Mu-opioid Receptor Research Articles

Preparation, Evaluation and Optimization of Polymeric Nanoparticles of Eluxadoline for the Treatment of Irritable Bowel Syndrome

ABSTRACT Eluxadoline, exhibits potential as an irritable bowel syndrome treatment and management strategy. Eluxadoline, a muopioid receptor agonist and delta-opioid receptor antagonist, has shown efficacy in relieving IBS symptoms. However, its applications have been limited due to low water solubility, which causes a low dissolution rate and oral bioavailability. In this study, we aimed to enhance the therapeutic efficacy of eluxadoline by formulating polymeric nanoparticles of eluxadoline. Polymeric nanoparticles of chitosan loaded with eluxadoline were prepared using the ionic gelation method. The formulation parameters including concentration of chitosan, concentration of tripolyphosphate and volume of tripolyphosphate were systematically optimized using a Box-Behnken design to achieve nanoparticles (CNP1-CNP17) with optimal physicochemical properties. The optimized formulation based on particle size of (300.4 nm) with a low PDI value (0.305), zeta potential (41.2mV) and % entrapment efficacy (76.89%). The efficiency of the drug release from optimised formulation was studied in vitro by using a dialysis bag diffusion technique in buffer condition pH 6.8 intestinal condition and showed a sustained release behaviour with maximum drug release 80.23%. Thus, the findings demonstrated that the formulated chitosan-based polymeric nanoparticles present a promising strategy for efficiently delivering eluxadoline via oral administration, offering a potential solution for treating irritable bowel syndrome.

Opioid drug seeking after early-life adversity: a role for delta opioid receptors

Opioid use disorder (OUD) is associated with a history of early-life adversity (ELA), an association that is particularly strong in women. In a rodent model, we previously found that ELA enhances risk for opioid addiction selectively in females, but the mechanisms for this effect are unclear. Here, we show that ELA robustly alters cFos responses to opioid drugs in females’ nucleus accumbens (NAc) and basolateral amygdala (BLA), but not elsewhere. We further identify delta opioid receptors (DOR), which mature in the first week of life and thus later than kappa or mu opioid receptors, as a potential mediator of ELA's impacts on reward circuit functions. Accordingly, DOR mRNA in NAc was persistently reduced in adult females with ELA history. Moreover, pharmacological stimulation of NAc DORs increased opioid demand in control females (recapitulating the ELA phenotype), while blocking DORs in ELA females conversely reduced high-effort drug consumption, simulating the control rearing phenotype. These findings support a role for NAc DORs in mediating ELA-induced opioid vulnerability. In contrast, BLA neurons expressing DOR protein do not overlap heroin- responsive cells in ELA rats, arguing against a direct relationship of BLA DORs to heroin's addiction-relevant actions in the brain. Together, these results suggest a novel and selective role for NAc DORs in contributing to enduring, ELA-provoked vulnerability to OUD.

Endomorphin-2 (Endo2) and substance P (SubP) co-application attenuates SubP-induced excitation and alters frequency plasticity in neonatal rat in vitro preparations

Substance P (SubP) and endomorphin-2 (Endo2) are co-localized presynaptically in vesicles of neurons adjacent to inspiratory rhythm-generating pre-Botzinger Complex (preBotC) neurons but the effects of co-released SubP and Endo2 on respiratory motor control are not known. To address this question, SubP alone or a combination of SubP and Endo2 (SubP/Endo2) were bath-applied in a sustained (15-min) or intermittent (5-min application, 5-min washout, x3) pattern at 10–100 nM to neonatal rat brainstem-spinal cord preparations. During neuropeptide application, SubP/Endo2 co-applications generally attenuated SubP-induced increases in burst frequency and decreases in burst amplitude. With respect to frequency plasticity (long-lasting increase in burst frequency 60 min post-neuropeptide application), SubP-induced frequency plasticity was increased with sustained SubP/Endo2 co-applications at 20 and 100 nM. Intermittent SubP/Endo2 co-applications tended to decrease the level of frequency plasticity induced by intermittent SubP alone applications. SubP/Endo2 co-applications revealed potentially new functions for neurokinin-1 (NK1R) and mu-opioid (MOR) receptors on respiratory rhythm-generating medullary neurons.

