Analysis of Molecular Assembly of Synaptic Molecules during Development

发育过程中突触分子的分子组装分析

• 批准号: 12470001
• 负责人: OKABE Shigeo
• 金额: $ 9.22万
• 依托单位: Tokyo Medical and Dental University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 2000
• 资助国家: 日本
• 起止时间: 2000 至 2002
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-12470001/
• 关键词: synapse; hippocampus; fluorescence microscopy; postsynaptic density; dendritic spine; green fluorescent protein(GFP); intracellular calcium

Transition of excitatory synapses from dendritic shafts to spines takes place during the period of 10-20 days in culture of hippocampal pyramidal neurons. This culture system provides a good model for the study of formation and remodeling of synaptic structure. To analyze dynamic properties of multiple postsynaptic density (PSD) proteins, we generated green fluorescent protein (GFP)-tagged PSD proteins, such as PSD-95 and PSD-Zip45 (also known as Homer Ic), together with presynaptic marker synaptophysin. The first set of experiments aimed at revealing time-course of morphogenesis of dendritic spines, assembly of PSD proteins, and accumulation of synaptic vesicles at single synaptic junctions. We performed dual-wavelength, time-lapse fluorescence microscopy of living hippocampal neurons and revealed temporal correlation among these three events. Furthermore, synaptogenesis was a rapid process, which took place less than an hour. In the second research project we analyzed dynamic redistr … More ibution of PSD-Zip45 after stimulation of cultured neurons. In contrast to the stable nature of PSD-95 after stimulation, PSD-Zip45 changed its distribution bidirectionally after increase of the intracellular calcium. This result indicates the presence of molecular mechanism that drives PSD-Zip45-specific translocation by the neuronal activity. In the third set of experiments we generated transgenic mice expressing either GFP-tagged PSD-95 or PSD-Zip45 to achieve stable expression of probe molecules in the hippocampus. Using cultured neurons isolated from the transgenic hippocampus, we characterized slow remodeling of PSD-95 and PSD-Zip45 clusters for >1 week. The results indicate the presence of cAMP-dependent signaling system that regulates the average density of PSD clusters within a single cell. In summary, these three sets of experiments revealed novel molecular dynamics of PSD proteins in living neurons and regulation of the distribution of PSD proteins by complex signaling system that can be differentially activated by various neuronal activities. Less

在10-20天的海马锥体神经元培养期间，在10-20天的时间内，兴奋突触过渡到脊柱发生。该培养系统为研究突触结构的形成和重塑提供了一个良好的模型。为了分析多种突触后密度（PSD）蛋白的动态特性，我们生成了绿色荧光蛋白（GFP）标记的PSD蛋白，例如PSD-95和PSD-ZIP45（也称为Homer IC），以及突触前标记突触蛋白。第一组实验旨在揭示树突状刺的形态发生，PSD蛋白组装以及在单个合成连接处的突触蔬菜的积累。我们对活海马神经元进行了双波长，延时荧光显微镜，并揭示了这三个事件之间的暂时相关性。此外，突触发生是一个快速的过程，发生不到一个小时。在第二个研究项目中，我们分析了动态重新分配……刺激培养的神经元后，PSD-ZIP45的更多ibution。与刺激后PSD-95的稳定性相反，PSD-ZIP45在增加细胞内钙后双向改变了其分布。该结果表明存在通过神经元活性驱动PSD-ZIP45特异性易位的分子机制。在第三组实验中，我们生成了表达GFP标记的PSD-95或PSD-ZIP45的转基因小鼠，以实现海马中探针分子的稳定表达。使用从转基因海马分离的培养神经元，我们表征了PSD-95和PSD-ZIP45簇的慢速重塑，> 1周。结果表明存在依赖CAMP的信号系统，该信号系统调节单个单元内PSD簇的平均密度。总而言之，这三组实验揭示了PSD蛋白在活神经元中的新分子动力学以及通过复杂信号系统对PSD蛋白分布的调节，这些动态可以通过各种神经元活性来不同地激活。较少的

项目成果

Ebihara, T., Komiya, Y., Izumi-Nakaseko, H., Adachi-Akahane, S., Okabe, S., and Okamura, Y.: "Coexpression of a Ca_v1.2 protein lacking N-terminus and the first domain specifically suppresses L-type calcium channel activity"FEBS Letters. 529. 203-207 (200

Ebihara, T.、Komiya, Y.、Izumi-Nakaseko, H.、Adachi-Akahane, S.、Okabe, S. 和 Okamura, Y.：“缺少 N 末端的 Ca_v1.2 蛋白与第一个蛋白的共表达

Ebihara, T., Kawabata, I., Usui, S., Sobue, K., S.Okabe: "Synchronized formation and remodeling of postsynaptic densities : long-term visualization of hippocampal neurons expressing postsynaptic density proteins tagged with GFP"Journal of Neuroscience. (i

Ebihara, T.、Kawabata, I.、Usui, S.、Sobue, K.、S.Okabe：“突触后密度的同步形成和重塑：表达 GFP 标记的突触后密度蛋白的海马神经元的长期可视化”杂志

Enomoto, M., Shinomiya, K., S.Okabe: "Migration and differentiation of neural progenitor cells from two different regions of embryonic central nervous system after transplantation into the intact spinal cord"Europian journal of Neuroscience. (in press).

Enomoto, M.、Shinomiya, K.、S.Okabe：“移植到完整脊髓后胚胎中枢神经系统两个不同区域的神经祖细胞的迁移和分化”欧洲神经科学杂志。

Okabe, S., Miwa, A., H.Okado: "Spine formation and correlated assembly of presynaptic and postsynaptic molecules"Journal of Neuroscience. 21. 6105-6114 (2001)

Okabe, S.、Miwa, A.、H.Okado：“脊柱形成和突触前和突触后分子的相关组装”神经科学杂志。

Umeda, T. and S. Okabe.: "Visualizing synapse formation and remodeling : recent advances in real-time imaging of CNS synapses"Neuroscience Research. 40. 291-300 (2001)

Umeda, T. 和 S. Okabe.：“可视化突触形成和重塑：中枢神经系统突触实时成像的最新进展”神经科学研究。