µ-Opioid receptor transcriptional variants in the murine forebrain and spinal cord.

Oprm1, the gene encoding the µ-opioid receptor, has multiple reported transcripts, with a variable 3' region and many alternative sequences encoding the C-terminus of the protein. The functional implications of this variability remain mostly unexplored, though a recurring notion is that it could be exploited by developing selective ligands with improved clinical profiles. Here, we comprehensively examined Oprm1 transcriptional variants in the murine central nervous system, using long-read RNAseq as well as spatial and single-cell transcriptomics. The results were validated with RNAscope in situ hybridization. We found a mismatch between transcripts annotated in the mouse genome (GRCm38/mm10) and the RNA-seq results. Sequencing data indicated that the primary Oprm1 transcript has a 3' terminus located on chr10:6,860,027, which is âˆ¼ 9.5 kilobases downstream of the longest annotated exon 4 end. Long-read sequencing confirmed that the final Oprm1 exon included a 10.2 kilobase long 3' untranslated region, and the presence of the long variant was unambiguously confirmed using RNAscope in situ hybridization in the thalamus, striatum, cortex and spinal cord. Conversely, expression of the Oprm1 reference transcript or alternative transcripts of the Oprm1 gene was absent or close to the detection limit. Thus, the primary transcript of the Oprm1 mouse gene is a variant with a long 3' untranslated region, which is homologous to the human OPRM1 primary transcript and encodes the same conserved C-terminal amino acid sequence.

Structural basis of µ-opioid receptor targeting by a nanobody antagonist.

The µ-opioid receptor (µOR), a prototypical G protein-coupled receptor (GPCR), is the target of opioid analgesics such as morphine and fentanyl. Due to the severe side effects of current opioid drugs, there is considerable interest in developing novel modulators of µOR function. Most GPCR ligands today are small molecules, however biologics, including antibodies and nanobodies, represent alternative therapeutics with clear advantages such as affinity and target selectivity. Here, we describe the nanobody NbE, which selectively binds to the µOR and acts as an antagonist. We functionally characterize NbE as an extracellular and genetically encoded µOR ligand and uncover the molecular basis for µOR antagonism by determining the cryo-EM structure of the NbE-µOR complex. NbE displays a unique ligand binding mode and achieves µOR selectivity by interactions with the orthosteric pocket and extracellular receptor loops. Based on a ß-hairpin loop formed by NbE that deeply protrudes into the µOR, we design linear and cyclic peptide analogs that recapitulate NbE's antagonism. The work illustrates the potential of nanobodies to uniquely engage with GPCRs and describes lower molecular weight µOR ligands that can serve as a basis for therapeutic developments.

An emerging multi-omic understanding of the genetics of opioid addiction.

Opioid misuse, addiction, and associated overdose deaths remain global public health crises. Despite the tremendous need for pharmacological treatments, current options are limited in number, use, and effectiveness. Fundamental leaps forward in our understanding of the biology driving opioid addiction are needed to guide development of more effective medication-assisted therapies. This Review focuses on the omics-identified biological features associated with opioid addiction. Recent GWAS have begun to identify robust genetic associations, including variants in OPRM1, FURIN, and the gene cluster SCAI/PPP6C/RABEPK. An increasing number of omics studies of postmortem human brain tissue examining biological features (e.g., histone modification and gene expression) across different brain regions have identified broad gene dysregulation associated with overdose death among opioid misusers. Drawn together by meta-analysis and multi-omic systems biology, and informed by model organism studies, key biological pathways enriched for opioid addiction-associated genes are emerging, which include specific receptors (e.g., GABAB receptors, GPCR, and Trk) linked to signaling pathways (e.g., Trk, ERK/MAPK, orexin) that are associated with synaptic plasticity and neuronal signaling. Studies leveraging the agnostic discovery power of omics and placing it within the context of functional neurobiology will propel us toward much-needed, field-changing breakthroughs, including identification of actionable targets for drug development to treat this devastating brain disease.

