Requirement of new protein synthesis of a transcription factor for memory consolidation: Paradoxical changes in mRNA and protein levels of C/EBP
Requirement of new protein synthesis of a transcription factor for memory consolidation: Paradoxical changes in mRNA and protein levels of C/EBP
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DOI：
10.1016/j.jmb.2005.12.009
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发表时间：
2006-02-24
影响因子：
5.6
通讯作者：
Ito, E
中科院分区：
文献类型：
--
作者：
Hatakeyama, D;Sadamoto, H;Ito, E
一些特定的转录因子是跨物种记忆巩固所必需的。然而，目前还不清楚是否只有这些保守的转录因子的组成型表达形式的激活参与记忆巩固或其从头合成也发生在学习后。这个问题仍然没有答案，部分原因是缺乏一种有效的方法来确定单个神经元中特定mRNA的拷贝数，这使得在细胞水平上检测新的转录。在这里，我们应用了一个新开发的协议，单细胞定量实时聚合酶链反应（qRT-PCR）的单个神经元在联想学习中发挥重要作用。具体而言，我们研究了一个高度保守的转录因子，CCAAT/增强子结合蛋白（C/EBP）的mRNA和蛋白质表达水平的变化，在配对的B2运动神经元的池塘蜗牛滞流。这些颊神经元参与摄食行为的运动控制，在条件性味觉厌恶（CTA）中具有潜在的重要作用。单细胞qRT-PCR显示CTA训练后记忆巩固过程中B2运动神经元中B2C/EBP mRNA拷贝数显著减少。相比之下，等电聚焦和免疫印迹的提取物的颊神经节显示，翻译和磷酸化水平的EBP/EBP显着增加记忆巩固。C/EBP样免疫反应的B2运动神经元，这是在颊神经节的主要免疫阳性成分，也显着增加记忆巩固过程中，这表明在颊神经节的蛋白质水平增加的主要来源是B2运动神经元。因此，L. stagnalis涉及在控制进食的神经网络中快速合成和磷酸化的C/EBP以及快速分解的C/EBP mRNA，这表明所有这些过程在C/EBP巩固记忆的功能中发挥作用。(c)2005爱思唯尔有限公司保留所有权利。
Some specific transcription factors are essential for memory consolidation across species. However, it is still unclear whether only the activation of constitutively expressed forms of these conserved transcription factors is involved in memory consolidation or their de novo synthesis also occurs after learning. This question has remained unanswered partly because of the lack of an efficient method for the determination of copy numbers of particular mRNAs in single neurons, which allows the detection of new transcription at the cellular level. Here we applied a newly developed protocol of single-cell quantitative real-time polymerase chain reaction (qRT-PCR) to single neurons playing an important role in associative learning. Specifically, we examined the changes in the mRNA and protein expression levels of a highly conserved transcription factor, CCAAT/enhancer binding protein (C/EBP), in the paired B2 motoneurons of the pond snail Lymnaea stagnalis. These buccal neurons are involved in the motor control of feeding behavior, with a potentially important role in conditioned taste aversion (CTA). Single-cell qRT-PCR revealed a significant decrease in LymC/EBP mRNA copy numbers in the B2 motoneurons during memory consolidation after CTA training. By contrast, isoelectric focusing and immunoblotting of extracts of the buccal ganglia showed that translation and phosphorylation levels of LymC/EBP significantly increased during memory consolidation. The C/EBP-like immunoreactivity in the B2 motoneurons, which are the major immunopositive component in the buccal ganglia, also significantly increased during memory consolidation, suggesting that the main source of increase in the level of protein in the buccal ganglia are the B2 motoneurons. Thus, early memory consolidation after CTA learning in L. stagnalis involves both the rapid synthesis and phosphorylation of LymC/EBP as well as the rapid breakdown of LymC/EBP mRNA in the neural network controlling feeding, suggesting that all of these processes play a role in the function of C/EBP in memory consolidation. (c) 2005 Elsevier Ltd. All rights reserved.