Viral tracing of genetically defined neural circuitry

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，目的是创造新的神经回路示踪剂。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相对较好地表征了，但突触通信仍然知之甚少。这种电路的解剖需要神经元示踪剂，这种示踪剂有能力标记突触连接的细胞。历史上，神经示踪剂如小麦胚芽凝集素（WGA）、纳米粒和荧光染料都有助于这项研究。这些试剂有固有的稀释限制和可疑的示踪特异性。这些问题可以通过创建一个遗传模型来解决，在这个遗传模型中，电路中的每个细胞都会产生示踪剂，或者使用病毒，这是一种自我复制的实体。利用小鼠视网膜，我的目标是开发能够标记体内不同电路类型的新追踪方法。我将首先开发一种工具，允许在选定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，一种含有ASLV-A细胞外结构域的改良狂犬病毒将使用该工具靶向特异性表达TVA的无毛细胞。这些细胞的突触连通性可以通过生理分析来证实。最后，使用狂犬病毒糖蛋白作为模板，我将设计一个逆行示踪剂，永久地和特异性地标记整个电路。含有Cre和GFP，它将具有类似于病毒跨突触示踪剂的功能，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检查谱系追踪的新工具和两种新颖的、不同的方法来追踪中枢神经系统中的神经元回路，这将提供神经回路中的突触连接信息，并将揭示相对简单但优雅的视网膜回路。修复与异常神经回路相关的问题，如记忆丧失、失明和神经再生，需要对这些回路的正常活动有基本的了解。因此，通过创建一个工具来更好地解剖电路，我的目标是帮助阐明认知计算的浩瀚和与异常电路功能相关的病理。

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.