Developing Genetic Reagents for the Dissection of Dopaminergic Circuitry

开发用于解剖多巴胺能回路的遗传试剂

• 批准号: 8892534
• 负责人: Christopher John Potter
• 金额: $ 24.3万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-02-01 至 2017-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8892534
• 关键词: Adult; Animal Model; Animals; Arousal; Attention; Behavior; Biological Neural Networks; Brain; Caenorhabditis elegans; Carboxy-Lyases; Cells; Communities; Complex; Computers; Coupled; Courtship; Databases; Development; Disease; Dissection; Dopa; Dopamine; Drosophila genus; Drosophila melanogaster; Drug Addiction; Engineering; Enhancers; Exhibits; Functional disorder; Generations; Genes; Genetic; Genetic Enhancer Element; Genetic Models; Genetic Techniques; Genomics; Goals; Human; Hyperactive behavior; Image; Individual; Label; Lead; Learning; Link; Locomotion; Mammals; Maps; Membrane; Memory; Mental Depression; Mental disorders; Methods; Modeling; Mus; Names; Neurologic; Neuromodulator; Neurons; Neurophysiology - biologic function; Neurotransmitters; Other Genetics; Parkinson Disease; Pattern; Reagent; Reporter; Restless Legs Syndrome; Rewards; Schizophrenia; Signal Transduction; Sleep; Staging; System; Techniques; Therapeutic Intervention; Time; Tracer; Transgenic Organisms; Tyrosine 3-Monooxygenase; Work; addiction; base; combinatorial; dopamine transporter; dopaminergic neuron; fly; genetic manipulation; genetic resource; in vivo; insight; man; nervous system disorder; neural circuit; novel; pleasure; public health relevance; research study; tool

 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.

 描述（由适用提供）：多巴胺（DA）是一种关键的神经递质，由秀丽隐杆线虫构成，它调节了各种各样的行为，包括学习和记忆，求爱行为，寻求奖励，寻求奖励和睡眠/唤醒节奏。 DA Neurocircut的功能障碍反过来导致帕金森氏病，抑郁和吸毒等疾病。然而，DA神经环路如何促进这些行为或疾病并不理解。这项工作的目的是开发，表征和利用新型遗传剂在果蝇中解剖DA神经元网络。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，可以推动许多在哺乳动物中发现的相同DA依赖性行为。此外，果蝇对神经元电路的分析高度处理，因为它们的生成时间很短，遗传技术和资源良好，并且可以很容易地保持大量。在以前的工作中，我们产生了新型的转基因飞行线，使我们能够操纵DA神经元的不同子集。使用这些线，我们确定了一对DA神经元，该神经元通过投射到并直接抑制促进睡眠的电路来促进唤醒。此外，我们最近开发了一种新型的遗传方法，即跨膜伴侣的夹具（细胞标记）），该方法仅基于连通性模式，可以识别，形态表征和功能操纵。在这里，我们建议生成新型的转基因DA驱动线，该驱动器将用于蝇脑中DA神经元网络的识别，表征和连通性映射。首先，我们将基于来自DA细胞中不同基因的基因组增强子生成新的DA转基因蝇线。其次，我们将筛选已建立的GAL4线以在DA细胞的子集中表达。通过使用组合遗传相交方法，这些蝇线将共同产生约22,600个不同的标记模式，其中包含小型DA神经元的小亚集。这些线将通过1）识别和命名单个DA细胞以及使用计算机跟踪技术结合注册到标准的大脑模型来标记投影模式来识别和命名单个DA细胞，以促进DA神经元的功能分析，以促进DA神经元的功能分析。第四，通过使用夹具方法系统地绘制这些DA神经元的连通性，我们将开发果蝇中DA神经元的详细模型。了解果蝇功能中的DA回路如何调节不同的行为，将为包括人类在内的哺乳动物的相关机制提供见解，从而为基于电路的特定神经和精神病疾病的基于电路的治疗干预措施奠定了基础。