Opioid Receptor Gene Research Articles

Solid Tumor Opioid Receptor Expression and Oncologic Outcomes: Analysis of the Cancer Genome Atlas and Genotype Tissue Expression Project.

BackgroundOpioid receptors are expressed not only by neural cells in the central nervous system, but also by many solid tumor cancer cells. Whether perioperative opioids given for analgesia after tumor resection surgery might inadvertently activate tumor cells, promoting recurrence or metastasis, remains controversial. We analysed large public gene repositories of solid tumors to investigate differences in opioid receptor expression between normal and tumor tissues and their association with long–term oncologic outcomes.MethodsWe investigated the normalized gene expression of µ, κ, δ opioid receptors (MOR, KOR, DOR), Opioid Growth Factor (OGFR), and Toll-Like 4 (TLR4) receptors in normal and tumor samples from twelve solid tumor types. We carried out mixed multivariable logistic and Cox regression analysis on whether there was an association between these receptors’ gene expression and the tissue where found, i.e., tumor or normal tissue. We also evaluated the association between tumor opioid receptor gene expression and patient disease–free interval (DFI) and overall survival (OS).ResultsWe retrieved 8,780 tissue samples, 5,852 from tumor and 2,928 from normal tissue, of which 2,252 were from the Genotype Tissue Expression Project (GTEx) and 672 from the Cancer Genome Atlas (TCGA) repository. The Odds Ratio (OR) [95%CI] for gene expression of the specific opioid receptors in the examined tumors varied: MOR: 0.74 [0.63–0.87], KOR: 1.27 [1.17–1.37], DOR: 1.66 [1.48–1.87], TLR4: 0.29 [0.26–0.32], OGFR: 2.39 [2.05–2.78]. After controlling all confounding variables, including age and cancer stage, there was no association between tumor opioid receptor expression and long–term oncologic outcomes.ConclusionOpioid receptor gene expression varies between different solid tumor types. There was no association between tumor opioid receptor expression and recurrence. Understanding the significance of opioid receptor expression on tumor cells remains elusive.

Exploring Pharmacological Functions of Alternatively Spliced Variants of the Mu Opioid Receptor Gene, Oprm1, via Gene-Targeted Animal Models.

The mu opioid receptor has a distinct place in the opioid receptor family, since it mediates the actions of most opioids used clinically (e.g., morphine and fentanyl), as well as drugs of abuse (e.g., heroin). The single-copy mu opioid receptor gene, OPRM1, goes through extensive alternative pre-mRNA splicing to generate numerous splice variants that are conserved from rodents to humans. These OPRM1 splice variants can be classified into three structurally distinct types: (1) full-length 7 transmembrane (TM) carboxyl (C)-terminal variants; (2) truncated 6TM variants; and (3) single TM variants. Distinct pharmacological functions of these splice variants have been demonstrated by both in vitro and in vivo studies, particularly by using several unique gene-targeted mouse models. These studies provide new insights into our understanding of the complex actions of mu opioids with regard to OPRM1 alternative splicing. This review provides an overview of the studies that used these gene-targeted mouse models for exploring the functional importance of Oprm1 splice variants.

Exploring the Mechanism of Yiqi Qingre Ziyin Method in Regulating Neuropeptide Expression for the Treatment of Atrophic Rhinitis.

