In Vitro Functional Profiling of Fentanyl and Nitazene Analogs at the µ-Opioid Receptor Reveals High Efficacy for Gi Protein Signaling.

Novel synthetic opioids (NSOs), including both fentanyl and non-fentanyl analogs that act as µ-opioid receptor (MOR) agonists, are associated with serious intoxication and fatal overdose. Previous studies proposed that G-protein-biased MOR agonists are safer pain medications, while other evidence indicates that low intrinsic efficacy at MOR better explains the reduced opioid side effects. Here, we characterized the in vitro functional profiles of various NSOs at the MOR using adenylate cyclase inhibition and ß-arrestin2 recruitment assays, in conjunction with the application of the receptor depletion approach. By fitting the concentration-response data to the operational model of agonism, we deduced the intrinsic efficacy and affinity for each opioid in the Gi protein signaling and ß-arrestin2 recruitment pathways. Compared to the reference agonist [d-Ala2,N-MePhe4,Gly-ol5]enkephalin, we found that several fentanyl analogs were more efficacious at inhibiting cAMP production, whereas all fentanyl analogs were less efficacious at recruiting ß-arrestin2. In contrast, the non-fentanyl 2-benzylbenzimidazole (i.e., nitazene) analogs were highly efficacious and potent in both the cAMP and ß-arrestin2 assays. Our findings suggest that the high intrinsic efficacy of the NSOs in Gi protein signaling is a common property that may underlie their high risk of intoxication and overdose, highlighting the limitation of using in vitro functional bias to predict the adverse effects of opioids. In addition, the extremely high potency of many NSOs now infiltrating illicit drug markets further contributes to the danger posed to public health.

Delayed estrogen actions diminish food consumption without changing food approach, motor activity, or hypothalamic activation elicited by corticostriatal µ-opioid signaling.

Mu-opioid receptor (µ-OR) signaling in forebrain sites including nucleus accumbens (Acb) and ventromedial prefrontal cortex (vmPFC) modulates reward-driven feeding and may play a role in the pathophysiology of disordered eating. In preclinical models, intra-Acb or intra-vmPFC µ-OR stimulation causes overeating and vigorous responding for food rewards. These effects have been studied mainly in male animals, despite demonstrated sex differences and estrogen modulation of central reward systems. Hence, the present study investigated sex differences and estrogen modulation of intra-Acb and intra-vmPFC µ-OR-driven feeding behaviors. First, the dose-related effects of intra-Acb and intra-vmPFC infusions of the µ-OR-selective agonist, DAMGO, were compared among intact female, ovariectomized (OVX) female, and intact male rats. The DAMGO feeding dose-effect function was flattened in intact females relative to the robust, dose-dependent effects observed in OVX females and intact males. Thus, in intact females, intra-Acb DAMGO failed to elevate food intake relative to vehicle, while intra-vmPFC DAMGO elevated food intake, but to a smaller degree compared to males and OVX females. Next, to explore the possible role of estrogen in mediating the diminished DAMGO response observed in intact females, OVX rats were given intra-Acb or intra-vmPFC infusions of DAMGO either immediately after a subcutaneous injection of 17-beta-estradiol 3-benzoate (EB; 5 µg/0.1 mL) or 24 h after EB injection. Intra-Acb DAMGO effects were not changed at the immediate post-EB time point. At the delayed post-EB timepoint, significant lordosis was noted and the duration of intra-Acb DAMGO-driven feeding bouts was significantly reduced, with no change in the number of bouts initiated, locomotor hyperactivity, or Fos immunoreactivity in hypothalamic feeding and arousal systems. Similarly, EB failed to alter the motor-activational effects of intra-vmPFC DAMGO while reducing feeding. These findings indicate that delayed, presumably genomically mediated estrogen actions modulate the µ-OR-generated motivational state by reducing consummatory activity while sparing goal-approach and general arousal/activity. The results additionally suggest that EB regulation of consummatory activity occurs outside of forebrain-µ-OR control of hypothalamic systems.

The rostromedial tegmental nucleus RMTg is not a critical site for ethanol-induced motor activation in rats.

