Functional modular networks identify the pivotal genes associated with morphine addiction and potential drug therapies.

BACKGROUND: Chronic morphine usage induces lasting molecular and microcellular adaptations in distinct brain areas, resulting in addiction-related behavioural abnormalities, drug-seeking, and relapse. Nonetheless, the mechanisms of action of the genes responsible for morphine addiction have not been exhaustively studied. METHODS: We obtained morphine addiction-related datasets from the Gene Expression Omnibus (GEO) database and screened for Differentially Expressed Genes (DEGs). Weighted Gene Co-expression Network Analysis (WGCNA) functional modularity constructs were analyzed for genes associated with clinical traits. Venn diagrams were filtered for intersecting common DEGs (CDEGs). Gene Ontology (GO) enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis for functional annotation. Protein-protein interaction network (PPI) and CytoHubba were used to screen for hub genes. Potential treatments for morphine addiction were figured out with the help of an online database. RESULTS: Sixty-five common differential genes linked to morphine addiction were identified, and functional enrichment analysis showed that they were primarily involved in ion channel activity, protein transport, the oxytocin signalling pathway, neuroactive ligand-receptor interactions, and other signalling pathways. Based on the PPI network, ten hub genes (CHN2, OLIG2, UGT8A, CACNB2, TIMP3, FKBP5, ZBTB16, TSC22D3, ISL1, and SLC2A1) were checked. In the data set GSE7762, all of the Area Under Curve (AUC) values for the hub gene Receiver Operating Characteristic (ROC) curves were greater than 0.8. We also used the DGIdb database to look for eight small-molecule drugs that might be useful for treating morphine addiction. CONCLUSIONS: The hub genes are crucial genes associated with morphine addiction in the mouse striatum. The oxytocin signalling pathway may play a vital role in developing morphine addiction.

Chronic morphine intoxication reduces binding of HuD to BDNF long 3'-UTR, while morphine withdrawal stimulates BDNF expression in the frontal cortex of male Wistar rats.

BACKGROUND: Brain-derived neurotrophic factor (BDNF) mediates opiate dependence phenomenon. In the brain of morphine dependent animals BDNF level is controlled transcriptionally, however, post-transcriptional mechanisms of BDNF regulation in this context remain unknown. Regulation of mRNA by binding of specific proteins to the 3'-untranslated region (3'-UTR) is one of such mechanisms. Among RNA-binding proteins neuronal Hu antigen D (HuD) is the best characterized positive regulator of BDNF, however its involvement in opiate dependence remains obscure. We suggested that HuD binding to the BDNF 3'-UTR may be linked to changes in BDNF expression induced by morphine. The aim of this study was to investigate potential association of HuD with BDNF 3'-UTR in relation to BDNF expression (Exon- and 3'-UTR-specific mRNA variants and protein level) in the frontal cortex and midbrain of male Wistar rats after chronic morphine intoxication and spontaneous withdrawal in dependent animals. RESULTS: After chronic morphine intoxication but not during morphine withdrawal HuD binding to the long BDNF 3'-UTR in the frontal cortex decreased as compared with the corresponding control group, however after intoxication BDNF expression did not change. The level of BDNF Exon I as well as mature BDNF polypeptide increased in the frontal cortex upon morphine withdrawal, while no changes in HuD binding could be detected. CONCLUSION: Thus, contrary to the assumption, HuD-BDNF 3'-UTR interaction and BDNF expression in the frontal cortex differentially change in a manner dependent on the context of morphine action.

Mice with high FGF21 serum levels had a reduced preference for morphine and an attenuated development of acute antinociceptive tolerance and physical dependence.

