Genetically engineered anterograde monosynaptic viral tracers for multi-species neural circuit analysis

项目摘要 跨突触病毒示踪剂的开发是大脑倡议的重要组成部分。目前， 缺乏高信号强度和低毒性的基于病毒的顺行单突触示踪工具是一个空白 在神经科学方面。单纯疱疹病毒(HSV)1型129株(H129)是最有前途的病毒治疗工具 顺行神经元追踪。然而，当前版本的转基因H129病毒受到以下限制 毒力和毒性高，标记信号弱，需要免疫染色才能检测，并且依赖于时间 分布在多个突触上。还有一个令人担忧的问题是顺行的方向性 H129重组子的繁殖，因为它们可能会逆行繁殖。该领域的调查人员一直在 积极开发改进版本的顺行病毒示踪剂，但进展有限。我们 形成了一个由病毒学家和系统神经学家组成的强大的跨学科协作团队 高信号强度、毒性小或无毒性的顺行单突触重组H129示踪剂的研制 用于多物种神经电路分析。我们已发表的工作和初步数据证实了该方法的可行性和 拟议研究的关键方法。我们将利用我们已有的人工细菌 基于染色体(BAC)的重组H129载体的快速生成和精确控制系统 H129有效载荷。我们有一个合理的计划来降低病毒毒性，增强标签信号并产生变种 承载不同的功能有效载荷。我们的总体目标是创建一套新的安全、有效和有效的 顺行导向的病毒载体允许在特定的单突触投射靶点中有效地标记 神经元类型。这些新工具将产生广泛的影响，使光学成像、生理记录、 以及定义的顺行投影网络的活动操纵。为了快速共享资源，我们将创建 通过UCI病毒研究中心的服务平台将新的分子工具传播给 神经科学界。

Project Summary The development of trans-synaptic viral tracers is an important component of the BRAIN Initiative. At present, the lack of viral-based anterograde monosynaptic tracing tools with high signal strength and low toxicity is a gap in neuroscience. Herpes simplex virus (HSV) type 1 strain 129 (H129) is the most promising viral tool for anterograde neuronal tracing. However, current versions of genetically modified H129 viruses are limited by high virulence and toxicity, weak label signals that require immunostaining for detection, and time-dependent spread across multiple synapses. There is also a concern of the directional specificity of anterograde propagation of H129 recombinants, as they may propagate retrogradely. Investigators in the field have been working actively to develop improved versions of anterograde viral tracers, but progress has been limited. We have formed a strong interdisciplinary collaborative team composed of virologists and systems neuroscientists to develop anterograde monosynaptic recombinant H129 tracers with high signal strength and little or no toxicity for multi-species neural circuit analysis. Our published work and preliminary data establish the feasibility and key methodologies for the proposed research. We will capitalize on our established bacterial artificial chromosome (BAC) based system for rapid generation of recombinant H129 vectors and precise control of the H129 payload. We have a sound plan to reduce viral toxicity, enhance label signals and generate variants carrying different functional payloads. Our overall goal is to create a new set of safe, effective and validated anterograde-directed viral vectors that allow efficient labeling in monosynaptic projection targets of specific neuron types. These new tools will have a broad impact by enabling optical imaging, physiological recording, and activity manipulation of defined anterograde projection networks. For rapid resource sharing, we will create a service platform through the UCI Center for Virus Research to disseminate the new molecular tools to the neuroscience community.