RATIONALE: Opioid drugs indirectly activate dopamine (DA) neurons in the ventral tegmental area (VTA) through a disinhibition mechanism mediated by mu opioid receptors (MORs) present both on the GABA projection neurons located in the medial tegmental nucleus/tail of the VTA (RMTg/tVTA) and on the VTA GABA interneurons. It is well demonstrated that ethanol, like opioid drugs, provokes VTA DA neuron disinhibition by interacting (through its secondary metabolite, salsolinol) with MORs present in VTA GABA interneurons, but it is not known whether ethanol could disinhibit VTA DA neurons through the MORs present in the RMTg/tVTA. OBJECTIVES: The objective of the present study was to determine whether ethanol, directly microinjected into the tVTA/RMTg, is also able to induce VTA DA neurons disinhibition. METHODS: Disinhibition of VTA DA neurons was indirectly assessed through the analysis of the motor activity of rats. Cannulae were placed into the tVTA/RMTg to perform microinjections of DAMGO (0.13 nmol), ethanol (150 or 300 nmol) or acetaldehyde (250 nmol) in animals pre-treated with either aCSF or the irreversible antagonist of MORs, beta-funaltrexamine (beta-FNA; 2.5 nmol). After injections, spontaneous activity was monitored for 30 min. RESULTS: Neither ethanol nor acetaldehyde directly administered into the RMTg/tVTA were able to increase the locomotor activity of rats at doses that, in previous studies performed in the posterior VTA, were effective in increasing motor activities. However, microinjections of 0.13 nmol of DAMGO into the tVTA/RMTg significantly increased the locomotor activity of rats. These activating effects were reduced by local pre-treatment of rats with beta-FNA (2.5 nmol). CONCLUSIONS: The tVTA/RMTg does not appear to be a key brain region for the disinhibiting action of ethanol on VTA DA neurons. The absence of dopamine in the tVTA/RMTg extracellular medium, the lack of local ethanol metabolism or both could explain the present results.

New Insights into the Opioid Analgesic Profile of cis-(-)-N-Normetazocine-derived Ligands.

In this work, we report on the in vitro and in vivo pharmacological properties of LP1 analogs to complete the series of structural modifications aimed to generate compounds with improved analgesia. To do that, the phenyl ring in the N-substituent of our lead compound LP1 was replaced by an electron-rich or electron-deficient ring and linked through a propanamide or butyramide spacer at the basic nitrogen of the (-)-cis-N-normetazocine skeleton. In radioligand binding assays, compounds 3 and 7 were found to display nanomolar binding affinity for the µ opioid receptor (MOR) (Ki = 5.96 ± 0.08 nM and 1.49 ± 0.24 nM, respectively). In the mouse vas deferens (MVD) assay, compound 3 showed an antagonist effect against DAMGO ([D-Ala2, N-MePhe4, Gly-ol]-enkephalin), a highly selective MOR prototype agonist, whereas compound 7 produced naloxone reversible effect at MOR. Moreover, compound 7, as potent as LP1 and DAMGO at MOR, was able to reduce thermal and inflammatory pain assessed by the mouse tail-flick test and rat paw pressure thresholds (PPTs) measured by a Randall-Selitto test.

Opioids Induce Bidirectional Synaptic Plasticity in a Brainstem Pain Center in the Rat.

Opioids are powerful analgesics commonly used in pain management. However, opioids can induce complex neuroadaptations, including synaptic plasticity, that ultimately drive severe side effects, such as pain hypersensitivity and strong aversion during prolonged administration or upon drug withdrawal, even following a single, brief administration. The lateral parabrachial nucleus (LPBN) in the brainstem plays a key role in pain and emotional processing; yet, the effects of opioids on synaptic plasticity in this area remain unexplored. Using patch-clamp recordings in acute brainstem slices from male and female Sprague Dawley rats, we demonstrate a concentration-dependent, bimodal effect of opioids on excitatory synaptic transmission in the LPBN. While a lower concentration of DAMGO (0.5 µM) induced a long-term depression of synaptic strength (low-DAMGO LTD), abrupt termination of a higher concentration (10 µM) induced a long-term potentiation (high-DAMGO LTP) in a subpopulation of cells. LTD involved a metabotropic glutamate receptor (mGluR)-dependent mechanism; in contrast, LTP required astrocytes and N-methyl-D-aspartate receptor (NMDAR) activation. Selective optogenetic activation of spinal and periaqueductal gray matter (PAG) inputs to the LPBN revealed that, while LTD was expressed at all parabrachial synapses tested, LTP was restricted to spino-parabrachial synapses. Thus, we uncovered previously unknown forms of opioid-induced long-term plasticity in the parabrachial nucleus that potentially modulate some adverse effects of opioids. PERSPECTIVE: We found a previously unrecognized site of opioid-induced plasticity in the lateral parabrachial nucleus, a key region for pain and emotional processing. Unraveling opioid-induced adaptations in parabrachial function might facilitate the identification of new therapeutic measures for addressing adverse effects of opioid discontinuation such as hyperalgesia and aversion.

