the dopaminergic system. An RLS autopsy study reveals decreases in endogenous opioids, β-endorphin and perhaps Met-enkephalin in the thalamus of RLS patients. A total opioid receptor knock-out (mu, delta and kappa) and a mu-opioid receptor knock-out mouse model of RLS show circadian motor changes akin to RLS and, although both models show sensory changes, the mu-opioid receptor knock mouse shows circadian sensory changes closest to those seen in idiopathic RLS. Both models show changes in striatal dopamine, anaemia and low serum iron. However, only in the total receptor knock-out mouse do we see the decreases in serum ferritin that are normally found in RLS. There are also decreases in serum iron when wild-type mice are administered a mu-opioid receptor blocker. In addition, the mu-opioid receptor knock-out mouse also shows increases in striatal zinc paralleling similar changes in RLS. Adrenocorticotropic hormone and α-melanocyte stimulating hormone are derived from pro-opiomelanocortin as is β-endorphin. However, they cause RLS-like symptoms and periodic limb movements when injected intraventricularly into rats. These results collectively suggest that an endogenous opioid deficiency is pathogenetic to RLS and that an altered melanocortin system may be causal to RLS as well.

The Role of Mu Opioid Receptors in High Fat Diet-Induced Reward and Potentiation of the Rewarding Effect of Oxycodone.

Excessive high fat diet (HFD) consumption can induce food addiction, which is believed to involve the communication between the hypothalamus and mesolimbic dopaminergic neurons, originating in the ventral tegmental area (VTA) and projecting to the nucleus accumbens (NAc). These brain areas are densely populated with opioid receptors, raising the possibility that these receptors, and particularly mu opioid receptors (MORs), are involved in rewards elicited by palatable food. This study sought to investigate the involvement of MORs in HFD-induced reward and if there is any difference between male and female subjects in this response. We also assessed if exposure to HFD would alter the rewarding action of oxycodone, a relatively selective MOR agonist. The place conditioning paradigm was used as an animal model of reward to determine if short-time (STC, 2 h) or long-time (LTC, 16 h) conditioning with HFD induces reward or alters the rewarding action of oxycodone. Male and female C57BL/6J mice as well as MOR knockout and their wildtype littermates of both sexes were tested for basal place preference on day 1 and then conditioned with an HFD in one chamber and a regular chow diet (RCD) in another chamber for 2 h on alternate days. Three sets of STC were used, followed by a set of LTC. Each set of conditioning consisted of two conditioning with RCD and two conditioning with HFD. Mice were tested for place preference after each set of STC and again after LTC. Controls were conditioned with RCD in both conditioning chambers. Following the last place preference test, mice were treated with oxycodone and conditioned in the HFD-paired chamber and with saline in the RCD-paired chamber for one hour once a day to explore the possibility if the HFD could alter oxycodone reward. The result showed that HFD induced conditioned place preference (CPP) in male but not female subjects. However, oxycodone conditioning elicited reward in both male and female mice of the HFD group but not the control group, showing that prior conditioning with HFD potentiated the rewarding action of oxycodone. The latter response was mediated via MORs, as it was blunted in MOR knockout mice. Similarly, HFD-induced CPP was blunted in male MOR knockout mice, suggesting sexual dimorphism in this response.

6-O-(2-[18F]Fluoroethyl)-6-O-Desmethyl-Diprenorphine ([18F]FE-DPN) Preferentially Binds to Mu Opioid Receptors In Vivo.

6-O-(2-[18F]Fluoroethyl)-6-O-desmethyl-diprenorphine ([18F]FE-DPN) is regarded as a non-selective opioid receptor radiotracer. Here, we report the first characterization of [18F]FE-DPN synthesized from the novel precursor, 6-O-(2-tosyloxyethoxy)-6-O-desmethyl-3-O-trityl-diprenorphine (TE-TDDPN), using a one-pot, two-step nucleophilic radiosynthesis to image opioid receptors in rats and mice using positron emission tomography. We also show that [18F]FE-DPN and [3H]DPN exhibit negligible brain uptake in mu opioid receptor (MOR) knockout mice. Taken together with prior findings, our results suggest that [18F]FE-DPN and [3H]DPN preferentially bind to MOR in rodents in vivo.

