Opioid Gene Expression Research Articles

Voluntary wheel running access produces opposite effects in male and female rats on both palatable diet consumption and associated ventral striatal opioid- and dopamine-related gene expression.

The relationship between physical activity levels and feeding behaviors has been a focus of preclinical research for decades, yet this interaction has only recently been explored for potential sex differences. The aim of the present study was to isolate sex-dependent effects of voluntary wheel running (RUN) vs. sedentary locked wheel (SED) home cage conditions on palatability-driven feeding behavior using a 2-diet choice task between standard chow and a high-fat diet. The sex-dependent effects of physical activity on feeding behavior were examined following a within-subject novel reversal design of physical activity conditions (i.e., RUN > SED > RUN), to assess temporal sensitivity of the interaction. Following the final 2 weeks of reestablished and sustained RUN vs. SED conditions in separate groups of both males and females, reward-related opioid and dopamine gene expression within the nucleus accumbens (Acb) brain region were analyzed. Results demonstrated that the initial RUN > SED transition led to sex-dependent effects of SED condition, as males increased, and females decreased their high fat consumption, compared to their respective high fat consumption during previous RUN condition phase. Following reintroduction to the RUN condition, males decreased, and females increased their high fat consumption, compared to their separate SED control group. Last, sex-dependent shifts in ventral striatal opioid- and dopamine-related gene expression were observed to parallel the behavioral effects. The major findings of the study reveal that SED and RUN home cage conditions shift palatability-driven feeding in the opposite direction for males and females, these effects are sensitive to reversal, and these sex-dependent feeding behaviors track sex-dependent changes to critical reward-related gene expression patterns in the Acb. Considering the present high rates of sedentary behavior and obesity, furthering our understanding of the interaction between physical activity (or lack thereof) and feeding behavior should be a priority, especially in the context of these divergent sex-dependent outcomes.

Abstract 2201: Effect of opioid and cannabinoid receptors gene expression on survival of patients with colorectal cancer

Abstract Introduction: Colorectal cancer (CRC) is one of the leading causes of cancer death in the world. CRC patients are often treated with opioid (e.g., morphine, or synthetic opioid piritramide) analgesia for pain, nausea, and side effects of chemotherapy. These analgesics act through opioid and cannabinoid receptors, which pathways are involved in tumor progression and metastases and thus can negatively affect the survival of patients. In our previous studies, we analyzed the presence of circulating tumor cells (CTCs) in two groups of CRC patients. One group received morphine analgesia (MA) and second piritramide analgesia (PA). We found that MA associated with increased CTCs levels and significantly shorter cancer-specific survival (CSS). These findings lead to further analysis of the opioid and cannabinoid receptors tumor gene expressions. We expected that the different expression of specific receptors in tumor tissue makes a difference between the effects of MA and PA on patients´ survival. Methods: Gene expression of opioid receptors µ (OPRM), κ (OPRK), δ (OPRD), nociceptin (OPRL) and cannabinoid receptors 1 and 2 (CB1, CB2) was analyzed in RNA purified from tumor tissues in 131 patients with CRC. Expression of markers was detected using real-time RT-PCR on LightCycler 1536 from Roche. Β-actin gene expression was used for gene expression normalization. Specific cut-off values were calculated for each marker using maxstat R software, ver. 3.3.1. Relationship between expression of receptors in tumor tissue and patients’ survival was analyzed using COX regression, Kruskal-Wallis/ANOVA test, and Kaplan-Meier method. Results: In total, 118 patients (45 females and 73 males, average age 68 years), of clinical stage-III were analyzed. It was found that MA in CRC patients was associated with significantly shorter cancer-specific survival (CSS; p=0,027). The univariate survival analysis revealed that CRC patients with high OPRM tumor tissue gene expression had significantly longer overall survival (OS; p=0.017) and patients with CB2 gene expression had significantly longer disease-free survival (DFS; p=0,029). All other tested opioid receptors expressions had no statistically significant effect on patients’ survival. Conclusion: The diverse opioid receptors’ expressions on tumor and immune cells can affect the survival of cancer patients treated with opioid analgesia. Morphine perioperative analgesia seems to shorten the CCS in CRC patients in our study. However, further research is required to elucidate the effect of opioids on different cell populations. Acknowledgement: This study was supported by Ministry of Health of the Czech Republic [NV18-03-00470]; Palacky University Olomouc [LF 2022_012]; European Regional Development Fund [ENOCHCZ.02.1.01/0.0/0.0/16_019/0000868) and European Union - Next Generation EU (EXCELES LX22NPO5102). Citation Format: Monika Vidlarova, Josef Srovnal, Emil Berta, Sona Gurska, Alona Rehulkova, Pavla Kourilova, Marian Hajduch. Effect of opioid and cannabinoid receptors gene expression on survival of patients with colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2201.