Atrophic rhinitis (AR) is a chronic disease that causes severe structural changes to the nasal mucosa leading to squamous epithelial metaplasia. However, treatment regarding AR remains a major challenge. We used network pharmacology and molecular docking methods to explore the potential mechanisms of the Yiqi Qingre Ziyin method to modulate neuropeptides in the treatment of AR. The active ingredients of the Yiqi Qingre Ziyin method and their targets of action were obtained from the Traditional Chinese Medicine Systematic Pharmacology Database Analysis Platform (TCMSP). Disease targets for AR were obtained from four databases: GeneCards, PharmGKB, DrugBank, and Online Mendelian Inheritance in Man (OMIM). A total of 59 active ingredients, 39 potential targets, and 76 relevant neuropeptides were obtained after deduplication. We constructed target interaction networks with the STRING database. Gene Ontology (GO) enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis were performed on the 14 potential target proteins. We used Cytoscape software to construct the “drug-active ingredient-potential target” and “ingredient-target-pathway” networks of the Yiqi Qingre Ziyin method for treating AR. Molecular docking results suggest that dipeptidyl peptidase 4 (DPP4), opioid receptor gene d1 (OPRD1), and opioid receptor m1 (OPRM1) are key targets for the Yiqi Qingre Ziyin method. Therefore, this study proposed a potential mechanism for the treatment of AR by affecting the expression of neuropeptide-related genes (including DPP4, OPRD1, and OPRM1), which may potentially improve the immune microenvironment of the nasal mucosa.

Acute Inflammation Induces Neuroendocrine and Opioid Receptor Genes Responses in the Seabass Dicentrarchus labrax Brain.

Simple SummaryIt is generally accepted (in mammals and in teleost fish, too) that stressful conditions affect the performance of an immune response. What is still far from being known is at what extend does an immune process affects the neuroendocrine system. Vaccination for instance, is nowadays a common practice in aquaculture and little is known about its physiological implications other than immunization. Here is a first approach to the study of the European seabass’ brain gene expression patterns in response to a peripheral inflammatory process. Genes related to the stress response were focused, along with those related to the opioid system. Increased expression of certain genes suggests the activation of a stress response triggered by inflammatory signals. Additionally, contrasting expression patterns of the same gene (increased vs decreased) in the different brain regions (as well as the time needed for changes to happen) point at different functions. These results clearly show the reactivity of different brain responses to an immune response, highlighting the importance of further studies on downstream implications (behavior, feeding, welfare, reproduction). In fish, as observed in mammals, any stressful event affects the immune system to a larger or shorter extent. The neuroendocrine-immune axis is a bi-directional network of mobile compounds and their receptors that are shared between both systems (neuroendocrine and immune) and that regulate their respective responses. However, how and to what extent immunity modulates the neuroendocrine system is not yet fully elucidated. This study was carried out to understand better central gene expression response patterns in a high-valued farmed fish species to an acute peripheral inflammation, focusing on genes related to the hypothalamus-pituitary-interrenal axis and the opioid system. European seabass, Dicentrarchus labrax, were intra-peritoneally injected with either Freund’s Incomplete Adjuvant to induce a local inflammatory response or Hanks Balances Salt Solution to serve as the control. An undisturbed group was also included to take into account the effects due to handling procedures. To evaluate the outcomes of an acute immune response, fish were sampled at 4, 24, 48, and 72 h post-injection. The brain was sampled and dissected for isolation of different regions: telencephalon, optic tectum, hypothalamus, and pituitary gland. The expression of several genes related to the neuroendocrine response was measured by real-time PCR. Data were statistically analyzed by ANOVA and discriminant analyses to obtain these genes’ responsiveness for the different brain regions. Serotonergic receptors were upregulated in the telencephalon, whereas the optic tectum inhibited these transcription genes. The hypothalamus showed a somewhat delayed response in which serotonin and glucocorticoid receptors were concerned. Still, the hypothalamic corticotropin-releasing hormone played an important role in differentiating fish undergoing an inflammatory response from those not under such conditions. Opioid receptors gene expression increased in both the hypothalamus and the telencephalon, while in the optic tectum, most were downregulated. However, no changes in the pituitary gland were observed. The different brain regions under immune stimulation demonstrated clear, distinct responses regarding gene transcription rates as well as the time period needed for the effect to occur. Further, more integrative studies are required to associate functions to the evaluated genes more safely and better understand the triggering mechanisms.