Cyclic Glycopeptide Analogs of Endomorphin-1 Provide Highly Effective Antinociception in Male and Female Mice.

Opioids acting at the mu opioid receptor (MOR) remain the most effective treatment for moderate to severe pain, but their use is limited by serious side effects. We have shown that a cyclized analog of endomorphin-1 provided pain relief comparable to that of morphine with reduction or absence of several side effects, including abuse liability. Glycosylation can promote penetration of cellular barriers. Here we developed cyclic glycopeptide endomorphin (glycoEM) analogs as drug candidates for potent and long-lasting analgesia. The analogs were assessed in receptor binding and functional assays and for blood-brain barrier penetration by microdialysis and MS. Two of the analogs showed MOR selectivity and more potent and longer lasting antinociception than morphine in male and female mice. Comparable antinociception occurred at A2 doses 5-fold lower (20-fold on a molar basis) than morphine doses. The results support further study of the glycoEMs for clinical application.

Effect of chronic delivery of the NOP/MOR partial agonist AT-201 and NOP antagonist J-113397 on heroin relapse in a rat model of opioid maintenance

RationaleThe opioid crisis persists despite availability of effective opioid agonist maintenance treatments (methadone and buprenorphine). Thus, there is a need to advance novel medications for the treatment of opioid use and relapse.ObjectivesWe recently modeled maintenance treatment in rats and found that chronic delivery of buprenorphine and the mu opioid receptor (MOR) partial agonist TRV130 decreases relapse to oxycodone seeking and taking. In contrast, chronic delivery of the buprenorphine analog BU08028 had mixed effects on different heroin relapse-related measures. Here, we tested the effect of the mixed nociceptin (NOP) receptor/MOR partial agonist AT-201 and the NOP receptor antagonist J-113397 on different heroin relapse-related measures.MethodsWe trained male and female rats to self-administer heroin (6-h/d, 14-d) in context A and then implanted osmotic minipumps containing AT-201 (0, 3.8, or 12 mg/kg/d) or J-113397 (0, 12.6, or 40 mg/kg/d). Next, we tested the effect of chronic delivery of the compounds on (1) incubation of heroin seeking in a non-drug context B, (2) extinction responding reinforced by heroin-associated discrete cues in context B, (3) context A-induced reinstatement of heroin seeking, and (4) reacquisition of heroin self-administration in context A.ResultsIn females, AT-201 modestly increased reacquisition of heroin self-administration and J-113397 modestly decreased incubation of heroin seeking. The compounds had no effect on the other relapse-related measures in females, and no effect on any of the measures in males.ConclusionThe NOP/MOR partial agonist AT-201 and the NOP antagonist J-113397 did not mimic buprenorphine’s inhibitory effects on relapse in a rat model of opioid maintenance treatment.

Mu-Opioid Receptor (MOR) Dependence of Pain in Chemotherapy-Induced Peripheral Neuropathy.

We recently demonstrated that transient attenuation of Toll-like receptor 4 (TLR4) in dorsal root ganglion (DRG) neurons, can both prevent and reverse pain associated with chemotherapy-induced peripheral neuropathy (CIPN), a severe side effect of cancer chemotherapy, for which treatment options are limited. Given the reduced efficacy of opioid analgesics to treat neuropathic, compared with inflammatory pain, the cross talk between nociceptor TLR4 and mu-opioid receptors (MORs), and that MOR and TLR4 agonists induce hyperalgesic priming (priming), which also occurs in CIPN, we determined, using male rats, whether (1) antisense knockdown of nociceptor MOR attenuates CIPN, (2) and attenuates the priming associated with CIPN, and (3) CIPN also produces opioid-induced hyperalgesia (OIH). We found that intrathecal MOR antisense prevents and reverses hyperalgesia induced by oxaliplatin and paclitaxel, two common clinical chemotherapy agents. Oxaliplatin-induced priming was also markedly attenuated by MOR antisense. Additionally, intradermal morphine, at a dose that does not affect nociceptive threshold in controls, exacerbates mechanical hyperalgesia (OIH) in rats with CIPN, suggesting the presence of OIH. This OIH associated with CIPN is inhibited by interventions that reverse Type II priming [the combination of an inhibitor of Src and mitogen-activated protein kinase (MAPK)], an MOR antagonist, as well as a TLR4 antagonist. Our findings support a role of nociceptor MOR in oxaliplatin-induced pain and priming. We propose that priming and OIH are central to the symptom burden in CIPN, contributing to its chronicity and the limited efficacy of opioid analgesics to treat neuropathic pain.

