CDNA LIBRARIES PREPARED FROM PURE NEURONAL PROCESSES

这一建议的具体目的是从神经元突起建立cDNA文库，以研究mRNAs在树突和/或轴突中的定位。许多研究表明，mRNAs定位于神经元突起并调节这些mRNAs的翻译是神经元基因表达的重要机制。为了开始研究这种形式的基因表达，我们将创建分离的神经元突起的准备工作，开发从这些准备工作中收集的严格有限的mRNA来创建cDNA文库的方法，并开发分析在基础状态和刺激状态下神经元突起中的mRNA物种的表达和积累的方法。我们将使用培养的海兔感觉神经元和培养的小鼠海马神经元。海兔神经元的独特优势是可以手动从培养的神经元中取出细胞体和胶质细胞，留下纯粹的神经元突起作为构建cDNA文库的起始材料。啮齿动物海马神经元可以培养在细胞膜上，使神经元轴突和树突能够穿透细胞膜中的孔洞，从而与细胞体分离。利用这些准备，我们将：1.开发纯神经元突起的准备2.开发从这些神经元突起创建cDNA文库的方法3.开发测试转录产物在未刺激和刺激的神经元突起中的表达和局部积累的方法。

The specific aim of this proposal is to create cDNA libraries from neuronal processes in order to study the localization of mRNAs to dendrites and/or axons. A number of studies have indicated that localization of mRNAs to neuronal processes and regulated translation of these mRNAs serves as an important mechanism of gene expression in neurons. To begin to study this form of gene expression, we will create preparations of isolated neuronal processes, develop methodologies for creating cDNA libraries from the severely limited amount of mRNA collectable from these preparations, and develop methodologies for analyzing the representation and accumulation of mRNA species in neuronal processes in both basal and stimulated states. We will use cultured Aplysia sensory neurons and cultured mouse hippocampal neurons. The unique advantage of Aplysia neurons is that cell bodies and glia can be manually removed from cultured neurons, leaving pure neuronal processes as a starting material for cDNA library construction. Rodent hippocampal neurons can be cultured on membranes such that the neuronal axons and dendrites can penetrate through the pores in the membrane and can therefore be separated from cell bodies. Using these preparations we will: 1. Develop preparations of pure neuronal processes 2. Develop methodologies for creating cDNA libraries from these neuronal processes 3. Develop methodologies to test the representation and localized accumulation of transcripts in unstimulated and stimulated neuronal processes.