BRAIN CONNECTS: Synaptic resolution whole-brain circuit mapping of molecularly defined cell types using a barcoded rabies virus

摘要 单细胞转录组学彻底改变了我们对神经元多样性的理解， 跨大脑区域和物种的分子细胞类型的表征。我们和其他人开创了多- 模式技术，如Patch-seq和空间转录组学，将分子定义的细胞类型与其 生理学，细胞形态学和解剖学特征，但我们仍然缺乏高通量，具有成本效益的方法 它可以提供整个哺乳动物大脑的全面的突触分辨率接线图， 这些连接体与分子定义的细胞类型。 我们建议进一步开发和验证狂犬病条形码相互作用检测，然后进行测序 （RaBID-seq），以实现高通量，可扩展和具有成本效益的全脑突触水平的映射， 映射神经元的必要性和转录组学特征。我们优化了狂犬病病毒的生产， 包装，以实现包含超过170万个独特条形码的条形码库，两个订单的mag， 与先前的研究相比，Nitude更高，足以将输入映射到数千个突触后神经元， 单一动物。然而，这项技术仍然面临着一些实验和计算挑战，以实现 它的全部潜力。在目标1中，我们将解决当将RaBID-seq扩展到 研究全脑，密集标记的电路：初始感染和传播的随机性，毒性和潜在的 当许多创始细胞被标记时，多突触事件。此外，我们将开发一种新的Ra变体- bies具有一个可进化的条形码，可以消除单突触与多突触传播的歧义， 密集标记。在目标2中，我们将对RaBID-seq连接体与其他金标准技术进行基准测试 使用多序列测序和空间转录组学测量连接性。在目标3中，我们将开发新的算法 使用图神经网络从条形码病毒数据集重建单突触连接组，评估 这些算法的鲁棒性在不同的实验参数下在silico，并测试是否进化 条形码可以改善单突触回路重建。如果成功，这些研究将建立RaBID-seq 作为一种可扩展的，具有成本效益的工具，用于全脑连接映射，可以整合转录组细胞类型 在单细胞分辨率下的突触水平布线图。 通过将突触连接问题简化为条形码测序问题，我们的方法 有可能显著提高通量，降低成本，并提供与转录组的直接联系 每个映射的细胞。RaBID-seq将把全脑电路映射变成一个常规实验， 在任何实验室进行适度的资源，使之有可能探索电路之间的差异治疗 条件，疾病状态，性别之间，以及整个生命周期。我们还将在这两个方面生成试点数据， 小鼠和人类切片培养物，以证明该工具在物种间的实用性。

ABSTRACT Single-cell transcriptomics has revolutionized our understanding of neuronal diversity and enabled high-throughput characterization of molecular cell types across brain areas and species. We and others have pioneered multi- modal technologies such as Patch-seq and spatial transcriptomics to link molecularly-defined cell types with their physiology, cytomorphology, and anatomical features, but we still lack high-throughput, cost-effective methods that can provide comprehensive synaptic resolution wiring diagrams of entire mammalian brains and integrate these connectomes with molecularly defined cell types. We propose to further develop and validate Rabies Barcode Interaction Detection followed by sequencing (RaBID-seq) to enable high-throughput, scalable, and cost-effective mapping of brain-wide synaptic-level con- nectivity and transcriptomic profiling of the mapped neurons. We have optimized rabies virus production and packaging to achieve barcoded libraries containing more than 1.7 million unique barcodes, two orders of mag- nitude higher compared to prior studies, enough to map the inputs to thousands of post-synaptic neurons in a single animal. However, this technology still faces several experimental and computational challenges to realize its full potential. In Aim 1, we will address three potential challenges that may arise when scaling RaBID-seq to study brain-wide, densely labeled circuits: stochasticity of initial infection and spread, toxicity, and the potential for polysynaptic events when many founder cells are labeled. In addition, we will develop a new variant of Ra- bies featuring an evolvable barcode that can disambiguate monosynaptic vs polysynaptic spread in the setting of dense labeling. In Aim 2, we will benchmark RaBID-seq connectomes against other gold standard techniques measuring connectivity using multipatch-seq and spatial transcriptomics. In Aim 3, we will develop new algorithms using graph neural networks to reconstruct monosynaptic connectomes from barcoded viral datasets, assess the robustness of these algorithms under different experimental parameters in silico, and test whether an evolvable barcode can improve monosynaptic circuit reconstruction. If successful, these studies will establish RaBID-seq as a scalable, cost-effective tool for brain-wide connectivity mapping that can integrate transcriptomic cell types with their synaptic-level wiring diagram at single-cell resolution. By reducing the problem of synaptic connectivity into a problem of barcode sequencing, our approach has the potential to dramatically increase throughput, decrease costs and provide a direct link to the transcriptome of each mapped cell. RaBID-seq will transform brain-wide circuit mapping into a routine experiment that can be performed in any lab with modest resources, making it possible to explore how circuits differ between treatment conditions, in disease states, between the sexes, and across the lifespan. We will also generate pilot data in both mice and human slice cultures to demonstrate the utility of this tool across species.