Focal mu-opioid receptor activation promotes neuroinflammation and microglial activation in the mesocorticolimbic system: Alterations induced by inflammatory pain.

Microglia participates in the modulation of pain signaling. The activation of microglia is suggested to play an important role in affective disorders that are related to a dysfunction of the mesocorticolimbic system (MCLS) and are commonly associated with chronic pain. Moreover, there is evidence that mu-opioid receptors (MORs), expressed in the MCLS, are involved in neuroinflammatory events, although the way by which they do it remains to be elucidated. In this study, we propose that MOR pharmacological activation within the MCLS activates and triggers the local release of proinflammatory cytokines and this pattern of activation is impacted by the presence of systemic inflammatory pain. To test this hypothesis, we used in vivo microdialysis coupled with flow cytometry to measure cytokines release in the nucleus accumbens and immunofluorescence of IBA1 in areas of the MCLS on a rat model of inflammatory pain. Interestingly, the treatment with DAMGO, a MOR agonist locally in the nucleus accumbens, triggered the release of the IL1α, IL1ß, and IL6 proinflammatory cytokines. Furthermore, MOR pharmacological activation in the ventral tegmental area (VTA) modified the levels of IBA1-positive cells in the VTA, prefrontal cortex, the nucleus accumbens and the amygdala in a dose-dependent way, without impacting mechanical nociception. Additionally, MOR blockade in the VTA prevents DAMGO-induced effects. Finally, we observed that systemic inflammatory pain altered the IBA1 immunostaining derived from MOR activation in the MSCLS. Altogether, our results indicate that the microglia-MOR relationship could be pivotal to unravel some inflammatory pain-induced comorbidities related to MCLS dysfunction.

In vivo photopharmacology with a caged mu opioid receptor agonist drives rapid changes in behavior.

Photoactivatable drugs and peptides can drive quantitative studies into receptor signaling with high spatiotemporal precision, yet few are compatible with behavioral studies in mammals. We developed CNV-Y-DAMGO-a caged derivative of the mu opioid receptor-selective peptide agonist DAMGO. Photoactivation in the mouse ventral tegmental area produced an opioid-dependent increase in locomotion within seconds of illumination. These results demonstrate the power of in vivo photopharmacology for dynamic studies into animal behavior.

Synergistic interaction between DAMGO-NH2 and NOP01 in peripherally acting antinociception in two mouse models of formalin pain.

The most commonly used opioid analgesics are limited by their severe side-effects in the clinical treatment of pain. Preliminary reports indicate that the combination of classical opioids and N/OFQ receptor (NOP) ligands may be an effective strategy to reduce unwanted side-effects and improve antinociception. But the interaction of these two receptor ligands in pain regulation at the peripheral level remains unclear. In this study, the antinociception of a designed amide analogue of the mu opioid receptor (MOP) peptide agonist DAMGO, DAMGO-NH2, and its antinociceptive interaction with the peripherally limited NOP peptide agonist NOP01 was investigated in two mouse models of formalin pain. Our results showed that DAMGO-NH2 acted as a MOP agonist in in vitro functional assays. Moreover, local subcutaneous or intraplantar injection of DAMGO-NH2 exerted dose-related antinociception in both phases of the formalin orofacial and intraplantar pain, which could be mediated by the classical opioid receptor. Peripheral but not central pretreatment with the peripherally restricted opioid antagonist naloxone methiodide inhibited local DAMGO-NH2-induced antinociception, supporting the involvement of the peripheral opioid receptor in local DAMGO-NH2-induced antinociception. Furthermore, co-administration of the inactive doses of DAMGO-NH2 and NOP01 produced effective antinociception. More importantly, isobolographic analysis indicates that the combination of DAMGO-NH2 and NOP01 elicited supra-additive antinociception in these two models of formalin pain. In addition, the combination of DAMGO-NH2 and NOP01 did not change motor function of mice in rotarod test. In conclusion, these data suggest that peripheral DAMGO-NH2 and particularly its combination therapy with NOP01 may be effective for pain management.