The Role of the Mu Opioid Receptors of the Medial Prefrontal Cortex in the Modulation of Analgesia Induced by Acute Restraint Stress in Male Mice.

Mu opioid receptors (MORs) represent a vital mechanism related to the modulation of stress-induced analgesia (SIA). Previous studies have reported on the gamma-aminobutyric acid (GABA)ergic "disinhibition" mechanisms of MORs on the descending pain modulatory pathway of SIA induced in the midbrain. However, the role of the MORs expressed in the medial prefrontal cortex (mPFC), one of the main cortical areas participating in pain modulation, in SIA remains completely unknown. In this study, we investigated the contributions of MORs expressed on glutamatergic (MORGlut) and GABAergic (MORGABA) neurons of the medial prefrontal cortex (mPFC), as well as the functional role and activity of neurons projecting from the mPFC to the periaqueductal gray (PAG) region, in male mice. We achieved this through a combination of hot-plate tests, c-fos staining, and 1 h acute restraint stress exposure tests. The results showed that our acute restraint stress protocol produced mPFC MOR-dependent SIA effects. In particular, MORGABA was found to play a major role in modulating the effects of SIA, whereas MORGlut seemed to be unconnected to the process. We also found that mPFC-PAG projections were efficiently activated and played key roles in the effects of SIA, and their activation was mediated by MORGABA to a large extent. These results indicated that the activation of mPFC MORGABA due to restraint stress was able to activate mPFC-PAG projections in a potential "disinhibition" pathway that produced analgesic effects. These findings provide a potential theoretical basis for pain treatment or drug screening targeting the mPFC.

Predictive Model for Opioid Use Disorder in Chronic Pain: A Development and Validation Study.

There are several questionnaires for the challenge of anticipating opioid use disorder (OUD). However, many are not specific for chronic non-cancer pain (CNCP) or have been developed in the American population, whose sociodemographic factors are very different from the Spanish population, leading to scarce translation into clinical practice. Thus, the aim of this study is to prospectively validate a predictive model for OUD in Spanish patients under long-term opioids. An innovative two-stage predictive model was developed from retrospective (n = 129) and non-overlapping prospective (n = 100) cohorts of real-world CNCP outpatients. All subjects used prescribed opioids for 6 or more months. Sociodemographic, clinical and pharmacological covariates were registered. Mu-opioid receptor 1 (OPRM1, A118G, rs1799971) and catechol-O-methyltransferase (COMT, G472A, rs4680) genetic variants plus cytochrome P450 2D6 (CYP2D6) liver enzyme phenotypes were also analyzed. The model performance and diagnostic accuracy were calculated. The two-stage model comprised risk factors related to OUD (younger age, work disability and high daily opioid dose) and provided new useful information about other risk factors (low quality of life, OPRM-G allele and CYP2D6 extreme phenotypes). The validation showed a satisfactory accuracy (70% specificity and 75% sensitivity) for our predictive model with acceptable discrimination and goodness of fit. Our study presents the results of an innovative model for predicting OUD in our setting. After external validation, it could represent a change in the paradigm of opioid treatment, helping clinicians to better identify and manage the risks and reduce the side effects and complications.

Atp1a2 and Kcnj9 are candidate genes underlying sensitivity to oxycodone-induced locomotor activation and withdrawal-induced anxiety-like behaviors in C57BL/6 substrains.

