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BRAIN计划的重要组成部分。目前， 缺乏具有高信号强度和低毒性的基于病毒的顺行单突触追踪工具是一个缺口 在神经科学中。单纯疱疹病毒（HSV）1型毒株129（H129）是最有前途的病毒工具， 顺行神经元追踪然而，目前的转基因H129病毒版本受到以下限制： 高毒力和毒性，需要免疫染色检测的弱标记信号，以及时间依赖性 分布在多个突触上也有一个关注的方向特异性顺行 H129重组体的繁殖，因为它们可以逆行繁殖。现场的调查人员已经 积极开发改进的顺行病毒示踪剂，但进展有限。我们 已经形成了一个强大的跨学科合作团队，由病毒学家和系统神经科学家组成 开发具有高信号强度和低毒性或无毒性的顺行单突触重组H129示踪剂 用于多物种神经回路分析我们已发表的工作和初步数据建立了可行性和 建议研究的主要方法。我们将利用我们现有的细菌人工 用于快速产生重组H129载体和精确控制H129表达的基于BAC的系统。 H129有效载荷。我们有一个健全的计划，以减少病毒毒性，增强标签信号和产生变异 携带不同功能的有效载荷。我们的总体目标是创建一套新的安全，有效和有效的 顺行定向的病毒载体，其允许在特异性的单突触投射靶点中进行有效标记， 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， 以及对已定义的顺行投射网络的活动操纵。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，向 神经科学社区

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.