Contribution of the Opioid System to the Antidepressant Effects of Fluoxetine

BackgroundSelective serotonin reuptake inhibitors such as fluoxetine have a limited treatment efficacy. The mechanism by which some patients respond to fluoxetine while others do not remains poorly understood, limiting treatment effectiveness. We have found the opioid system to be involved in the responsiveness to fluoxetine treatment in a mouse model for anxiety- and depressive-like behavior. MethodsWe analyzed gene expression changes in the dentate gyrus of mice chronically treated with corticosterone and fluoxetine. After identifying a subset of genes of interest, we studied their expression patterns in relation to treatment responsiveness. We further characterized their expression through in situ hybridization and the analysis of a single-cell RNA sequencing dataset. Finally, we behaviorally tested mu and delta opioid receptor knockout mice in the novelty suppressed feeding test and the forced swim test after chronic corticosterone and fluoxetine treatment. ResultsChronic fluoxetine treatment upregulates proenkephalin expression in the dentate gyrus, and this upregulation is associated with treatment responsiveness. The expression of several of the most significantly upregulated genes, including proenkephalin, is localized to an anatomically and transcriptionally specialized subgroup of mature granule cells in the dentate gyrus. We have also found that the delta opioid receptor contributes to some, but not all, of the behavioral effects of fluoxetine. ConclusionsThese data indicate that the opioid system is involved in the antidepressant effects of fluoxetine, and this effect may be mediated through the upregulation of proenkephalin in a subpopulation of mature granule cells.

Involvement of the Peripheral μ-Opioid Receptor in Tramadol-Induced Constipation in Rodents.

Tramadol is a weak opioid that produces analgesic effect via both the μ-opioid receptor (MOR) and non-opioid targets. Constipation is the most common opioid-related side effect in patients with cancer and non-cancer pain. However, the contribution of MOR to tramadol-induced constipation is unclear. Therefore, we used naldemedine, a peripherally acting MOR antagonist, and MOR-knockout mice to investigate the involvement of peripheral MOR in tramadol-induced constipation using a small intestinal transit model. A single dose of tramadol (3-100 mg/kg, per os (p.o.)) inhibited small intestinal transit dose-dependently in rats. Naldemedine (0.01-10 mg/kg, p.o.) blocked the inhibition of small intestinal transit induced by tramadol (30 mg/kg, p.o.) in rats. The transition rate increased dose-dependently over the range of naldemedine 0.01-0.3 mg/kg, and complete recovery was observed at 0.3-10 m/kg. Additionally, tramadol (30 and 100 mg/kg, subcutaneously (s.c.)) inhibited small intestinal transit in wild-type mice but not in MOR-knockout mice. These results suggest that peripheral MOR participates in tramadol-induced constipation.

Naltrexone is neuroprotective against traumatic brain injury in mu opioid receptor knockout mice

AimsNaltrexone is a mu opioid receptor (MOR) antagonist used to treat drug dependence in patients. Previous reports indicated that MOR antagonists reduced neurodegeneration and inflammation after brain injury. The purpose of this study was to evaluate the neuroprotective effect of naltrexone in cell culture and a mouse model of traumatic brain injury (TBI).MethodsThe neuroprotective effect of naltrexone was examined in primary cortical neurons co‐cultured with BV2 microglia. Controlled cortical impact (CCI) was delivered to the left cerebral cortex of adult male MOR wild‐type (WT) and knockout (KO) mice. Naltrexone was given daily for 4 days, starting from day 2 after lesioning. Locomotor activity was evaluated on day 5 after the CCI. Brain tissues were collected for immunostaining, Western, and qPCR analysis.ResultsGlutamate reduced MAP2 immunoreactivity (‐ir), while increased IBA1‐ir in neuron/BV2 co‐culture; both responses were antagonized by naltrexone. TBI significantly reduced locomotor activity and increased the expression of IBA1, iNOS, and CD4 in the lesioned cortex. Naltrexone significantly and equally antagonized the motor deficits and expression of IBA1 and iNOS in WT and KO mice. TBI‐mediated CD4 protein production was attenuated by naltrexone in WT mice, but not in KO mice.ConclusionNaltrexone reduced TBI‐mediated neurodegeneration and inflammation in MOR WT and KO mice. The protective effect of naltrexone involves non‐MOR and MOR mechanisms.