Opioid use disorder is heritable, yet its genetic etiology is largely unknown. C57BL/6J and C57BL/6NJ mouse substrains exhibit phenotypic diversity in the context of limited genetic diversity which together can facilitate genetic discovery. Here, we found C57BL/6NJ mice were less sensitive to oxycodone (OXY)-induced locomotor activation versus C57BL/6J mice in a conditioned place preference paradigm. Narrow-sense heritability was estimated at 0.22-0.31, implicating suitability for genetic analysis. Quantitative trait locus (QTL) mapping in an F2 cross identified a chromosome 1 QTL explaining 7-12% of the variance in OXY locomotion and anxiety-like withdrawal in the elevated plus maze. A second QTL for EPM withdrawal behavior on chromosome 5 near Gabra2 (alpha-2 subunit of GABA-A receptor) explained 9% of the variance. To narrow the chromosome 1 locus, we generated recombinant lines spanning 163-181 Mb, captured the QTL for OXY locomotor traits and withdrawal, and fine-mapped a 2.45-Mb region (170.16-172.61 Mb). Transcriptome analysis identified five, localized striatal cis-eQTL transcripts and two were confirmed at the protein level (KCNJ9, ATP1A2). Kcnj9 codes for a potassium channel (GIRK3) that is a major effector of mu opioid receptor signaling. Atp1a2 codes for a subunit of a Na+/K+ ATPase enzyme that regulates neuronal excitability and shows functional adaptations following chronic opioid administration. To summarize, we identified two candidate genes underlying the physiological and behavioral properties of opioids, with direct preclinical relevance to investigators employing these widely used substrains and clinical relevance to human genetic studies of opioid use disorder.

Buprenorphine-Naloxone for Opioid Use Disorder: Reduction in Mortality and Increased Remission.

As fentanyl has become more readily available, opioid-related morbidity and mortality in the United States has increased dramatically. Preliminary studies suggest that high-affinity, partial mu-opioid receptor agonists such as the combination product buprenorphine-naloxone may reduce mortality from overdose and promote remission. With the escalating prevalence of opioid use disorder (OUD), it is essential to evaluate the effectiveness of opioid agonists like buprenorphine-naloxone. This study examines mortality and remission rates for OUD patients prescribed buprenorphine-naloxone to determine the efficacy of this treatment toward these outcomes. We carried out a retrospective analysis using the US Collaborative Network database in TriNetX, examining de-identified medical records from nearly 92 million patients across 56 healthcare organizations. The study spanned the years from January 1, 2017-May 13, 2022. Cohort 1 included OUD patients who began buprenorphine-naloxone treatment within one-year post-diagnosis, while Cohort 2, the control group, consisted of OUD patients who were not administered buprenorphine. The study measured mortality and remission rates within a year of the index event, incorporating propensity score matching for age, gender, and race/ethnicity. Prior to propensity matching, we identified a total of 221,967 patients with OUD. Following exclusions, 61,656 patients treated with buprenorphine-naloxone showed 34% fewer deaths within one year of diagnosis compared to 159,061 patients who did not receive buprenorphine (2.6% vs 4.0%; relative risk [RR] 0.661; 95% confidence interval [CI] 0.627-0.698; P < 0.001). The remission rate was approximately 1.9 times higher in the buprenorphine-naloxone group compared to the control group (18.8% vs 10.1%; RR 1.862; 95% CI 1.812-1.914; P < 0.001). After propensity matching, the effect on mortality decreased but remained statistically significant (2.6% vs 3.0%; RR 0.868; 95% CI 0.813-0.927; P < 0.001) and the remission rate remained consistent (18.8% vs 10.4%; RR 1.812; 95% CI 1.750-1.876; P < 0.001). Number needed to treat for benefit was 249 for death and 12 for remission. Buprenorphine-naloxone was associated with significantly reduced mortality and increased remission rates for patients with opioid use disorder and should be used as a primary treatment. The recognition and implementation of treatment options like buprenorphine-naloxone is vital in alleviating the impact of OUD.

Buprenorphine: The Opioid that Cried 'Partial Agonist'.