Because of increased opioid misuse, there is a need to identify new targets for minimizing opioid tolerance, and physical and psychological dependence. Previous studies showed that fibroblast growth factor 21 (FGF21) decreased alcohol and sweet preference in mice. In this study, FGF21-transgenic (FGF21-Tg) mice, expressing high FGF21 serum levels, and wildtype (WT) C57BL/6J littermates were treated with morphine and saline to determine if differences exist in their physiological and behavioral responses to opioids. FGF21-Tg mice displayed reduced preference for morphine in the conditioned place preference assay compared to WT littermates. Similarly, FGF21-Tg mice had an attenuation of the magnitude and rate of acute morphine antinociceptive tolerance development, and acute and chronic morphine physical dependence, but exhibited no change in chronic morphine antinociceptive tolerance. The ED50 values for morphine-induced antinociception in the 55 °C hot plate and the 55 °C warm-water tail withdrawal assays were similar in both strains of mice. Likewise, FGF21-Tg and WT littermates had comparable responses to morphine-induced respiratory depression. Overall, FGF21-Tg mice had a decrease in the development of acute analgesic tolerance, and the development of physical dependence, and morphine preference. FGF21 and its receptor have therapeutic potential for reducing opioid withdrawal symptoms and craving, and augmenting opioid therapeutics for acute pain patients to minimize tolerance development.

The role of circTmeff-1 in incubation of context-induced morphine craving.

A progressive increase in drug craving following drug exposure is an important trigger of relapse. CircularRNAs (CircRNAs), key regulators of gene expression, play an important role in neurological diseases. However, the role of circRNAs in drug craving is unclear. In the present study, we trained mice to morphine conditioned place preference (CPP) and collected the nucleus accumbens (NAc) sections on abstinence day 1 (AD1) and day 14 (AD14) for RNA-sequencing. CircTmeff-1, which was highly expressed in the NAc core, was associated with incubation of context-induced morphine craving. The gain- and loss- of function showed that circTmeff-1 was a positive regulator of incubation. Simultaneously, the expression of miR-541-5p and miR-6934-3p were down-regulated in the NAc core during the incubation period. The dual luciferase reporter, RNA pulldown, and fluorescence insitu hybridization assays confirmed that miR-541-5p and miR-6934-3p bind to circTmeff-1 selectively. Furthermore, bioinformatics and western blot analysis suggested that vesicle-associated membrane protein 1 (VAMP1) and neurofascin (NFASC), both overlapping targets of miR-541-5p and miR-6934-3p, were highly expressed during incubation. Lastly, AAV-induced down-regulation of circTmeff-1 decreased VAMP1 and NFASC expression and incubation of morphine craving. These findings suggested that circTmeff-1, a novel circRNA, promotes incubation of context-induced morphine craving by sponging miR-541/miR-6934 in the NAc core. Thus, circTmeff-1 represents a potential therapeutic target for context-induced opioid craving, following prolonged abstinence.

Peptide Mimetic of BDNF Loop 4 Blocks Behavioral Signs of Morphine Withdrawal Syndrome and Prevents the Increase in ΔFosB Level in the Striatum of Rats.

Activity of compound GSB-106, a low-molecular mimetic of loop 4 of the brain neurotrophic factor (BDNF), was studied in experimental morphine withdrawal syndrome simulated in outbred rats. Single and subchronic (5 intraperitoneal injections) administration of GSB-106 in a dose of 0.1 mg/kg significantly reduced the total index of morphine withdrawal syndrome by 55.2 and 45.6%, respectively. GSB-106 reduced the severity of some behavioral signs (piloerection, gnashing of teeth, wet-dog shaking, and runaway attempts), but had no effect on mechanical allodynia formed in the rats with dependence. Subchronic treatment with GSB-106 prevented the increase in the content of ΔFosB (product of early response gene) in the striatum induced by morphine withdrawal. The results confirmed the concept on the involvement of neurotrophins, specifically BDNF and its analogs, in the mechanisms associated with the formation of opiate dependence.

Circular RNA expression profiling in the nucleus accumbens: Effects of electroacupuncture treatment on morphine-induced conditioned place preference.