Novel prognostic value and potential utility of opioid receptor gene methylation in liquid biopsy for oral cavity cancer

Opioids are a class of recreational drugs and prescription medications that bind to a group of G-protein-coupled receptors known as opioid receptors (ORs). ORs are involved in the development of many types of cancer; however, their role in head and neck squamous cell carcinoma (HNSCC) is complex and poorly understood. Here, we analyzed the methylation status of five OR genes in verification (301 HNSCC primary samples) and validation (five circulating tumor DNA [ctDNA] samples) studies using quantitative methylation-specific PCR (Q-MSP). OPRL1 and OPRM1 methylation levels were significantly higher in HNSCC tissues than in corresponding normal tissues from the same individuals (P = 0.001 and P < 0.001, respectively). In Kaplan-Meier estimate and multivariate Cox proportional hazard analyses, two genes (OPRL1 and OPRM1) were significantly associated with increased recurrence in the methylation group with oral cavity cancer. Furthermore, a validation study of ctDNA demonstrated that OPRL1 genes exhibited predictive performance as emerging biomarkers and were each capable of discriminating the plasma from tumor-free individuals. We characterized the relationship between OR gene methylation status and outcomes in oral cavity cancer. Our results highlight the potential utility of ctDNA methylation-based detection in the clinical management of oral cavity cancer.

Alternative Pre-mRNA Splicing of the Mu Opioid Receptor Gene, OPRM1: Insight into Complex Mu Opioid Actions.

Most opioid analgesics used clinically, including morphine and fentanyl, as well as the recreational drug heroin, act primarily through the mu opioid receptor, a class A Rhodopsin-like G protein-coupled receptor (GPCR). The single-copy mu opioid receptor gene, OPRM1, undergoes extensive alternative splicing, creating multiple splice variants or isoforms via a variety of alternative splicing events. These OPRM1 splice variants can be categorized into three major types based on the receptor structure: (1) full-length 7 transmembrane (TM) C-terminal variants; (2) truncated 6TM variants; and (3) single TM variants. Increasing evidence suggests that these OPRM1 splice variants are pharmacologically important in mediating the distinct actions of various mu opioids. More importantly, the OPRM1 variants can be targeted for development of novel opioid analgesics that are potent against multiple types of pain, but devoid of many side-effects associated with traditional opiates. In this review, we provide an overview of OPRM1 alternative splicing and its functional relevance in opioid pharmacology.

Opioid Addiction and Opioid Receptor Dimerization: Structural Modeling of the OPRD1 and OPRM1 Heterodimer and Its Signaling Pathways.

Opioid addiction is a complex phenomenon with genetic, social, and other components. Due to such complexity, it is difficult to interpret the outcome of clinical studies, and thus, mutations found in individuals with these addictions are still not indisputably classified as opioid addiction-causing variants. Here, we computationally investigated two such mutations, A6V and N40D, found in the mu opioid receptor gene OPRM1. The mutations are located in the extracellular domain of the corresponding protein, which is important to the hetero-dimerization of OPRM1 with the delta opioid receptor protein (OPRD1). The hetero-dimerization of OPRD1–OPRM1 affects the signaling pathways activated by opioids and natural peptides and, thus, could be considered a factor contributing to addiction. In this study, we built four 3D structures of molecular pathways, including the G-protein signaling pathway and the β-arrestin signaling pathway of the heterodimer of OPRD1–OPRM1. We also analyzed the effect of mutations of A6V and N40D on the stability of individual OPRM1/OPRD1 molecules and the OPRD1–OPRM1 heterodimer with the goal of inferring their plausible linkage with opioid addiction. It was found that both mutations slightly destabilize OPRM1/OPRD1 monomers and weaken their association. Since hetero-dimerization is a key step for signaling processes, it is anticipated that both mutations may be causing increased addiction risk.

Modulation of OPRM1 Alternative Splicing by Morphine and HIV-1 Nef.

