Developing Genetic Reagents for the Dissection of Dopaminergic Circuitry

 描述(申请人提供)：多巴胺(DA)是一种关键的神经递质，从线虫到人类都是如此，它调节着各种各样的行为，包括学习和记忆、求爱行为、寻求奖励和睡眠/醒来节奏。DA神经回路的功能障碍反过来又会导致帕金森氏症、抑郁症和药物成瘾等疾病。然而，DA神经回路如何对这些行为或紊乱起作用还不是很清楚。这项工作的目标是开发、表征和利用新型遗传试剂来解剖黑腹果蝇的DA神经网络。果蝇是进行这些研究的理想系统，因为果蝇只有~250个DA神经元来驱动许多与哺乳动物相同的DA依赖行为。此外，苍蝇非常容易进行神经回路分析，因为它们的世代时间短，遗传技术和资源发达，而且很容易大量维持。在之前的工作中，我们创造了新的转基因果蝇品系，使我们能够操纵DA神经元的不同子集。利用这些线，我们识别了一对DA神经元，它们通过投射到并直接抑制促进睡眠的回路来促进觉醒。此外，我们最近开发了一种新的遗传方法，CLAMP(细胞跨膜伙伴标记)，它允许仅基于连接模式来识别、形态特征和功能操纵神经元。在这里，我们建议建立新的转基因DA驱动系，用于识别、表征和绘制苍蝇脑中DA神经网络的连接图。首先，基于在DA细胞中表达的不同基因的基因组增强子，我们将产生新的DA转基因果蝇品系。其次，我们将筛选已建立的Gal4系在DA细胞亚群中的表达。通过使用组合遗传交叉方法，这些苍蝇品系将共同产生约22,600个不同的标记模式，其中包含DA神经元的小亚集。这些线路将向科学界提供，以促进对DA神经网络的功能分析。第三，我们将通过1)识别和命名单个DA细胞和2)使用计算机跟踪技术结合注册到标准大脑模型来标记投影模式，来创建飞行大脑中DA神经元的综合数据库。第四，通过使用钳夹方法系统地映射这些DA神经元的连接，我们将建立一个详细的果蝇DA神经网络模型。了解果蝇DA回路如何调节不同的行为，将有助于深入了解包括人类在内的哺乳动物的相关机制，从而为特定神经和精神疾病的基于回路的治疗干预奠定基础。

 DESCRIPTION (provided by applicant): Dopamine (DA) is a critical neurotransmitter, conserved from C. elegans to man, that regulates a wide variety of behaviors, including learning and memory, courtship behaviors, reward-seeking, and sleep/wake rhythms. Dysfunction of DA neural circuits in turn contributes to disorders such as Parkinson's disease, depression, and drug addiction. Yet how DA neural circuits contribute to these behaviors or disorders is not well-understood. The goal of this work is to develop, characterize, and utilize novel genetic reagents for the dissection of the DA neural network in Drosophila melanogaster. Drosophila is an ideal system to carry out these studies, as fruit flies have only ~250 DA neurons to drive many of the same DA-dependent behaviors found in mammals. Moreover, flies are highly tractable to neural circuit analyses, as they have a short generation time, well- developed genetic techniques and resources, and can be easily maintained in large numbers. In previous work, we generated novel transgenic fly lines that allowed us to manipulate distinct subsets of DA neurons. Using these lines, we identified a single pair of DA neurons that promote arousal by projecting to and directly inhibiting a sleep-promoting circuit. In addition, we have recently developed a novel genetic method, CLAMP (Cell Labeling Across Membrane Partners), which allows for identification, morphological characterization, and functional manipulation of neurons based solely on connectivity patterns. Here, we propose to generate novel transgenic DA driver lines, which will be used for the identification, characterization, and connectivity mapping of the DA neural network in the fly brain. First, we will generate new DA transgenic fly lines, based on the genomic enhancers from different genes that express in DA cells. Second, we will screen established Gal4 lines for expression in subsets of DA cells. By using a combinatorial genetic intersectional approach, these fly lines will collectively generate ~22,600 distinct labeling patterns containing small subsets of DA neurons. These lines will be made available to the scientific community to facilitate functional analyses of the DA neural network. Third, we will create a comprehensive database of DA neurons in the fly brain by 1) identifying and naming individual DA cells and 2) by using computer tracing techniques combined with registration to a standard brain model to label projection patterns. Fourth, by using the CLAMP method to systematically map the connectivity of these DA neurons, we will develop a detailed model of the DA neural network in Drosophila. Understanding how DA circuits in Drosophila function to regulate different behaviors would provide insights into related mechanisms in mammals, including humans, and thus set the stage for circuit-based therapeutic interventions for specific neurological and psychiatric diseases.