健康与遗传心理学研究室知识库

研究目的:药物与伴药线索/环境之间形成连接是成瘾行为长期存在以及用药线索/环境能诱发复吸的根本原因。这种连接的形成依赖于奖赏记忆加工的核心脑区内的基因转录发生长时程调控。DNA甲基转移酶(DNMT)催化发生的DNA甲基化通过诱导靶基因的转录沉默导致核团脑区功能长期改变，近些年新发现该过程可能被10-11易位酶(TET)催化的DNA主动去甲基化反向调控。揭示TET与DNMT共同调控药物一线索/环境形成连接的机制，有助于理解成瘾行为长期维持的表观遗传调控基础。

研究方案:为了研究DNMT和TET在药物一线索的条件化连接中的作用及机制，本研究以大鼠吗啡自身给药行为的获得作为药物一线索/环境之间形成连接的模型，进行如下实验:1)明确大鼠吗啡自身给药过程中DNMT和TET表达改变的时间规律、关键脑区及亚型特异性;2)明确关键亚型的DNMT和TET在吗啡自身给药行为获得中分别发挥的调控作用;3>初步探索DNMT和TET在此过程中调控的下游靶基因。

研究结果:1)吗啡自身给药训练过程中，海马CA1中的DNMT3 a而非DNMT3b表达持续上调，TET3的表达在早期上调，后期恢复。TET1的表达在早期不变，后期下调。NAc shell中DNMT和TET的表达水平均未显著改变。在被动接受吗啡注射的yoke组以及糖精水自身给药训练组中，CA1或NAc shell内DNMT或TET表达均没有显著变化。2) CA1内注射DNMT抑制剂_5-aza，或者敲减DNMT3 a，都可以抑制吗啡自身给药的获得。在CA1内过表达TET1

一定程度上抑制吗啡自身给药的获得，而敲减TET 1对吗啡自身给药的获得没有显著影响。敲减CA1中的TET3增强吗啡自身给药早期的获得。3)敲减DNMT3 a后，Ntrk2和乃Cpl cb的mRNA表达水平显著升高。过表达TET1后Camk2a的mRNA水平显著降低。

结论:本研究发现CA1中的DNMT和TET通过不同的靶基因共同调控阿片类药物的操作性学习过程，且发挥的作用相反。提示DNA甲基化修饰可能是成瘾行为形成的关键表观遗传调控因子。临床上使用阿片药物的同时抑制DNA甲基化或者促进DNA去甲基化，可能成为预防成瘾的新思路。

Rationale and objective: The association between drug and drug related cues has a pivotal role in long-lasting drug related behavior and cue-induced relapse. The formation of the drug-cue association needs long-term regulation of genes transcription in reward memory processing brain regions. A deal of studies have highlighted the importance of DNA methylation, the most stable chromatin modification catalyzed by DNA methyltransferases (DNMTs), in drug induced synaptic plasticity via transcriptional silence. Recent studies also showed that active DNA demethylation can be catalysed by ten-eleven translocation (TET) enzyme family. Therefore, uncovering the mechanisms that drug-cue association is possibly regulated by both DNMT and TET contributes to understand the transcriptional regulation of long-lasting drug behavior.

Methods: In order to study the mechanisms that DNMT and TET regulated drug-cue association, we used morphine self-administration (SA) as drug-cue operant conditioning model. (1)We explored the brain region specificity, enzyme subtype and time course of DNMT and TET expression during morphine SA. (2) We verified the important regulatory role of the key subtypes of DNMT and TET during morphine SA. (3)We preliminary explored the expression of target genes which was regulated by DNMT and TET during morphine SA.

Results: We found that: (1)The expression of DNMT3a, not DNMT3b, in the hippocampal CA1 was up-regulated during morphine SA. The expression of TET1 in the CA1 was down-regulated at the later phase of morphine SA, while the expression of TET3 in the CA1 was up-regulated at the early phase of morphine SA. The expression of DNMT and TET were not changed in the NAc shell. The expression of DNMT and TET were not changed ether in saccharin SA rats or yoked morphine rats which get morphine contingently. (2) Microinjection of DNMT inhibitor 5-aza daily into the CA1 or specifically knocking-down DNMT3a before SA training inhibited the acquisition of morphine SA; Over-expression TET1 in the CA1 also inhibited the acquisition of morphine SA to some extent, while knocking-down TET1 did not affect the acquisition. Knocking-down TET3 in the CA1 promoted the acquisition of morphine SA in the early phase. (3) We observed an up-regulation of the mRNA level of Ntrk2 and Ppplcb in DNMT3a knock-down group after 7 days morphine SA. The mRNA level of Camk2a was down regulated in TET1 over-expression group after 7 days SA.

Conclusion: These results showed that both DNMT and TET in the CA1 regulated the operant learning of opiate via regulating the expression of different target genes, which suggested that the DNA epigenetic modification may be the key regulator in the acquisition of drug addiction related behavior. Combined using DNMT inhibitors or agonist of TET with opiate in clinical treatment may be one of the new ideas for preventing the addiction.