Clinically used opioids, such as morphine, activate the mu opioid receptor (MOR) encoded by Opioid Receptor Mu 1 (OPRM1) gene. Examination of the opioid receptor genes showed that the human OPRM1 pre-mRNA undergoes extensive alternative splicing events and capable of expressing 21 isoforms. However, characterization of OPRM1 signaling is generalized, and only one isoform (MOR-1) has been extensively studied. Compounding this issue is the increasing significance of intravenous drug abuse in HIV neuropathogenesis. Here, we investigated the molecular impact of morphine and HIV-1 on regulation of OPRM1 pre-mRNA splicing in in vitro and in vivo models. Our results suggested that morphine treatment specifically induces the alternative splicing of MOR-1X isoform among the other isoforms analyzed in neuronal cells. Interestingly, alternative splicing and expression of MOR-1X isoform was also induced in postmortem brain tissues obtained from people with HIV (PWH). Additionally, treatment of control rats with morphine induced alternative splicing of MOR-1X in the brain regions involved in the reward pathways. More interestingly, HIV-1 transgenic (HIV-1Tg) rats, showed an additive induction of MOR-1X isoform with the exposure to morphine. To further assess the possible role of HIV secretory proteins in alternative splicing of OPRM1 gene, we analyzed the impact of HIV-1 Tat, gp120 and Nef proteins on alternative splicing of MOR-1X isoform. While the Tat and gp120 had no visible effects, treatment of neurons with Nef induced MOR-1X alternative splicing that was comparable to treatment with morphine. Altogether, our results suggest that HIV-1 may alter MOR isoform expression with Nef protein by amplifying the rate of MOR-1X alternative splicing induced by morphine.

Intraoperative opioid exposure, tumour genomic alterations, and survival differences in people with lung adenocarcinoma

BackgroundOpioids have been linked to worse oncologic outcomes in surgical patients. Studies in certain cancer types have identified associations between survival and intra-tumoural opioid receptor gene alterations, but no study has investigated whether the tumour genome interacts with opioid exposure to affect survival. We sought to determine whether intraoperative opioid exposure is associated with recurrence-specific survival and overall survival in early-stage lung adenocarcinoma, and whether selected tumour genomics are associated with this relationship. Associations between ketamine and dexmedetomidine and outcomes were also studied. MethodsSurgical patients (N=740) with pathological stage I–III lung adenocarcinoma and next-generation sequencing data were retrospectively reviewed from a prospectively maintained database. ResultsOn multivariable analysis, ketamine administration was protective for recurrence-specific survival (hazard ratio = 0.44, 95% confidence interval 0.24–0.80; P=0.007), compared with no adjunct. Higher intraoperative oral morphine milligram equivalents were significantly associated with worse overall survival (hazard ratio=1.09/10 morphine milligram equivalents, 95% confidence interval 1.02–1.17; P=0.010). Significant interaction effects were found between morphine milligram equivalents and fraction genome altered and morphine milligram equivalents and CDKN2A, such that higher fraction genome altered or CDKN2A alterations were associated with worse overall survival at higher morphine milligram equivalents (P=0.044 and P=0.052, respectively). In contrast, alterations in the Wnt (P=0.029) and Hippo (P=0.040) oncogenic pathways were associated with improved recurrence-specific survival at higher morphine milligram equivalents, compared with unaltered pathways. ConclusionsIntraoperative opioid exposure is associated with worse overall survival, whereas ketamine exposure is associated with improved recurrence-specific survival in patients with early-stage lung adenocarcinoma. This is the first study to investigate tumour-specific genomic interactions with intraoperative opioid administration to modify survival associations.

