Relation between change in animal behavior and change in transcription factor in a single neuron

基因表达在每个细胞中受到不同的调控，即使这些细胞包括在同一组织中。因此，我们需要测量单个细胞中的mRNAs的数量，以更好地了解转录机制。因此，我们开发了一种方法来确定单个细胞内mRNAs的确切拷贝数。我们首先分离了Lymneea stagnalis的大脑巨细胞，因为这个神经元在学习的摄食行为的记忆巩固过程中扮演着关键角色。然后，我们用实时定量聚合酶链式反应测定了环状AMP反应元件结合蛋白(CREB)的mRNAs的拷贝数。接下来，我们仍然不知道CREB的组成性表达形式是否足够，还是新合成的形式是突触增强所必需的。此外，如果需要新合成的形式，我们必须确定CREB从其mRNA翻译的时间。我们应用RNA干扰和实时定量聚合酶链式反应的方法对大脑巨细胞CREB进行了检测。大脑巨细胞使用CREB级联来增强对神经元的突触，如B1运动神经元。将CREB激活型(CREB1)或抑制型(CREB2)的siRNA注入大鼠大脑巨细胞，检测B1运动神经元记录到的EPSP波幅的变化。同时观察脑巨细胞CREB1和CREB2mRNAs表达的变化。注射CREB1siRNA后15min，EPSP波幅受到抑制，而在注射CREB2siRNA后60min，EPSP波幅增强。在后者中，实时定量聚合酶链式反应证实了CREB2基因表达的减少。我们的结果表明，在记忆巩固的突触增强中，CREB的从头合成形式在数十分钟内是必需的。

Gene expression is differently regulated in every cell even though the cells are included in the same tissue. For this reason, we need to measure the amount of mRNAs in a single cell to understand transcription mechanism better. We therefore developed a procedure for determining the exact copy numbers of mRNAs within a single cell. We first isolated the cerebral giant cell of Lymnaea stagnalis as this neuron plays a key role in the process of memory consolidation of a learned behavior of feeding. We then determined the copy numbers of mRNAs for the cyclic AMP-responsive element binding proteins (CREBs) by quantitative real-time PCR. Next, we still do not know whether the constitutively expressed forms of CREB are enough or the newly synthesized forms are required for the synaptic enhancement. In addition, if the newly synthesized forms are needed, we must determine the time for translation of CREB from its mRNA. We applied the methods of RNA interference and real-time PCR to CREB in the cerebral giant cells. The cerebral giant cells employ a CREB cascade for the synaptic enhancement to neurons such as the B1 motoneurons. We injected the siRNA of the activator type of CREB (CREB1) or the repressor type (CREB2) into the cerebral giant cells and examined the changes in amplitude of EPSP recorded in the B1 motoneurons. The changes in the amounts of CREB1 and CREB2 mRNAs were also examined in the cerebral giant cells. The EPSP amplitude was suppressed 15 min after injection of CREB1 siRNA, whereas that was augmented 60 min after injection of CREB2 siRNA. In the latter case, the decrease in the amount of CREB2 mRNA was confirmed by real-time PCR. Our results showed that the de novo synthesized forms of CREB are required within tens of minutes for the synaptic enhancement in memory consolidation.