Novel Molecular Tools for Transsynaptic Circuit Analysis

用于突触电路分析的新型分子工具

• 批准号: 7229842
• 负责人: HAIG S KESHISHIAN
• 金额: $ 17.55万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-03-01 至 2009-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7229842
• 关键词: Behavior; Brain; Carrier Proteins; Cells; Chimeric Proteins; Chromosome Pairing; Development; Disease; Drosophila genus; Gene Activation; Gene Expression; Goals; Green Fluorescent Proteins; Injury; Label; Maps; Methods; Molecular; Molecular Genetics; Neurons; Neurosciences; Pattern; Plant Lectins; Property; Proteins; Public Health; Reporter Genes; Signal Transduction; Structure; Synapses; System; Tetanus Toxin; Tracer; Transcription Coactivator; Transcriptional Activation; Transgenic Organisms; barley lectin; base; experience; improved; in vivo; neural circuit; novel; relating to nervous system; research study; response; tool; vector

DESCRIPTION (Provided by the Applicant): A major goal of neuroscience is to characterize neuronal connectivity in the CNS in order to understand the mechanisms that govern the development and function of neural circuits. This goal requires new tools for the direct labeling of neural circuits, using molecular genetic approaches for trans-synaptic transfer. A further goal is to generate molecular constructs that control gene expression within all the cells of specific neural circuits. This project will advance us toward these goals. There are three specific objectives: First, to generate novel transgenic lines where transsynaptic labeling constructs are expressed in vivo in specific targeted neurons. The constructs are based on both plant lectins (WGA and BL) and the tetanus toxin C chain (TTC), molecules that cross synapses. The carrier proteins will convey various cargos, such as GFP and RFP, across synapses in either a forward or reverse direction. Second, we will identify the essential domains of these carrier proteins through structure-function analysis, generating novel fusion proteins with improved carrier properties. Finally, we will develop a method for controlling gene expression within specific neural circuits, which we term "Inducible Transsynaptic Expression with Amplification" (ITEM). This entails generating molecules for transsynaptic transcriptional activation, based on fusions between known transsynaptic carriers and the GAL4::VP16 transcriptional activator. With ITEM, gene expression in a specific neural circuit is achieved when the transcriptional activator is transferred sequentially from cell to cell. By placing the transsynaptic transcriptional activator under UAS control, the molecule activates its own expression in recipient cells, resulting in amplification of the signal as it passes from cell to cell. The ITEM method will be a powerful tool for mapping neural connectivity, as well as for experimentally controlling the development or function of neural circuits. Although the studies will be pioneered in Drosophila, successful transsynaptic reporter gene activation would provide a proof of principle that could then be adapted for higher systems. Relevance to public health: In this project we will create new tools for looking at how nerve cells form connections with one another. These will help us understand how different patterns of connections give rise to different behaviors, and determine how nerve cells modify their connections in response to experience or to changes in brain function caused by injury or disease.

描述（由申请人提供）：神经科学的一个主要目标是表征中枢神经系统中的神经元连接，以了解控制神经回路发育和功能的机制。这一目标需要新的工具来直接标记神经回路，使用分子遗传学方法进行跨突触转移。进一步的目标是在特定神经回路的所有细胞中产生控制基因表达的分子结构。这个项目将推动我们实现这些目标。有三个特定的目标：首先，产生新的转基因系，其中跨突触标记结构在体内特定的靶向神经元中表达。该结构基于植物凝集素（WGA和BL）和破伤风毒素C链（TTC），这是交叉突触的分子。载体蛋白将以正向或反向的方式在突触间传递各种货物，如GFP和RFP。其次，我们将通过结构-功能分析确定这些载体蛋白的基本结构域，生成具有改进载体特性的新型融合蛋白。最后，我们将开发一种在特定神经回路中控制基因表达的方法，我们称之为“可诱导的跨突触表达扩增”（ITEM）。这需要基于已知的跨突触载体和GAL4::VP16转录激活因子之间的融合产生跨突触转录激活分子。在ITEM中，当转录激活子在细胞间依次传递时，基因在特定神经回路中的表达得以实现。通过将跨突触转录激活因子置于UAS控制下，该分子激活其自身在受体细胞中的表达，导致信号在细胞间传递时被放大。ITEM方法将成为绘制神经连通性以及实验控制神经回路发育或功能的有力工具。虽然这项研究将首先在果蝇身上进行，但成功的跨突触报告基因激活将为随后适用于更高系统的原理提供证据。与公共卫生相关：在这个项目中，我们将创造新的工具来观察神经细胞是如何形成相互联系的。这将帮助我们了解不同的连接模式如何产生不同的行为，并确定神经细胞如何根据经验或损伤或疾病引起的脑功能变化修改它们的连接。