Addictive Drugs To Induce Gene Expression Regulation And Its Impact On Drug Reward Behavior Research

Drug addiction (drug abuse) is a chronic, relapsed brain disease characterized by continuing, compulsive, uncontrolled drug use behavior. Accumulated studies have shown that alteration of gene expression by addictive drugs is the most important mechanism lay in the drug abuse in the key reward brain region. So, it is particularly important to elucidate the regulatory mechanism of gene expression.MiRNAs are small non-coding regulatory single-stranded RNA molecules, which regarded as an important regulator of gene expression. By the process of recognizing target gene’s3’untranslated region (UTR), miRNAs can form a silencing complex with Dicer and AGO. Then, the complex will be brought inhibition or instability to their target mRNA, resulting in the short-or long-term suppressed expression. In recent years, many experimental evidences reveal important function of miRNAs in neural cells. However, the role of miRNAs in drug reward behavior was still unknown.Epigenetic mechanism controls gene transcription, indicating that complex mechanisms regulate the accessibility of genes to the transcriptional machinery. It is regarded that increased histone acetylation is associated with DNA relaxation and elevated transcriptional activity, whereas decreased acetylation, induced by histone deacetylases (HDACs) or other mechanism, results in tighter DNA coiling and gene silencing. Nowadays, it is reported that histone acetylation plays a critical role in cocaine-induced neuronal and behavioral plasticity. However, research has not completely clarified the epigenetic mechanism in the different phases of drug self-administration including the initial, reinforcement, incubation and reinstatement stages.To study the effect of miRNAs or histone acetylation of nucleus accumbens (NAc) mediated gene regulation in drug addiction, we have adopted heroin conditioned place preference model and heroin self-administration model, which are widely applied in the current drug addiction research, to detect the influence of miRNA or histone acetylation on drug-induced reward behaviors. Our results are shown as follow:1. Regulation of heroin reward behavior by NAc miR-218Chronic heroin administration alters miRNA precursor levels in NAc Rats were treated daily with heroin or saline for14consecutive days, and the levels of precursor of76miRNAs that expressed in NAc, were determined by real-time PCR. The precursors of miR-103, miR-136, miR-20a, miR-21, and miR-298were significantly up-regulated, while miR-218, miR-221, miR-351, miR-382, and miR-383precursors were down-regulated in the NAc in chronic heroin administration group. These data indicate that chronic heroin treatment alters miRNAs level in NAc.Chronic heroin administration down regulates miR218level in NAc In parallel with the change in miR-218precursor in NAc induced by chronic drug treatment, significant reduction of the functional form, mature miR-218level was significantly reduced following chronic heroin treatment, but not a single injection of heroin. These data indicate that chronic heroin treatment down regulates miR-218level in NAc.Construction and identification of miR-218-Lv anti-miR-218-Lv To investigate the role of miR-218in heroin reward in NAc, we first constructed and identified efficiacy of miR-218-Lv anti-miR-218-Lv. We found that infusion of miR-218-Lv into NAc results in higher miR-218level in NAc significantly.The efficiacy of anti-miR-218-Lv is confirmed by detection of miR-218target gene’s protein level after anti-miR-218-Lv infusion into NAc. These data shows that construction of miR-218-Lv and anti-miR-218-Lv is successfully.MiR-218in NAc inhibits heroin-induced self-administration and CPP The rats overexpressing miR-218with miR-218-Lv in the NAc displayed a significant decrease in heroin preference as compared with the shGal-lv control group, and the rats which were knockdown miR-218with anti-miR-218-Lv in the NAc spent more time in heroin-paired chamber. In heroin SA, overexpression of miR-218in NAc decreases heroin intake and knockdown of miR-218increases