Morphine-induced mechanical hypersensitivity in mice requires δ receptors, β-arrestin2, and c-Src activity.

Morphine diminishes pain, but its long-term use is compromised by tolerance and hyperalgesia. Studies implicate δ receptors, β-arrestin2 and Src kinase in tolerance. We examined whether these proteins are also involved in morphine-induced hypersensitivity (MIH). A common pathway for tolerance and hypersensitivity may provide a single target to guide improved analgesic approaches. We examined mechanical sensitivity using automated von Frey in wild-type (WT) and transgenic male and female C57Bl/6mice before and after hind paw inflammation by complete Freund's adjuvant (CFA). CFA-evoked hypersensitivity ceased on day 7 in WT but persisted for the 15-day testing period in μ-/- . Recovery was delayed until day 13 in δ-/- . We explored the expression of opioid genes in the spinal cord using quantitative RT-PCR. Restoration to basal sensitivity in WT occurred with increased δ expression. By contrast, κ expression was reduced, while μ remained unchanged. Daily morphine reduced hypersensitivity in WT on day 3 compared to controls; however, hypersensitivity recurred on day 9 and beyond. By contrast, WT had no recurrence of hypersensitivity in the absence of daily morphine. We used β-arrestin2-/- , δ-/- and Src inhibition by dasatinib in WT to establish whether these approaches, which diminish tolerance, also attenuate MIH. While none of these approaches affected CFA-evoked inflammation or acute hypersensitivity, all caused sustained morphine anti-hypersensitivity, abolishing MIH. Like morphine tolerance, MIH in this model requires δ receptors, β-arrestin2 and Src activity. Our findings suggest that MIH is caused by a tolerance-induced reduction in endogenous opioid signalling. KEY POINTS: Morphine is effective for treating severe acute pain, but tolerance and hypersensitivity often develop during its use in treating chronic pain. It is unclear whether these detrimental effects share similar mechanisms; if so, it might be possible to develop a single approach to minimise both phenomena. Mice deficient in μ receptors, δ receptors or β-arrestin2 and wild type mice treated with the Src inhibitor dasatinib exhibit negligible morphine tolerance. We show that these same approaches also prevent the development of morphine-induced hypersensitivity during persistent inflammation. This knowledge identifies strategies, such as the use of Src inhibitors, which may mitigate tolerance and morphine induced hyperalgesia.

Differential Regulation of Tachykinin and Opioid System Gene Expression in Brain and Immune Cells of Chronic Binge Alcohol-Treated Simian Immunodeficiency Virus-Infected Macaques.