Electroacupuncture (EA) has been developed on the basis of traditional Chinese acupuncture. EA can suppress craving in opioid addicts and opioid-seeking responses in rodents. However, the molecular mechanism of EA on the rewarding properties of morphine and craving responses is not known. Here, we have applied a conditioned place preference paradigm in mice to measure morphine-induced rewarding effects along with EA treatment. Circular RNAs (circRNAs) can function as micro RNA (miRNA) sponges to effectively regulate gene expression levels. CircRNA profiling within the nucleus accumbens (NAc) was performed in EA-treated and sham-treated mice. Following RNAseq, data were analyzed by gene ontology (GO) and Kyoto Encyclopedia Genes and Genomes (KEGG) tools. We identified 112 significantly differentially expressed circRNAs, including 51 that were up-regulated and 61 that were down-regulated. Our bioinformatics analyses show that these differentially expressed circRNAs map into pathways that are mainly involved with renin secretion and the cGMP-PKG signaling. We further constructed a circRNA-miRNA network that predicts the potential roles of the differentially expressed circRNAs and the interaction of circRNAs with miRNAs. Our secondary sequencing and bioinformatics analysis in the NAc after EA treatment on morphine-induced CPP provides putative novel targets on molecular mechanisms involved in morphine reinforcement and possibly craving.

The Mechanisms Involved in Morphine Addiction: An Overview.

Opioid use disorder is classified as a chronic recurrent disease of the central nervous system (CNS) which leads to personality disorders, co-morbidities and premature death. It develops as a result of long-term administration of various abused substances, along with morphine. The pharmacological action of morphine is associated with its stimulation of opioid receptors. Opioid receptors are a group of G protein-coupled receptors and activation of these receptors by ligands induces significant molecular changes inside the cell, such as an inhibition of adenylate cyclase activity, activation of potassium channels and reductions of calcium conductance. Recent data indicate that other signalling pathways also may be involved in morphine activity. Among these are phospholipase C, mitogen-activated kinases (MAP kinases) or ß-arrestin. The present review focuses on major mechanisms which currently are considered as essential in morphine activity and dependence and may be important for further studies.

‬The expression of purinergic P2X4 and P2X7 receptors in selected mesolimbic structures during morphine withdrawal in rats.

Morphine is one of the most potent analgesics used in medicine and it's long-term use is associated with the risk of the state of dependence. The cessation of chronic morphine administration leads to withdrawal signs which are associated with neurotransmitter dysregulations within mesolimbic system. Adenosine 5'-triphosphate (ATP) and purinergic system play an important role in the activity of central nervous system (CNS). Purinergic receptors are widely distributed in neurons and glial cells throughout the CNS taking part in integration of functional activity between neurons, glial and vascular cells. In the present study the mRNA and protein expression of purinergic P2X4 and P2X7 receptors in selected mesolimbic structures (striatum, hippocampus and prefrontal cortex) during morphine withdrawal in rats was investigated by RT-PCR and Western Blot analysis. Two experimental models of morphine withdrawal were studied: single and repeated morphine withdrawal. We demonstrated that expression of P2X4 and P2X7 receptors was altered during morphine withdrawal period in rats. These alterations were varied in particular mesolimbic areas depending on the scheme of morphine administration. Our results extend the current knowledge on morphine withdrawal and for the first time high-light interactions between purinergic system and morphine withdrawal. It seems, the purinergic system may be a new, valuable tool in searching for a new strategy of management of opioid dependence.

Engineered endomorphin-2 gene: A novel therapy for improving morphine reinstatement in CPP model of rats by using deficient adenovirus as the vector.

Optimal therapeutics to deal with high relapse rates when discontinued is urgent for opioid dependence treatments. Endogenous endomorphin-2 (EM2) level in the central nervous system (CNS) down-regulates obviously after sustained morphine exposure, which suggested that to up-regulate the EM2 level could be a novel method for reinstatement. But the clinical applications of EM2 through conventional administration are limited owing to its short half-life. In our study, we engineered an EM2 gene to achieve the sustained release of EM-2 in CNS by utilizing a signal peptide of mouse growth factor for out-secreting EM2 and a deficient adenovirus as the vector. By intrathecally injecting engineering EM2 gene, a sustained increase of EM2 concentration in the cerebral spinal fluid (CSF) was observed along with a reduction of CPP scores. Also, the activation of astrocytes was suppressed in the hippocampus. In summary, this study provides evidence and reference for using intraspinal gene therapy with a combination of mouse growth factor and EM2 to treat morphine reinstatement.

Comparative Assessment of the Effectiveness of Noncompetitive NMDA Receptor Antagonists Amantadine and Hemantane in Morphine Withdrawal Syndrome Model.