Biased signaling at mu opioid receptor full‐length C‐terminal splice variants

Acting on the samee G-protein coupled receptor, various ligands produce distinct activities by differentially activating downstream signaling pathways, a phenomenon referred to as biased agonism or functional selectivity. The original mu opioid receptor (MOR-1) has largely been studied in biased agonism mainly through G protein and β-arrestin2 signaling pathways. Mu opioid-induced G protein activation is often associated with its analgesic action, while mu opioid-induced β-arrestin recruitment is commonly linked to the side effects related to traditional opiates. However, there exist multiple full-length 7 transmembrane (7TM) C-terminal variants generated by alternative 3′ splicing of the mu opioid receptor gene, OPRM1, which is conserved from rodent to human. These 7TM C-terminal variants have identical receptor structures including the ligand binding pocket except for a variable intracellular C-terminal sequence. Several in vitro studies indicated marked differences among the 7TM C-terminal variants in mu agonist-induced G protein activation, receptor phosphorylation, internalization, desensitization, and post-endocytic sorting. Using a set of gene targeting mouse models in which a specific C-terminus was truncated at translational level, we demonstrated the functional importance of the C-terminal tails in various morphine actions. In the current study, we examined the role of different 7TM C-terminal variants on mu agonist-induced G protein activation and β-arrestin2 recruitment, which were used to determine signaling bias. The results showed that mu agonists induced significant differences in G protein activation and β-arrestin recruitment among various C-terminal variants. Especially, exon 7-associated 7TM variants displayed greater β-arrestin2 bias for most mu agonists compared to exon 4-associated 7TM MOR-1. Our finding provides new insights into mu agonist-induced biased signaling in vivo where these 7TM C-terminal variants are co-expressed.

Topical administration of the kappa opioid receptor agonist nalfurafine suppresses corneal neovascularization and inflammation

Corneal neovascularization (CNV) causes higher-order aberrations, corneal edema, ocular inflammation, and corneal transplant rejection, thereby decreasing visual acuity. In this study, we investigated the effects of topical administration of the kappa opioid receptor agonist nalfurafine (TRK-820) on CNV. To induce CNV, intrastromal corneal sutures were placed on the corneal stroma of BALB/c mice for 2 weeks. Nalfurafine (0.1 µg/2 μL/eye) was topically administered to the cornea once or twice daily after CNV induction. The CNV score, immune cell infiltration, and mRNA levels of angiogenic and pro-inflammatory factors in neovascularized corneas were evaluated using slit-lamp microscopy, immunohistochemistry, flow cytometry, and polymerase chain reaction. The mRNA expression of the kappa opioid receptor gene Oprk1 was significantly upregulated following CNV induction. Topical administration of nalfurafine twice daily significantly suppressed CNV and lymphangiogenesis, as well as reduced the mRNA levels of angiogenic and pro-inflammatory factors in the neovascularized corneas. Moreover, nalfurafine administration twice daily reduced the numbers of infiltrating leukocytes, neutrophils, macrophages, and interferon-γ-producing CD4+ T cells in the neovascularized corneas. In this study, we demonstrated that topical administration of nalfurafine suppressed local CNV in a mouse model along with the activation of KOR, suggesting that nalfurafine may prevent and control CNV in humans.

Hippocampal mu opioid receptors are modulated following cocaine self-administration in rat.

Cocaine addiction is a complex pathology induced by long-term brain changes. Understanding the neurochemical changes underlying the reinforcing effects of this drug of abuse is critical for reducing the societal burden of drug addiction. The mu opioid receptor plays a major role in drug reward. This receptor is modulated by chronic cocaine treatment in specific brain structures, but few studies investigated neurochemical adaptations induced by voluntary cocaine intake. In this study, we investigated whether intravenous cocaine-self administration (0.33mg/kg/injection, fixed-ratio 1 [FR1], 10days) in rats induces transcriptional and functional changes of the mu opioid receptor in reward-related brain regions. Epigenetic processes with histone modifications were examined for two activating marks, H3K4Me3, and H3K27Ac. We found an increase of mu opioid receptor gene expression along with a potentiation of its functionality in hippocampus of cocaine self-administering animals compared to saline controls. Chromatin immunoprecipitation followed by qPCR revealed no modifications of the histone mark H3K4Me3 and H3K27Ac levels at mu opioid receptor promoter. Our study highlights the hippocampus as an important target to further investigate neuroadaptive processes leading to cocaine addiction.