People living with HIV have a high likelihood of at-risk alcohol use and are at increased risk for neurocognitive decline. The underlying mechanisms involved in HIV-associated neurocognitive disorder (HAND) are not completely understood. Previously, we showed that chronic binge alcohol (CBA) administration produced behavioral deficits in non antiretroviral therapy (ART)-treated simian immunodeficiency virus (SIV)-infected macaques. Moreover, we observed that CBA/SIV enhanced neuroinflammatory gene expression and attenuated growth factor signaling in the frontal cortex (FC) and basal ganglia, effects that were partially ameliorated by ART. We hypothesized that the neuroinflammatory and growth factor changes observed could be associated with alterations in opioid, tachykinin, and endocannabinoid gene expression. Furthermore, we proposed that gene expression patterns in peripheral blood mononuclear cells (PBMCs) could serve as an indicator of expression changes in the brain (FC). We examined gene expression patterns of opioid, tachykinin, and endocannabinoid systems in FC and PBMCs isolated from CBA/SIV macaques. Expression of targeted genes as determined by reverse transcription-quantitative polymerase chain reaction was analyzed in relation to CBA, ART, plasma, and brain viral loads (PVL and BVL, respectively) and compared with baseline (PBMC) or FC from SIV- controls. FC expression of ORM1, POMC, and TACR1 was negatively associated with PVL (p = .03, .002, .05 respectively). FC expression of TAC1 was positively associated with CBA exposure (p = .05). PBMC expression of DAGLA was positively associated with CBA exposure; but negatively associated with combined CBA/ART exposure (p = .03). Our findings reflect the complex interactions of SIV, CBA, and ART in modulating opioid and tachykinin system gene expression. Contrary to our prediction, results did not reveal parallel changes (in magnitude or direction) in PBMC and FC gene expression. Further studies are warranted to determine the relevance of these transcriptional changes in modulating HAND-related behaviors resulting from at-risk alcohol use and HIV/SIV exposure.

Molecular examination of the endogenous opioid system in rhesus macaque monkeys with self-injurious behavior.

Self-injurious behavior (SIB) can lead to serious injury and occurs in approximately 1%-4% of the adult population, with higher incidences in adolescent and institutionalized populations, as well as in children with developmental disorders such as Autism. SIB also spontaneously occurs in a low percentage of captive monkeys. Rhesus macaque (Macaca mulatta) monkeys are evolutionarily and physiologically similar to humans, share 93% genetic sequence similarity to humans, and have long been used as testing subjects for vaccine and clinical trials. Previous studies hypothesized that altered endogenous opioid expression occurs in the brains of individuals and animals that self-injure. We examined the regional mRNA expression of opioid signaling genes in sixteen rhesus macaques that exhibited SIB and eight sex- and age- matched controls. The brain regions examined are linked to reward reinforcement and stress adaptation including the hypothalamus, orbital frontal cortex, nucleus accumbens, hippocampus, caudate, and the amygdala. We found decreased μ-opioid receptor (OPRM1) in the amygdala of monkeys with SIB, and reduced prodynorphin (PDYN) in the hypothalamus. Our data suggest dysfunction in the regulation of opioid peptide precursors and calls for further investigation of the endogenous opioid system in SIB.

Central and cardiac stress resilience consistently linked to integrated immuno-neuroendocrine responses across stress models in male mice.

Stress resilience, and behavioural and cardiovascular impacts of chronic stress, are theorised to involve integrated neuro-endocrine/inflammatory/transmitter/trophin signalling. We tested for this integration, and whether behaviour/emotionality, together with myocardial ischaemic tolerance, are consistently linked to these pathways across diverse conditions in male C57Bl/6 mice. This included Restraint Stress (RS), 1h restraint/day for 14 days; Chronic Unpredictable Mild Stress (CUMS), seven stressors randomised over 21 days; Social Stress (SS), 35 days social isolation with brief social encounters in final 13 days; and Control conditions (CTRL; un-stressed mice). Behaviour was assessed via open field (OFT) and sucrose preference (SPT) tests, and neurobiology from frontal cortex (FC) and hippocampal transcripts. Endocrine factors, and function and ischaemic tolerance in isolated hearts, were also measured. Model characteristics ranged from no behavioural or myocardial changes with homotypic RS, to increased emotionality and cardiac ischaemic injury (with apparently distinct endocrine/neurobiological profiles) in CUMS and SS models. Highly integrated expression of HPA axis, neuro-inflammatory, BDNF, monoamine, GABA, cannabinoid and opioid signalling genes was confirmed across conditions, and consistent/potentially causal correlations identified for (i) locomotor activity (noradrenaline, ghrelin; FC Crhr1, Tnfrsf1b, Il33, Nfkb1, Maoa, Gabra1; hippocampal Il33); (ii) thigmotaxis (adrenaline, leptin); (iii) anxiety-like behaviour (adrenaline, leptin; FC Tnfrsf1a; hippocampal Il33); (iv) depressive-like behaviour (ghrelin; FC/hippocampal s100a8); and (v) cardiac stress-resistance (noradrenaline, leptin; FC Il33, Tnfrsf1b, Htr1a, Gabra1, Gabrg2; hippocampal Il33, Tnfrsf1a, Maoa, Drd2). Data support highly integrated pathway responses to stress, and consistent adipokine, sympatho-adrenergic, inflammatory and monoamine involvement in mood and myocardial disturbances across diverse conditions.

