Novel Molecular Tools for Transsynaptic Circuit Analysis

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，以便了解支配神经回路发展和功能的机制。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。另一个目标是产生控制特定神经回路所有细胞内基因表达的分子结构。这个项目将推动我们朝着这些目标前进。有三个具体的目标：第一，建立新的转基因系，其中跨突触标记构建物在体内特定的靶向神经元中表达。这些结构是基于植物凝集素(WGA和BL)和破伤风毒素C链(TTC)，这两个分子跨越突触。载体蛋白将以正向或反向通过突触运送各种货物，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本质结构域，产生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，我们将其称为“带有扩增的诱导性跨突触表达”(Item)。这需要根据已知的跨突触载体与GAL4：：VP16转录激活子之间的融合，产生跨突触转录激活的分子。有了Item，当转录激活物在细胞之间顺序转移时，特定神经回路中的基因表达就实现了。通过将跨突触转录激活因子置于UAS控制下，该分子激活了自身在受体细胞中的表达，导致信号在细胞之间传递时被放大。项法将成为绘制神经连接性图以及实验控制神经回路的发展或功能的有力工具。虽然这些研究将在果蝇中率先进行，但成功的跨突触报告基因激活将提供一个原理证明，然后可以适应更高的系统。与公共健康相关：在这个项目中，我们将创建新的工具来研究神经细胞如何相互连接。这些将帮助我们了解不同的连接模式如何导致不同的行为，并确定神经细胞如何修改它们的连接，以应对经验或因损伤或疾病导致的大脑功能变化。

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.