Spinal endomorphins attenuate burn-injury pain in male mice by inhibiting p38 MAPK signaling pathway through the mu-opioid receptor

Burn injury is one of the main causes of mortality worldwide and frequently associated with severe and long-lasting pain that compromises the quality of patient life. Several studies have shown that the mu-opioid system plays an important role in burn pain relief. In this study, we investigated the spinal antinociception induced by the endogenous mu-opioid receptor (MOR) agonists endomorphins and explored their mechanisms of actions in burn injury-induced pain model. Our results showed that intrathecal injection of endomorphin-1 and -2 dose-dependently attenuated mechanical allodynia and thermal hyperalgesia via the mu-opioid receptor in mice on day 3 after burn injury, which was consistent with the data obtained from the mu-opioid receptor knockout mice. Western blot showed that the phosphorylation levels of extracellular signal-regulated kinase1/2 (ERK1/2) and p38 mitogen-activated protein kinase (p38 MAPK) in ipsilateral spinal cord tissues were significantly up-regulated after burn injury. Intrathecal injection of endomorphins selectively inhibited the activation of p38 MAPK on day 3 after burn injury via the mu-opioid receptor. Further studies found that repeated application of the specific p38 MAPK inhibitor SB203580 dose-dependently inhibited burn-injury pain, as well as the activation of spinal p38 MAPK. Taken together, our present study demonstrates that intrathecal injection of endomorphins attenuates burn-injury pain in male mice by affecting the spinal activation of p38 MAPK via the mu-opioid receptor.

β-endorphin differentially contributes to food anticipatory activity in male and female mice undergoing activity-based anorexia.

Anorexia nervosa (AN) has a lifetime prevalence of up to 4% and a high mortality rate (~5–10%), yet little is known regarding the etiology of this disease. In an attempt to fill the gaps in knowledge, activity‐based anorexia (ABA) in rodents has been a widely used model as it mimics several key features of AN including severely restricted food intake and excessive exercise. Using this model, a role for the hypothalamic proopiomelanocortin (POMC) system has been implicated in the development of ABA as Pomc mRNA is elevated in female rats undergoing the ABA paradigm. Since the Pomc gene product α‐MSH potently inhibits food intake, it could be that elevated α‐MSH might promote ABA. However, the α‐MSH receptor antagonist SHU9119 does not protect against the development of ABA. Interestingly, it has also been shown that female mice lacking the mu opioid receptor (MOR), the primary receptor activated by the Pomc‐gene‐derived opioid β‐endorphin, display blunted food anticipatory behavior (FAA), a key feature of ABA. Thus, we hypothesized that the elevation in Pomc mRNA observed during ABA may lead to increased β‐endorphin concentrations and MOR activation to promote ABA. Further, given the known sex differences in AN and ABA, we hypothesized that MORs may contribute differentially in male and female mice. Using wild‐type and MOR knockout mice of both sexes, a MOR antagonist and careful analysis of food anticipatory behavior and β‐endorphin levels, we found 1) increased Pomc mRNA levels in both female and male mice that underwent ABA, 2) increased β‐endorphin in female mice that underwent ABA, and 3) blunted FAA in both sexes in response to MOR genetic deletion yet blunted FAA only in males in response to MOR antagonism. The results presented provide support for both hypotheses and suggest that it may be the β‐endorphin resulting from increased Pomc transcription that supports the development of some features of ABA.

Mu opioid receptor knockout mouse: Phenotypes with implications on restless legs syndrome.

Restless legs syndrome (RLS) is characterized by an irresistible need to move the legs while sitting or lying at night with insomnia as a frequent consequence. Human RLS has been associated with abnormalities in the endogenous opioid system, the dopaminergic system, the iron regulatory system, anemia, and inflammatory and auto-immune disorders. Our previous work indicates that mice lacking all three subtypes of opioid receptors have a phenotype similar to that of human RLS. To study the roles of each opioid receptor subtype in RLS, we first used mu opioid receptor knockout (MOR KO) mice based on our earlier studies using postmortem brain and cell culture. The KO mice showed decreased hemoglobin, hematocrit, and red blood cells (RBCs), with an appearance of microcytic RBCs indicating anemia. Together with decreased serum iron and transferrin, but increased ferritin levels, the anemia is similar to that seen with chronic inflammation in humans. A decreased serum iron level was also observed in the wildtype mice treated with an MOR antagonist. Iron was increased in the liver and spleen of the KO mice. Normal circadian variations in the dopaminergic and serotoninergic systems were absent in the KO mice. The KO mice showed hyperactivity and increased thermal sensitivity in wakefulness primarily during what would normally be the sleep phase similar to that seen in human RLS. Deficits in endogenous opioid system transmission could predispose to anemia of inflammation and loss of circadian variations in dopaminergic or serotonergic systems, thereby contributing to an RLS-like phenotype.