Activities of noncompetitive NMDA receptor antagonists (aminoadamantane derivatives) were assessed in random-bred rats with modeled morphine withdrawal syndrome. A single intraperitoneal injection of hemantane (10 or 20 mg/kg) significantly and dose-dependently moderated some behavioral symptoms (teeth-chattering, ptosis, and vocalization) and reduced total score of morphine withdrawal syndrome. In morphine-abstinent rats, hemantane partially prevented the decrease in the thresholds of tactile sensitivity, but had no effect on locomotor activity and body weight loss. Under conditions of morphine withdrawal, intraperitoneal injection of amantadine (10 or 20 mg/kg) decreased motor activity and promoted body weight loss in parallel with the development of mechanical allodynia, but had no effect on the total withdrawal score. Comparison of aminoadamantane derivatives by behavioral and physiological parameters demonstrated the advantage of hemantane during morphine abstinence indicating the need of its study as a promising anti-addiction remedy.

The role of IRAS/Nischarin involved in the development of morphine tolerance and physical dependence.

Morphine is a potent opioid analgesic used to alleviate moderate or severe pain, but the development of drug tolerance and dependence limits its use in pain management. Our previous studies showed that the candidate protein for I1 imidazoline receptor, imidazoline receptor antisera-selected (IRAS)/Nischarin, interacts with µ opioid receptor (MOR) and modulates its trafficking. However, there is no report of the effect of IRAS on morphine tolerance and physical dependence. In the present study, we found that IRAS knockout (KO) mice showed exacerbated analgesic tolerance and physical dependence compared to wild-type (WT) mice by chronic morphine treatment. Chronic morphine treatment down-regulated the expression of MOR in spinal cord of IRAS KO mice, while had no significant effect on MOR expression in WT mice. We observed the compensatory increase of cAMP accumulation in spinal cord after morphine tolerance, and this change was more significant in KO mice than WT mice. Furthermore, KO mice showed more elevation in the phosphorylation of AMPA receptor GluR1-S845 than WT mice, while the total expression of GluR1 remained unchanged after morphine dependence. Altogether, these data suggest that IRAS may play an important role in the development of morphine tolerance and physical dependence in vivo through modulating MOR expression, as well as AMPA GluR1-S845 phosphorylation, which might be one of the mechanisms underlying the development of opiate addiction.

Dysregulation of Dopaminergic Regulatory Factors TH, Nurr1, and Pitx3 in the Ventral Tegmental Area Associated with Neuronal Injury Induced by Chronic Morphine Dependence.

The ventral tegmental area (VTA), a critical portion of the mesencephalic dopamine system, is thought to be involved in the development and maintenance of addiction. It has been proposed that the dopaminergic regulatory factors TH, Nurr1, and Pitx3 are crucial for determining the survival and maintenance of dopaminergic neurons. Thus, the present study investigated whether abnormalities in these dopaminergic regulatory factors in the VTA were associated with neuronal injury induced by chronic morphine dependence. Rat models with different durations of morphine dependence were established. Thionine staining was used to observe morphological changes in the VTA neurons. Immunohistochemistry and western blot were used to observe changes in the expression of the dopaminergic regulatory proteins TH, Nurr1, and Pitx3. Thionine staining revealed that prolonged morphine dependence resulted in dopaminergic neurons with edema, a lack of Nissl bodies, and pyknosis. Immunohistochemistry showed that the number of TH⁺, Nurr1⁺, and Pitx3⁺ cells, and the number of TH⁺ cells expressing Nurr1 or Pitx3, significantly decreased in the VTA after a long period of morphine dependence. Western blot results were consistent with the immunohistochemistry findings. Chronic morphine exposure resulted in abnormalities in dopaminergic regulatory factors and pathological changes in dopaminergic neurons in the VTA. These results suggest that dysregulation of dopaminergic regulatory factors in the VTA are associated with neuronal injury induced by chronic morphine dependence.

Expression Levels of the Tyrosine Hydroxylase Gene and Histone Modifications Around its Promoter in the Locus Coeruleus and Ventral Tegmental Area of Rats during Forced Abstinence from Morphine.