Endogenous opioid receptor system mediates costly altruism in the human brain.

Functional neuroimaging studies suggest that a large-scale brain network transforms others' pain into its vicarious representation in the observer, potentially modulating helping behavior. However, the neuromolecular basis of individual differences in vicarious pain and helping is poorly understood. We investigated the role of the endogenous µ-opioid receptor (MOR) system in altruistic costly helping. MOR density was measured using [11C]carfentanil. In a separate fMRI experiment, participants could donate money to reduce a confederate's pain from electric shocks. Participants were generally willing to help, and brain activity was observed in amygdala, anterior insula, anterior cingulate cortex (ACC), striatum, primary motor cortex, primary somatosensory cortex and thalamus when witnessing others' pain. Haemodynamic responses were negatively associated with MOR availability in emotion circuits. However, MOR availability positively associated with the ACC and hippocampus during helping. These findings suggest that the endogenous MOR system modulates altruism in the human brain.

The µ-opioid receptor differentiates two distinct human nociceptive populations relevant to clinical pain.

The shortfall in new analgesic agents is a major impediment to reducing reliance on opioid medications for control of severe pain. In both animals and man, attenuating nociceptive transmission from primary afferent neurons with a µ-opioid receptor agonist yields highly effective analgesia. Consequently, deeper molecular characterization of human nociceptive afferents expressing OPRM1, the µ-opioid receptor gene, is a key component for advancing analgesic drug discovery and understanding clinical pain control. A co-expression matrix for the µ-opioid receptor and a variety of nociceptive channels as well as δ- and κ-opioid receptors is established by multiplex in situ hybridization. Our results indicate an OPRM1-positive population with strong molecular resemblance to rodent peptidergic C-nociceptors associated with tissue damage pain and an OPRM1-negative population sharing molecular characteristics of murine non-peptidergic C-nociceptors. The empirical identification of two distinct human nociceptive populations that differ profoundly in their presumed responsiveness to opioids provides an actionable translational framework for human pain control.

Discovery of cyanoguanidine derivatives as biased µ-opioid receptor agonists.

Opioid agonists, including morphine and its derivatives, have historically been utilized in conventional pain relief therapies. However, the morphine-like side effects associated with these compounds have constrained their broader application in clinical environments. Fortunately, novel compounds that selectively activate µ-opioid receptors (MOR) without activating the ß-arrestin2 pathway, such as PZM21 and TRV130, demonstrate the potential to mitigate side effects while maintaining analgesic efficacy. In this study, we structurally modified PZM21 to get a series of compounds with a 2-cyanoguanidine scaffold, the majority of which display significant analgesic effects. Notably, Compound I-11 exhibited an analgesic effect comparable to that of morphine and selectively activates µ-opioid receptors while avoiding the activation of the ß-arrestin2 pathway. Our work not only introduces a novel biased µ-opioid receptor agonist but also serves as a valuable reference for the further optimization of PZM21.

Field and laboratory perspectives on fentanyl and carfentanil decontamination.

Abuse of the highly toxic compound fentanyl and its analogues is increasing, raising serious public health concerns due to their potency and availability. Therefore, there is a need for decontamination methodologies to safely remove fentanyl to avoid harmful exposure. In this study, the efficacy of commercial and in-house synthesized decontamination agents (Dahlgren Decon, RSDL (Reactive Skin Decontamination Lotion), FAST-ACT (First applied sorbent treatment against chemical threats), GDS2000, alldecont MED, bleach, Domestos Spray Bleach, Effekt Klor, MgO, TiO2-nanodiamond, and CeO2) were evaluated for the degradation of fentanyl and carfentanil under controlled laboratory conditions and on wooden floor surfaces. Liquid chromatography/mass spectrometry analysis showed that oxidative decontamination agents were the most effective, with N-oxides identified as major degradation products. The physiological effects of these N-oxides were also investigated regarding their ability to activate the µ-opioid receptor and their metabolism in human liver microsomes. The results provide empirical evidence that complements prior research findings on the degradation of fentanyl and carfentanil using a variety of decontamination agents.

