Laboratory of Cellular Pharmacology, Graduate School of Pharmaceutical Sciences, Nagoya University, Nagoya, Japan.
Laboratory of Neural Information Processing, Institute for Advanced Research, Nagoya University, Nagoya, Japan.
Front Neural Circuits. 2020 Jan 10;13:77. doi: 10.3389/fncir.2019.00077. eCollection 2019.
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 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.
神经回路相互连接，组成大规模网络，这些网络产生感知、认知、记忆和行为。神经系统中的信息既通过平行的、独立的回路进行处理，也通过混合的回路进行处理。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。用糖蛋白（G）基因缺失的狂犬病病毒载体（RVΔG）进行单突触回路追踪，是研究神经回路结构和功能的有力方法。用外来包膜 EnvA 对 RVΔG 进行假型化，允许在表达 TVA 的细胞中表达荧光蛋白、遗传编码传感器或光遗传工具等转基因，TVA 是 EnvA 的同源受体。用狂犬病病毒糖蛋白（RV-G）进行反式互补，可使直接连接到表达 TVA 和 RV-G 的起始神经元的输入神经元进行突触后标记。然而，在单个动物中，仅用 EnvA/TVA 介导的 RV 追踪系统，同时对多个细胞群体的神经元连接及其在混合回路中的相互作用进行映射仍然具有挑战性。为了克服这一限制，我们通过优化不同的病毒包膜（oEnvX）及其相应的受体（oTVX），对 RVΔG 回路追踪进行了多重化。基于源自禽肉瘤白血病病毒（ASLV）的 EnvB/TVB 和 EnvE/DR46-TVB 系统，我们开发了具有较低或较高亲和力的优化 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 TVE 受体（oTVE-H）和其嵌合包膜 oEnvE。我们证明了 oEnvA/oTVA、oEnvB/oTVB 和 oEnvE/oTVE 系统之间 RVΔG 感染的独立性，并证明了从靶向皮质或皮质下区域的两类不同的第 5 层神经元中进行多重回路追踪的概念验证。我们还成功地用多重 RVΔG 追踪标记了外侧膝状体核对皮质-皮质第 5 层神经元和小鼠 V1 抑制性神经元的共同输入。这些 oEnvA/oTVA、oEnvB/oTVB 和 oEnvE/oTVE 系统允许对不同的回路进行差异标记，以揭示大脑中通过独立回路进行并行处理以及通过回路之间的相互作用进行整合处理的机制。