BACKGROUND: Epigenetic mechanisms such as histone modifications may be involved in the structural and behavioral changes associated with addiction. We studied whether morphine-induced changes in mRNA levels of the catecholamine biosynthesis enzyme, tyrosine hydroxylase (TH), are associated with histone modifications around the promoter of this gene in the locus coeruleus (LC) and ventral tegmental area (VTA) of rats. METHODS: Dependence was induced in rats by intraperitoneal injections of morphine for 11 days. The animals were killed 2 h (chronic morphine), 24 h and 7 days (spontaneous withdrawal) after the last injection of morphine. RESULTS: Analysis of our real-time quantitative reverse transcription PCR results by 1-way ANOVA showed significant upregulation (5.13 ± 0.39 folds) of LC levels of the TH transcript 24 h after the last injection of morphine to rats, when compared with 2 h and 7 days time points. Chronic morphine and morphine abstinence failed to cause any significant changes in the levels of TH mRNA in the VTA after cessation of morphine. Consistently, chromatin immunoprecipitation real-time quantitative PCR assays revealed that 24 h after the last injection of morphine, levels of H3 acetylation were significantly increased (4.12 ± 0.38 folds) at the promoter of the TH gene in the LC but not in the VTA. Our data also showed that histone H3 trimethylation failed to change around the TH gene promoter either in the VTA or in the LC after morphine abstinence. CONCLUSIONS: Results of the present study, for the first time, demonstrate the involvement of histone H3 acetylation in the regulation of TH gene expression in the LC of rats during forced abstinence from morphine.

Selection and Validation of Reference Genes for RT-PCR Expression Analysis of Candidate Genes Involved in Morphine-Induced Conditioned Place Preference Mice.

The expression of reference genes should be constitutively stable under the experimental conditions, so determining stable reference genes is critical for obtaining reliable results in gene expression studies. Morphine addiction persistently influences neurotransmitters and signal transduction systems, which may negatively alter behavioral responses at the cellular levels and interfere the expression of reference genes. In order to research morphine dependence, animal models are commonly used in physiology, pathology, and therapeutics field since human trials have many limitations. Therefore, it is necessary to select stable reference genes in standardized animal model. The objective of this study is to find out a set of optimal reference genes to standardize the gene expression of morphine-induced conditioned place preference (CPP) mice. During the process, eight reference genes were chosen. Then, the stability of their expression in two different brain tissues (Caudate Putamen and Hippocampus) was tested in two developmental stages (puberty and adult) under two treatments (physiological saline as control and morphine). Based on two algorithm-based methods (geNorm and NormFinder), which can rank and assess the stability of expression of eight reference genes, thereby quantifying the transcriptional levels of these genes by high sensitive, specific, and accurate real-time quantitative reverse transcription PCR (RT-qPCR) assays.

Adult rat morphine exposure changes morphine preference, anxiety, and the brain expression of dopamine receptors in male offspring.

Addiction to drugs, including opioids is the result of an interplay between environmental and genetic factors. It has been shown that the progeny of addict people is at higher risk for drug addiction. However, the mechanisms of such trans-generational effects of drugs are not so clear. Here we have evaluated the effects of parental morphine consumption on anxiety, morphine preference, and mRNA expression of dopamine receptors in F1 and F2 male offspring. Morphine was chronically administered to adult male and female Wistar rats followed by 14-day abstinence before mating. Morphine preference and anxiety-like behavior in the offspring were measured by two-bottle-choice paradigm and elevated-plus maze, respectively. Real-time PCR was used to measure the mRNA expression level of dopamine receptors in the striatum, nucleus accumbens, prefrontal cortex, and hippocampus of F1 animals. The results indicated that F1 but not the F2 male progeny of morphine-exposed parents had a greater preference for morphine, and more anxiety-like behavior compared to the offspring of saline-treated parents. In F1 male progeny of morphine-treated parents, D1 and D5 dopamine receptors were significantly increased in the prefrontal cortex and nucleus accumbens. D5 and D2 receptors were decreased in the hippocampus. D4 dopamine receptor was augmented in striatum and hippocampus and decreased in the prefrontal cortex. Adulthood exposure to chronic morphine in male and female rats before conception leads to higher morphine preference and increased anxiety in F1 but not F2 male progeny. Alterations of dopamine receptor expression in the reward system may be one mechanism responsible for observed changes in F1 offspring.