Although buprenorphine use has increased dramatically over the past decade, its unique pharmacologic and pharmacokinetic profile often leads to misconceptions about its overall utility and has created a drastic underrepresentation in patients with chronic non-can- cer pain. A common misnomer associated with buprenorphine is because of 'partial agonist' activity, it exhibits a plateauing of typical opioid-related side effects (including respiratory depression, constipation, euphoria, and hypogonadal axis suppression), but additionally it must exhibit a plateauing effect of overall analgesic potential. However, novel downstream molecular and cellular mechanisms offer new insights that help support the clinical potential that buprenorphine's analgesic actions may not have a ceiling, like its side effect profile. This interactive symposium will provide an enhanced review of the evolving research that helps unravel the complexity around buprenorphine's varying pharmacologic effects including actions on various opioid receptors, promiscuity to elicit varying actions on mu-opioid receptors coupled with different isoforms of G~ subunits, role in the intracellular recruitment of beta-arrestin, binding to different splice variants of mu-opioid receptors, and greater spinal versus supraspinal activity. The final half of this symposium will be designed to substantiate evidence with various human clinical trial data to further support buprenorphine's place on the analgesic ladder.

Mu opioid receptors gate the locus coeruleus pain generator.

The locus coeruleus (LC) plays a paradoxical role in chronic pain. Although largely known as a potent source of endogenous analgesia, increasing evidence suggests injury can transform the LC into a chronic pain generator. We sought to clarify the role of this system in pain. Here, we show optogenetic inhibition of LC activity is acutely antinociceptive. Following long-term spared nerve injury, the same LC inhibition is analgesic - further supporting its pain generator function. To identify inhibitory substrates that may naturally serve this function, we turned to endogenous LC mu opioid receptors (LC-MOR). These receptors provide powerful LC inhibition and exogenous activation of LC-MOR is antinociceptive. We therefore hypothesized that endogenous LC-MOR-mediated inhibition is critical to how the LC modulates pain. Using cell type-selective conditional knockout and rescue of LC-MOR receptor signaling, we show these receptors bidirectionally regulate thermal and mechanical hyperalgesia - providing a functional gate on the LC pain generator.

Gabapentinoids Increase the Potency of Fentanyl and Heroin and Decrease the Potency of Naloxone to Antagonize Fentanyl and Heroin in Rats Discriminating Fentanyl.

Despite a significant decrease in the number of prescriptions for opioids, the opioid crisis continues, fueled in large part by the availability of the phenylpiperidine mu opioid receptor (MOR) agonist fentanyl. In contrast, the number of prescriptions for and the off-label use of gabapentinoids (gabapentin and pregabalin) has increased dramatically, with gabapentinoids commonly detected in opioid overdose victims. Although gabapentinoids can decrease the potency of the opioid receptor antagonist naloxone to reverse heroin-induced hypoventilation in male rats, the specificity and nature of interaction between gabapentinoids and MOR agonists and any potential sex difference in those interactions are not well characterized. Gabapentinoids were studied in female and male rats discriminating fentanyl (0.0032 mg/kg, i.p.) or cocaine (3.2 mg/kg, i.p.). Alone, neither gabapentin nor pregabalin significantly increased fentanyl- or cocaine-appropriate responding. In rats discriminating fentanyl, each gabapentinoid dose-dependently shifted the fentanyl and heroin discrimination dose-effect functions to the left, whereas naloxone dose-dependently shifted the fentanyl and heroin discrimination dose-effect functions to the right. Each gabapentinoid (100 mg/kg) significantly decreased the potency of naloxone to antagonize the discriminative stimulus effect of fentanyl or heroin. In contrast, each gabapentinoid dose-dependently shifted the cocaine and d-methamphetamine discrimination dose-effect functions to the right. There were no significant sex differences in this study. These results suggest that gabapentinoids impact the misuse of opioids, the co-use of opioids and stimulant drugs, and the increasing number of overdose deaths in individuals using opioids, stimulant drugs, and gabapentinoids in mixtures. SIGNIFICANCE STATEMENT: The number of prescriptions for and the off-label use of gabapentinoids (gabapentin and pregabalin) has increased dramatically, with gabapentinoids commonly detected in opioid overdose victims. This study reports that in rats gabapentinoids increase the potency of fentanyl and heroin to produce discriminative stimulus effects while decreasing the potency of naloxone to antagonize those effects of fentanyl and heroin. These results can help guide policies for regulating gabapentinoids and treating opioid misuse and overdose.

