Analysis of differentially expressed genes depending on the synaptic long-term potentiation

我们已经开发了一种用于鸡分离的鸡大脑神经元的培养系统，在该系统中，谷氨酸能突触发生随着胚胎等效天数（胚胎年龄和体外天数的总和）的传递而进行。一般来说，谷氨酸能突触包含两种类型的嗜离子性谷氨酸受体，n-甲基- d -天冬氨酸受体（NMDARs）和非nmda受体。然而，我们发现这些受体在突触位点的比例受突触后神经元发育阶段的控制。接下来，我们证实了在该培养体系中短暂暴露于Mg^<2+>培养基后，可以诱导长期增强（LTP）。NMDARs的特异性拮抗剂和蛋白质和RNA合成抑制剂抑制了这种增强作用。在增强的神经元中，突触传递在突触后微兴奋电流的频率上增加，但在振幅上没有增加。此外，在条件培养基中，一种促进增强的扩散分子(s)似乎更多地参与其中。然后，我们建立了四种分离差异表达cdna的方法。首先尝试了亲和素-生物素结合的减法和差分显示系统，但发现这两种方法都存在高水平的假阳性。其次，我们尝试了抑制减法杂交（SSH）。SSH方法通过结合杂交步骤克服了mPNA丰度差异的问题。我们通过northern blot分析证明了该方法的有效性。最后，我们介绍了一种新的方法，即限制性内切标记cDNA扫描（RLCS），该方法可以定量地同时显示许多cDNA物种的二维凝胶点。在RLCS中，直接将mRNA转化为cDNA，无需PCR扩增，排除假阳性，获得的凝胶点具有较高的重复性。使用后两种方法，我们现在分离依赖于LTP的差异表达基因。少

We have developed a system for the culture of chick dissociated chick cerebral neurons in which glutamatergic synaptogenesis proceeds with the passages of embryonic equivalent days (the sum of the embryonic age and days in vitro). In general, glutamatergic synapses contain two types of ionotropic glutamate receptor, N-metyl-D-aspartate receptors (NMDARs) and non-NMDA receptors. Whereas, we showed that the ratio of these receptors at synaptic sites was controlled by the developmental stage of postsynaptic neurons. Next, we confirmed that long-term potentiation (LTP) was induced in this culture system after brief exposure to Mg^<2+> -free medium. The potentiation was inhibited by a specific antagonist of NMDARs and by inhibitors of protein and RNA synthesis. In the potentiated neurons, synaptic transmission increased in terms of the frequency of miniature excitatory postsynaptic currents but not in terms of amplitude. In addition, a diffusible molecule (s) that promoted the potentiation … More appeatred to be involved in conditioned medium. Then, we have established four methods to isolate differentially expressed cDNAs. At first, the subtraction method accomplished by avidin-biotin binding and the differential display system were attempted, however, both methods were found out to have a high level of false positives. Secondarily, we tried the suppression subtractive hybridization (SSH). The SSH method overcomes the problem of differences in mPNA abundance by incorporating a hybridization step. We demonstrate the effectiveness of this method by northern blot analysis. Finally, we have introduced a new method, designated restriction landmark cDNA scanning (RLCS), which displays many cDNA species quantitatively and simultaneously as two-dimensional gel spots. In RLCS,mRNA is simply converted to cDNA without PCR amplification, false positives were excluded and the gel spots were obtained with higher reproducibility. Using the latter two methods, we are now isolating differentially expressed genes depending on the LTP. Less