Impact of OPRM1 (Mu-opioid Receptor Gene) A112G Polymorphism on Dual Oxycodone and Cocaine Self-administration Behavior in a Mouse Model.

The use of mu-opioid receptor (MOP-r) agonists such as oxycodone together with cocaine is prevalent, and deaths attributed to using these combinations have increased. RATIONALE: It is unknown if functional single nucleotide polymorphisms (SNPs), such as the OPRM1 (MOP-r gene) SNP A118G, can predispose individuals to more dual opioid and psychostimulant intake. The dual self-administration (SA) of MOP-r agonists and cocaine has not been thoroughly examined, especially with regard to neurobiological changes. OBJECTIVES: We examined oxycodone SA and subsequent dual oxycodone and cocaine SA in male and female A112G (A/G and G/G, heterozygote and homozygote, respectively) mice, models of human A118G carriers, versus wild-type (A/A) mice. METHODS: Adult male and female A/G, G/G and A/A mice self-administered oxycodone (0.25 mg/kg/infusion, 4hr/session, FR 1.) for 10 consecutive days (sessions 1-10). Mice then self-administered cocaine (2 hr) following oxycodone SA (4 hr, as above) in each session for a further 10 consecutive days (sessions 11-20). Message RNA transcripts of 24 reward-related genes were examined in the dorsal striatum. RESULTS: Male and female A/G and G/G mice had greater oxycodone SA than A/A mice did in the initial 10 days and in the last 10 sessions. Further, A/G and G/G mice showed greater cocaine intake than A/A mice. Dorsal striatal mRNA levels of Pdyn, Fkbp5, Oprk1, and Oprm1 were altered following oxycodone and cocaine SA. CONCLUSIONS: These studies demonstrated that this functional genetic variation in Oprm1 affected dual opioid and cocaine SA and altered specific gene expression in the striatum.

Multi-trait genome-wide association study of opioid addiction: OPRM1 and beyond.

Opioid addiction (OA) is moderately heritable, yet only rs1799971, the A118G variant in OPRM1, has been identified as a genome-wide significant association with OA and independently replicated. We applied genomic structural equation modeling to conduct a GWAS of the new Genetics of Opioid Addiction Consortium (GENOA) data together with published studies (Psychiatric Genomics Consortium, Million Veteran Program, and Partners Health), comprising 23,367 cases and effective sample size of 88,114 individuals of European ancestry. Genetic correlations among the various OA phenotypes were uniformly high (rg > 0.9). We observed the strongest evidence to date for OPRM1: lead SNP rs9478500 (p = 2.56 × 10-9). Gene-based analyses identified novel genome-wide significant associations with PPP6C and FURIN. Variants within these loci appear to be pleiotropic for addiction and related traits.

Analyses of polymorphisms of intron 2 of OPRK1 (kappa-opioid receptor gene) in association with opioid and cocaine dependence diagnoses in an African-American population.