The effect of opioid receptor gene polymorphism (A118G) on postoperative tramadol consumption after gynecological surgery performed with a pfannenstiel incision

Introduction: The analgesic efficacy and side effects of opioid medications show great inter-individual differences. Genetic studies have indicated that this difference is considerably associated with the relationship between opioid and receptor. Therefore, in this study it was aimed to investigate the effect of A118G polymorphism on postoperative tramadol consumption and opioid-related side-effects after gynecological surgery performed. Material and Methods: Evaluation was made of 80 patients with I-II ASA status, scheduled for gynecological surgery performed with a pfannenstiel incision under general anesthesia. Genomic DNA was extracted from the blood samples. After surgery, all of the patients were equipped with an intravenous Tramadol patient-controlled analgesia device and tramadol consumption was measured. Pain scores were measured with a numerical rating scale. All assessments were performed prior to gene analysis. In order to detect the genotype for A118G single point mutation, Polymerase Chain Reaction (PCR) and Restriction Fragment Length Polymorphism(RFLP) methods were used. Results: The study included 80 patients were included. Of these, 60(75%) patients were detected to have homozygous 118AA(AA) genotype and 20(25%) patients to have heterozygous 118AG(AG). No patients with homozygous 118GG(GG mutant) genotype were detected. Patients were divided into 2 separate groups based on their genotypes. The postoperative total tramadol consumption and pain score in the group carrying the AG heterozygous allele were significantly higher than in the group carrying the AA homozygous allele. No statistically significant difference was detected between the groups in respect of side-effects. Conclusions: A118G polymorphism detected in the µ-opioid receptor gene has an effect on postoperative tramadol consumption.

Genetic susceptibility of opioid receptor genes polymorphism to drug addiction: A candidate-gene association study

BackgroundLike other complex diseases including drug addiction, genetic factors can interfere with the disease. In this study, three opioid genes (OPRM1, OPRD1, and OPRK1) were examined for an association with drug addiction among Jordanian males.MethodsThe study involved 498 addicts, in addition to 496 healthy controls and all from Arab descent.ResultsThe findings in this study showed that rs1799971 of the OPRM1 gene was in association with drug addiction for both alleles and genotypes with P-values = 0.002 and 0.01, respectively. In addition, a significant association between the dominant model (A/A vs G/A-G/G) of rs1799971 (OPRM1) and drug addiction (P-value = 0.003, OR = 1.59 (1.17–2.15)) was detected. Moreover, a genetic haplotype (AGGGCGACCCC) of theOPRM1 gene revealed a significant association with drug addiction (P-value = 0.01, OR = 1.56 (1.15–2.12)). We also found that the age of addicts, smoking, and marital status with genetic variants within OPRM1, OPRD1, and OPRK1 genes may be implicated in drug addiction risk.ConclusionWe propose that rs1799971 of the OPRM1gene is a genetic risk factor for drug addiction among Jordanian males.

Variation of the human mu-opioid receptor (OPRM1) gene predicts vulnerability to frustration

Understanding the emotional reaction to loss, or frustration, is a critical problem for the field of mental health. Animal models of loss have pointed to the opioid system as a nexus of frustration, physical pain, and substance abuse. However, few attempts have been made to connect the results of animal models of loss to human behavior. Allelic differences in the human mu opioid receptor gene, notably the A118G single nucleotide polymorphism, have been linked to individual differences in pain sensitivity, depressive symptoms, and reward processing. The present study explored the relationship between A118G and behavior in two frustrating tasks in humans. Results showed that carriers of the mutant G-allele were slower to recover behavior following a reward downshift and abandoned a frustrating task earlier than those without the mutation. Additionally, G-carriers were more sensitive to physical pain. These results highlight the overlap between frustration and pain, and suggest that genetic variation in opioid tone may contribute to individual differences in vulnerability and resilience following emotional disturbances.

Sex-dependent effects of an Hnrnph1 mutation on fentanyl addiction-relevant behaviors but not antinociception in mice.

