Barcoded Rabies In Situ Connectomics for high-throughput reconstruction of neural circuits

Sequencing of oligonucleotide barcodes holds promise as a high-throughput approach for reconstructing synaptic connectivity at scale. Rabies viruses can act as a vehicle for barcode transmission, thanks to their ability to spread between synaptically connected cells. However, applying barcoded rabies viruses to map synaptic connections in vivo has proved challenging. Here, we develop Barcoded Rabies In Situ Connectomics (BRISC) for high-throughput connectivity mapping in the mouse brain. To ensure that the majority of post-synaptic "starter" neurons are uniquely labeled with distinct barcode sequences, we first generated libraries of rabies viruses with sufficient diversity to label >1000 neurons uniquely. To minimize the probability of barcode transmission between starter neurons, we developed a strategy to tightly control their density. We then applied BRISC to map inputs of single neurons in the primary visual cortex (V1). Using in situ sequencing, we read out the expression of viral barcodes in rabies-infected neurons, while preserving spatial information. We then matched barcode sequences between starter and presynaptic neurons, mapping the inputs of 385 neurons and identifying 7,814 putative synaptic connections. The resulting connectivity matrix revealed layer- and cell-type-specific local connectivity rules and topographic organization of long-range inputs to V1. These results show that BRISC can simultaneously resolve the synaptic connectivity of hundreds of neurons while preserving spatial information, enabling reconstruction of neural circuits at an unprecedented scale. UMI-tagged amplicon sequencing of barcoded rabies genome plasmid and barcoded rabies viral library samples, collected from purified supercoiled plasmid preps and concentrated pseudotyped rabies virus preps respectively.

寡核苷酸条形码测序（oligonucleotide barcode sequencing）作为一种高通量技术，有望实现大规模突触连接的重建。狂犬病毒可作为条形码传递的载体，因其能够在突触连接的细胞间传播。然而，利用携带条形码的狂犬病毒在活体中绘制突触连接图谱仍面临诸多挑战。在此研究中，我们开发了条形码狂犬病毒原位连接组学（Barcoded Rabies In Situ Connectomics，BRISC）技术，用于小鼠脑内的高通量连接图谱绘制。为确保大多数突触后“起始”神经元能被独特的条形码序列唯一标记，我们首先构建了具有足够多样性的狂犬病毒文库，可实现对超过1000个神经元的唯一标记。为降低起始神经元间发生条形码传递的概率，我们开发了一种可严格控制其密度的策略。随后，我们将BRISC技术应用于小鼠初级视觉皮层（primary visual cortex，V1）中单神经元输入连接的图谱绘制。我们利用原位测序（in situ sequencing）技术，在保留空间信息的前提下，读取狂犬病毒感染神经元中病毒条形码的表达情况。随后我们对起始神经元与突触前神经元的条形码序列进行匹配，完成了385个神经元的输入连接图谱绘制，并鉴定出7814个潜在突触连接。所得的连接矩阵揭示了具有皮层分层和细胞类型特异性的局部连接规则，以及投射至V1的远距离输入的地形组织模式。上述结果表明，BRISC技术可在保留空间信息的同时，解析数百个神经元的突触连接，从而实现前所未有的规模的神经环路重建。本研究对携带条形码的狂犬病毒基因组质粒样本与携带条形码的狂犬病毒文库样本分别进行了唯一分子标识符（Unique Molecular Identifier，UMI）标记的扩增子测序，两类样本分别取自纯化超螺旋质粒制备物与浓缩假型狂犬病毒制备物。