Detection of molecular alterations in methamphetamine-activated Fos-expressing neurons from a single rat dorsal striatum using fluorescence-activated cell sorting (FACS)
Detection of molecular alterations in methamphetamine-activated Fos-expressing neurons from a single rat dorsal striatum using fluorescence-activated cell sorting (FACS)
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DOI：
10.1111/jnc.12381
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发表时间：
2014-01-01
影响因子：
4.7
通讯作者：
Hope, Bruce T.
中科院分区：
文献类型：
--
作者：
Liu, Qing-Rong;Rubio, Francisco J.;Hope, Bruce T.
甲基苯丙胺和其他药物激活大脑中所有神经元的一小部分。我们之前开发了一种基于荧光激活细胞分选（FACS）的方法来表征在表达神经活动标记物Fos的激活神经元中选择性诱导的分子变化。然而，这种方法需要汇集许多大鼠的样本。我们现在描述了一种改良的基于FACS的方法，以表征分子改变Fos表达背侧纹状体神经元从一个单一的大鼠使用多重预扩增策略。Fos和NeuN（神经元标记物）免疫组织化学表明，在急性甲基苯丙胺注射（5 mg/kg，i. p.）而少于0.5%的神经元被盐水注射激活。我们使用FACS将NeuN标记的神经元分离为Fos阳性和Fos阴性神经元，并使用RT-qPCR评估来自少至5个Fos阳性神经元的mRNA表达。甲基苯丙胺诱导3-20倍增加的即时早期基因弧，homer-2，c-fos，fosB，及其异构体（德尔塔fosB和一个新的异构体德尔塔fosB-2）在Fos阳性，而不是Fos阴性神经元。立即早期基因mRNA的诱导是10倍低或不存在时，在单背侧纹状体匀浆的未分选样品进行评估。我们改进的方法使得研究复杂成瘾模型中药物或药物相关线索激活的神经元中独特的分子改变成为可能。
Methamphetamine and other drugs activate a small proportion of all neurons in the brain. We previously developed a fluorescence-activated cell sorting (FACS)-based method to characterize molecular alterations induced selectively in activated neurons that express the neural activity marker Fos. However, this method requires pooling samples from many rats. We now describe a modified FACS-based method to characterize molecular alterations in Fos-expressing dorsal striatal neurons from a single rat using a multiplex pre-amplification strategy. Fos and NeuN (a neuronal marker) immunohistochemistry indicate that 5-6% of dorsal striatum neurons were activated 90 min after acute methamphetamine injections (5 mg/kg, i.p.) while less than 0.5% of neurons were activated by saline injections. We used FACS to separate NeuN-labeled neurons into Fos-positive and Fos-negative neurons and assessed mRNA expression using RT-qPCR from as little as five Fos-positive neurons. Methamphetamine induced 3-20-fold increases of immediate early genes arc, homer-2, c-fos, fosB, and its isoforms (Delta fosB and a novel isoform Delta fosB-2) in Fos-positive but not Fos-negative neurons. Immediate early gene mRNA induction was 10-fold lower or absent when assessed in unsorted samples from single dorsal striatum homogenates. Our modified method makes it feasible to study unique molecular alterations in neurons activated by drugs or drug-associated cues in complex addiction models.