Dendritic Protein Synthesis in Hippocampal Neurons

海马神经元的树突状蛋白合成

• 批准号: 6460937
• 负责人: ERIN M SCHUMAN
• 金额: $ 22.63万
• 依托单位: CALIFORNIA INSTITUTE OF TECHNOLOGY
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-06-01 至 2007-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6460937
• 关键词: animal tissue; biological signal transduction; brain derived neurotrophic factor; confocal scanning microscopy; dendritic cells; electrophysiology; gene expression; glutamate receptor; green fluorescent proteins; hippocampus; long term potentiation; messenger RNA; nerve /myelin protein; neural plasticity; neural transmission; neurons; nucleic acid inhibitor; photolysis; protein biosynthesis; protein localization; protein structure function; synapses; synapsins; tissue /cell culture; transfection /expression vector

DESCRIPTION (provided by applicant): Many studies of behavioral and synaptic plasticity have demonstrated that long-lasting changes in synaptic transmission and behavior require both gene transcription and mRNA translation. To understand long-term information storage, the problem is to determine how the potentiated synapses of a single neuron become selectively modified during the long-lasting phases of synaptic plasticity. That is, how do the products of transcription and/or translation reach the modified synaptic sites without affecting the unmodified sites within the same neuron? We hypothesize that proteins synthesized locally, in dendrites, may allow for this specificity. In order to visualize dendritic protein synthesis directly, we have developed a series of reporter GFP constructs that possess a dendritic localization domain in their mRNA. Thus, the GFP message is routed to the dendrite and can be translated locally. Using this fluorescent reporter and time-lapse confocal microscopy, we have shown that BDNF, a growth factor implicated in synaptic plasticity, can stimulate dendritic protein synthesis in cultured hippocampal neurons. To extend these initial findings, we will ask whether long-term potentiation (LTP) or metabotropic-receptor-dependent long-term depression (LTD) results in dendritic protein synthesis in hippocampal slices. We will re-examine the protein synthesis dependence of LTP by examining whether protein synthesis inhibitors applied after LTP induction can affect the level of synaptic potentiation. We will also attempt to spatially restrict the area of protein synthesis inhibitor to the dendrite using a caged protein synthesis inhibitor. To determine the specificity of synthesis and destination of dendritically synthesized proteins, we will determine the spatial domain over which dendritic protein synthesis occurs when it is stimulated in a local, restricted area. We will examine this by local application of agonists in cultured neurons coupled with time-lapse imaging of the entire dendrite. In hippocampal slices we will selectively stimulate and induce plasticity in spatially distinct axon populations that make synapses with CA1 neurons. Lastly, we will determine the cellular mechanisms underlying dendritic protein synthesis.

描述（由申请人提供）： 许多关于行为和突触可塑性的研究表明， 突触传递和行为的长期变化需要两个基因 转录和mRNA翻译。了解长期信息 存储，问题是要确定如何增强突触的一个单一的 神经元在突触的长期持续阶段中被选择性地修饰， 可塑性也就是说，转录和/或翻译的产物如何 到达修饰的突触位点而不影响内部未修饰的位点， 同一个神经元？我们假设蛋白质是局部合成的， 树突，可能允许这种特异性。为了观察树突状细胞 蛋白质合成直接，我们已经开发了一系列的报告GFP 在其mRNA中具有树突定位结构域的构建体。因此，在本发明中， GFP消息被路由到树突并且可以被本地翻译。使用 这种荧光报告和延时共聚焦显微镜，我们已经表明， BDNF是一种与突触可塑性有关的生长因子， 培养海马神经元树突状蛋白的合成。扩展这些 初步研究结果，我们将问是否长时程增强（LTP）或 代谢型受体依赖性长期抑制（LTD）导致树突状细胞 海马切片中的蛋白质合成。我们会重新检查蛋白质 通过检测蛋白质合成抑制剂是否 在LTP诱导后应用可影响突触增强的水平。我们 还将尝试在空间上限制蛋白质合成抑制剂的区域， 用笼状蛋白质合成抑制剂将其转移到树突上确定 树突状合成蛋白质的合成和目的地的特异性， 我们将确定树突状蛋白合成的空间域 当它在一个局部的、受限制的区域受到刺激时就会发生。我们将对此进行研究 通过在培养的神经元中局部应用激动剂， 整个树突的成像。在海马切片中，我们将选择性地 刺激和诱导空间上不同轴突群体的可塑性， 与CA 1神经元形成突触。最后，我们将确定细胞 树突状蛋白合成的潜在机制。