Purpose

Repeated methamphetamine (METH) administration in mice readily produces behavioural sensitization, but the underlying mechanisms remain elusive. The present research aimed to identify new targets affecting METH-induced behavioural sensitization.

Methods

We first established a mouse model of METH-induced behavioural sensitization. To characterize the animal model, we performed behavioural tests at different stages of behavioural sensitization and simultaneously detected changes in several neurotransmitters in the prefrontal cortex (PFC). Next, we perfromed RNA sequencing (RNA-seq) to screen new targets, which were subsequently and verified by RT-PCR and western blot. Finally, we confirmed the roles of the new targets in METH-induced behavioural sensitization by injection of overexpressed lentiviruses and further detected related protein levels by western blot and histological changes by haematoxylin and eosin (HE) staining.

Results

We successfully established a mouse model of METH-induced behavioural sensitization. The locomotor activities of the mice changed at different stages of sensitization, accompanied by changes in the levels of DA, 5-HT, GABA and glutamate. For RNA-seq analysis, we chose Fas as target, meanwhile, we chose GIT1 as target through literature. The detection of gene expression by RT-PCR indicated that METH-sensitized mice exhibited decreased levels of Fas, MEK1 and CREB and increased levels of Erk1/2 in the PFC. Western blot analysis revealed decreased Fas, GIT1, MEK1 and phosphorylated CREB levels and increased phosphorylated Erk1/2 levels in METH-sensitized mice. Injection of Fas and GIT1 injection showed that overexpression of Fas and GIT1 inhibited the induction of METH sensitization and reversed the changes in neurotransmitter levels and related protein levels, including MEK1, phosphorylated CREB and phosphorylated Erk1/2, in METH-sensitized mice. Overexpression of Fas and GIT1 also reduced histological lesions induced by METH.

Conclusion

The findings indicated that the development of behavioural sensitization to METH may be mediated by Fas and GIT1 through the MEK1-Erk1/2-CREB pathway.

中文翻译：

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前额叶皮层中的 Fas 和 GIT1 信号传导介导小鼠对甲基苯丙胺的行为敏化。

目的

在小鼠中重复施用甲基苯丙胺 (METH) 很容易产生行为致敏，但潜在的机制仍然难以捉摸。本研究旨在确定影响 METH 诱导的行为敏化的新目标。

方法

我们首先建立了 METH 诱导的行为致敏的小鼠模型。为了表征动物模型，我们在行为敏化的不同阶段进行了行为测试，并同时检测了前额叶皮层 (PFC) 中几种神经递质的变化。接下来，我们进行了 RNA 测序 (RNA-seq) 以筛选新的目标，随后通过 RT-PCR 和蛋白质印迹进行验证。最后，我们通过注射过表达的慢病毒证实了新靶点在 METH 诱导的行为敏化中的作用，并通过蛋白质印迹进一步检测了相关蛋白质水平，并通过苏木精和伊红 (HE) 染色进一步检测了组织学变化。

结果

我们成功建立了 METH 诱导的行为致敏小鼠模型。小鼠的运动活动在不同致敏阶段发生变化，伴随着DA、5-HT、GABA和谷氨酸水平的变化。对于 RNA-seq 分析，我们选择 Fas 作为目标，同时，我们通过文献选择 GIT1 作为目标。RT-PCR检测基因表达表明，METH致敏小鼠PFC中Fas、MEK1和CREB水平降低，Erk1/2水平升高。蛋白质印迹分析显示，在 METH 致敏小鼠中，Fas、GIT1、MEK1 和磷酸化 CREB ​​水平降低，磷酸化 Erk1/2 水平增加。Fas和GIT1注射液的注射表明Fas和GIT1的过表达抑制了METH致敏小鼠的METH致敏的诱导，并逆转了METH致敏小鼠神经递质水平和相关蛋白水平的变化，包括MEK1、磷酸化CREB和磷酸化Erk1/2。Fas 和 GIT1 的过度表达也减少了 METH 诱导的组织学损伤。

结论

研究结果表明，对 METH 的行为敏感的发展可能是由 Fas 和 GIT1 通过 MEK1-Erk1/2-CREB ​​途径介导的。