RATIONALE: The dynorphin/kappa-opioid receptor (KOR) system (encoded by PDYN and OPRK1 genes respectively) is highly regulated by repeated exposure to drugs of abuse, including mu-opioid agonists and cocaine. These changes in the dynorphin/KOR system can then influence the rewarding effects of these drugs of abuse. Activation of the dynorphin/KOR system is also thought to have a role in the pro-addictive effects of stress. Recent in vitro assays showed that the OPRK1 intron 2 may function as a genomic enhancer in the regulation KOR expression, and contains a glucocorticiod-responsive sequence site. We hypothesize that SNPs in intron 2 of OPRK1 are associated with categorical opioid or cocaine dependence diagnoses, as well as with dimensional aspects of drug use (i.e., magnitude of drug exposure). METHODS: This study includes 577 subjects ≥ 18 years old, with African ancestry (AA) from the USA. They were divided into three groups: 152 control subjects, 142 persons with lifetime opioid dependence diagnosis (OD), and 283 subjects with lifetime cocaine dependence diagnosis (CD). Five SNPs (rs16918909, rs7016778, rs997917, rs6473797, rs10111937) that span 10 Kb nucleotides in intron 2 of OPRK1 were used for the association analyses. Genotyping was performed with the Smokescreen® array or sequencing of PCR-amplified DNA fragments. Association analyses for OD and CD diagnoses and the OPRK1 intron 2 alleles were carried out with Fisher's exact test. The Kreek-McHugh-Schluger-Kellogg (KMSK) scales were used for dimensional measure of maximum exposure to specific drugs, using Mann-Whitney tests. RESULTS: Two SNPs, rs997917 and rs10111937 showed point-wise significant allelic association (p < 0.05) with CD diagnosis, and rs10111937 showed a point-wise significance in association with OD. None of these single SNP associations with categorical diagnoses were significant after correction for multiple testing (pcorr > 0.05). However, significant associations of several genotype patterns (diplotypes) were found with cocaine dependence, but none for opioid dependence. The most significant genotype pattern with cocaine dependence diagnosis occurred for rs6473797 and rs10111937 (pcorr = 0.036, odds ratio = 1.92, FDR < 0.05), and survived correction for multiple testing. Dimensional analyses with KMSK scores show that persons with either rs997917 or rs10111937 variants had greater exposure to cocaine, compared to those with prototype allele (Mann-Whitney tests, point-wise). CONCLUSIONS: This study provides additional support of potential importance of regulatory regions of intron 2 of the OPRK1 gene in development of cocaine and opioid dependence diagnoses, in a population with African-American ancestry.

Genotype Pattern Mining for Pairs of Interacting Variants Underlying Digenic Traits.

Some genetic diseases ("digenic traits") are due to the interaction between two DNA variants, which presumably reflects biochemical interactions. For example, certain forms of Retinitis Pigmentosa, a type of blindness, occur in the presence of two mutant variants, one each in the ROM1 and RDS genes, while the occurrence of only one such variant results in a normal phenotype. Detecting variant pairs underlying digenic traits by standard genetic methods is difficult and is downright impossible when individual variants alone have minimal effects. Frequent pattern mining (FPM) methods are known to detect patterns of items. We make use of FPM approaches to find pairs of genotypes (from different variants) that can discriminate between cases and controls. Our method is based on genotype patterns of length two, and permutation testing allows assigning p-values to genotype patterns, where the null hypothesis refers to equal pattern frequencies in cases and controls. We compare different interaction search approaches and their properties on the basis of published datasets. Our implementation of FPM to case-control studies is freely available.

OPRD1 SNPs associated with opioid addiction are cis-eQTLs for the phosphatase and actin regulator 4 gene, PHACTR4, a mediator of cytoskeletal dynamics.

Several OPRD1 intronic variants were associated with opioid addiction (OD) in a population-specific manner. This follow-up study aims to further characterize the OPRD1 haplotype pattern of the risk variants in different populations and apply in silico analysis to identify potential causal variants. A population-specific haplotype pattern was revealed based on six OPRD1 eQTL SNPs and five common haplotypes were identified in a sample of European ancestry (CEU). A European-specific haplotype ('Hap 3') that includes SNPs previously associated with OD and is tagged by SNP rs2236861 is more common in subjects with OD. It is quite common (10%) in CEU but is absent in the African sample (YRI) and extends upstream of OPRD1. SNP rs2236857 is most probably a non-causal variant in LD with the causal SNP/s in a population-specific manner. The study provides an explanation for the lack of association in African Americans, despite its high frequency in this population. OD samples homozygous for 'Hap 3' were reanalyzed using a denser coverage of the region and revealed at least 25 potentially regulatory SNPs in high LD. Notably, GTEx data indicate that some of the SNPs are eQTLs for the upstream phosphatase and actin regulator 4 (PHACTR4), in the cortex, and others are eQTLs for OPRD1 and the upstream lncRNA ENSG00000270605, in the cerebellum. The study highlights the limitation of single SNP analysis and the sensitivity of association studies of OPRD1 to a genetic background. It proposes a long-range functional connection between OPRD1 and PHACTR4. PHACTR4, a mediator of cytoskeletal dynamics, may contribute to drug addiction by modulating synaptic plasticity.