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.

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.

Endogenous mu-opioid modulation of social connection in humans: a systematic review and meta-analysis.

Social bonding, essential for health and survival in all social species, depends on mu-opioid signalling in non-human mammals. A growing neuroimaging and psychopharmacology literature also implicates mu-opioids in human social connectedness. To determine the role of mu-opioids for social connectedness in healthy humans, we conducted a preregistered ( https://osf.io/x5wmq ) multilevel random-effects meta-analysis of randomised double-blind placebo-controlled opioid antagonist studies. We included data from 8 publications and 2 unpublished projects, totalling 17 outcomes (N = 455) sourced from a final literature search in Web of Science, Scopus, PubMed and EMBASE on October 12, 2023, and through community contributions. All studies used naltrexone (25-100 mg) to block the mu-opioid system and measured social connectedness by self-report. Opioid antagonism slightly reduced feelings of social connectedness (Hedges' g [95% CI) = -0.20] [-0.32, -0.07]. Results were highly consistent within and between studies (I2 = 23%). However, there was some indication of bias in favour of larger effects among smaller studies (Egger's test: B = -2.16, SE = 0.93, z = -2.33, p = 0.02), and publication bias analysis indicated that the effect of naltrexone might be overestimated. The results clearly demonstrate that intact mu-opioid signalling is not essential for experiencing social connectedness, as robust feelings of connectedness are evident even during full pharmacological mu-opioid blockade. Nevertheless, antagonism reduced measures of social connection, consistent with a modulatory role of mu-opioids for human social connectedness. The modest effect size relative to findings in non-human animals, could be related to differences in measurement (subjective human responses versus behavioural/motivation indices in animals), species specific neural mechanisms, or naltrexone effects on other opioid receptor subtypes. In sum, these results help explain how mu-opioid dysregulation and social disconnection can contribute to disability, and conversely-how social connection can buffer risk of ill health.

Intrathecal administration of MCRT produced potent antinociception in chronic inflammatory pain models via µ-δ heterodimer with limited side effects.

An important goal in the opioid field is to discover effective analgesic drugs with minimal side effects. MCRT demonstrated potent antinociceptive effects with limited side effects, making it a promising candidate. However, its pharmacological properties and how it minimizes side effects remain unknown. Various mouse pain and opioid side effect models were used to evaluate the antinociceptive properties and safety at the spinal level. The targets of MCRT were identified through cAMP measurement, isolated tissue assays, and pharmacological experiments. Immunofluorescence was employed to visualize protein expression. MCRT displayed distinct antinociceptive effects between acute and chronic inflammatory pain models due to its multifunctional properties at the µ opioid receptor (MOR), µ-δ heterodimer (MDOR), and neuropeptide FF receptor 2 (NPFFR2). Activation of NPFFR2 reduced MOR-mediated antinociception, leading to bell-shaped response curves in acute pain models. However, activation of MDOR produced more effective antinociception in chronic inflammatory pain models. MCRT showed limited tolerance and opioid-induced hyperalgesia in both acute and chronic pain models and did not develop cross-tolerance to morphine. Additionally, MCRT did not exhibit addictive properties, gastrointestinal inhibition, and effects on motor coordination. Mechanistically, peripheral chronic inflammation or repeated administration of morphine and MCRT induced an increase in MDOR in the spinal cord. Chronic administration of MCRT had no apparent effect on microglial activation in the spinal cord. These findings suggest that MCRT is a versatile compound that provides potent antinociception with minimal opioid-related side effects. MDOR could be a promising target for managing chronic inflammatory pain and addressing the opioid crisis.

µ-Opioid Receptor Modulation of the Glutamatergic/GABAergic Midbrain Inputs to the Mouse Dorsal Hippocampus.