Deletion of mu opioid receptors reduces palatable solution intake in a mouse model of binge eating.

Binge eating in humans is driven by hedonic properties of food, suggesting that brain reward systems may contribute to this behaviour. We examined the role of mu opioid receptors (MOP) in binge eating by examining sweet solution intake in mice with genetic deletion of the MOP. Wildtype and MOP knockout mice had 4 hours access to food in the home cage combined with limited (4 hours) access to sucrose (17.1% w/v) or saccharin (0.09% w/v), or continuous (24 hours) access to sucrose. Only limited access groups exhibited binge intake, measured as increased solution consumption during the first hour. Knockout mice consumed less solution and food during the first hour as well as less food each day compared with wildtype mice. Limited access groups consumed more food and gained more weight than continuous access groups, and the effect was magnified in saccharin-consuming mice. Indeed, the increased food consumption in animals given limited access to saccharin was so excessive that caloric intake of this group was significantly higher than either of the sucrose groups (limited or continuous access). Within this group, females consumed more food per bodyweight than males, highlighting important sex differences in feeding behaviours under restricted access schedules.

The role of opioids in cancer progression.

The role of opioids in cancer progression.

Activation of Kappa Opioid Receptor Regulates the Hypothermic Response to Calorie Restriction and Limits Body Weight Loss.

Mammals maintain a nearly constant core body temperature (Tb) by balancing heat production and heat dissipation. This comes at a high metabolic cost that is sustainable if adequate calorie intake is maintained. When nutrients are scarce or experimentally reduced such as during calorie restriction (CR), endotherms can reduce energy expenditure by lowering Tb [1-6]. This adaptive response conserves energy, limiting the loss of body weight due to low calorie intake [7-10]. Here we show that this response is regulated by the kappa opioid receptor (KOR). CR is associated with increased hypothalamic levels of the endogenous opioid Leu-enkephalin, which is derived from the KOR agonist precursor dynorphin [11]. Pharmacological inhibition of KOR, but not of the delta or the mu opioid receptor subtypes, fully blocked CR-induced hypothermia and increased weight loss during CR independent of calorie intake. Similar results were seen with DIO mice subjected to CR. In contrast, inhibiting KOR did not change Tb in animals fed ad libitum (AL). Chemogenetic inhibition of KOR neurons in the hypothalamic preoptic area reduced the CR-induced hypothermia, whereas chemogenetic activation of prodynorphin-expressing neurons in the arcuate or the parabrachial nucleus lowered Tb. These data indicate that KOR signaling is a pivotal regulator of energy homeostasis and can affect body weight during dieting by modulating Tb and energy expenditure.

The opioid receptor positive allosteric modulator BMS‐986187 enhances some, but not all, delta‐opioid receptor mediated behaviors

Activation of the delta opioid receptor (DOR) by orthosteric agonists produces antinociception, antihyperalgesia, antidepressant‐like effects, and convulsions in animal models. Positive allosteric modulators (PAMs) bind to a receptor site distinct from the orthosteric binding site and can augment the activity of the endogenous system. Therefore, a DOR PAM could be an effective strategy for eliciting potential therapeutic effects without convulsions. To evaluate this hypothesis, we investigated the ability of the opioid receptor PAM BMS‐986187 to elicit DOR‐mediated behaviors in mice alone or in combination with the DOR orthosteric agonist SNC80 and evaluated the potential contribution of multiple opioid receptor subtypes to the observed behavioral effects. Antidepressant‐like effects were evaluated in the mouse forced swim test and convulsions were evaluated by continuous observation. The contribution of DORs and mu‐opioid receptors (MORs) was evaluated through pharmacological and genetic manipulation of the receptor subtypes. BMS‐986187 alone produced antidepressant‐like effects without convulsions. These antidepressant‐like effects were blocked by the DOR antagonist naltrindole and absent in DOR knockout mice, indicating a DOR‐mediated effect. The antidepressant‐like effects of BMS‐986187 were enhanced in MOR knockout mice and following betaFNA pretreatment, suggesting that activation of MORs functionally inhibited DOR‐mediated antidepressant‐like effects. BMS‐986187 increased the potency of the orthosteric DOR agonist SNC80 to decrease immobility in the forced swim test but not to elicit convulsions. In addition, SNC80, but not BMS‐986187, enhanced the convulsive effects of pentylenetetrazol. Overall, these findings suggest that BMS‐986187 has activity at DORs and MORs in vivo and enhances some, but not all, behavioral effects of DOR agonists.Support or Funding InformationThis work was funded in part by a Research Starter Grant from the PhRMA Foundation.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