Effect of bovine beta-casomorphins on rat pancreatic beta cells (RIN-5F) under glucotoxic stress

Beta-casomorphins (BCMs) are the bio-active peptides having opioid properties which are formed by the proteolytic digestion of β-caseins in milk. BCM-7 forms when A1 milk is digested in the small intestine due to a histidine at the 67th position in β-casein, unlike A2 milk, which has proline at this position and produces BCM-9. BCM-7 has further degraded into BCM-5 by the dipeptidyl peptidase-IV (DPP-IV) enzyme in the intestine. The opioid-like activity of BCM-7 is responsible for eliciting signaling pathways which enable a wide range of physiological effects. The aim of our study was to find out the differential role of BCMs (BCM-7, BCM-9 and BCM-5) on pancreatic β-cell proliferation, insulin secretion, and opioid peptide binding receptors from β-cells (RIN-5F cell line) in normal (5.5 mM) and high glucose (27.5 mM) concentrations. Our results showed that BCM-7/9/5 did not affect β-cell viability, proliferation, and insulin secretion at normal glucose level. However, at higher glucose concentration, BCMs significantly protected β-cells from glucotoxicity but did not affect the insulin secretion. Interestingly, in the presence of Mu-opioid peptide receptor antagonist CTOP, BCMs did not protect β-cells from glucotoxicity. The results suggest that BCMs protect β-cells from glucotoxicity via non-opioid mediated pathways because BCMs did not modulate the gene expression of the mu, kappa and delta opioid peptide receptors.

Tianeptine-involved emergency department visits, fatal overdoses, and substance seizures in Tennessee, 2021–2023

BackgroundTianeptine is an antidepressant that acts as an agonist to the mu-opioid receptor and enhances serotonin reuptake. Tianeptine has been legally sold as an antidepressant in some countries but is not approved for any medical use by the U.S. Food and Drug Administration (FDA). Tianeptine is not a federally controlled substance, but became a schedule II substance in Tennessee on July 1, 2022. This publication aims to describe the prevalence of tianeptine-involved emergency department visits, fatal overdoses, and substance seizures in Tennessee from 2021 to 2023. MethodsWe conducted a study to examine the prevalence of tianeptine-involved emergency department visits and fatal overdoses in Tennessee using data for 2021 to 2023 from the Tennessee Electronic Surveillance System for the Early Notification of Community based Epidemics (ESSENCE) database and the Tennessee State Unintentional Drug Overdose Reporting System (SUDORS). Substance seizure data from National Forensic Laboratory Information System (NFLIS) are included. ResultsOur search of ESSENCE, SUDORS, and NFLIS yielded 50 tianeptine-involved emergency department visits, 6 tianeptine-involved fatal overdoses, and 19 tianeptine substance seizures respectively. Demographic information is provided for the emergency department visits and tianeptine-involved fatal overdoses. Discharge diagnosis and clinical symptomology information are provided for the emergency department visits. ConclusionEmergency department visits and fatal overdoses involving tianeptine have occurred in Tennessee despite tianeptine becoming a schedule II substance. Among emergency department visits, tianeptine use is most commonly associated with gastrointestinal and psychological symptoms. All fatal cases where tianeptine was detected involved other substances, suggesting tianeptine plays a role in polysubstance use.

Synthetic opioids have disrupted conventional wisdom for treating opioid overdose