Modulation of miR-139-5p on chronic morphine-induced, naloxone-precipitated cAMP overshoot in vitro.

Chronic exposure to morphine can produce tolerance, dependence and addiction, but the underlying neurobiological basis is still incompletely understood. c-Jun, as an important component of the activator protein-1 transcription factor, is supposed to take part in regulating gene expression in AC/cAMP/PKA signaling. MicroRNA (miRNA) has emerged as a critical regulator of neuronal functions. Although a number of miRNAs have been reported to regulate the µ-opioid receptor expression, there has been no report about miRNAs to regulate chronic morphine-induced, naloxone-precipitated cAMP overshoot. Our results showed that chronic morphine pretreatment induced naloxone-precipitated cAMP overshoot in concentration- and time-dependent manners in HEK 293/µ cells. Chronic morphine pretreatment alone elevated both c-Jun protein and miR-139-5p expression levels, while dramatically artificial elevation of miR-139-5p inhibited c-Jun at the translational level. Furthermore, dramatically artificial upregulation of intracellular miR-139-5p limited chronic morphine-induced, naloxone-precipitated cAMP overshoot. These findings suggested that miR-139-5p was involved in regulating chronic morphine-induced, naloxone-precipitated cAMP overshoot in a negative feedback manner through its target c-Jun, which extends our understanding of neurobiological mechanisms underlying morphine dependence and addiction.

Role of the guanine nucleotide binding protein, Gαo, in the development of morphine tolerance and dependence.

RATIONALE: The use of morphine and other opioids for chronic pain is limited by the development of analgesic tolerance and physical dependence. Morphine produces its effects by activating the µ opioid receptor, which couples to Gαi/o-containing heterotrimeric G proteins. Evidence suggests that the antinociceptive effects of morphine are mediated by Gαo. However, the role of Gαo in the development of morphine tolerance and dependence is unknown. OBJECTIVE: The objective of the study is to evaluate the contribution of Gαo to the development of morphine tolerance and dependence in mice. METHODS: 129S6 mice lacking one copy of the Gαo gene (Gαo +/-) were administered morphine acutely or chronically. Mice were examined for tolerance to the antinociceptive action of morphine using the 52 °C hot plate as the nociceptive stimulus and for dependence by evaluating the severity of naltrexone-precipitated withdrawal. Wild-type littermates of the Gαo +/- mice were used as controls. Changes in µ receptor number and function were determined in midbrain and hindbrain homogenates using radioligand binding and µ agonist-stimulated [35S]GTPγS binding, respectively. RESULTS: Following either acute or chronic morphine treatment, all mice developed antinociceptive tolerance and physical dependence, regardless of genotype. With chronic morphine treatment, Gαo +/- mice developed tolerance faster and displayed more severe naltrexone-precipitated withdrawal in some behaviors than did wild-type littermates. Morphine tolerance was not associated with changes in µ receptor number or function in brain homogenates from either wild-type or Gαo +/- mice. CONCLUSIONS: These data suggest that the guanine nucleotide binding protein Gαo offers some protection against the development of morphine tolerance and dependence.

Reduced dosing and liability in methadone maintenance treatment by targeting oestrogen signal for morphine addiction.