TouchScreen-based phenotyping: altered stimulus/reward association and lower perseveration to gain a reward in mu opioid receptor knockout mice

While the contribution of Mu Opioid Receptors (MORs) to hedonic aspects of reward processing is well-established, the notion that these receptors may also regulate motivation to gain a reward, and possibly other related cognitive dimensions, has been less investigated. The prefrontal cortex (PFC) is a critical site for these processes. Our previous functional magnetic resonance imaging study found alterations of functional connectivity (FC) in reward/aversion networks in MOR knockout mice. Here we pursued voxelwise seed-based FC analyses using the same dataset with a focus on the PFC. We observed significant reduction of PFC FC in mutant mice, predominantly with the nucleus accumbens, supporting the notion of altered reward-driven top-down controls. We tested motivation for palatable food in a classical operant self-administration paradigm, and found delayed performance for mutant mice. We then evaluated motivational and cognitive abilities of MOR knockout mice in TouchScreen-based behavioral tests. Learning was delayed and stimulus/reward association was impaired, suggesting lower hedonic reward value and reduced motivation. Perseverative responses were decreased, while discriminatory behavior and attention were unchanged, indicative of increased inhibitory controls with otherwise intact cognitive performance. Together, our data suggest that MORs contribute to enhance reward-seeking and facilitate perseverative behaviors. The possibility that MOR blockade could reduce maladaptive compulsivity deserves further investigation in addiction and self-control disorder research.

Mu opioid receptors in GABAergic neurons of the forebrain promote alcohol reward and drinking.

Mu opioid receptors (MORs) are widely distributed throughout brain reward circuits and their role in drug and social reward is well established. Substantial evidence has implicated MOR and the endogenous opioid system in alcohol reward, but circuit mechanisms of MOR-mediated alcohol reward and intake behavior remain elusive, and have not been investigated by genetic approaches. We recently created conditional knockout (KO) mice targeting the Oprm1 gene in GABAergic forebrain neurons. These mice (Dlx-MOR KO) show a major MOR deletion in the striatum, whereas receptors in midbrain (including the Ventral Tegmental Area or VTA) and hindbrain are intact. Here, we compared alcohol-drinking behavior and rewarding effects in total (MOR KO) and conditional KO mice. Concordant with our previous work, MOR KO mice drank less alcohol in continuous and intermittent two-bottle choice protocols. Remarkably, Dlx-MOR KO mice showed reduced drinking similar to MOR KO mice, demonstrating that MOR in the forebrain is responsible for the observed phenotype. Further, alcohol-induced conditioned place preference was detected in control but not MOR KO mice, indicating that MOR is essential for alcohol reward and again, Dlx-MOR KO recapitulated the MOR KO phenotype. Taste preference and blood alcohol levels were otherwise unchanged in mutant lines. Together, our data demonstrate that MOR expressed in forebrain GABAergic neurons is essential for alcohol reward-driven behaviors, including drinking and place conditioning. Challenging the prevailing VTA-centric hypothesis, this study reveals another mechanism of MOR-mediated alcohol reward and consumption, which does not necessarily require local VTA MORs but rather engages striatal MOR-dependent mechanisms.

Morphine-induced hyperalgesia involves mu opioid receptors and the metabolite morphine-3-glucuronide

Opiates are potent analgesics but their clinical use is limited by side effects including analgesic tolerance and opioid-induced hyperalgesia (OIH). The Opiates produce analgesia and other adverse effects through activation of the mu opioid receptor (MOR) encoded by the Oprm1 gene. However, MOR and morphine metabolism involvement in OIH have been little explored. Hence, we examined MOR contribution to OIH by comparing morphine-induced hyperalgesia in wild type (WT) and MOR knockout (KO) mice. We found that repeated morphine administration led to analgesic tolerance and hyperalgesia in WT mice but not in MOR KO mice. The absence of OIH in MOR KO mice was found in both sexes, in two KO global mutant lines, and for mechanical, heat and cold pain modalities. In addition, the morphine metabolite morphine-3beta-D-glucuronide (M3G) elicited hyperalgesia in WT but not in MOR KO animals, as well as in both MOR flox and MOR-Nav1.8 sensory neuron conditional KO mice. M3G displayed significant binding to MOR and G-protein activation when using membranes from MOR-transfected cells or WT mice but not from MOR KO mice. Collectively our results show that MOR is involved in hyperalgesia induced by chronic morphine and its metabolite M3G.