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.

Heme oxygenase-1 in the spinal cord plays crucial roles in the analgesic effects of pregabalin and gabapentin in a spared nerve-injury mouse model

IntroductionNeuropathic pain remains one of the most intractable types of pain; although calcium channel α2δ ligands, such as pregabalin and gabapentin, are classified as first-line drugs, they have only modest efficacy. Heme oxygenase-1 (HO-1) signaling attenuates glial activation during neuropathic pain. Thus, this study aimed to investigate the effects of the blood–brain barrier (BBB)-permeable HO-1 inhibitor, tin protoporphyrin IX (SnPP), or the BBB-impermeable HO-1 inhibitor, zinc (II) protoporphyrin IX (ZnPP), on the analgesic efficacy of pregabalin and gabapentin. Additionally, we examined the effects of co-administration of SnPP with pregabalin or gabapentin on the expression of glial markers or other genes. MethodsNeuropathic pain was induced by spared nerve injury (SNI) of the sciatic nerve. The mechanical threshold was tested using the von Frey filaments. The expression of spinal glial markers or other genes was examined using reverse transcription polymerase chain reaction. ResultsSystemic HO-1 inhibition reversed the mechanical antiallodynic effects of pregabalin and gabapentin, although peripheral HO-1 inhibition did not alter the mechanical antiallodynic effects of either pregabalin or gabapentin. Intrathecal injection of SnPP or ZnPP abolished the mechanical antiallodynic effects of pregabalin and gabapentin. Pregabalin and gabapentin increased HO-1, arginase-1, and endogenous opioid precursor preproenkephalin gene expression and decreased the expression of glial markers, interleukin-1β, and inducible nitric oxide synthase. ConclusionsThis study suggests that spinal HO-1 plays a crucial role in the analgesic effects of calcium channel α2δ ligands through the attenuation of glial activation and endogenous opioid release.

Left-Right Side-Specific Neuropeptide Mechanism Mediates Contralateral Responses to a Unilateral Brain Injury.

Neuropeptides are implicated in control of lateralized processes in the brain. A unilateral brain injury (UBI) causes the contralesional sensorimotor deficits. To examine whether opioid neuropeptides mediate UBI induced asymmetric processes we compared effects of opioid antagonists on the contralesional and ipsilesional hindlimb responses to the left-sided and right-sided injury in rats. UBI induced hindlimb postural asymmetry (HL-PA) with the contralesional hindlimb flexion, and activated contralesional withdrawal reflex of extensor digitorum longus (EDL) evoked by electrical stimulation and recorded with EMG technique. No effects on the interossei (Int) and peroneaus longus (PL) were evident. The general opioid antagonist naloxone blocked postural effects, did not change EDL asymmetry while uncovered cryptic asymmetry in the PL and Int reflexes induced by UBI. Thus, the spinal opioid system may either mediate or counteract the injury effects. Strikingly, effects of selective opioid antagonists were the injury side-specific. The μ-antagonist β-funaltrexamine (FNA) and κ-antagonist nor-binaltorphimine (BNI) reduced postural asymmetry after the right but not left UBI. In contrast, the δ-antagonist naltrindole (NTI) inhibited HL-PA after the left but not right-side brain injury. The opioid gene expression and opioid peptides were lateralized in the lumbar spinal cord, and coordination between expression of the opioid and neuroplasticity-related genes was impaired by UBI that together may underlie the side-specific effects of the antagonists. We suggest that mirror-symmetric neural circuits that mediate effects of left and right brain injury on the contralesional hindlimbs are differentially controlled by the lateralized opioid system.