Association of Serotonin Transporter (SERT) Polymorphisms with Opioid Dependence and Dimensional Aspects of Cocaine Use in a Caucasian Cohort of Opioid Users.

INTRODUCTION: A functional tandem repeat polymorphism in the promoter of the serotonin transporter (SERT) gene (SLC6A4) has been studied for association to neuropsychiatric conditions, including substance use disorders. Short (S) forms of this repeat result in reduced transcription, and presumably greater synaptic levels of serotonin, which are involved in opioid and cocaine-induced reward. Dual exposure to heroin and cocaine is a common pattern of poly-drug use and is associated with considerable morbidity. We hypothesize that SLC6A4 variants are associated with cocaine exposure in subjects with an opioid dependence diagnosis (OD), and also in non-dependent opioid users (NOD). Other single nucleotide polymorphisms (SNPs) of SLC6A4 may also be likewise associated. MATERIALS AND METHODS: This study determined whether variants of the SLC6A4 promoter repeats and two intronic SNPs, rs16965628 and rs2066713, are associated with categorical diagnoses of opioid dependence (DSM-IV criteria) and with dimensional aspects of cocaine use, in a Caucasian cohort (n=591). Three groups of subjects were examined: (1) 276 subjects with opioid dependence diagnosis (OD); (2) 163 subjects who had used opioids for non-medical reasons but never had an opioid dependence diagnosis (NOD); (3) 152 healthy controls (HC). RESULTS: Aside from high exposure to heroin in the OD group, relatively high exposure to cocaine was detected in both OD and NOD groups. The SERT repeat genotype (classified as "long-long" [LL] versus "short-long" plus "short-short" [SL+SS]) was not associated with categorical opioid dependence diagnoses. A nominally significant association was identified with the [SL+SS] genotype of SLC6A4 and cocaine KMSK scores ≥"cutpoint" for a cocaine dependence diagnosis (p=0.026). The [SL+SS] genotype was associated with more rapid cocaine escalation than the LL genotype. No significant associations of rs16965628 and rs2066713 SNPs were found overall. CONCLUSION: The functional SERT promoter tandem repeat genotype may be associated to heavy cocaine exposure and more rapid escalation of cocaine use, in persons with and without opioid dependence diagnosis.

Polymorphisms in Stress-Related Genes Are Associated with Reduced Cocaine Abuse and Longer Retention in Methadone Maintenance Treatment for Opioid Use Disorder.

BACKGROUND: As CRH-binding protein (CRHBP) SNP rs1500 was associated with reduced cocaine abuse after 1 year in methadone maintenance treatment (MMT) for heroin addiction, we evaluated the association of additional 28 selected SNPs, in 17 stress-related genes, with MMT outcome. METHODS: The distribution of genotypes of each SNP by cocaine abuse after 1 year in MMT was assessed under the dominant and recessive models using χ2. Cumulative retention (up to 26.5 years) was studied using Kaplan-Meier analyses. Logistic regression and Cox model were used for multivariate analyses. RESULTS: Of a nonselective sample of 404 patients, 25 patients with <50% Europeans/Middle Eastern ancestry were excluded. Of the remaining 379 patients, 330 (87.1%) stayed at least 1 year in treatment. Four SNPs were associated with cocaine abuse after 1 year in MMT. A lower proportion of cocaine abusers was found in the groups of subjects with the following genotypes: arginine vasopressin (AVP) SNP rs2282018 CC, CRHBP rs7728378 TT, galanin rs3136541 TT/TC, and neuropeptide Y receptor Y1 (NPY1R) rs4518200 AA. The following independent variables were associated with lack of cocaine in urine after 1 year (multivariate analyses): CRHBP rs7728378 TT, NPY1R rs4518200 AA, no cocaine in urine on admission, as well as opiate and benzodiazepine use after 1 year in MMT. Cumulative retention (n = 379) was longer in carriers of AVP rs2282018 CC (13.7 years, 95% CI 11.1-16.2) versus TT/TC genotypes (10.5, 95% CI 9.4-11.5) (p = 0.0230) Conclusions: The study suggests that a reduction in cocaine abuse and longer retention among MMT patients is mediated in part by variants in stress-related genes and is a step toward precision medicine.

