Multiplex Neural Circuit Tracing With G-Deleted Rabies Viral Vectors
Multiplex Neural Circuit Tracing With G-Deleted Rabies Viral Vectors
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DOI：
10.3389/fncir.2019.00077
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发表时间：
2020-01
影响因子：
3.5
通讯作者：
Toshiaki Suzuki;Nao Morimoto;A. Akaike;Fumitaka Osakada
中科院分区：
文献类型：
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作者：
Toshiaki Suzuki;Nao Morimoto;A. Akaike;Fumitaka Osakada
神经回路相互连接以组织产生感知、认知、记忆和行为的大规模网络。神经系统中的信息通过平行的独立回路和混合回路进行处理。分析回路之间的相互作用对于阐明大脑的功能尤其不可或缺。糖蛋白（G）基因缺失的狂犬病病毒载体（RVΔG）单突触通路追踪是研究神经通路结构和功能的有效途径。RVΔG与外源包膜EnvA的假型化允许在表达TVA（EnvA的同源受体）的细胞中表达转基因，如荧光蛋白、遗传编码的传感器或光遗传学工具。与狂犬病病毒糖蛋白（RV-G）的反式互补使得能够跨突触标记直接连接到表达TVA和RV-G的起始神经元的输入神经元。然而，它仍然具有挑战性的同时映射来自多个细胞群的神经元连接和它们之间的相互作用的混合电路之间的EnvA/TVA介导的RV示踪系统在一个单一的动物。为了克服这一局限性，我们通过优化不同的病毒包膜（oEnvX）及其相应的受体（oTVX）来多重RVΔG回路追踪。基于来源于禽肉瘤白血病病毒（ASLV）的EnvB/TVB和EnvE/DR 46-TVB系统，我们开发了具有较低或较高亲和力的优化的TVB受体（oTVB-L或oTVB-H）和嵌合包膜oEnvB，以及具有较高亲和力的优化的TVE受体（oTVE-H）及其嵌合包膜oEnvE。我们证明了oEnvA/oTVA、oEnvB/oTVB和oEnvE/oTVE系统之间RVΔG感染的独立性，以及针对其他皮质或皮质下区域的两种不同类别的第5层神经元的多路电路追踪的体内概念验证。我们还成功地用RVΔG多重示踪技术标记了小鼠V1区皮质-皮质第5层神经元和抑制性神经元的外侧膝状体核共同输入。这些oEnvA/oTVA、oEnvB/oTVB和oEnvE/oTVE系统允许不同回路的差异标记，以揭示通过独立回路进行并行处理和通过大脑回路之间的相互作用进行集成处理的潜在机制。
Neural circuits interconnect to organize large-scale networks that generate perception, cognition, memory, and behavior. Information in the nervous system is processed both through parallel, independent circuits and through intermixing circuits. Analyzing the interaction between circuits is particularly indispensable for elucidating how the brain functions. Monosynaptic circuit tracing with glycoprotein (G) gene-deleted rabies viral vectors (RVΔG) comprises a powerful approach for studying the structure and function of neural circuits. Pseudotyping of RVΔG with the foreign envelope EnvA permits expression of transgenes such as fluorescent proteins, genetically-encoded sensors, or optogenetic tools in cells expressing TVA, a cognate receptor for EnvA. Trans-complementation with rabies virus glycoproteins (RV-G) enables trans-synaptic labeling of input neurons directly connected to the starter neurons expressing both TVA and RV-G. However, it remains challenging to simultaneously map neuronal connections from multiple cell populations and their interactions between intermixing circuits solely with the EnvA/TVA-mediated RV tracing system in a single animal. To overcome this limitation, here, we multiplexed RVΔG circuit tracing by optimizing distinct viral envelopes (oEnvX) and their corresponding receptors (oTVX). Based on the EnvB/TVB and EnvE/DR46-TVB systems derived from the avian sarcoma leukosis virus (ASLV), we developed optimized TVB receptors with lower or higher affinity (oTVB-L or oTVB-H) and the chimeric envelope oEnvB, as well as an optimized TVE receptor with higher affinity (oTVE-H) and its chimeric envelope oEnvE. We demonstrated independence of RVΔG infection between the oEnvA/oTVA, oEnvB/oTVB, and oEnvE/oTVE systems and in vivo proof-of-concept for multiplex circuit tracing from two distinct classes of layer 5 neurons targeting either other cortical or subcortical areas. We also successfully labeled common input of the lateral geniculate nucleus to both cortico-cortical layer 5 neurons and inhibitory neurons of the mouse V1 with multiplex RVΔG tracing. These oEnvA/oTVA, oEnvB/oTVB, and oEnvE/oTVE systems allow for differential labeling of distinct circuits to uncover the mechanisms underlying parallel processing through independent circuits and integrated processing through interaction between circuits in the brain.