Novel Receptor-Binding-Based Assay for the Detection of Opioids in Human Urine Samples.

Consumption of opioids is a growing global health problem. The gold standard for drugs of abuse screening is immunochemical assays. However, this method comes with some disadvantages when screening for a wide variety of opioids. Detection of the binding of a compound at the human µ-opioid receptor (MOR) offers a promising alternative target. Here, we set up a urine assay to allow for detection of compounds that bind at the MOR, thus allowing the assay to be utilized as a screening tool for opioid intake. The assay is based on the incubation of MOR-containing cell membranes with the selective MOR-ligand DAMGO and urine. After filtration, the amount of DAMGO in the eluate is analyzed by liquid chromatography tandem mass spectroscopy (LC-MS/MS). The absence of DAMGO in the eluate corresponds to a competing MOR ligand in the urine sample, thus indicating opiate/opioid intake by the suspect. Sensitivity and specificity were determined by the analysis of 200 consecutive forensic routine casework urine samples. A pronounced displacement of DAMGO was observed in 29 of the 35 opiate/opioid-positive samples. Detection of fentanyl intake proved to be the most challenging aspect. Applying a cut-off value of, e.g., 10% DAMGO binding would lead to a sensitivity of 83% and a specificity of 95%. Consequently, the novel assay proved to be a promising screening tool for opiate/opioid presence in urine samples. The nontargeted approach and possible automation of the assay make it a promising alternative to conventional methods.

Analgesic tolerance and cross-tolerance to the bifunctional opioid/neuropeptide FF receptors agonist EN-9 and µ-opioid receptor ligands at the supraspinal level in mice.

The chimeric peptide EN-9 was reported as a κ-opioid/neuropeptide FF receptors bifunctional agonist that modulated chronic pain with no tolerance. Many lines of evidence have shown that the effect of the κ-opioid receptor is mediated by not only its specific activation but also downstream events participation, especially interaction with the µ-opioid receptor pathway in antinociception and tolerance on most occasions. The present study investigated the acute and chronic cross-tolerance of EN-9 with µ-opioid receptor agonist EM-2, DAMGO, and morphine after intracerebroventricularly (i.c.v) injection in the mouse tail-flick test. In the acute tolerance test, EN-9 showed symmetrical acute cross-tolerance to DAMGO but no cross-tolerance to EM2. In the chronic tolerance test, EN-9 had no tolerance after eight days of repeated administration. However, EN-9 illustrated complete cross-tolerance to morphine and symmetrical cross-tolerance to EM2. In addition, inhibition of NPFF receptor could induce the tolerance development of EN-9. These findings indicated that supraspinal EN-9-induced antinociception contains additional components, which are mediated by the downstream µ-opioid receptor pathway both in acute and chronic treatment, whereas the subtypes of µ-opioid receptor or NPFF system pathway involved in antinociceptive effects induced by EN-9 are complex. Identifying the receptor mechanism could help design preferable bifunctional opioid compounds.

Brief Opioid Exposure Paradoxically Augments Primary Afferent Input to Spinal Excitatory Neurons via α2δ-1-Dependent Presynaptic NMDA Receptors.

