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节，即使这些细胞包含在同一组织中。出于这个原因，我们需要测量单个细胞中mRNA的数量，以更好地了解转录机制。因此，我们开发了一种用于确定单个细胞内mRNA的确切拷贝数的方法。我们首先分离出的大脑巨细胞的滞流，因为这个神经元在记忆巩固的过程中起着关键作用的学习行为的喂养。然后，我们通过定量实时PCR确定了环AMP反应元件结合蛋白（CREB）的mRNA拷贝数。接下来，我们仍然不知道CREB的组成型表达形式是否足够，或者新合成的形式是否需要突触增强。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。本研究应用RNA干扰和实时荧光定量PCR技术对脑巨细胞CREB进行了研究。脑巨细胞采用CREB级联反应来增强神经元如B1运动神经元的突触。将CREB激活型（CREB 1）或阻遏型（CREB 2）的siRNA分别注入脑巨细胞，观察B1运动神经元EPSP波幅的变化。CREB 1和CREB 2 mRNA的量的变化也在大脑巨细胞中进行了检查。注射CREB 1 siRNA后15 min，EPSP振幅被抑制，而注射CREB 2 siRNA后60 min，EPSP振幅被增强。在后一种情况下，通过实时PCR证实CREB 2 mRNA的量减少。我们的研究结果表明，CREB的重新合成形式需要在几十分钟内的突触增强记忆巩固。

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.