Variants of opioid genes and response to treatment of opioid use disorder with buprenorphine-naloxone versus extended-release naltrexone in Caucasians.

Background: Sublingual buprenorphine-naloxone (BUP-NX), an FDA-approved treatment for opioid use disorder (OUD), combines buprenorphine (a partial mu/kappa agonist) with naloxone (a mu/ kappa antagonist). Extended-release injection naltrexone (XR-NTX; a mu receptor antagonist and kappa receptor partial agonist) is also an FDA-approved treatment for OUD. However, while some patients respond well to these medications, many others leave treatment and relapse. Objectives: Determine whether gene variants in the opioid gene system are associated with better or worse treatment response. Methods: In a 24-week, multisite, randomized, comparative effectiveness trial of daily, sublingual self-administration of BUP-NX versus monthly injection of XR-NTX conducted in the National Drug Abuse Clinical Trials Network, DNA was collected and four opioid gene variants were evaluated: (1) mu opioid receptor 118A>G; (2) 68-bp repeat in prodynorphin; (3) prodynorphin SNP rs910080; and (4) kappa opioid receptor SNP rs6473797. In non-Hispanic Caucasians (N = 334), two outcomes measures were assessed: received first dose (yes/no) and received last dose (yes/no). Separate logistic regressions were used to model each outcome measure as a function of treatment (XR-NTX vs BUP-NX), each gene variant, and their interaction. Results: There were no significant main effects of gene variant on receiving first dose or last dose. There were also no significant gene variant by treatment interactions. Conclusions: The outcome of treatment of OUD with medications is likely a complex function of multiple factors, including environmental, psychosocial, and possibly genetic, such that major effects of genetic variants may be unlikely.

Further evidence for the association of GAL, GALR1 and NPY1R variants with opioid dependence.

Aim: Heroin addiction is a chronic, relapsing disease that has genetic and environmental, including drug-induced, contributions. Stress influences the development of addictions. This study was conducted to determine if variants in stress-related genes are associated with opioid dependence (OD). Patients & methods: One hundred and twenty variants in 26 genes were analyzed in 597 Dutch subjects. Patients included 281 OD in methadone maintenance with or without heroin-assisted treatment and 316 controls. Results: Twelve SNPs in seven genes showed a nominally significant association with OD. Experiment-wise significant associations (p < 0.05) were found for three SNP pairs, through an interaction effect: NPY1R/GAL rs4691910/rs1893679, NPY1R/GAL rs4691910/rs3136541 and GALR1/GAL rs9807208/rs3136541. Conclusion: This study lends more evidence to previous reports of association of stress-related variants with heroin dependence.

A 3' UTR SNP rs885863, a cis-eQTL for the circadian gene VIPR2 and lincRNA 689, is associated with opioid addiction.

There is a reciprocal relationship between the circadian and the reward systems. Polymorphisms in several circadian rhythm-related (clock) genes were associated with drug addiction. This study aims to search for associations between 895 variants in 39 circadian rhythm-related genes and opioid addiction (OUD). Genotyping was performed with the Smokescreen® array. Ancestry was verified by principal/MDS component analysis and the sample was limited to European Americans (EA) (OUD; n = 435, controls; n = 138). Nominally significant associations (p < 0.01) were detected for several variants in genes encoding vasoactive intestinal peptide receptor 2 (VIPR2), period circadian regulator 2 (PER2), casein kinase 1 epsilon (CSNK1E), and activator of transcription and developmental regulator (AUTS2), but no signal survived correction for multiple testing. There was intriguing association signal for the untranslated region (3' UTR) variant rs885863 in VIPR2, (p = .0065; OR = 0.51; 95% CI 0.31-0.51). The result was corroborated in an independent EA OUD sample (n = 398, p = 0.0036; for the combined samples). Notably, this SNP is an expression quantitative trait locus (cis-eQTL) for VIPR2 and a long intergenic non-coding RNA, lincRNA 689, in a tissue-specific manner, based on the Genotype-Tissue Expression (GTEx) project. Vasoactive intestinal peptide (VIP) is an important peptide of light-activated suprachiasmatic nucleus cells. It regulates diverse physiological processes including circadian rhythms, learning and memory, and stress response. This is the first report of an association of a VIPR2 variant and OUD. Additionally, analysis of combinations of single nucleotide polymorphisms (SNPs) genotypes revealed an association of PER2 SNP rs80136044, and SNP rs4128839, located 41.6 kb downstream of neuropeptide Y receptor type 1 gene, NPY1R (p = 3.4 × 10-6, OR = 11.4, 95% CI 2.7-48.2). The study provides preliminary insight into the relationship between genetic variants in circadian rhythm genes and long non-coding RNA (lncRNAs) in their vicinity, and opioid addiction.

