建立灵敏、减毒、级数可控的顺行跨突触VSV病毒示踪系统

Understanding the wiring diagrams of brain network is the basis to reveal the principle of the brain function and to resolve the mechanism of brain diseases. Trans-synaptic tracing with neurotropic viruses has become one of the most effective technologies to dissect the neural circuits. Although, the retrograde transsynaptic tracing with recombinant Rabies and Pseudorabies virus has been broadly applied in neuroscience, the use of the anterograde trans-ssynaptic tracing was limited for the lack of effective viral tools. The recombinant vesicular stomatitis virus has shown high efficiency in anterograde trans-synaptic tracing, which also has various features, such as simple genomic structure, clear molecular mechanism, safety and high expression of exogenous gene, which are key points needed to developing a fine anterograde trans-synaptic tracer. However, there are still several defects that have to be overcome, such as high neurotoxicity, low efficiency of trans-monosynaptic labeling, hard to recovery et al. This project will intend to develop new viral tools that could anterograde trace in neural circuit in the way of trans-synaptic-step controllable based on the modified version of VSV and the helper AAV system. Meanwhile, this project will intend to establish a set of strategies for anterograde and trans-mono/multi synaptic tracing and to dissect the neural circuit of the olfactory system in transgenic mice.

解析大脑神经网络是认识大脑工作原理和揭示脑疾病发生发展机理的前提。基于嗜神经病毒发展而来的跨突触追踪技术是揭示大脑神经网络结构的最有效手段之一。目前，针对输入神经网络的结构解析已有较成熟的工具病毒系统，而针对输出神经网络的结构解析尚缺乏有效工具和方法。近年发现可顺行示踪的水泡性口炎病毒(vesicular stomatitis virus, VSV)具备基因组小、分子机制明确、操作安全、外源基因表达丰度高等优势，具备改造成更高效的顺行跨突触工具病毒的潜质。但现有VSV存在细胞毒性大、跨单突触效率低、拯救困难等问题。对此，本项目拟利用反向遗传学等技术对VSV进行减毒改造，在此基础上建立灵敏、级数可控的顺行跨多/单级突触的VSV库，及控制VSV特异性感染和跨突触的辅助AAV库。利用该新型工具病毒系统，结合转基因小鼠，建立一套顺行跨突触示踪神经环路的方法，并用于解析嗅觉系统的输出/整合网络。

解析大脑神经网络是认识大脑工作机制的前提，也是诊治脑疾病的关键。基于嗜神经病毒发展而来的跨突触追踪技术是揭示大脑神经网络结构的最有效手段之一。目前，针对输入神经网络的结构解析已有较成熟的工具病毒系统，而针对输出神经网络的结构解析尚缺乏好的工具和方法。近年来发现的可顺行跨突触的VSV重组病毒具有基因结构简单、分子机制明确、操作安全、外源基因表达丰度高等优势，具备了改造成更高效的顺行跨突触工具病毒的潜质，但仍存在细胞毒性大、跨单突触效率低、拯救难度大等问题。针对这些问题，本项目拟利用反向遗传学等技术对VSV进行进一步的减毒改造，在此基础上建立灵敏、级数可控的顺行跨多级或单级突触的VSV工具病毒系列，以及控制VSV感染和跨突触的辅助病毒系列。利用该新型工具病毒系统，结合转基因小鼠，建立一套顺行跨突触示踪神经网络的方法，并用于解析嗅觉系统的输出网络。