Chronic stress differentially altersmRNAexpression of opioid peptides and receptors in the dorsal hippocampus of female and male rats

Chronic immobilization stress (CIS) results in sex-dependent changes in opioid peptide levels and receptor subcellular distributions within the rat dorsal hippocampus, which are paralleled with an inability for males to acquire conditioned place preference (CPP) to oxycodone. Here, RNAScope in situ hybridization was used to determine the expression of hippocampal opioid peptides and receptors in unstressed (US) and CIS estrus female and male adult (∼2.5 months old ) Sprague Dawley rats. In all groups, dentate granule cells expressed PENK and PDYN; additionally, numerous interneurons expressed PENK. OPRD1 and OPRM1 were primarily expressed in interneurons, and to a lesser extent, in pyramidal and granule cells. OPRK1-was expressed in sparsely distributed interneurons. There were few baseline sex differences: US females compared to US males had more PENK-expressing and fewer OPRD1-expressing granule cells and more OPRM1-expressing CA3b interneurons. Several expression differences emerged after CIS. Both CIS females and males compared to their US counterparts had elevated: (1) PENK-expressing dentate granule cells and interneurons in CA1 and CA2/3a; (2) OPRD1 probe number and cell expression in CA1, CA2/3a and CA3b and the dentate gyrus; and (3) OPRK1-expressing interneurons in the dentate hilus. Also, CIS males compared to US males had elevated: (1) PDYN expression in granule cells; (2) OPRD1 probe and interneuron expression in CA2/3a; (3) OPRM1 in granule cells; and (4) OPRK1 interneuron expression in CA2/3a. The sex-specific changes in hippocampal opioid gene expression may impact network properties and synaptic plasticity processes that may contribute to the attenuation of oxycodone CPP in CIS males.

Selective alterations in endogenous opioid system genes expression in rats selected for high ethanol intake during adolescence

Historically, the roots of alcoholism have been linked to either environment or heredity. However, the interaction between these factors is still largely unexplored. The evidence supports a link between alcohol consumption and the endogenous opioid system. We here studied the opioid genes expression in male and female Wistar rats derived from a short-term breeding program which selected -- at adolescence -- for high (ADHI line) or low (ADLO line) ethanol drinking. Specifically, in this work we analyzed central opioid gene expression in the rats of the second filial generation (S2-ADLO and S2-ADHI). Selective downregulation of pronociceptin (Pnoc) and its receptor (Oprl1) mRNA levels were observed in the prefrontal cortex of male S2-ADHI rats when compared to S2-ADLO, and for Oprl1 also in the nucleus accumbens. An increase in gene expression was instead observed for pro-opiomelanocortin (Pomc) in the nucleus accumbens of S2-ADHI males when compared to S2-ADLO, as well as for mu opioid receptor (Oprm1) but in females. The differences in mRNA levels may be due to the different alcohol consumption between the two groups of rats or may represent pre-existing differences between them. Moreover, we show a sex-specific modulation of the expression of these genes, thus pointing out the importance of sex on ethanol responses. The results might lead to more specific and effective pharmacological treatments for alcoholism.