Sex Differences in Neuroplasticity- and Stress-Related Gene Expression and Protein Levels in the Rat Hippocampus Following Oxycodone Conditioned Place Preference.

Prescription opioid abuse is a serious public health issue. Recently, we showed that female and male Sprague-Dawley rats acquire conditioned place preference (CPP) to the mu opioid receptor agonist oxycodone. Anatomical analysis of the hippocampus from these rats unveiled sex differences in the opioid system in a way that would support excitation and opiate associative learning processes especially in females. In this study, we examined the expression and protein densities of opioid, plasticity, stress and related kinase and signaling molecules in the hippocampus of female and male rats following oxycodone CPP. Oxycodone CPP females have: a) increases in ARC (activity regulated cytoskeletal-associated protein)-immunoreactivity (ir) in CA3 pyramidal cells; b) decreases in Npy (neuropeptide Y) gene expression in the medial hippocampus but higher numbers of NPY-containing hilar interneurons compared to males; c) increases in Crhr2 (corticotropin releasing factor receptor 2) expression in CA2/3; d) increases in Akt1 (AKT serine/threonine kinase 1) expression in medial hippocampus; and e) decreases in phosphorylated MAPK (mitogen activated protein kinase)-ir in CA1 and dentate gyrus. Oxycodone CPP males have: a) increases in Bdnf (brain derived-neurotrophic factor) expression, which is known to be produced in granule cells, relative to females; b) elevated Mapk1 expression and pMAPK-ir in the dentate hilus which harbors newly generated granule cells; and c) increases in CRHR1-ir in CA3 pyramidal cell soma. These sex-specific changes in plasticity, stress and kinase markers in hippocampal circuitry parallel previously observed sex differences in the opioid system after oxycodone CPP.

Association of variants of prodynorphin promoter 68-bp repeats in caucasians with opioid dependence diagnosis: Effect on age trajectory of heroin use.

The dynorphin/kappa opioid receptor (Dyn/KOR) system is involved in reward processing and dysphoria/anhedonia. Exposure to mu-opioid receptor agonists such as heroin increases expression of the prodynorphin gene (PDYN) in the brain. In this study in a Caucasian cohort, we examined the association of the functional PDYN 68-bp repeat polymorphism with opioid use disorders. In this case-control study, 554 subjects with Caucasian ancestry (142 healthy controls, 153 opioid-exposed, but never opioid dependent, NOD, and 259 with an opioid dependence diagnosis, OD) were examined for association of the PDYN 68-bp repeats with the diagnosis of opioid dependence (DSM-IV criteria), with a dimensional measure of heroin exposure (KMSK scale), and age trajectory parameters of heroin use (age of heroin first use, and age of onset of heaviest use). The PDYN 68-bp repeat genotype (classified as: "short-short" [SS], "long-long" [LL], and "short-long" [SL], based on the number of repeats) was not associated with categorical opioid dependence diagnoses. However, the LL genotype was associated with later age of first heroin use than the SS + SL genotype (19 versus 18 years; p < 0.01). This was also confirmed by a significant positive correlation between the number of repeats and the age of first use of heroin, in volunteers with OD (Spearman r = 0.16; p = 0.01). This suggests that the functional PDYN 68-bp repeat genotype is associated with the age of first use of heroin in Caucasians diagnosed with opioid dependence.

VMAT2 gene (SLC18A2) variants associated with a greater risk for developing opioid dependence.