We used virus-mediated anterograde and retrograde tracing, optogenetic modulation, immunostaining, in situ hybridization, and patch-clamp recordings in acute brain slices to study the release mechanism and µ-opioid modulation of the dual glutamatergic/GABAergic inputs from the ventral tegmental area and supramammillary nucleus to the granule cells of the dorsal hippocampus of male and female mice. In keeping with previous reports showing that the two transmitters are released by separate active zones within the same terminals, we found that the short-term plasticity and pharmacological modulation of the glutamatergic and GABAergic currents are indistinguishable. We further found that glutamate and GABA release at these synapses are both virtually completely mediated by N- and P/Q-type calcium channels. We then investigated µ-opioid modulation of these synapses and found that activation of µ-opioid receptors (MORs) strongly inhibits the glutamate and GABA release, mostly through inhibition of presynaptic N-type channels. However, the modulation by MORs of these dual synapses is complex, as it likely includes also a disinhibition due to downmodulation of local GABAergic interneurons which make direct axo-axonic contacts with the dual glutamatergic/GABAergic terminals. We discuss how this opioid modulation may enhance LTP at the perforant path inputs, potentially contributing to reinforce memories of drug-associated contexts.

Functional differences in the mu opioid receptor SNP 118A>G are dependent on receptor splice-variant and agonist-specific recruitment of ß-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 ß-arrestin association with MOR r generated data that tested this prediction. Consistent with this in silico prediction, we show changes in morphine-mediated ß-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.

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.

Inhibition of Dorsal Root Ganglia Transient Receptor Potential Ankyrin 1 Upregulation Contributes to the Protective Effect of Morphine Against Gastric Mucosal Damage Induced by Water-Immersion Restraint Stress.

The pathogenesis mechanism of acute gastric mucosal lesions (AGML) is still unclear; further exploration is urgently needed to find a new therapeutic target. This study aimed to investigate whether morphine might regulate the expression and function of transient receptor potential ankyrin 1 (TRPA1) through a cyclic adenosine monophosphate/protein kinase A (cAMP/PKA)-dependent pathway, thereby alleviating gastric mucosal lesions caused by water-immersion restraint stress (WIRS). Rats were administered with intrathecal morphine, TRPA1 antagonist (HC-030031), µ-opioid receptor antagonist, or protein kinase A inhibitor (H-89), respectively, before WIRS. After 6 hours of WIRS, microscopic lesions, hematoxylin and eosin staining, and transmission electron microscopy were applied to assess the damage of the gastric mucosa. Real-time polymerase chain reaction, Western blot, and enzyme-linked immunosorbent assay were conducted to detect the levels of TRPA1 and substance P (SP) in the dorsal root ganglia (DRG) and gastric tissues. In addition, immunofluorescence was used to explore the possible co-expression of TRPA1 and µ-opioid receptors in the DRG. The results indicated that WIRS upregulated TRPA1 and SP in gastric mucosa, and HC-030031 or H-89 could alleviate gastric mucosal lesions caused by WIRS (P < .0001). Morphine was found to suppress both WIRS-induced gastric mucosal lesions (P < .0001) and the upregulation of TRPA1 (P = .0086) and SP (P = .0013). Both TRPA1 and SP play important roles in the pathogenesis of WIRS-induced AGML. Exogenous gastroprotective strategies reduce elevated levels of TRPA1 via the cAMP/PKA-dependent pathway. Inhibition of TRPA1 upregulation in the DRG is critical for intrathecal morphine preconditioning-induced gastric protection.

Detection of N-desethyl etonitazene in a drug checking sample: Chemical analysis and pharmacological characterization of a recent member of the 2-benzylbenzimidazole "nitazene" class.