Treatment with opioids not only inhibits nociceptive transmission but also elicits a rebound and persistent increase in primary afferent input to the spinal cord. Opioid-elicited long-term potentiation (LTP) from TRPV1-expressing primary afferents plays a major role in opioid-induced hyperalgesia and analgesic tolerance. Here, we determined whether opioid-elicited LTP involves vesicular glutamate transporter-2 (VGluT2) or vesicular GABA transporter (VGAT) neurons in the spinal dorsal horn of male and female mice and identified underlying signaling mechanisms. Spinal cord slice recordings revealed that µ-opioid receptor (MOR) stimulation with DAMGO initially inhibited dorsal root-evoked EPSCs in 87% VGluT2 neurons and subsequently induced LTP in 49% of these neurons. Repeated morphine treatment increased the prevalence of VGluT2 neurons displaying LTP with a short onset latency. In contrast, DAMGO inhibited EPSCs in 46% VGAT neurons but did not elicit LTP in any VGAT neurons even in morphine-treated mice. Spinal superficial laminae were densely innervated by MOR-containing nerve terminals and were occupied by mostly VGluT2 neurons and few VGAT neurons. Furthermore, conditional Grin1 knockout in dorsal root ganglion neurons diminished DAMGO-elicited LTP in lamina II neurons and attenuated hyperalgesia and analgesic tolerance induced by repeated treatment with morphine. In addition, DAMGO-elicited LTP in VGluT2 neurons was abolished by protein kinase C inhibition, gabapentin, Cacna2d1 knockout, or disrupting the α2δ-1-NMDA receptor interaction with an α2δ-1 C terminus peptide. Thus, brief MOR stimulation distinctively potentiates nociceptive primary afferent input to excitatory dorsal horn neurons via α2δ-1-coupled presynaptic NMDA receptors, thereby causing hyperalgesia and reducing analgesic actions of opioids.SIGNIFICANCE STATEMENT Opioid drugs are potent analgesics for treating severe pain and are commonly used during general anesthesia. However, opioid use often induces pain hypersensitivity, rapid loss of analgesic efficacy, and dose escalation, which can cause dependence, addiction, and even overdose fatality. This study demonstrates for the first time that brief opioid exposure preferentially augments primary sensory input to genetically identified glutamatergic excitatory, but not GABAergic/glycinergic inhibitory, neurons in nociceptive dorsal horn circuits. This opioid-elicited synaptic plasticity is cell type specific and mediated by protein kinase C-dependent and α2δ-1-dependent activation of NMDA receptors at primary sensory nerve terminals. These findings elucidate how intraoperative use of opioids for preemptive analgesia paradoxically aggravates postoperative pain and increases opioid consumption and suggest new strategies to improve opioid analgesic efficacy.

Biased Activation Mechanism Induced by GPCR Heterodimerization: Observations from µOR/δOR Dimers.

GPCRs regulate multiple intracellular signaling cascades. Biasedly activating one signaling pathway over the others provides additional clinical utility to optimize GPCR-based therapies. GPCR heterodimers possess different functions from their monomeric states, including their selectivity to different transducers. However, the biased signaling mechanism induced by the heterodimerization remains unclear. Motivated by the issue, we select an important GPCR heterodimer (µOR/δOR heterodimer) as a case and use microsecond Gaussian accelerated molecular dynamics simulation coupled with potential of mean force and protein structure network (PSN) to probe mechanisms regarding the heterodimerization-induced constitutive ß-arrestin activity and efficacy change of the agonist DAMGO. The results show that only the lowest energy state of the µOR/δOR heterodimer, which adopts a slightly outward shift of TM6 and an ICL2 conformation close to the receptor core, can selectively accommodate ß-arrestins. PSN further reveals important roles of H8, ICL1, and ICL2 in regulating the constitutive ß-arrestin-biased activity for the apo µOR/δOR heterodimer. In addition, the heterodimerization can allosterically alter the binding mode of DAMGO mainly by means of W7.35. Consequently, DAMGO transmits the structural signal mainly through TM6 and TM7 in the dimer, rather than TM3 similar to the µOR monomer, thus changing the efficacy of DAMGO from a balanced agonist to the ß-arrestin-biased one. On the other side, the binding of DAMGO to the heterodimer can stabilize µOR/δOR heterodimers through a stronger interaction of TM1/TM1 and H8/H8, accordingly enhancing the interaction of µOR with δOR and the binding affinity of the dimer to the ß-arrestin. The agonist DAMGO does not change main compositions of the regulation network from the dimer interface to the transducer binding pocket of the µOR protomer, but induces an increase in the structural communication of the network, which should contribute to the enhanced ß-arrestin coupling. Our observations, for the first time, reveal the molecular mechanism of the biased signaling induced by the heterodimerization for GPCRs, which should be beneficial to more comprehensively understand the GPCR bias signaling.

Pharmacological characterization of novel synthetic opioids: Isotonitazene, metonitazene, and piperidylthiambutene as potent µ-opioid receptor agonists.