Morphine worsens the severity and prevents pancreatic regeneration in mouse models of acute pancreatitis

BackgroundOpioids such as morphine are widely used for the management of pain associated with acute pancreatitis. Interestingly, opioids are also known to affect the immune system and modulate inflammatory pathways...

Spinal or supraspinal phosphorylation deficiency at the MOR C-terminus does not affect morphine tolerance in vivo

The development of tolerance to morphine, one of the most potent analgesics, in the management of chronic pain is a significant clinical problem and its mechanisms are poorly understood. Morphine exerts its pharmacological effects via the μ-opioid receptor (MOR). Tolerance is highly connected to G-protein-coupled receptors (GPCR) phosphorylation and desensitization increase. Because morphine desensitization previously has been shown to be MOR phosphorylation- and ß-arrestin2-independent (in contrast to agonists such as fentanyl), we examined the contribution of phosphorylation of the entire C-terminus to the development of antinociceptive tolerance to the partial (morphine) and full (fentanyl) MOR agonists in vivo. In MOR knockout (MORKO) mice, we delivered via lentivirus the genes encoding the wild-type MOR (WTMOR) or a phosphorylation-deficient MOR (Cterm(-S/T)MOR) in which all of the serine and threonine residues were mutated to alanine into the ventrolateral periaqueductal grey matter (vlPAG) or lumbar spinal cord (SC), structures that are involved in nociception. We compared the analgesic ED50 in WTMOR- and Cterm(-S/T)MOR-expressing MORKO mice before and after morphine or fentanyl tolerance was induced. Morphine acute antinociception was partially restored in WTMOR- or Cterm(-S/T)MOR-transferred MORKO mice. Fentanyl acute antinociception was observed only in MORKO mice with the transgenes expressed in the SC. Morphine antinociceptive tolerance was not affected by expressing Cterm(-S/T)MOR in the vlPAG or SC of MORKO mice. Fentanyl-induced tolerance in MORKO mice expressing WTMOR or Cterm(-S/T)MOR, is greater than morphine-induced tolerance. Thus, MOR C-terminus phosphorylation does not appear to be critical for morphine tolerance in vivo.

Interacting Cannabinoid and Opioid Receptors in the Nucleus Accumbens Core Control Adolescent Social Play.

Social play behavior is a highly rewarding, developmentally important form of social interaction in young mammals. However, its neurobiological underpinnings remain incompletely understood. Previous work has suggested that opioid and endocannabinoid neurotransmission interact in the modulation of social play. Therefore, we combined behavioral, pharmacological, electrophysiological, and genetic approaches to elucidate the role of the endocannabinoid 2-arachidonoylglycerol (2-AG) in social play, and how cannabinoid and opioid neurotransmission interact to control social behavior in adolescent rodents. Systemic administration of the 2-AG hydrolysis inhibitor JZL184 or the opioid receptor agonist morphine increased social play behavior in adolescent rats. These effects were blocked by systemic pretreatment with either CB1 cannabinoid receptor (CB1R) or mu-opioid receptor (MOR) antagonists. The social play-enhancing effects of systemic morphine or JZL184 treatment were also prevented by direct infusion of the CB1R antagonist SR141716 and the MOR antagonist naloxone into the nucleus accumbens core (NAcC). Searching for synaptic correlates of these effects in adolescent NAcC excitatory synapses, we observed that CB1R antagonism blocked the effect of the MOR agonist DAMGO and, conversely, that naloxone reduced the effect of a cannabinoid agonist. These results were recapitulated in mice, and completely abolished in CB1R and MOR knockout mice, suggesting that the functional interaction between CB1R and MOR in the NAcC in the modulation of social behavior is widespread in rodents. The data shed new light on the mechanism by which endocannabinoid lipids and opioid peptides interact to orchestrate rodent socioemotional behaviors.