Aim: To determine if selected serotonergic and noradrenergic gene variants are associated with heroin addiction. Subjects & methods: A total of 126 variants in 19 genes in subjects with Dutch European ancestry from The Netherlands. Subjects included 281 opioid-dependent volunteers in methadone maintenance or heroin-assisted treatment, 163 opioid-exposed but not opioid-dependent volunteers who have been using illicit opioids but never became opioid-dependent and 153 healthy controls. Results: Nominal associations were indicated for 20 variants in six genes including an experiment-wise significant association from the combined effect of three SLC18A2 SNPs (rs363332, rs363334 and rs363338) with heroin dependence (pfinal = 0.047). Conclusion: Further studies are warranted to confirm and elucidate the role of these variants in the vulnerability to opioid addiction.

Genetic Variant in the CRH-binding Protein Gene (CRHBP) is Associated With Cessation of Cocaine Use in Methadone Maintenance Patients With Opioid Addiction.

OBJECTIVES: We have previously shown associations between 4 genetic variants in opioid and stress-related genes (OPRM1, NPYR1/NPYR5, NR3C1, and CRHBP) and prolonged abstinence from heroin without methadone maintenance treatment (MMT). We currently assessed the associations between these variants and MMT patients' characteristics. METHODS: A non-selective group of 351 patients who stayed at least 1 year in their first admission to MMT were genotyped and their characteristics and substance in urine on admission and after 1 year were studied. RESULTS: The proportions of patients with both cocaine and benzodiazepine abuse were reduced significantly after 1 year in MMT; however, cocaine abuse cessation was significantly associated with the non-carriers of the CRHBP (corticotrophin releasing hormone binding protein) SNP rs1500 minor C allele (GG genotype) (P = 0.0009, PBonferroni = 0.0221). More carriers of the 2 C alleles (CC genotype) than carriers of the GC and GG genotypes abused cocaine on admission (32.3% vs 19.7%, respectively, P = 0.0414, recessive model), and more of the C allele carriers (GC and CC genotypes) than non-carriers (GG genotype) abused cocaine after 1 year in MMT (25.7% vs 15.8%, respectively, P = 0.0334, dominant model). Abusers of benzodiazepine were more prevalent among carriers of the C allele compared with non-carriers on admission (60.6% vs 45.9%, respectively, P = 0.0080, dominant model), as well as after 1 year in MMT (50.9% vs 39.1%, respectively, P = 0.0362). CONCLUSIONS: Reduction in cocaine abuse among MMT patients may be mediated by a genetic effect in a stress-related gene (CRHBP SNP rs1500 minor C allele). Evaluations of larger samples, additional SNPs, and different populations are needed to support these findings.

Chronic Oxycodone Self-administration Altered Reward-related Genes in the Ventral and Dorsal Striatum of C57BL/6J Mice: An RNA-seq Analysis.

Prescription opioid abuse, for example of oxycodone, is a pressing public health issue. This study focuses on how chronic oxycodone self-administration (SA) affects the reward pathways in the mouse brain. In this study, we tested the hypothesis that the expression of reward-related genes in the ventral and dorsal striatum, areas involved in different aspects of opioid addiction models, was altered within 1 h after chronic oxycodone SA, using transcriptome-wide sequencing (RNA-seq). Based on results from earlier human genetic and rodent preclinical studies, we focused on a set of genes that may be associated with the development of addictive diseases and the rewarding effect of drugs of abuse, primarily in the opioid, stress response and classical neurotransmitter systems. We found that 32 transcripts in the ventral striatum, and 7 in the dorsal striatum, were altered significantly in adult mice that had self-administered oxycodone (n = 5) for 14 consecutive days (4 h/day) compared with yoked saline controls (n = 5). The following 5 genes in the ventral striatum showed experiment-wise significant changes: proopiomelanocortin (Pomc) and serotonin 5-HT-2A receptor (Htr2a) were upregulated; serotonin receptor 7 (Htr7), galanin receptor1 (Galr1) and glycine receptor 1 (Glra1) were downregulated. Some genes detected by RNA-seq were confirmed by quantitative polymerase chain reaction (qPCR). Conclusion: A RNA-seq study shows that chronic oxycodone SA alters the expression of several reward-related genes in the dorsal and ventral striatum. These results suggest potential mechanisms underlying neuronal adaptation to chronic oxycodone self-exposure, of relevance to our mechanistic understanding of prescription opioid abuse.