Early exposure to environmental enrichment modulates the effects of prenatal ethanol exposure upon opioid gene expression and adolescent ethanol intake

Prenatal ethanol exposure (PEE) promotes ethanol consumption in the adolescent offspring accompanied by the transcriptional regulation of kappa opioid receptor (KOR) system genes. This study analysed if environmental enrichment (EE, from gestational day 20 to postnatal day 26) exerts protective effects upon PEE-modulation of gene expression, ethanol intake and anxiety responses. Pregnant rats were exposed to PEE (0.0 or 2.0 g/kg ethanol, gestational days 17–20) and subsequently the dam and offspring were reared under EE or standard conditions. PEE upregulated KOR mRNA levels in amygdala (AMY) and prodynorphin (PDYN) mRNA levels in ventral tegmental area (VTA) with the latter effect associated with lower DNA methylation at the gene promoter. These effects were normalized by exposure to EE. PEE modulated BDNF mRNA levels in VTA and Nucleus accumbens (AcbN), and EE mitigated the changes in AcbN. EE induced a protective effect on ethanol intake and preference, an effect more noticeable in males than in females, and in prenatal vehicle-treated (PV) than in PEE rats. The male offspring drank significantly less ethanol than the female offspring. The latter result suggests that the protective effect of EE on ethanol drinking may only emerge at lower levels of drinking. In the dams, PEE induced an upregulation of PDYN and KOR in AcbN. PDYN gene expression was normalized by exposure to EE. These results suggest that EE is a promising treatment to inhibit the effects of PEE. The results confirm that PEE effects are mediated by alterations in the transcriptional regulation of KOR system genes.

Downregulation of the neuronal opioid gene expression concomitantly with neuronal decline in dorsolateral prefrontal cortex of human alcoholics

Molecular changes in cortical areas of addicted brain may underlie cognitive impairment and loss of control over intake of addictive substances and alcohol. Prodynorphin (PDYN) gives rise to dynorphin (DYNs) opioid peptides which target kappa-opioid receptor (KOR). DYNs mediate alcohol-induced impairment of learning and memory, while KOR antagonists block excessive, compulsive-like drug and alcohol self-administration in animal models. In human brain, the DYN/KOR system may undergo adaptive changes, which along with neuronal loss, may contribute to alcohol-associated cognitive deficit. We addressed this hypothesis by comparing the expression levels and co-expression (transcriptionally coordinated) patterns of PDYN and KOR (OPRK1) genes in dorsolateral prefrontal cortex (dlPFC) between human alcoholics and controls. Postmortem brain specimens of 53 alcoholics and 55 controls were analyzed. PDYN was found to be downregulated in dlPFC of alcoholics, while OPRK1 transcription was not altered. PDYN downregulation was confined to subgroup of subjects carrying C, a high-risk allele of PDYN promoter SNP rs1997794 associated with alcoholism. Changes in PDYN expression did not depend on the decline in neuronal proportion in alcoholics, and thereby may be attributed to transcriptional adaptations in alcoholic brain. Absolute expression levels of PDYN were lower compared to those of OPRK1, suggesting that PDYN expression is a limiting factor in the DYN/KOR signaling, and that the PDYN downregulation diminishes efficacy of DYN/KOR signaling in dlPFC of human alcoholics. The overall outcome of the DYN/KOR downregulation may be disinhibition of neurotransmission, which when overactivated could contribute to formation of alcohol-related behavior.

Differential effects of left and right neuropathy on opioid gene expression in lumbar spinal cord

The endogenous opioid system (EOS) controls the processing of nociceptive stimuli and is a pharmacological target for opioids. Alterations in expression of the EOS genes under neuropathic pain condition may account for low efficacy of opioid drugs. We here examined whether EOS expression patterns are altered in the lumbar spinal cord of the rats with spinal nerve ligation (SNL) as a neuropathic pain model. Effects of the left- and right-side SNL on expression of EOS genes in the ipsi- and contralateral spinal domains were analysed. The SNL-induced changes were complex and different between the genes; between the dorsal and ventral spinal domains; and between the left and right sides of the spinal cord. Prodynorphin (Pdyn) expression was upregulated in the ipsilateral dorsal domains by each the left and right-side SNL, while changes in expression of μ-opioid receptor (Oprm1) and proenkephalin (Penk) genes were dependent on the SNL side. Changes in expression of the Pdyn and κ-opioid receptor (Oprk1) genes were coordinated between the ipsi- and contralateral sides. Withdrawal response thresholds, indicators of mechanical allodynia correlated negatively with Pdyn expression in the right ventral domain after right side SNL. These findings suggest multiple roles of the EOS gene products in spinal sensitization and changes in motor reflexes, which may differ between the left and right sides.

