Viral tracing of genetically defined neural circuitry

基因定义的神经回路的病毒追踪

• 批准号: 7809977
• 负责人: KEVIN T BEIER
• 金额: $ 3.24万
• 依托单位: HARVARD MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-02-01 至 2013-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7809977
• 关键词: Action Potentials; Amacrine Cells; Blindness; Cell Culture Techniques; Cells; Chimeric Proteins; Cognitive; Communication; Detection; Dissection; Engineering; Extracellular Domain; Fluorescent Dyes; Genetic; Genetic Models; Genetic Recombination; Goals; In Vitro; Investigation; Label; Light; Memory Loss; Metabolic; Methods; Mus; Neurobiology; Neurons; Optic Nerve; Pathology; Photons; Physiological; Population; Rabies virus; Reagent; Recording of previous events; Research; Retina; Site; Specificity; Synapses; Testing; Tissue Microarray; Tracer; Viral; Viral Proteins; Virus; Virus Diseases; Wheat Germ Agglutinins; cell type; design; ganglion cell; in vivo; neural circuit; novel; rabies virus glycoprotein G; relating to nervous system; tool; virology

DESCRIPTION (provided by applicant): My doctoral dissertation research integrates virology, genetics, and neurobiology with the aim of creating novel neural circuitry tracers. While general information flow in the retina from photon detection to action potentials through the optic nerve has been relatively well characterized, synaptic communication remains rather poorly understood. The dissection of this circuitry requires neuronal tracers that have the ability to label cells that are synaptically connected. Historically, neural tracers such as wheat germ agglutinin (WGA), nanobeads, and fluorescent dyes have aided this investigation. These reagents have the inherent limitations of dilution and questionable tracer specificity. These problems can be obviated either by creating a genetic model in which each cell in a circuit produces the tracer, or by using viruses, which are self- replicating entities. Using the murine retina, I aim to develop new tracing methods capable of labeling distinct circuit types in vivo. I will first develop a tool that permits viral Infection in small populations of a chosen cell type. Employing the specificity of the TVA/ASLV-A Interaction used by Wickersham and colleagues, a modified rabies virus containing the ASLV-A extracellular domain will be used to target specific TVA-expressing amacrine cells using this tool. The synaptic connectivity of these cells can be verified by physiological analyses. Lastly, using the rabies virus glycoprotein as a template, I will design a retrograde tracer that labels entire circuits permanently and specifically. Containing both Cre and GFP, it will have capabilities similar to viral transsynaptic tracers without the deleterious effects of metabolic alterations. This research will provide neuroscientists with a new tool for examining lineage tracing and two novel, distinct methods for tracing neuronal circuits in the CNS that will provide synaptic connectivity information in neural circuits, and will shed light on the relatively simple yet elegant circuitry of the retina. Fixing problems associated with aberrant neural circuitry, such as memory loss, blindness, and neurodeneratlon requires a fundamental understanding of the normal activites of these circuits. Therefore, by creating a tool to better dissect circuitry, my goal is to help elucidate the vastness of cognitive computation and the pathologies associated with abnormal circuitry function.

描述（由申请人提供）：我的博士论文研究整合了病毒学、遗传学和神经生物学，旨在创造新型神经回路示踪剂。虽然视网膜中从光子检测到通过视神经的动作电位的一般信息流已经得到相对较好的表征，但突触通讯仍然知之甚少。对该电路的剖析需要能够标记突触连接的细胞的神经元示踪剂。从历史上看，小麦胚芽凝集素 (WGA)、纳米珠和荧光染料等神经示踪剂曾帮助过这项研究。这些试剂具有稀释的固有局限性和令人怀疑的示踪剂特异性。这些问题可以通过创建一个遗传模型来避免，其中电路中的每个细胞都会产生示踪剂，或者通过使用病毒（一种自我复制的实体）。我的目标是利用小鼠视网膜开发新的追踪方法，能够在体内标记不同的电路类型。我将首先开发一种工具，允许在选定的细胞类型的小群体中进行病毒感染。利用 Wickersham 及其同事使用的 TVA/ASLV-A 相互作用的特异性，包含 ASLV-A 胞外结构域的改良狂犬病病毒将用于使用该工具来靶向表达 TVA 的特定无长突细胞。这些细胞的突触连接可以通过生理分析来验证。最后，我将使用狂犬病病毒糖蛋白作为模板，设计一种逆行示踪剂，永久且特异性地标记整个电路。它同时含有 Cre 和 GFP，具有与病毒突触示踪剂类似的功能，而不会产生代谢改变的有害影响。这项研究将为神经科学家提供一种用于检查谱系追踪的新工具，以及两种用于追踪中枢神经系统中神经元回路的新颖、独特的方法，这些方法将提供神经回路中的突触连接信息，并将揭示相对简单而优雅的视网膜回路。解决与异常神经回路相关的问题，例如记忆丧失、失明和神经退化，需要对这些回路的正常活动有基本的了解。因此，通过创建一种工具来更好地剖析电路，我的目标是帮助阐明大量的认知计算以及与异常电路功能相关的病理学。