NIDA Intramural Research Program, NIH/DHHS, Baltimore, Maryland, USA.
J Neurochem. 2014 Jan;128(1):173-85. doi: 10.1111/jnc.12381. Epub 2013 Aug 21.
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 (ΔfosB and a novel isoform ΔfosB-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. Methamphetamine and other drugs activate a small proportion of all neurons in the brain. We here report an improved method to characterize molecular alterations induced selectively in activated neurons that express the neural activity marker Fos. We used FACS along with targeted PCR pre-amplification to assess acute methamphetamine-induced gene expression from as few as 5 Fos-expressing neurons from a single rat dorsal striatum. Methamphetamine induced 3-20-fold increases of immediate early genes (IEGs) in Fos-positive but not Fos-negative neurons. Targeted PCR pre-amplification makes it feasible to study unique molecular alterations in neurons activated by drugs or drug-associated cues in complex addiction models.
冰毒和其他毒品会激活大脑中一小部分神经元。我们之前开发了一种基于荧光激活细胞分选（FACS）的方法，用于鉴定选择性地在表达神经活动标志物 Fos 的激活神经元中诱导的分子变化。然而，这种方法需要从许多大鼠中收集样本。现在，我们描述了一种改良的基于 FACS 的方法，使用多重预扩增策略，从单个大鼠的 Fos 表达背侧纹状体神经元中鉴定分子变化。Fos 和 NeuN（神经元标志物）免疫组织化学表明，急性冰毒注射（5mg/kg，ip）90 分钟后，背侧纹状体中有 5-6%的神经元被激活，而生理盐水注射仅激活不到 0.5%的神经元。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 从少至 5 个 Fos 阳性神经元中评估 mRNA 表达。冰毒诱导 Fos 阳性神经元中即时早期基因 arc、 Homer-2、c-fos、fosB 及其同种型（ΔfosB 和一种新型同种型ΔfosB-2）的 3-20 倍增加，但 Fos 阴性神经元中无此变化。在从单个背侧纹状体匀浆的未分选样品中评估时，即时早期基因 mRNA 诱导降低了 10 倍或消失。我们的改良方法使得在复杂成瘾模型中，研究药物或与药物相关的线索激活的神经元中的独特分子变化成为可能。