Recent trends of opioid abuse and related fatalities have highlighted the critical role of Novel Synthetic Opioids (NSOs). We studied the µ-opioid-like properties of isotonitazene (ITZ), metonitazene (MTZ), and piperidylthiambutene (PTB) using different approaches. In vitro studies showed that ITZ and MTZ displayed a higher potency in both rat membrane homogenates (EC50:0.99 and 19.1 nM, respectively) and CHO-MOR (EC50:0.71 and 10.0 nM, respectively) than [D-Ala2, NMe-Phe4, Gly-ol5]-enkephalin (DAMGO), with no difference in maximal efficacy (Emax) between DAMGO and NSOs. ITZ also has higher affinity (Ki:0.06 and 0.05 nM) at the MOR than DAMGO in both systems, whilst MTZ has higher affinity in CHO-MOR (Ki=0.23 nM) and similar affinity in rat cerebral cortex (Ki = 0.22 nM). PTB showed lower affinity and potency than DAMGO. In vivo, ITZ displayed higher analgesic potency than fentanyl and morphine (ED50:0.00156, 0.00578, 2.35 mg/kg iv, respectively); ITZ (0.01 mg/kg iv) and MTZ (0.03 mg/kg iv) reduced behavioral activity and increased dialysate dopamine (DA) in the NAc shell (max. about 200% and 170% over basal value, respectively. Notably, ITZ elicited an increase in DA comparable to that of higher dose of morphine (1 mg/kg iv), but higher than the same dose of fentanyl (0.01 mg/kg iv). In silico, induced fit docking (IFD) and metadynamic simulations (MTD) showed that binding modes and structural changes at the receptor, ligand stability, and the overall energy score of NSOs were consistent with the results of the biological assays.

Coincident Regulation of PLCß Signaling by Gq-Coupled and µ-Opioid Receptors Opposes Opioid-Mediated Antinociception.

Pain management is an important problem worldwide. The current frontline approach for pain management is the use of opioid analgesics. The primary analgesic target of opioids is the µ-opioid receptor (MOR). Deletion of phospholipase Cß3 (PLCß3) or selective inhibition of Gßγ regulation of PLCß3 enhances the potency of the antinociceptive effects of morphine suggesting a novel strategy for achieving opioid-sparing effects. Here we investigated a potential mechanism for regulation of PLC signaling downstream of MOR in human embryonic kidney 293 cells and found that MOR alone could not stimulate PLC but rather required a coincident signal from a Gq-coupled receptor. Knockout of PLCß3 or pharmacological inhibition of its upstream regulators, Gßγ or Gq, ex vivo in periaqueductal gray slices increased the potency of the selective MOR agonist [D-Ala2, N-Me-Phe4, Gly5-ol]-enkephalin acetate salt in inhibiting presynaptic GABA release. Finally, inhibition of Gq- G protein-coupled receptor coupling in mice enhanced the antinociceptive effects of morphine. These data support a model where Gq and Gßγ-dependent signaling cooperatively regulate PLC activation to decrease MOR-dependent antinociceptive potency. Ultimately, this could lead to identification of new non-MOR targets that would allow for lower-dose utilization of opioid analgesics. SIGNIFICANCE STATEMENT: Previous work demonstrated that deletion of phospholipase Cß3 (PLCß3) in mice potentiates µ-opioid receptor (MOR)-dependent antinociception. How PLCß3 is regulated downstream of MOR had not been clearly defined. We show that PLC-dependent diacylglycerol generation is cooperatively regulated by MOR-Gßγ and Gq-coupled receptor signaling through PLCß3 and that blockade of either Gq-signaling or Gßγ signaling enhances the potency of opioids in ex vivo brain slices and in vivo. These results reveal potential novel strategies for improving opioid analgesic potency and safety.

Age-related changes in peripheral nociceptor function.

Pain and pain management in the elderly population is a significant social and medical problem. Pain sensation is a complex phenomenon that typically involves activation of peripheral pain-sensing neurons (nociceptors) which send signals to the spinal cord and brain that are interpreted as pain, an unpleasant sensory experience. In this work, young (4-5 months) and aged (26-27 months) Fischer 344 x Brown Norway (F344xBN) rats were examined for nociceptor sensitivity to activation by thermal (cold and heat) and mechanical stimulation following treatment with inflammatory mediators and activators of transient receptor potential (TRP) channels. Unlike other senses that decrease in sensitivity with age, sensitivity of hindpaw nociceptors to thermal and mechanical stimulation was not different between young and aged F344xBN rats. Intraplantar injection of bradykinin (BK) produced greater thermal and mechanical allodynia in aged versus young rats, whereas only mechanical allodynia was greater in aged rats following injection of prostaglandin E2 (PGE2). Intraplantar injection of TRP channel activators, capsaicin (TRPV1), mustard oil (TRPA1) and menthol (TRPM8) each resulted in greater mechanical allodynia in aged versus young rats and capsaicin-induced heat allodynia was also greater in aged rats. A treatment-induced allodynia that was greater in young rats was never observed. The anti-allodynic effects of intraplantar injection of kappa and delta opioid receptor agonists, salvinorin-A and D-Pen2,D-Pen5]enkephalin (DPDPE), respectively, were greater in aged than young rats, whereas mu opioid receptor agonists, [D-Ala2, N-MePhe4, Gly-ol]-enkephalin (DAMGO) and morphine, were not effective in aged rats. Consistent with these observations, in primary cultures of peripheral sensory neurons, inhibition of cAMP signaling in response to delta and kappa receptor agonists was greater in cultures derived from aged rats. By contrast, mu receptor agonists did not inhibit cAMP signaling in aged rats. Thus, age-related changes in nociceptors generally favor increased pain signaling in aged versus young rats, suggesting that changes in nociceptor sensitivity may play a role in the increased incidence of pain in the elderly population. These results also suggest that development of peripherally-restricted kappa or delta opioid receptor agonists may provide safer and effective pain relief for the elderly.

