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

根据突触长时程增强差异表达基因的分析

基本信息

批准号：
07808075
负责人：
UYEDA Atsuko
金额：
$ 1.28万
依托单位：
Osaka University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1995
资助国家：
日本
起止时间：
1995 至 1996
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-07808075/
关键词：
long-term potentiation cerebral neuron Dissociated culture Differential Display system Subtraction method Suppression subtractive hybridization Restriction landmark cDNA scanning RLCS法

项目摘要

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

我们已经开发了一种用于培养小鸡分离的小鸡脑神经元的系统，其中，神经元突触发生随着胚胎等效天数（胚胎年龄和体外天数的总和）的传代而进行。一般来说，谷氨酸能突触含有两种类型的离子型谷氨酸受体，N-甲基-D-天冬氨酸受体（NMDAR）和非NMDA受体。然而，我们发现这些受体在突触部位的比例受突触后神经元发育阶段的控制。接下来，我们证实了在短暂暴露于无Mg^<2+>培养基后，在该培养系统中诱导了长时程增强（LTP）。这种增强作用被NMDARs的特异性拮抗剂和蛋白质和RNA合成的抑制剂所抑制。在增强的神经元，突触传递增加的频率方面的微型兴奋性突触后电流，但不是在振幅方面。此外，促进增强的可扩散分子 ...更多信息似乎参与了条件培养基。然后，我们建立了四种分离差异表达cDNA的方法。首先，尝试了通过亲和素-生物素结合完成的消减方法和差异显示系统，然而，发现这两种方法具有高水平的假阳性。其次，我们尝试了抑制性消减杂交（SSH）。SSH方法通过结合杂交步骤克服了mPNA丰度差异的问题。我们通过北方印迹分析证明了这种方法的有效性。最后，我们介绍了一种新的方法，指定的限制性标志cDNA扫描（RLCS），它显示了许多cDNA物种的定量和同时作为二维凝胶点。在RLCS中，mRNA被简单地转化为cDNA而无需PCR扩增，排除了假阳性，并且获得了具有更高重复性的凝胶斑点。使用后两种方法，我们现在正在分离依赖于LTP的差异表达基因。少