Viral tracing of genetically defined neural circuitry

描述(申请人提供)：我的博士论文研究融合了病毒学、遗传学和神经生物学，目的是创造新的神经电路示踪剂。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了相对较好的描述，但突触通信仍然知之甚少。对这一回路的解剖需要具有标记突触连接细胞的能力的神经元示踪剂。历史上，神经示踪剂，如小麦胚芽凝集素(WGA)、纳米棒和荧光染料帮助了这项研究。这些试剂具有稀释性和可疑的示踪剂特异性的固有限制。这些问题可以通过创建一个遗传模型来避免，在这个模型中，电路中的每个细胞都会产生示踪剂，或者通过使用病毒来避免，病毒是自我复制的实体。利用小鼠视网膜，我的目标是开发新的跟踪方法，能够标记体内不同的电路类型。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，含有ASLV-A胞外区的改良狂犬病病毒将被用于靶向表达TVA的特定无长突细胞。这些细胞的突触连接可以通过生理学分析得到验证。最后，使用狂犬病病毒糖蛋白作为模板，我将设计一种逆行示踪剂，永久地和特定地标记整个电路。同时含有CRE和GFP的它将具有类似于病毒跨突触示踪剂的能力，而不会受到代谢变化的有害影响。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，以及两种新的、不同的方法来追踪中枢神经系统中的神经元回路，这两种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，如记忆力丧失、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，通过创造一个更好地剖析电路的工具，我的目标是帮助阐明认知计算的广泛性以及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.