The emergence of 2-benzylbenzimidazole "nitazene" opioids is stirring up the recreational synthetic opioid market. Many nitazene analogues act as potent agonists at the µ­opioid receptor (MOR), as demonstrated in various in vitro and in vivo studies. Severe intoxication and overdose deaths associated with nitazene analogues are increasingly being reported. Nitazene opioids are classified as a public health threat, stressing the need for close monitoring of new developments on the recreational drug market. This study reports on the detection of N-desethyl etonitazene in a sample handed in by a recreational drug user at a Swiss drug checking service in August 2023. The person bought the sample through an internet source where it was stated to contain isotonitazene. Chemical analyses were conducted to characterize the sample, i.e. nuclear magnetic resonance (NMR), capillary electrophoresis (CE), and high-resolution mass spectrometry (HRMS). The sample was additionally investigated using two different in vitro MOR activation assays. NMR and high-performance liquid chromatography (HPLC) coupled to HRMS confirmed the presence of N-desethyl etonitazene at a high purity and in the absence of isotonitazene and etonitazene. N-Desethyl nitazene analogues have been detected before as metabolites of isotonitazene and etonitazene. However, as first seen with N-desethyl isotonitazene, they are now emerging as standalone drugs. The applied bioassays demonstrated increased efficacy and approximately 6-9-fold higher potency of N-desethyl etonitazene at MOR compared to fentanyl. N-Desethyl etonitazene showed EC50 values of 3.35 nM and 0.500 nM in the ß-arrestin 2 recruitment and Aequoscreen® assays, respectively. The opioid activity present in the collected sample was additionally evaluated using the bioassays and showed good overlap with the reference standard, in line with the analytical purity assessment. This demonstrates the potential of these bioassays to provide a rapid opioid activity assessment of authentic samples. The emergence of other N-desethyl nitazene analogues must be considered during forensic and clinical toxicology casework, to avoid misclassification of intake of such analogues as metabolites. Finally, drug checking services enable the close monitoring of market developments and trends and are of great value for early warning and harm reduction purposes.

Elucidating the harm potential of brorphine analogues as new synthetic opioids: Synthesis, in vitro, and in vivo characterization.

The emergence of new synthetic opioids (NSOs) has added complexity to recreational opioid markets worldwide. While NSOs with diverse chemical structures have emerged, brorphine currently remains the only NSO with a piperidine benzimidazolone scaffold. However, the emergence of new generations of NSOs, including brorphine analogues, can be anticipated. This study explored the pharmaco-toxicological, opioid-like effect profile of brorphine alongside its non-brominated analogue (orphine) and three other halogenated analogues (fluorphine, chlorphine, iodorphine). In vitro, radioligand binding assays in rat brain tissue indicated that all analogues bind to the µ-opioid receptor (MOR) with nM affinity. While analogues with smaller-sized substituents showed the highest MOR affinity, further in vitro characterization via two cell-based (HEK 293T) MOR activation (ß-arrestin 2 and mini-Gαi recruitment) assays indicated that chlorphine, brorphine, and iodorphine were generally the most active MOR agonists. None of the compounds showed significant in vitro biased agonism compared to hydromorphone. In vivo, we investigated the effects of intraperitoneal (IP) administration of the benzimidazolones (0.01-15 mg/kg) on mechanical and thermal antinociception in male CD-1 mice. Chlorphine and brorphine overall induced the highest levels of antinociception. Furthermore, the effects on respiratory changes induced by a fixed dose (15 mg/kg IP) of the compounds were investigated using non-invasive plethysmography. Fluorphine-, chlorphine-, and brorphine-induced respiratory depressant effects were the most pronounced. For some compounds, pretreatment with naloxone (6 mg/kg IP) could not reverse respiratory depression. Taken together, brorphine-like piperidine benzimidazolones are opioid agonists that have the potential to cause substantial harm to users should they emerge as NSOs. This article is part of the Special Issue on "Novel Synthetic Opioids (NSOs)".

Antimigraine activity of Asarinin by OPRM1 pathway with multifaceted impacts through network analysis.