Oprm1 A112G, a single nucleotide polymorphism, alters expression of stress-responsive genes in multiple brain regions in male and female mice.

BACKGROUND: OPRM1 A118G, a functional human mu-opioid receptor (MOR) polymorphism, is associated with drug dependence and altered stress responsivity in humans as well as altered MOR signaling. MOR signaling can regulate many cellular processes, including gene expression, and many of the long-term, stable effects of drugs and stress may stem from changes in gene expression in diverse brain regions. A mouse model bearing an equivalent polymorphism (Oprm1 A112G) was previously generated and studied. Mice homozygous for the G112 allele show differences in opioid- and stress-related phenotypes. APPROACH: The current study examines the expression of 24 genes related to drug and stress responsivity in the caudoputamen, nucleus accumbens, hypothalamus, hippocampus, and amygdala of drug-naïve, stress-minimized, male and female mice homozygous for either the G112 variant allele or the wild-type A112 allele. RESULTS: We detected nominal genotype-dependent changes in gene expression of multiple genes. We also detected nominal sex-dependent as well as sex-by-genotype interaction effects on gene expression. Of these, four genotype-dependent differences survived correction for multiple testing: Avp and Gal in the hypothalamus and Oprl1 and Cnr1 in the hippocampus. CONCLUSIONS: Changes in the regulation of these genes by mu-opioid receptors encoded by the G112 allele may be involved in some of the behavioral and molecular consequences of this polymorphism observed in mice.

Sex differences after chronic stress in the expression of opioid-, stress- and neuroplasticity-related genes in the rat hippocampus.

Opioid peptides and their receptors re-organize within hippocampal neurons of female, but not male, rats following chronic immobilization stress (CIS) in a manner that promotes drug-related learning. This study was conducted to determine if there are also sex differences in gene expression in the hippocampus following CIS. Adult female and male rats were subjected to CIS (30 min/day) for 10 days. Twenty-four hours after the last stressor, the rats were euthanized, the brains were harvested and the medial (dentate gyrus/CA1) and lateral (CA2/CA3) dorsal hippocampus were isolated. Following total RNA isolation, cDNA was prepared for gene expression analysis using a RT2 Profiler PCR expression array. This custom designed qPCR expression array contained genes for opioid peptides and receptors, as well as genes involved in stress-responses and candidate genes involved in synaptic plasticity, including those upregulated following oxycodone self-administration in mice. Few sex differences are seen in hippocampal gene expression in control (unstressed) rats. In response to CIS, gene expression in the hippocampus was altered in males but not females. In males, opioid, stress, plasticity and kinase/signaling genes were all down-regulated following CIS, except for the gene that codes for corticotropin releasing hormone, which was upregulated. Changes in opioid gene expression following chronic stress were limited to the CA2 and CA3 regions (lateral sample). In conclusion, modest sex- and regional-differences are seen in expression of the opioid receptor genes, as well as genes involved in stress and plasticity responses in the hippocampus following CIS.

Oxycodone Self-Administration Induces Alterations in Expression of Integrin, Semaphorin and Ephrin Genes in the Mouse Striatum.