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.

The standardized Withania somnifera Dunal root extract alters basal and morphine-induced opioid receptor gene expression changes in neuroblastoma cells

BackgroundBehavioral studies demonstrated that the administration of Withania somnifera Dunal roots extract (WSE), prolongs morphine-elicited analgesia and reduces the development of tolerance to the morphine’s analgesic effect; however, little is known about the underpinning molecular mechanism(s). In order to shed light on this issue in the present paper we explored whether WSE promotes alterations of μ (MOP) and nociceptin (NOP) opioid receptors gene expression in neuroblastoma SH-SY5Y cells.MethodsA range of WSE concentrations was preliminarily tested to evaluate their effects on cell viability. Subsequently, the effects of 5 h exposure to WSE (0.25, 0.50 and 1.00 mg/ml), applied alone and in combination with morphine or naloxone, on MOP and NOP mRNA levels were investigated.ResultsData analysis revealed that morphine decreased MOP and NOP receptor gene expression, whereas naloxone elicited their up-regulation. In addition, pre-treatment with naloxone prevented the morphine-elicited gene expression alterations. Interestingly, WSE was able to: a) alter MOP but not NOP gene expression; b) counteract, at its highest concentration, morphine-induced MOP down-regulation, and c) hamper naloxone-induced MOP and NOP up-regulation.ConclusionPresent in-vitro data disclose novel evidence about the ability of WSE to influence MOP and NOP opioid receptors gene expression in SH-SY5Y cells. Moreover, our findings suggest that the in-vivo modulation of morphine-mediated analgesia by WSE could be related to the hindering of morphine-elicited opioid receptors down-regulation here observed following WSE pre-treatment at its highest concentration.

Neuronal Expression of Opioid Gene is Controlled by Dual Epigenetic and Transcriptional Mechanism in Human Brain.

Molecular mechanisms that define patterns of neuropeptide expression are essential for the formation and rewiring of neural circuits. The prodynorphin gene (PDYN) gives rise to dynorphin opioid peptides mediating depression and substance dependence. We here demonstrated that PDYN is expressed in neurons in human dorsolateral prefrontal cortex (dlPFC), and identified neuronal differentially methylated region in PDYN locus framed by CCCTC-binding factor binding sites. A short, nucleosome size human-specific promoter CpG island (CGI), a core of this region may serve as a regulatory module, which is hypomethylated in neurons, enriched in 5-hydroxymethylcytosine, and targeted by USF2, a methylation-sensitive E-box transcription factor (TF). USF2 activates PDYN transcription in model systems, and binds to nonmethylated CGI in dlPFC. USF2 and PDYN expression is correlated, and USF2 and PDYN proteins are co-localized in dlPFC. Segregation of activatory TF and repressive CGI methylation may ensure contrasting PDYN expression in neurons and glia in human brain.

The expression of opioid genes in non-classical reward areas depends on early life conditions and ethanol intake

The young brain is highly sensitive to environmental influences that can cause long-term changes in neuronal function, possibly through altered gene expression. The endogenous opioid system continues to mature after birth and because of its involvement in reward, an inadequate maturation of this system could lead to enhanced susceptibility for alcohol use disorder. Recent studies show that the classical reward areas nucleus accumbens and ventral tegmental area are less affected by early life stress whereas endogenous opioids in non-classical areas, e.g. dorsal striatum and amygdala, are highly responsive. The aim was to investigate the interaction between early life conditions and adult voluntary ethanol intake on opioid gene expression. Male Wistar rats were exposed to conventional rearing, 15, or 360min of daily maternal separation (MS) postnatal day 1–21, and randomly assigned to ethanol or water drinking postnatal week 10–16. Rats exposed to early life stress (MS360) had increased opioid receptor gene (Oprm1, Oprd1 and Oprk1) expression in the dorsal striatum. Ethanol drinking was associated with lower striatal Oprd1 and Oprk1 expression solely in rats exposed to early life stress. Furthermore, rats exposed to early life stress had high inherent Pomc expression in the amygdala but low expression after ethanol intake. Thus, adverse events early in life induced changes in opioid gene expression and also influenced the central molecular response to ethanol intake. These long-term consequences of early life stress can contribute to the enhanced risk for excessive ethanol intake and alcohol use disorder seen after exposure to childhood adversity.

