Highly multiplexed circuit mapping using barcoded rabies viruses and in situ sequencing.

摘要 了解神经回路的功能需要彻底研究两个回路 元素：细胞类型和连接性。轴突示踪与高通量 基因条形码神经元的DNA测序使得能够同时表征 神经元及其投射区域的解剖学和分子特征。但是，没有高- 目前存在的吞吐量工具允许我们映射突触前和突触后之间的连接， 突触后神经元，同时识别映射神经元的细胞类型。 在这里，我们提出了一个家庭的新的测序为基础的方法，使用条形码狂犬病 病毒实现两个主要目标：第一，允许投射神经元的表征 在大脑中的每一个地方都投射到多个目标区域;第二，允许同时 将输入映射到转录组学上不同的神经元亚型。我们会联合收割机我们的好- 建立了具有逆行标记（Retro-seq）的单细胞RNA-seq（scRNA-seq）管道， 狂犬病条形码检测，以进行高度多重的细胞类型表征， 神经元投射到大脑中的许多目标区域中的每一个。此外，我们将适应 我们的条形码解剖结构通过测序（BARseq）管道解析，以表征空间 这些投射神经元的分布和细胞类型（Retro-BARseq）。进一步建立 研究神经回路的解剖学和转录组学特征的方法 通过使用scRNA-seq/BARseq的基于狂犬病的逆行跨突触回路追踪揭示 （TransR-seq和TransR-BARseq）。最后，我们将努力达到单神经元水平 通过应用优化的狂犬病条形码在野生型小鼠中进行连接体追踪和测序 病毒来实现单个神经元的独特条形码。 该项目的成功将导致一套高通量，高分辨率的方法， 通过结合条形码狂犬病病毒和组学对连接体进行测序， 揭示了给定神经元内神经元的连接性和转录组学身份， 电路.这些技术，其中大部分应该适用于任何哺乳动物物种， 将提供神经元回路的吞吐量和分辨率的数量级增加 分析，对神经科学的许多分支产生了变革性的影响。

ABSTRACT Understanding the function of neural circuits requires thorough investigation of two circuit elements: cell types and connectivity. The combination of axonal tracing with high-throughput DNA sequencing of genetically barcoded neurons has enabled the simultaneous characterization of anatomical and molecular identities of neurons and their projection fields. However, no high- throughput tools currently exist that allow us to map connections between presynaptic and postsynaptic neurons while identifying the cell types of mapped neurons. Here we propose to apply a family of novel sequencing-based approaches using barcoded rabies viruses to achieve two main goals: first, to allow characterization of projection neurons everywhere in the brain that project to multiple target regions; and second, to allow simultaneous mapping of the inputs to transcriptomically-distinct subtypes of neurons. We will combine our well- established single-cell RNA-seq (scRNA-seq) pipeline with retrograde labeling (Retro-seq) for rabies barcode detection in order to conduct highly multiplexed cell type characterization of neurons projecting to each of the many target areas across the brain. Furthermore, we will adapt our barcoded anatomy resolved by sequencing (BARseq) pipeline to characterize the spatial distribution and cell types of those projection neurons (Retro-BARseq). We will further establish the methodology to investigate the anatomical and transcriptomic features of neural circuitry revealed by rabies-based retrograde trans-synaptic circuit tracing using scRNA-seq/BARseq (TransR-seq and TransR-BARseq). Finally, we will strive to reach single-neuron-level connectome tracing and sequencing in wild-type mice by applying the optimized barcoded rabies viruses to achieve unique barcoding of individual neurons. Success of the project will result in a set of high-throughput, high-resolution methods for sequencing the connectome by combining barcoded rabies viruses with omics, therefore revealing both the connectivity and transcriptomic identities of neurons within a given neural circuit. These techniques, the majority of which should be applicable to any mammalian species, will provide orders of magnitude increases in both throughput and resolution of neuronal circuit analysis, with a transformative impact on many branches of neuroscience.