Oxycodone is one a commonly used medication for pain, and is also a widely abused prescription opioid, like other short-acting MOPr agonists. Neurochemical and structural adaptations in brain following chronic MOPr-agonist administration are thought to underlie pathogenesis and persistence of opiate addiction. Many axon guidance molecules, such as integrins, semaphorins, and ephrins may contribute to oxycodone-induced neuroadaptations through alterations in axon-target connections and synaptogenesis, that may be implicated in the behaviors associated with opiate addiction. However, little is known about this important area. The aim of this study is to investigate alterations in expression of selected integrin, semaphorin, ephrins, netrin, and slit genes in the nucleus accumbens (NAc) and caudate putamen (CPu) of mice following extended 14-day oxycodone self-administration (SA), using RNAseq. Methods: Total RNA from the NAc and CPu were isolated from adult male C57BL/6J mice within 1 h after the last session of oxycodone in a 14-day self-administration paradigm (4h/day, 0.25 mg/kg/infusion, FR1) or from yoked saline controls. Gene expressions were examined using RNA sequencing (RNA-Seq) technology. RNA-Seq libraries were prepared using Illumina's TruSeq® Stranded Total RNA LT kit. The reads were aligned to the mouse reference genome (version mm10) using STAR. DESeq2 was applied to the counts of protein coding genes to estimate the fold change between the treatment groups. False Discovery Rate (FDR) q < 0.1 were used to select genes that have a significant expression change. For selection of a subset of genes related to axon guidance pathway, REACTOME was used. Results: Among 38 known genes of the integrin, semaphorin, and ephrin gene families, RNA-seq data revealed up-regulation of six genes in the NAc: heterodimer receptor, integrins Itgal, Itgb2, and Itgam, and its ligand semaphorin Sema7a, two semaphorin receptors, plexins Plxnd1 and Plxdc1. There was down-regulation of eight genes in this region: two integrin genes Itga3 and Itgb8, semaphorins Sema3c, Sema4g, Sema6a, Sema6d, semaphorin receptor neuropilin Nrp2, and ephrin receptor Epha3. In the CPu, there were five differentially expressed axon guidance genes: up-regulation of three integrin genes, Itgal, Itgb2, Itga1, and down-regulation of Itga9 and ephrin Efna3 were thus observed. No significant alterations in expression of Netrin-1 or Slit were observed. Conclusion: We provide evidence for alterations in the expression of selective axon guidance genes in adult mouse brain following chronic self-administration of oxycodone. Further examination of oxycodone-induced changes in the expression of these specific axon guidance molecules and integrin genes in relation to behavior may provide new insights into development of addiction to oxycodone.

A non-coding CRHR2 SNP rs255105, a cis-eQTL for a downstream lincRNA AC005154.6, is associated with heroin addiction.

Dysregulation of the stress response is implicated in drug addiction; therefore, polymorphisms in stress-related genes may be involved in this disease. An analysis was performed to identify associations between variants in 11 stress-related genes, selected a priori, and heroin addiction. Two discovery samples of American subjects of European descent (EA, n = 601) and of African Americans (AA, n = 400) were analyzed separately. Ancestry was verified by principal component analysis. Final sets of 414 (EA) and 562 (AA) variants were analyzed after filtering of 846 high-quality variants. The main result was an association of a non-coding SNP rs255105 in the CRH (CRF) receptor 2 gene (CRHR2), in the discovery EA sample (Pnominal = .00006; OR = 2.1; 95% CI 1.4-3.1). The association signal remained significant after permutation-based multiple testing correction. The result was corroborated by an independent EA case sample (n = 364). Bioinformatics analysis revealed that SNP rs255105 is associated with the expression of a downstream long intergenic non-coding RNA (lincRNA) gene AC005154.6. AC005154.6 is highly expressed in the pituitary but its functions are unknown. LincRNAs have been previously associated with adaptive behavior, PTSD, and alcohol addiction. Further studies are warranted to corroborate the association results and to assess the potential relevance of this lincRNA to addiction and other stress-related disorders.

Genetic variations in genes of the stress response pathway are associated with prolonged abstinence from heroin.

AIM: This study assesses whether genetic variants in stress-related genes are associated with prolonged abstinence from heroin in subjects that are not in long-term methadone treatment. METHODS: Frequencies of 117 polymorphisms in 30 genes were compared between subjects with history of heroin addiction, either without agonist treatment (n = 129) or in methadone maintenance treatment (n = 923). RESULTS: SNP rs1500 downstream of CRHBP and an interaction of SNPs rs10482672 (NR3C1) and rs4234955 (NPY1R/NPY5R) were significantly associated with prolonged abstinence without agonist treatment. CONCLUSION: This study suggests that variability in stress-related genes may contribute to the ability of certain subjects to remain in prolonged abstinence from heroin, possibly due to higher resilience to stress.