Dendritic Protein Synthesis in Hippocampal Neurons

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这两个基因 转录和信使核糖核酸翻译。了解长期信息 存储，问题是如何确定单个突触的增强 神经元在突触的长时间阶段变得选择性地改变 可塑性。也就是说，转录和/或翻译的产物是如何 到达修改的突触位置而不影响其中的未修改的位置 是同一个神经元吗？我们假设本地合成的蛋白质，在 树突，可能允许这种特异性。为了可视化树枝状结构 蛋白质直接合成，我们开发了一系列报告绿色荧光蛋白 在其mRNA中具有树突状定位结构域的构建物。因此， GFP消息被路由到树枝状结构，并且可以在本地翻译。vbl.使用 这个荧光报告器和延时共聚焦显微镜，我们已经展示了 脑源性神经营养因子，一种与突触可塑性有关的生长因子，可以刺激 培养的海马神经元中树突状蛋白的合成。要扩展这些功能 最初的发现，我们会问长时程增强(LTP)或 代谢性受体依赖型长期抑郁(LTD)导致树突状细胞 海马片的蛋白质合成。我们会重新检查蛋白质 检测蛋白质合成抑制物对LTP合成的依赖性 LTP诱导后应用可影响突触增强水平。我们 也将尝试在空间上限制蛋白质合成抑制物的区域 用笼子里的蛋白质合成抑制剂将其转化为树突。要确定 树枝状合成蛋白的合成和目的地的特异性， 我们将确定树突状蛋白质合成的空间域 当它在局部的受限区域受到刺激时发生。我们将对此进行研究 在培养的神经元上局部应用激动剂结合时间推移 整个树突的成像。在海马区切片中，我们将选择性地 刺激和诱导空间上不同的轴突群体的可塑性 与CA1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。

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.