More than 90 % of opioid overdose deaths in North America are now caused by synthetic opioids, and while they are not as prevalent in the European illicit drug market, there are indications that they may become so in the near future. Multiple publications have argued that neither higher doses of naloxone nor more potent opioid receptor antagonists are needed to reverse a synthetic opioid overdose. However, the unique physicochemical properties of synthetic opioids result in a very rapid onset of respiratory depression compared to opium-based molecules, reducing the margin of opportunity to reverse an overdose. While intravenous administration rapidly delivers the high naloxone concentrations needed to reverse a synthetic opioid overdose, this option is often unavailable to first responders. A translational mechanistic model of opioid overdose developed by the FDA’s Division of Applied Regulatory Science provides an unbiased approach to evaluate the effectiveness of overdose reversal strategies. Reports using this model demonstrated the naloxone tools (2mg intramuscular and 4mg intranasal) used by many first responders can result in an unacceptable loss of life following a synthetic opioid (fentanyl, carfentanil) overdose. Moreover, sequential (2.5minutes between doses) administration of up to four doses of intranasal naloxone was no more effective at reducing the incidence of cardiac arrest (a surrogate endpoint for lethality) than a single dose, suggesting that attempts at titration may not provide the rapid absorption required to reverse a synthetic opioid overdose. This model was also used to compare the effectiveness of intranasal naloxone to intranasal nalmefene, a recently FDA-approved opioid receptor antagonist with a more rapid absorption and a higher affinity at mu-opioid receptors compared to intranasal naloxone. Intranasal nalmefene resulted in large and clinically meaningful reductions in the incidence of cardiac arrest compared to intranasal naloxone. Furthermore, simultaneous administration of four doses of intranasal naloxone was needed to reduce the incidence of cardiac arrest to levels approaching those produced by a single dose of intranasal nalmefene. These data are consistent with evidence that synthetics have indeed disrupted conventional wisdom in the treatment of opioid overdose.

Chronic morphine treatment induces sex- and synapse-specific cellular tolerance on thalamo-cortical mu opioid receptor signaling.

How cellular adaptations give rise to opioid analgesic tolerance to opioids like morphine is not well understood. For one, pain is a complex phenomenon comprising both sensory and affective components, largely mediated through separate circuits. Glutamatergic projections from the medial thalamus (MThal) to the anterior cingulate cortex (ACC) are implicated in processing of affective pain, a relatively understudied component of the pain experience. The goal of this study was to determine the effects of chronic morphine exposure on mu-opioid receptor (MOR) signaling on MThal-ACC synaptic transmission within the excitatory and feedforward inhibitory pathways. Using whole cell patch-clamp electrophysiology and optogenetics to selectively target these projections, we measured morphine-mediated inhibition of optically evoked postsynaptic currents in ACC layer V pyramidal neurons in drug-naïve and chronically morphine-treated mice. We found that morphine perfusion inhibited the excitatory and feedforward inhibitory pathways similarly in females but caused greater inhibition of the inhibitory pathway in males. Chronic morphine treatment robustly attenuated morphine presynaptic inhibition within the inhibitory pathway in males, but not females, and mildly attenuated presynaptic inhibition within the excitatory pathway in both sexes. These effects were not observed in MOR phosphorylation-deficient mice. This study indicates that chronic morphine treatment induces cellular tolerance to morphine within a thalamo-cortical circuit relevant to pain and opioid analgesia. Furthermore, it suggests this tolerance may be driven by MOR phosphorylation. Overall, these findings improve our understanding of how chronic opioid exposure alters cellular signaling in ways that may contribute to opioid analgesic tolerance.NEW & NOTEWORTHY Opioid signaling within the anterior cingulate cortex (ACC) is important for opioid modulation of affective pain. Glutamatergic medial thalamus (MThal) neurons synapse in the ACC and opioids, acting through mu opioid receptors (MORs), acutely inhibit synaptic transmission from MThal synapses. However, the effect of chronic opioid exposure on MThal-ACC synaptic transmission is not known. Here, we demonstrate that chronic morphine treatment induces cellular tolerance at these synapses in a sex-specific and phosphorylation-dependent manner.

Age- and sex-driven alterations in alcohol consumption patterns: Role of brain ethanol metabolism and the opioidergic system in the nucleus accumbens