Opioid gene expression changes and post-translational histone modifications at promoter regions in the rat nucleus accumbens after acute and repeated 3,4-methylenedioxy-methamphetamine (MDMA) exposure

The recreational drug of abuse 3,4-methylenedioxymethamphetamine (MDMA) has been shown to produce neurotoxic damage and long-lasting changes in several brain areas. In addition to the involvement of serotoninergic and dopaminergic systems, little information exists about the contribution of nociceptin/orphaninFQ (N/OFQ)-NOP and dynorphin (DYN)-KOP systems in neuronal adaptations evoked by MDMA. Here we investigated the behavioral and molecular effects induced by acute (8mg/kg) or repeated (8mg/kg twice daily for seven days) MDMA exposure.MDMA exposure affected body weight gain and induced hyperlocomotion; this latter effect progressively decreased after repeated administration. Gene expression analysis indicated a down-regulation of the N/OFQ system and an up-regulation of the DYN system in the nucleus accumbens (NAc), highlighting an opposite systems regulation in response to MDMA exposure.Since histone modifications have been strongly associated to the addiction-related maladaptive changes, we examined two permissive (acH3K9 and me3H3K4) and two repressive transcription marks (me3H3K27 and me2H3K9) at the pertinent opioid gene promoter regions. Chromatin immunoprecipitation assays revealed that acute MDMA increased me3H3K4 at the pN/OFQ, pDYN and NOP promoters. Following acute and repeated treatment a significant decrease of acH3K9 at the pN/OFQ promoter was observed, which correlated with gene expression results. Acute treatment caused an acH3K9 increase and a me2H3K9 decrease at the pDYN promoter which matched its mRNA up-regulation.Our data indicate that the activation of the DYNergic stress system together with the inactivation of the N/OFQergic anti-stress system contribute to the neuroadaptive actions of MDMA and offer novel epigenetic information associated with MDMA abuse.

Repeated nicotine exposure modulates prodynorphin and pronociceptin levels in the reward pathway

BackgroundNicotine dependence is maintained by neurobiological adaptations in the dopaminergic brain reward pathway with the contribution of opioidergic circuits. This study assessed the role of opioid peptides and receptors on the molecular changes associated with nicotine dependence. To this aim we analysed nicotine effects on opioid gene and receptor expression in the reward pathway in a nicotine sensitization model. MethodsSprague-Dawley rats received nicotine administrations for five days and locomotor activity assessment showed the development of sensitization. The mRNA expression of prodynorphin (pdyn), pronociceptin (pnoc) and the respective receptors was measured by quantitative PCR in the ventral midbrain (VM), the nucleus accumbens (NAc), the caudate-putamen (CPu), the pre-frontal cortex (PFCx), and the hippocampus. ResultsA significant positive effect of sensitization on pdyn mRNA levels was detected in the CPu. This effect was supported by a significant and selective correlation between the two parameters in this region. Moreover, chronic but not acute nicotine treatment significantly decreased pdyn mRNA levels in the NAc and increased expression in the PFCx. Pnoc mRNA was significantly increased in the VM and the PFCx after sub-chronic administration of nicotine, whereas no alterations were observed after acute treatment. No treatment associated changes were detected in κ-opioid receptor or nociceptin receptor mRNAs. ConclusionsThis experiment revealed an effect of nicotine administration that was distinguishable from the effect of nicotine sensitization. While several pnoc and pdyn changes were associated to nicotine administration, the only significant effect of sensitization was a significant increase in pdyn in the CPu.