Opioid Use Disorder (OUD) and opioid-related deaths remain a major public health concern in the United States. Both environmental and genetic factors influence risk for OUD. We previously identified Hnrnph1 as a quantitative trait gene underlying the stimulant, rewarding, and reinforcing properties of methamphetamine. Prior work shows that hnRNP H1, the RNA-binding protein encoded by Hnrnph1, post-transcriptionally regulates Oprm1 (mu opioid receptor gene)-the primary molecular target for the therapeutic and addictive properties of opioids. Because genetic variants can exert pleiotropic effects on behaviors induced by multiple drugs of abuse, in the current study, we tested the hypothesis that Hnrnph1 mutants would show reduced behavioral sensitivity to the mu opioid receptor agonist fentanyl. Hnrnph1 mutants showed reduced sensitivity to fentanyl-induced locomotor activity, along with a female-specific reduction in, and a male-specific induction of, locomotor sensitization following three, daily injections (0.2 mg/kg, i.p.). Hnrnph1 mutants also required a higher dose of fentanyl to exhibit opioid reward as measured via conditioned place preference (CPP). Male Hnrnph1 mutants showed reduced fentanyl reinforcement. Hnrnph1 mutants also showed reduced sucrose motivation, suggesting a reward deficit. No genotypic differences were observed in baseline thermal nociception, fentanyl-induced antinociception, physical or negative affective signs of opioid dependence, or in sensorimotor gating. In the context of our prior work, these findings suggest that Hnrnph1 dysfunction exerts a selective role in reducing the addiction liability to drugs of abuse (opioids and psychostimulants), which could provide new biological pathways to improve their therapeutic profiles.

Dissecting the Role of GABA Neurons in the VTA versus SNr in Opioid Reward.

Opioid reward has traditionally been thought to be mediated by GABA-induced disinhibition of dopamine (DA) neurons in the VTA. However, direct behavioral evidence supporting this hypothesis is still lacking. In this study, we found that the μ opioid receptor (MOR) gene, Oprm1, is highly expressed in GABA neurons, with ∼50% of GABA neurons in the substantia nigra pars reticulata (SNr), ∼30% in the VTA, and ∼70% in the tail of the VTA (also called the rostromedial tegmental nucleus) in male rats. No Oprm1 mRNA was detected in midbrain DA neurons. We then found that optogenetic inhibition of VTA DA neurons reduced intravenous heroin self-administration, whereas activation of these neurons produced robust optical intracranial self-stimulation in DAT-Cre mice, supporting an important role of DA neurons in opioid reward. Unexpectedly, pharmacological blockade of MORs in the SNr was more effective than in the VTA in reducing heroin reward. Optogenetic activation of VTA GABA neurons caused place aversion and inhibited cocaine, but not heroin, self-administration, whereas optogenetic activation of SNr GABA neurons caused a robust increase in heroin self-administration with an extinction pattern, suggesting a compensatory response in drug intake due to reduced heroin reward. In addition, activation of SNr GABA neurons attenuated heroin-primed, but not cue-induced, reinstatement of drug-seeking behavior, whereas inhibition of SNr GABA neurons produced optical intracranial self-stimulation and place preference. Together, these findings suggest that MORs on GABA neurons in the SNr play more important roles in opioid reward and relapse than MORs on VTA GABA neurons.SIGNIFICANCE STATEMENT Opioid reward has long been believed to be mediated by inhibition of GABA interneurons in the VTA that subsequently leads to disinhibition of DA neurons. In this study, we found that more μ opioid receptors (MORs) are expressed in GABA neurons in the neighboring SNr than in the VTA, and that pharmacological blockade of MORs in the SNr is more effective in reducing heroin reward than blockade of MORs in the VTA. Furthermore, optogenetic activation of VTA GABA neurons inhibited cocaine, but not heroin, self-administration, whereas activation of SNr GABA neurons inhibited heroin reward and relapse. These findings suggest that opioid reward is more likely mediated by stimulation of MORs in GABA afferents from other brain regions than in VTA GABA neurons.

Mu Opioids Induce Biased Signaling at the Full-Length Seven Transmembrane C-Terminal Splice Variants of the mu Opioid Receptor Gene, Oprm1.