the heroin consume. These data indicates that miR-218in NAc inhibits heroin-induced reward behavior.MiR-218in NAc does not alter sugar-induced self-administration and radial maze, o determine the effect of miR-218-Lv or anti-miR-218-Lv infusion into NAc, sugar-induced self-administration were performed in the rats overexpressing or inhibiting miR-218. The rats overexpressing miR-218with miR-218-Lv in the NAc consumed similar sugar in sugar-induced self-administration and radial maze, compared with the shGal-lv control group; similar results were also detected in the anti-miR-218-Lv rats. These results suggests that miR-218in NAc does not alter sugar-induced self-administration and radial maze behavior.2. Screening miR-218regulated genesScreening miR-218regulated genes To screen the regulated genes of miR-218in neuron, Affymetrix Rat Genome2302.0Array assay were performed to detect genes level of neurons, which were transfected with synthetic miR-218or miRNA mimic control. We found that overexpression of miR-218decrease1990genes in gene chip, including43genes in neuroactive ligand-receptor interaction pathways,37genes in calcium signaling pathways,20genes in axon guidance pathways,13genes in long-term potentiation pathways, suggesting the role of miR-218’in regulating synaptic plasticity. Furthermore, the down-regulation of genes like MeCP2, GluR2, GABRB3, DNMT3a, sema6b and NRXN1, was confirmed with real-time PCR. These data shows that miR-218regulate mRNA levels of MeCP2, DNMT3a, GluR2, GABRB3, Sema6b and NRXN1in neuron.miR-218targets GluR2and GABRB3To determine whether miR-218targets GluR2and GABRB3respectly, luciferase assays were applied.In vitro, we observed a phenomenon that miR-218decrease expression of luciferase,which were tagged with3’UTR of GluR2or GABRB3. Meanwhile, miR-218-Lv infusion decreased protein levels of GluR2and GABRB3in NAc, but anti-miR-218-Lv infusion increased protein levels of GluR2and GABRB3in NAc. These data indicates that GluR2and GABRB3are target genes of miR-218.Chronic heroin administration increase mRNA level of GluR2and GABRB3in NAc To determine the role of GluR2and GABRB3in the heroin induced behavior, real-time PCR were performed in NA of chronic heroin experienced rats or control chronic saline rats. The results indicate that GluR2and GABRB3mRNA levels were significantly increased in heroin exposed rats, as compared with the saline treated group and acute heroin treated group. And then we observed a strong correlation of heroin CPP score with GluR2mRNA level in NAc, but not in HIP.GIuR2level correlates with heroin CPP score in NAc The correlaton analysis were perfermed between heroin CPP scor and NAc or HIP mRNA level of GluR2and GABRB3. The results show a strong correlation of heroin CPP score with GluR2mRNA level in NAc, but not in HIP, not in GABRB3mRNA level.3. Mechanism of H3acetylation in NAc regulate Cocaine MotivationMotivation for cocaine is associated with H3acetylation in addiction related genes pomotor in the NAc shell. To investigate the association between the motivation for cocaine and H3acetylation in promoters of addiction-related genes in the NAc shell, chromatin immunoprecipitation (ChIP) assay was performed. The results shown that chronic-cocaine-induced gene transcriptional activation occurred primarily in the NAc shell and was predominantly modulated by the acetylation of H3. Furthermore, motivation for cocaine was correlated with the level of H3acetylation of NAc shell. These data indicated that drug-induced gene transcriptional activation in the NAc shell, modulated by H3acetylation, is a potential mechanism underlying the motivation for cocaine.Kockdown of CaMKⅡα in NAc shell decreases the motivation for cocaine. There is a strong correlation between motivation for the drug and CaMKⅡα mRNA level, but not CaMKⅡβ mRNA level. It is indicated that CaMKⅡα, but not CaMKⅡβ of NAc shell was essential for the maintenance of motivation in cocaine self-administration in lentivirus-mediated shRNA knockdown experiments. These data demonstrated that transcriptional regulation of genes, such as CaMKⅡα, plays a critical role in maintaining motivation for drug.