Methadone maintenance treatment (MMT) is the major tapering therapy for morphine addictive patients. There have gender differences reported in response to MMT. This study discovered that the estrogen-response element single nucleotide polymorphism (ERE-SNP; rs16974799, C/T) of cytochrome 2B6 gene (cyp2b6; methadone catabolic enzyme) responded differently to MMT dosing. Oestradiol was associated with high MMT dosing, high enantiomer (R- or S-) of 2-ethylidene-1,5-dimethyl-3,3-dipheny-pyrrolidine (EDDP; methadone metabolite) to methadone ratio and increased drug-seeking behaviour, implicating oestradiol-CYP-EDDP/methadone axis decreasing MMT efficacy. In mouse model, oestrogen mitigates methadone antinociceptive response, facilitates methadone catabolism and up-regulates methadone-associated metabolizing enzymes. Oestrogen also ablates chronic methadone administration-induced rewarding response. Mechanism dissection revealed the CC genotype of CYP2B6-ERE-SNP exerts higher ERE sequence alignment score, higher estrogenic response as compared to TT genotype. At last, preclinical study via targeting estrogen signal that tamoxifen (TMX; selective estrogen receptor modulator, SERM) could facilitate the tolerance phase rewarding response of methadone. Strikingly, TMX also reduces tapering/abstinence phases methadone liability in mice. In conclusion, this study demonstrates altering methadone metabolism through targeting estrogen signals might be able to free morphine addictive patients from the addiction of opioid replacement therapy. Therefore, the add-on therapy clinical trial introducing SERM in MMT regimen is suggested.

Alternatively spliced mu opioid receptor C termini impact the diverse actions of morphine.

Extensive 3' alternative splicing of the mu opioid receptor gene OPRM1 creates multiple C-terminal splice variants. However, their behavioral relevance remains unknown. The present study generated 3 mutant mouse models with truncated C termini in 2 different mouse strains, C57BL/6J (B6) and 129/SvEv (129). One mouse truncated all C termini downstream of Oprm1 exon 3 (mE3M mice), while the other two selectively truncated C-terminal tails encoded by either exon 4 (mE4M mice) or exon 7 (mE7M mice). Studies of these mice revealed divergent roles for the C termini in morphine-induced behaviors, highlighting the importance of C-terminal variants in complex morphine actions. In mE7M-B6 mice, the exon 7-associated truncation diminished morphine tolerance and reward without altering physical dependence, whereas the exon 4-associated truncation in mE4M-B6 mice facilitated morphine tolerance and reduced morphine dependence without affecting morphine reward. mE7M-B6 mutant mice lost morphine-induced receptor desensitization in the brain stem and hypothalamus, consistent with exon 7 involvement in morphine tolerance. In cell-based studies, exon 7-associated variants shifted the bias of several mu opioids toward ß-arrestin 2 over G protein activation compared with the exon 4-associated variant, suggesting an interaction of exon 7-associated C-terminal tails with ß-arrestin 2 in morphine-induced desensitization and tolerance. Together, the differential effects of C-terminal truncation illustrate the pharmacological importance of OPRM1 3' alternative splicing.

Methylation of glucocorticoid receptor gene promoter modulates morphine dependence and accompanied hypothalamus-pituitary-adrenal axis dysfunction.

Previous studies demonstrated that dysfunction of the hypothalamus-pituitary-adrenal (HPA) axis played an important role in morphine dependence. Nonetheless, the molecular mechanism underlying morphine-induced HPA axis dysfunction and morphine dependence remains unclear. In the current study, 5'-aza-2'-deoxycytidine (5-aza), an inhibitor of DNA methyltransferases (DNMTs), was used to examine the effects of glucocorticoid receptor (GR) promoter 17 methylation on chronic morphine-induced HPA axis dysfunction and behavioral changes in rats and the underlying mechanism. Our results showed that chronic but not acute morphine downregulated the expression of nuclear GR protein and GR exon 17 variant mRNA, and upregulated the methylation of GR 17 exon promoter in the hippocampus of rats. Meanwhile, 5-aza per se had no effect on observed molecular and behavior change. In contrast, pretreatment of 5-aza into rat hippocampus reversed chronic morphine-induced hypermethylation of GR 17 promoter and decrease in GR expression. Moreover, pretreatment of 5-aza attenuated chronic morphine-enhanced HPA axis reactivity and the naloxone-precipitated somatic signs in morphine-dependent rats. Our results suggest that chronic morphine induced hypermethylation of GR 17 promoter, which then downregulated the expression of hippocampal GR, and was thus involved in chronic morphine-induced dysfunction of the HPA axis and the modulation of morphine dependence. Moreover, chronic morphine-induced hypermethylation of GR 17 promoter may be at least partially due to the increase in hippocampal DNMT 1 expression and its binding at GR 17 promoter in the rat hippocampus. © 2016 Wiley Periodicals, Inc.