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快速、高通量生产改良的狂犬病病毒载体，用于特定、高效和通用的顺行性逆行标记。

Fast, high-throughput production of improved rabies viral vectors for specific, efficient and versatile transsynaptic retrograde labeling.

机构信息

Institute of Science and Technology Austria (ISTA), Klosterneuburg, Austria.

Department of Neurophysiology and Neuropharmacology, Vienna Medical University, Vienna, Austria.

出版信息

Elife. 2022 Aug 30;11:e79848. doi: 10.7554/eLife.79848.

DOI:10.7554/eLife.79848
PMID:36040301
原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC9477495/
Abstract

To understand the function of neuronal circuits, it is crucial to disentangle the connectivity patterns within the network. However, most tools currently used to explore connectivity have low throughput, low selectivity, or limited accessibility. Here, we report the development of an improved packaging system for the production of the highly neurotropic RVdG-CVS-N2c rabies viral vectors, yielding titers orders of magnitude higher with no background contamination, at a fraction of the production time, while preserving the efficiency of transsynaptic labeling. Along with the production pipeline, we developed suites of 'starter' AAV and bicistronic RVdG-CVS-N2c vectors, enabling retrograde labeling from a wide range of neuronal populations, tailored for diverse experimental requirements. We demonstrate the power and flexibility of the new system by uncovering hidden local and distal inhibitory connections in the mouse hippocampal formation and by imaging the functional properties of a cortical microcircuit across weeks. Our novel production pipeline provides a convenient approach to generate new rabies vectors, while our toolkit flexibly and efficiently expands the current capacity to label, manipulate and image the neuronal activity of interconnected neuronal circuits in vitro and in vivo.

摘要

为了理解神经元回路的功能，解析网络内的连接模式至关重要。然而，目前用于探索连接的大多数工具的通量低、选择性低或可及性有限。在这里，我们报告了一种改进的 RVdG-CVS-N2c 狂犬病病毒载体包装系统的开发，该系统可产生高神经亲和性、高滴度、无背景污染的病毒，生产时间缩短到原来的几分之一，同时保持转导效率。随着生产流水线的发展，我们开发了一系列“起始”AAV 和双顺反子 RVdG-CVS-N2c 载体，能够从广泛的神经元群中进行逆行标记，针对不同的实验需求进行定制。我们通过揭示小鼠海马结构中隐藏的局部和远端抑制性连接，以及通过数周时间对皮质微电路的功能特性进行成像，证明了新系统的强大功能和灵活性。我们的新型生产流水线为生成新的狂犬病病毒载体提供了一种便捷的方法，而我们的工具包则灵活高效地扩展了目前在体外和体内标记、操作和成像相互连接的神经元回路的神经元活动的能力。

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