Fentanyl induces autism-like behaviours in mice by hypermethylation of the glutamate receptor gene Grin2b.

BACKGROUND: Environmental factors contribute to autism spectrum disorder. Fentanyl, one of the most widely used opioid analgesics in anaesthesia, can induce neurotoxicity, but its role in autism remains unknown. We determined whether fentanyl induced autism-like behaviours in young mice and the underlying mechanisms. METHODS: Young male and female mice received fentanyl at postnatal days 6, 8, and 10, and performed behavioural tests, including three-chamber social preference, elevated plus maze, grooming behaviour, and open-field test, from postnatal days 30-32. Expression of Grin2b, the gene encoding the GluN2B subunit of the N-methyl-d-aspartate receptor, was assessed in the anterior cingulate cortex of male mice using fluorescence in situ hybridisation histochemistry. We used bisulfite target sequencing to determine Grin2b hypermethylation sites after fentanyl treatment. In the specific activation and rescue experiments, we injected the mu opioid receptor agonist [D-Ala,2 N-MePhe,4 Gly-ol]-enkephalin (DAMGO) or Grin2b overexpression lentivirus into the anterior cingulate cortex of male mice. RESULTS: Fentanyl induced autism-like behaviours in both young male and female mice, and downregulated Grin2b expression (0.49-fold [0.08] vs 1.00-fold [0.09]; P<0.01) and GluN2B protein amounts (0.38-fold [0.07] vs 1.00-fold [0.12]; P<0.01) in the anterior cingulate cortex through hypermethylation of Grin2b. The mu-opioid receptor antagonist naloxone and overexpression of Grin2b in anterior cingulate cortex attenuated the fentanyl-induced effects, whereas DAMGO injection into the anterior cingulate cortex induced autism-like behaviours. CONCLUSIONS: These data suggest that fentanyl induces autism-like behaviours in young mice via an epigenetic mechanism. Further research is required to determine possible clinical relevance to autism risk.

The rostral medial frontal cortex is crucial for engagement in consummatory behavior.

The medial frontal cortex (MFC) in rodents emits rhythmic activity that is entrained to the animal's licking cycle during consumption and encodes the value of consumed fluids. These signals are especially prominent in the rostral half of the MFC. This region is located above an orbitofrontal region where mu-opioid receptors regulate intake and reversible inactivation reduces behavioral measures associated with the incentive value and palatability of liquid sucrose. Here, we examined the effects of reversible inactivation and stimulation of mu-opioid receptors in rostral MFC on behavior in an incentive contrast licking task. Adult male rats licked to receive access to liquid sucrose, which alternated between high (16%) and low (4%) values over 30 s periods. Bilateral infusion of muscimol reduced the total number of licks over the 30 min test sessions, the time spent actively consuming sucrose, and the ratio of licks for the higher and lower value fluids. Inactivation did not alter licking frequency or variability or microstructural measures such as the duration of licking bouts that are classically associated with the palatability of a liquid reward. Infusions of [d-Ala2, N-Me-Phe4, Gly5-ol]-enkephalin (DAMGO; 1 µg/µL) at the same sites had inconsistent behavioral effects across different subjects. Our findings suggest that the rostral MFC has a distinct role in the control of consummatory behavior and contributes to persistent consumption and not to the expression of palatability. (PsycInfo Database Record (c) 2022 APA, all rights reserved).

