| objective: Heroin addiction is a chronic recurrent encephalopathy characterized by compulsive medication,which has become a major medical and social problem in recent years and has attracted wide attention at home and abroad.Effective,long-lasting therapies are still lacking.Therefore,this study aims to establish a model of conditioned place preference for heroin addiction,and to deeply explore the role of the NMDA receptor system in the heroin-dependent process with experimental methods such as behavior,morphology and molecular biology.methods:1.Establishment of the CPP model of heroin addiction: The SD rats were divided into three groups,saline control group(Control,22 rats),heroin-induced(Heroin,22 rats)group and heroin-induced withdrawal group(Withdrawal,22 rats).The heroin-induced CPP model was established in rats for 7 d,and the rats in Withdrawal group were stopped and were naturally withdrawn for 7 d.2.NAc transcriptome sequencing and ERK1/2,CREB m RNA and protein expression detection: After successfully establishing the CPP model according to the rat behavioral data,severed the brains of rats,stripped out the NAc according to the stereotactic map of rat brain tissue,conducted transcriptome sequencing,screened out the molecules related to heroin dependence by transcriptome sequencing results,and their expression levels in NAc were detected by real-time PCR and Western Blot experiments.3.Location relationship between GABA and NR2 B and NR2 C receptors in NAc and expression level of GABA protein: After the successful establishment of CPP model based on behavioral data,three groups of rats were extracted by left ventricular puncture and made into a continuous coronal section with a thickness of 20μm.The positional relationship between GABA and NR2 B and NR2 C and their protein expression levels in NAc were observed and detected by immunofluorescence and immunohistochemical staining,respectively.results:1.After 7 d of CPP training with continuous heroin administration,Heroin rats showed a significant increase in activity time and distance in the white box compared with Control group and the same group(P <0.01),indicating that the rat CPP model was successfully established after heroin induction;there was no significant difference in activity time in the Control group(P > 0.05),suggesting that the CPP model cannot be induced by saline.2.The Heroin group had 926 differential genes compared to the Control group;6528 and 3782 differential genes in the heroin and control groups,respectively;135common differential genes in the three groups;and Ptk2β were common highly expressed genes with BC and DC values.GO function enrichment results: Both Heroin and Withdrawal groups showed high expression of ERK1 and ERK2 cascades compared with Control group.At the CC level,glutamatergic synapses were significantly higher compared with the Heroin and Control groups.KEGG pathway enrichment results: the expression of calcium ion signaling pathway was significantly different compared with Heroin and Control groups(P <0.01).The differential expression of the MAPK signaling pathway between the Heroin and Control groups was statistically significant(P <0.01).3.RT-q PCR showed that ERK2 expression the highest in the Heroin group and the lowest in the withdrawal group.ERK1 did not change significantly in any of the three groups.Compared with the Control group,CREB was significantly higher in both the Heroin and Withdrawal groups,and the difference was statistically significant(P <0.05).Western Blot The results showed no significant difference in ERK1/2 and CREB protein expression between the groups.The expression of p-ERK1/2 and p-CREB was significantly increased in the Heroin and Withdrawal groups compared to the Control group(P <0.05)4.Immunofluorescence and immunofluostochemical staining showed that GABA,NR2 B and NR2 C,and GABA was vacuolar and co-expressed with NR2 B and NR2 C receptors;GABA in Heroin rats was significantly higher than that of Control and Withdrawal rats(P <0.01);GABA was the lowest in Withdrawal rats(P <0.01).Conclusion: The CPP system successfully established heroin-induced rat CPP model,and calcium ion signaling and MAPK signaling play important roles in rat nucleus accumbens at all stages of heroin induction;Ptk2β,three groups of common different genes,participated in the regulation of ERK1 and ERK2 cascade in GO functional biological processes.The alteration in ERK1/2 and CREB activity status induce the formation of heroin addiction and the reconsolidation of drug-related memories.The NMDA receptor subunits NR2 B and NR2 C regulate the generation of drug dependence and the occurrence of reignition through the regulation of GABA release.However,in the process of heroin addiction resurgence,the stimulation of the heroin-related environment can inhibit the release of GABA. |
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