pCREB-dependent transcriptional activation is a mediator of molecular neuroadaptations induced by methamphetamine self-administration in the rat dorsal striatum

Neuroplastic changes in the dorsal striatum participate in the transition from casual drug use to habitual and compulsive drug taking. These alterations might also play a critical role in the development of methamphetamine (METH) addiction. Nevertheless, the molecular substrates that underlie habitual METH consumption have yet to be elucidated. Therefore, in the present study, we examined the influence of METH self-administration on the expression of genes and proteins of interest, as potential substrates of METH-induced neuronal plasticity in the dorsal striatum. Rats self-administered METH (0.1 mg/kg/injection, i.v.) during 15 h sessions for 8 d and were euthanized after 2 h, 24 h, or 1 month of abstinence. Compared to yoked saline control, METH self-administration induced increases in the mRNA expression of the transcription factors, c-fos and fosb, the neurotrophic factor, Bdnf, and of the synaptic protein, synaptophysin (Syp) at 2 h after cessation of drug exposure. METH self-administration also caused changes in FosB, BDNF and TrkB protein levels, with increases at 2 and 24 h but decreases observed after 1 month of drug abstinence. Importantly, METH exposure caused increases in the levels of H3K4me3 and pCREB after 2 and 24 h of abstinence. Chromatin immunoprecipitation followed by qPCR was used to clarify the role of these proteins in the regulation of gene expression. We found that METH self-administration caused enrichment of pCREB, but not of H3K4me3, on the promoters of c-fos, fosb, Bdnf and Syp at 2 h after drug cessation. These data indicate that METH-induced activation of their transcription is mediated, in part, by pCREB-dependent epigenetic phenomena. Thus, METH self-administration might trigger epigenetic changes that caused alterations in the expression of genes and proteins serving as substrates for addiction-related synaptic plasticity.背侧纹状体的神经可塑性变化参与了从偶然药物使用到习惯性与强迫性服药的病理转变。此类改变可能在甲基苯丙胺（methamphetamine, METH）成瘾的发生发展中发挥关键作用。然而，介导习惯性甲基苯丙胺摄取的分子底物仍有待阐明。因此，本研究探讨了甲基苯丙胺自身给药行为对目标基因及蛋白表达的影响，以作为背侧纹状体中甲基苯丙胺诱导的神经元可塑性的潜在底物。大鼠接受15小时/次、共8天的甲基苯丙胺静脉自身给药（0.1 mg/kg/次），并分别在戒断2小时、24小时或1个月后实施安乐死。与配对盐水对照组相比，药物暴露结束后2小时，甲基苯丙胺自身给药可诱导转录因子c-fos、fosb，神经营养因子脑源性神经营养因子（brain-derived neurotrophic factor, Bdnf），以及突触蛋白突触素（synaptophysin, Syp）的mRNA表达水平升高。甲基苯丙胺自身给药还会改变FosB、脑源性神经营养因子（BDNF）及酪氨酸激酶受体B（tyrosine kinase B receptor, TrkB）的蛋白水平：在戒断2小时和24小时时表达上调，而在戒断1个月后则出现下调。值得注意的是，甲基苯丙胺暴露会在戒断2小时和24小时后升高组蛋白H3赖氨酸4三甲基化（histone H3 lysine 4 trimethylation, H3K4me3）与磷酸化环腺苷酸应答元件结合蛋白（phosphorylated cAMP response element-binding protein, pCREB）的水平。本研究采用染色质免疫共沉淀（chromatin immunoprecipitation, ChIP）联合实时定量聚合酶链式反应（quantitative real-time PCR, qPCR）的方法，明确这些蛋白在基因表达调控中的作用。研究发现，药物暴露结束后2小时，甲基苯丙胺自身给药会使pCREB而非H3K4me3在c-fos、fosb、Bdnf及Syp的启动子区域富集。上述数据表明，甲基苯丙胺诱导的上述基因转录激活，部分由依赖pCREB的表观遗传现象所介导。综上，甲基苯丙胺自身给药可能触发表观遗传改变，进而导致参与成瘾相关突触可塑性的基因及蛋白表达发生异常。