Alcohol consumption leads to significant neurochemical and neurobiological changes, contributing to the development of alcohol use disorders (AUDs), which exhibit sex- and age-dependent variations according to clinical data. However, preclinical studies often neglect these factors when investigating alcohol consumption patterns. In this study, we present data on male and female rats continuously exposed to a 20 % ethanol solution for one month. The animals were divided into two groups based on their age at the onset of drinking (8 and 12 weeks old). Interestingly, 12-week-old males consumed significantly less alcohol than both 12-week-old females and 8-week-old animals, indicating that alcohol consumption patterns vary with sex and age in our model. Additionally, to advance in the study of the neurobiological alterations induced by ethanol intake in the mesocorticolimbic system (MCLS) that may participate in its reinforcing properties and the maintenance of alcohol drinking behavior, we measured catalase activity—an enzyme involved in alcohol metabolism and related to ethanol reinforcement—in the nucleus accumbens (NAc) of these animals. Furthermore, we measured the levels of mu (MOR), kappa (KOR), delta (DOR), and nociceptin (NOP) opioid receptors in the NAc, as the endogenous opioidergic system plays a pivotal role in regulating the MCLS and alcohol reinforcement. MOR levels were lower in high alcohol-consuming groups (8-week-old males and all females). Both DOR and NOP levels decreased with age, whereas KOR levels remained unchanged. Our findings suggest that the age at onset of alcohol consumption critically influences alcohol intake, particularly in males. Additionally, females consistently showed higher alcohol intake regardless of age, highlighting inherent sex-specific differences. The dynamic changes in catalase activity and opioid receptor expression suggest the involvement of these factors in modulating alcohol consumption.

Key phosphorylation sites for robust β-arrestin2 binding at the MOR revisited

Desensitisation of the mu-opioid receptor (MOR) is proposed to underlie the initiation of opioid analgesic tolerance and previous work has shown that agonist-induced phosphorylation of the MOR C-tail contributes to this desensitisation. Moreover, phosphorylation is important for β-arrestin recruitment to the receptor, and ligands of different efficacies induce distinct phosphorylation barcodes. The C-tail 370TREHPSTANT379 motif harbours Ser/Thr residues important for these regulatory functions. 375Ser is the primary phosphorylation site of a ligand-dependent, hierarchical, and sequential process, whereby flanking 370Thr, 376Thr and 379Thr get subsequently and rapidly phosphorylated. Here we used GRK KO cells, phosphosite specific antibodies and site-directed mutagenesis to evaluate the contribution of the different GRK subfamilies to ligand-induced phosphorylation barcodes and β-arrestin2 recruitment. We show that both GRK2/3 and GRK5/6 subfamilies promote phosphorylation of 370Thr and 375Ser. Importantly, only GRK2/3 induce phosphorylation of 376Thr and 379Thr, and we identify these residues as key sites to promote robust β-arrestin recruitment to the MOR. These data provide insight into the mechanisms of MOR regulation and suggest that the cellular complement of GRK subfamilies plays an important role in determining the tissue responses of opioid agonists.

Functional differences in the mu opioid receptor SNP 118A>G are dependent on receptor splice-variant andagonist-specific recruitment of β-arrestin.

The OPRM1 gene codes for the mu opioid receptor (MOR) and polymorphisms are associated with complex patient clinical responses. The most studied single nucleotide polymorphism (SNP) in OPRM1 is adenine (A) substituted by guanine (G) at position 118 (118A>G, rs1799971) leading to a substitution of asparagine (Asn) for aspartic acid (Asp) at position 40 in the N terminus of the resulting protein. To date, no structural explanation for the associated clinical responses resulting from the 118A>G polymorphism has been proposed. We utilized computational modeling paired with functional cellular assays to predict unstructured N- and C-terminal regions of MOR-1. Using molecular docking and post-docking energy minimizations with morphine, we show that the extracellular substitution of Asn at position 40 alters the cytoplasmic C-terminal conformation, while leaving the G-protein binding interface unaffected. A real-time BRET assay measuring G-protein and β-arrestin association with MOR r generated data that tested this prediction. Consistent with this in silico prediction, we show changes in morphine-mediated β-arrestin association with receptor variants with little change in morphine-mediated G-protein association comparing MOR-1 wild type (WT) to MOR-1118A>G. We tested the system with different opioid agonists, the OPRM1 118A>G SNP, and different MOR splice variants (MOR-1 and MOR-1O). These results are consistent with the observation that patients with the 118A>G OPRM1 allele respond more readily to fentanyl than to morphine. In conclusion, the 118A>G substitution alters receptor responses to opioids through variable C-terminal domain movements that are agonist and splice variant dependent.