Development of a non-radioactive mass spectrometry-based binding assay at the µ-opioid receptor and its application for the determination of the binding affinities of 17 opiates/opioids as well as of the designer opioid isotonitazene and five further 2-benzylbenzimidazoles.

Radioactive ligand binding assays are the most commonly applied method for the determination of binding affinities of compounds at a particular receptor. While they are highly sensitive and high-throughput capable they come with major disadvantages due to the radioactive ligands utilized. Here we present the development of a mass-spectrometry-based binding assay for the determination of binding affinities at the human µ-opioid receptor using non-labelled DAMGO ([D-Ala2, N-MePhe4, Gly5-ol]-enkephalin). The runtime of the LC-MS/MS method was 5.5 min per data point and allowed for the highly sensitive detection of 38.5 fg DAMGO on column. The assay shows low non-specific binding and the equilibrium dissociation constant of DAMGO was 0.57 nM. The assay was applied to determine the Ki values of 17 opiates/opioids and the results were in good agreement with the data from radioactive receptor binding assays published in the literature. Additionally, the Ki value of six 2-benzylbenzimidazoles, including the widely abused designer opioid isotonitazene, were determined ranging from 0.654 to 72.9 nM. Consequently, the developed assay provides a suitable alternative to radioactive binding assays as it allows for a reliable and rapid determination of receptor binding affinities of e.g. newly emerging designer opioids.

Opioid receptor activation modulates the calcium current in the cochlear outer hair cells of the rat.

Previous works showed that opioid peptides are produced by olivocochlear efferent neurons, while cochlear hair cells express opioid receptors. It has been proposed that opioids protect the auditory system from damage by intense stimulation, although their use for therapeutic or illicit purposes links to hearing impairment. Therefore, it is relevant to study the effect of opioids in the auditory system to define their functional expression and mechanism of action. This study investigated the modulation of the Ca2+ currents by opioid peptides in the rat outer hair cells (OHC) using the whole-cell patch-clamp technique. The influence of agonists of the three opioid receptor subtypes (µ, δ, and κ) was studied. The κ opioid receptor agonist U-50488 inhibits the Ca2+ currents in a partially reversible form. Coincidently, norbinaltorphimine (a κ receptor antagonist) blocked the U-50488 inhibitory effect on the Ca2+ current. The δ and the µ opioid receptor agonists did not significantly affect the Ca2+ currents. These results indicate that the κ opioid receptor activation inhibits the Ca2+ current in OHC, modulating the intracellular Ca2+ concentration when OHCs depolarize. The modulation of the auditory function by opioids constitutes a relevant mechanism with a potential role in the physiopathology of auditory disturbances.

Modulation of the MOP Receptor (µ Opioid Receptor) by Imidazo[1,2-a]imidazole-5,6-Diones: In Search of the Elucidation of the Mechanism of Action.

The µ-opioid receptors belong to the family of G protein-coupled receptors (GPCRs), and their activation triggers a cascade of intracellular relays with the final effect of analgesia. Classical agonists of this receptor, such as morphine, are the main targets in the treatment of both acute and chronic pain. However, the dangerous side effects, such as respiratory depression or addiction, significantly limit their widespread use. The allosteric centers of the receptors exhibit large structural diversity within particular types and even subtypes. Currently, a considerable interest is aroused by the modulation of µ-opioid receptors. The application of such a technique may result in a reduction in the dose or even discontinuation of classical opiates, thus eliminating the side effects typical of this class of drugs. Our aim is to obtain a series of 1-aryl-5,6(1H)dioxo-2,3-dihydroimidazo[1,2-a]imidazole derivatives and provide more information about their activity and selectivity on OP3 (MOP, human mu opioid receptor). The study was based on an observation that some carbonyl derivatives of 1-aryl-2-aminoimidazoline cooperate strongly with morphine or DAMGO in sub-threshold doses, producing similar results to those of normal active doses. To elucidate the possible mechanism of such enhancement, we performed a few in vitro functional tests (involving cAMP and ß-arrestin recruitment) and a radioligand binding assay on CHO-K1 cells with the expression of the OP3 receptor. One of the compounds had no orthosteric affinity or intrinsic activity, but inhibited the efficiency of DAMGO. These results allow to conclude that this compound is a negative allosteric modulator (NAM) of the human µ-opioid receptor.