Development of novel transsynaptic tracers for use in the central nervous system

描述（由申请人提供）：中枢神经系统（CNS）神经元之间的连接映射是神经科学家的长期目标。这些连接的知识将使我们了解神经系统在健康和疾病中的功能。染料标记、生理记录、电子显微镜水平的解剖学和蛋白质的转移都被用来跟踪突触连接。嗜神经病毒也被利用，利用它们在神经元之间传播的能力。病毒示踪剂提供了更高通量的承诺，并且可以通过其基因转移能力将测定库扩展到作图连接之外。我们最近开发了水泡性口炎病毒（VSV）作为跨突触示踪剂。该病毒与先前开发的跨突触病毒、狂犬病病毒（RABV）和伪狂犬病病毒（PRV）相比具有几个优点。RABV是一种致命的病毒，这限制了它在实验室中的使用。PRV具有庞大而复杂的基因组，使得难以以直接的方式进行工程改造。VSV的基因组相对简单，与RABV非常相似。由于其快速生长至高滴度，因此具有良好的特性，并且在实验室中具有长期的安全记录。我们发现VSV可以在小鼠，猴子和其他多种物种（包括鸟类，两栖动物和鱼类）中跨突触传播。此外，我们发现我们可以创造VSV载体，可以特异性逆行或特异性顺行传播。方向性完全依赖于病毒包膜糖蛋白，由病毒G基因编码。我们建议创建一系列跨突触病毒示踪剂，这将对在不同生物体和神经系统不同区域工作的神经科学家有用。我们将首先探索使用VSV与不同的病毒G蛋白，应用我们所学到的设计特定的顺行和逆行示踪剂。我们希望扩大G蛋白的库，不仅可以与VSV一起使用，还可以与其他示踪剂一起使用。我们还建议开发一种替代无毒病毒作为示踪剂，使用我们已经为VSV开发的一些工具。最后，我们建议开发一种示踪剂，虽然基于病毒，但实际上不是病毒，而是一种将Cre重组酶移动穿过突触以映射连接的神经元的工具。这些替代策略提供了相对于VSV、RABV或PRV降低毒性的前景，以及可以与小鼠或其他模式生物的工程化品系组合的其他特征。

DESCRIPTION (provided by applicant): The mapping of connections among central nervous system (CNS) neurons is a longstanding goal of neuroscientists. Knowledge of the connections will inform our understanding of the function of the nervous system, in both health and disease. Dye labeling, physiological recordings, anatomy at the level of electron microscopy, and the transfer of proteins have all been used to track synaptic connections. Neurotropic viruses have also been used, taking advantage of their ability to spread among neurons. Viral tracers offer the promise of higher throughput, and can expand the repertoire of assays beyond mapping connections, via their gene transfer capabilities. We have recently developed vesicular stomatitis virus (VSV) as a transsynaptic tracer. This virus has several advantages over the previously developed transsynaptic viruses, rabies virus (RABV) and pseudorabies virus (PRV). RABV is a lethal virus, which limits its use in the laboratory. PRV has a large and complex genome, making it difficult to engineer in a straightforward manner. VSV has a relatively simple genome, very similar to that of RABV. It is very well characterized as it rapidly grows to high titr and has a long track record for safety in the laboratory. We found that VSV can transmit transsynaptically in mice, monkeys, and multiple other species, including birds, amphibians, and fish. Moreover, we found that we could create VSV vectors that could transmit specifically retrogradely or specifically anterogradely. The directionality was entirely dependent upon the viral envelope glycoprotein, encoded by a viral G gene. We propose to create a series of transsynaptic viral tracers that will be useful to neuroscientists working in different organisms and different areas of the nervous system. We will first explore the use of VSV with different viral G proteins, applying what we learn to the design of specific anterograde and retrograde tracers. We hope to expand the repertoire of G proteins that can be used not only with VSV, but with other tracers as well. We also propose to develop an alternative non-toxic virus for use as a tracer, using some of the tools that we have already developed for VSV. Finally, we propose to develop a tracer that, although based upon a virus, is actually not a virus, but a vehicle to move Cre recombinase across synapses to map connected neurons. These alternative strategies offer the promise of reduced toxicity, relative to VSV, RABV, or PRV, as well as other features that can be combined with engineered lines of mice, or other model organisms.