映射不同神经元类型的连通性为理解神经回路的结构和功能提供了基础。基于RNA条形码测序的高通量和低成本神经解剖学技术具有在细胞分辨率和全大脑范围内映射电路的潜力,但是现有的基于Sindbis病毒的技术只能使用顺行追踪方法绘制远程预测图。狂犬病病毒可以通过对投射神经元进行逆行标记或对遗传靶向的突触后神经元的直接输入进行单突触追踪来补充顺行追踪方法。然而,迄今为止,条形码狂犬病病毒仅用于绘制体内非神经元细胞相互作用和培养神经元的突触连接图。在这里,我们将条形码狂犬病病毒与单细胞和原位测序相结合,以在小鼠大脑中进行逆行标记和跨突触标记。我们使用单细胞RNA-seq对96个逆行标记的细胞和295个跨突触标记的细胞进行了测序,和4130个逆行标记的细胞和2914个原位跨突触标记的细胞。我们发现,使用单细胞RNA-seq和原位测序可以可靠地鉴定狂犬病病毒感染的细胞的转录组身份。通过将基因表达与从条形码测序推断的连通性相关联,我们从多个皮质区域中区分了远距离投射的皮质细胞类型,并确定了突触连接趋同或发散的细胞类型.原位测序与条形码狂犬病病毒的结合补充了现有的基于测序的神经解剖学技术,并为大规模绘制神经元类型的突触连接提供了潜在的途径。
在大脑中,信息通过复杂的轴突和树突网络从一个细胞传递到下一个细胞,这些轴突和树突在称为突触的连接处物理相互作用。映射出这个突触连接——也就是说,究竟哪些神经元通过突触连接-仍然是一个重大挑战。单突触追踪是一种强大的方法,它允许神经科学家通过利用通过突触在神经元之间传播的病毒来探索神经网络。尤其是狂犬病病毒。这种病原体仅从“突触后”传播到“突触前”神经元-从突触接收信息的细胞,回到发送它的那个。因此,狂犬病病毒的修饰变体可用于揭示与最初引入狂犬病病毒的神经元群连接的突触前细胞。然而,这种方法不允许科学家确定每个突触前细胞连接的突触后神经元。绕过这个问题的一种方法是将单突触追踪与RNA条形码相结合,以创建不同版本的改良狂犬病病毒,然后将其引入单独的神经元群体中。跟踪每个版本的传播使神经科学家能够准确地发现哪个突触前细胞向每个突触后神经元发出信号。到目前为止,这种方法已被用于检查实验室中生长的神经元的突触连接,但是将其应用于大脑中的神经元仍然很困难。作为回应,张,Jin等人。目的是证明依赖条形码狂犬病病毒的单突触追踪如何用于剖析小鼠大脑中的神经网络。首先,他们证实,利用原位RNA测序和单细胞RNA测序,可以准确检测出哪个版本的病毒已经传播到突触前神经元.接下来,他们描述了如何分析这些信息来建立潜在的神经网络模型,以及这项工作需要什么类型的额外实验。最后,他们使用这种方法来识别倾向于连接到相同突触后细胞的神经元,然后研究它们的共同点,展示了该技术如何更好地理解神经回路。总的来说,张的工作,Jin等人。提供了与基于条形码狂犬病病毒的单突触追踪实验相关的要求和限制的全面审查,以及未来如何优化该方法。这些信息将对在大脑中映射神经网络感兴趣的科学家产生广泛兴趣。
Mapping the connectivity of diverse neuronal types provides the foundation for understanding the structure and function of neural circuits. High-throughput and low-cost neuroanatomical techniques based on RNA barcode sequencing have the potential to map circuits at cellular resolution and a brain-wide scale, but existing Sindbis virus-based techniques can only map long-range projections using anterograde tracing approaches. Rabies virus can complement anterograde tracing approaches by enabling either retrograde labeling of projection neurons or monosynaptic tracing of direct inputs to genetically targeted postsynaptic neurons. However, barcoded rabies virus has so far been only used to map non-neuronal cellular interactions in vivo and synaptic connectivity of cultured neurons. Here we combine barcoded rabies virus with single-cell and in situ sequencing to perform retrograde labeling and transsynaptic labeling in the mouse brain. We sequenced 96 retrogradely labeled cells and 295 transsynaptically labeled cells using single-cell RNA-seq, and 4130 retrogradely labeled cells and 2914 transsynaptically labeled cells in situ. We found that the transcriptomic identities of rabies virus-infected cells can be robustly identified using both single-cell RNA-seq and in situ sequencing. By associating gene expression with connectivity inferred from barcode sequencing, we distinguished long-range projecting cortical cell types from multiple cortical areas and identified cell types with converging or diverging synaptic connectivity. Combining in situ sequencing with barcoded rabies virus complements existing sequencing-based neuroanatomical techniques and provides a potential path for mapping synaptic connectivity of neuronal types at scale.
In the brain, messages are relayed from one cell to the next through intricate networks of axons and dendrites that physically interact at junctions known as synapses. Mapping out this synaptic connectivity – that is, exactly which neurons are connected via synapses – remains a major challenge. Monosynaptic tracing is a powerful approach that allows neuroscientists to explore neural networks by harnessing viruses which spread between neurons via synapses, in particular the rabies virus. This pathogen travels exclusively from ‘postsynaptic’ to ‘presynaptic’ neurons – from the cell that receives a message at a synapse, back to the one that sends it. A modified variant of the rabies virus can therefore be used to reveal the presynaptic cells connecting to a population of neurons in which it has been originally introduced. However, this method does not allow scientists to identify the exact postsynaptic neuron that each presynaptic cell is connected to. One way to bypass this issue is to combine monosynaptic tracing with RNA barcoding to create distinct versions of the modified rabies virus, which are then introduced into separate populations of neurons. Tracking the spread of each version allows neuroscientists to spot exactly which presynaptic cells signal to each postsynaptic neuron. So far, this approach has been used to examine synaptic connectivity in neurons grown in the laboratory, but it remains difficult to apply it to neurons in the brain. In response, Zhang, Jin et al. aimed to demonstrate how monosynaptic tracing that relies on barcoded rabies viruses could be used to dissect neural networks in the mouse brain. First, they confirmed that it was possible to accurately detect which version of the virus had spread to presynaptic neurons using both in situ and single-cell RNA sequencing. Next, they described how this information could be analysed to build models of potential neural networks, and what type of additional experiments are required for this work. Finally, they used the approach to identify neurons that tend to connect to the same postsynaptic cells and then investigated what these have in common, showing how the technique enables a finer understanding of neural circuits. Overall, the work by Zhang, Jin et al. provides a comprehensive review of the requirements and limitations associated with monosynaptic tracing experiments based on barcoded rabies viruses, as well as how the approach could be optimized in the future. This information will be of broad interest to scientists interested in mapping neural networks in the brain.