Migraine is a debilitating neurological disorder impacting millions worldwide. Calcitonin Gene-Related Peptide (CGRP) has emerged as a key player in migraine pathophysiology, leading to the development of targeted therapies. This study reviews novel CGRP-targeted treatments, including monoclonal antibodies small molecule inhibitors/nutraceuticals and introduces Asarinin as a potential modulator of the pathway. Asarinin, a natural compound found in various plants, is examined for its pharmacological potential in migraine management. Pharmacokinetic assessments, toxicological modelling, molecular property analysis, and network pharmacology were conducted. Molecular docking and dynamics studies with CGRP reveal potential interactions, providing a foundation for understanding Asarinin's therapeutic effects. Asarinin's favourable pharmacokinetics, safety profile, and bioactivity, supporting its candidacy as a therapeutic agent. In-depth molecular docking studies with the CGRP receptor (PDB: 6ZHO) demonstrate strong binding affinity (- 10.3kcal/mol), while molecular dynamics simulations unveil the dynamic behavior of the Asarinin-CGRP complex, (- 10.53 kcal/mol) for Atogepant-CGRP complex. Network analysis highlights key proteins in migraine pathology, indicating Asarinin's potential efficacy. The groundwork for future investigations, suggests Asarinin as a promising candidate for migraine management by targeting OPRM1 pathway. The integration of diverse assessments provides a comprehensive understanding of Asarinin's potential and paves the way for further preclinical and clinical research.

Key phosphorylation sites for robust ß-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 ß-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 ß-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 ß-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.

Multiple layers of diversity govern the cell type specificity of GABAergic input received by mouse subicular pyramidal neurons.

The subiculum is a key region of the brain involved in the initiation of pathological activity in temporal lobe epilepsy, and local GABAergic inhibition is essential to prevent subicular-originated epileptiform discharges. Subicular pyramidal cells may be easily distinguished into two classes based on their different firing patterns. Here, we have compared the strength of the GABAa receptor-mediated inhibitory postsynaptic currents received by regular- vs. burst-firing subicular neurons and their dynamic modulation by the activation of µ opioid receptors. We have taken advantage of the sequential re-patching of the same cell to initially classify pyramidal neurons according to their firing patters, and then to measure GABAergic events triggered by the optogenetic stimulation of parvalbumin- and somatostatin-expressing interneurons. Activation of parvalbumin-expressing cells generated larger responses in postsynaptic burst-firing neurons whereas the opposite was observed for currents evoked by the stimulation of somatostatin-expressing interneurons. In all cases, events depended critically on ω-agatoxin IVA- but not on ω-conotoxin GVIA-sensitive calcium channels. Optogenetic GABAergic input originating from both parvalbumin- and somatostatin-expressing cells was reduced in amplitude following the exposure to a µ opioid receptor agonist. The kinetics of this pharmacological sensitivity was different in regular- vs. burst-firing neurons, but only when responses were evoked by the activation of parvalbumin-expressing neurons, whereas no differences were observed when somatostatin-expressing cells were stimulated. In conclusion, our results show that a high degree of complexity regulates the organizing principles of subicular GABAergic inhibition, with the interaction of pre- and postsynaptic diversity at multiple levels. KEY POINTS: Optogenetic stimulation of parvalbumin- and somatostatin-expressing interneurons (PVs and SOMs) triggers inhibitory postsynaptic currents (IPSCs) in both regular- and burst-firing (RFs and BFs) subicular pyramidal cells. The amplitude of optogenetically evoked IPSCs from PVs (PV-opto IPSCs) is larger in BFs whereas IPSCs generated by the light activation of SOMs (SOM-opto IPSCs) are larger in RFs. Both PV- and SOM-opto IPSCs critically depend on ω-agatoxin IVA-sensitive P/Q type voltage-gated calcium channels, whereas no major effects are observed following exposure to ω-conotoxin GVIA, suggesting no significant involvement of N-type channels. The amplitude of both PV- and SOM-opto IPSCs is reduced by the probable pharmacological activation of presynaptic µ opioid receptors, with a faster kinetics of the effect observed in PV-opto IPSCs from RFs vs. BFs, but not in SOM-opto IPSCs. These results help us understand the complex interactions between different layers of diversity regulating GABAergic input onto subicular microcircuits.