The biased signaling has been extensively studied in the original mu opioid receptor (MOR-1), particularly through G protein and β-arrestin2 signaling pathways. The concept that the G protein pathway is often linked to the therapeutic effect of the drug, while the β-arrestin pathway is associated to the side effects has been proposed to develop biased analgesic compounds with limited side-effects associated with traditional opiates. The mu opioid receptor gene, OPRM1, undergoes extensive alternative pre-mRNA splicing, generating multiple splice variants or isoforms that are conserved from rodent to human. One type of the Oprm1 splice variants are the full-length 7 transmembrane (7TM) C-terminal splice variants, which have identical receptor structures including entire binding pocket, but contain a different intracellular C-terminal tail resulted from 3' alternative splicing. Increasing evidence suggest that these full-length 7TM C-terminal variants play important roles in mu opioid pharmacology, raising questions regarding biased signaling at these multiple C-terminal variants. In the present study, we investigated the effect of different C-terminal variants on mu agonist-induced G protein coupling, β-arrestin2 recruitment, and ultimately, signaling bias. We found that mu agonists produced marked differences in G protein activation and β-arrestin2 recruitment among various C-terminal variants, leading to biased signaling at various level. Particularly, MOR-1O, an exon 7-associated variant, showed greater β-arrestin2 bias for most mu agonists than MOR-1, an exon 4-associated variant. Biased signaling of G protein-coupled receptors has been defined by evidences that different agonists can produce divergent signaling transduction pathways through a single receptor. Our findings that a single mu agonist can induce differential signaling through multiple 7TM splice variants provide a new perspective on biased signaling at least for Oprm1, which perhaps is important for our understanding of the complex mu opioid actions in vivo where all the 7TM splice variants co-exist.

Alternative Splicing of Opioid Receptor Genes Shows a Conserved Pattern for 6TM Receptor Variants

The opioid receptor (OPR) family comprises the mu-, delta-, and kappa-opioid, and nociceptin receptors that belong to the superfamily of 7-transmembrane spanning G protein-coupled receptors (GPCRs). The mu-opioid receptor is the main target for clinically used opioid analgesics, and its biology has been extensively studied. The N-terminally truncated 6TM receptors isoform produced through alternative splicing of the OPRM1 gene displays unique signaling and analgesic properties, but it is unclear if other OPRs have the same ability. In this study, we have built a comprehensive map of alternative splicing events that produce 6TM receptor variants in all the OPRs and demonstrated their evolutionary conservation. We then obtained evidence for their translation through ribosomal footprint analysis. We discovered that N-terminally truncated 6TM GPCRs are rare in the human genome and OPRs are overrepresented in this group. Finally, we also observed a significant enrichment of 6TM GPCR genes among genes associated with pain, psychiatric disorders, and addiction. Understanding the biology of 6TM receptors and leveraging this knowledge for drug development should pave the way for novel therapies.

Recovery profiles from reward downshift are correlated with operant licking maintained by alcohol, but not with genetic variation in the mu opioid receptor

After ten 5-min sessions of access to 32% sucrose, a reward downshift (RD) to 2% sucrose induces a transient rejection of the reward. Animals were segregated according to the speed of recovery from RD into Fast-recovery and Slow-recovery subgroups. Animals were subsequently trained in an operant licking (OL) task in which licking at an empty tube provided 10 s of access to a second tube containing 66% alcohol. Licking on the first tube was subjected to a progressive ratio (PR) schedule with a step of 4 licks. Fast-recovery animals (both males and females) licked to a higher ratio than Slow-recovery animals. Animals were also exposed to a well-lit open field (OF) for 20 min. Fast- and Slow-recovery males and females exhibited equal levels of activity in the OF. Tissue samples from tails were assessed for two well-known allelic variations of the human opioid receptor gene, OPRM1, known to affect mu opioid sensitivity: The C17T and A118G single nucleotide polymorphisms. There was no evidence of a relationship between genotype and behavior, suggesting that these genetic mechanisms in humans do not account for the individual differences in recovery from RD and OL for alcohol in rats.