High-throughput measurement of neuronal projections and synapses using Synapse-seq

摘要 大脑由数千种高度专业化的细胞类型组成，这些细胞类型形成了非常特殊的 彼此之间的突触连接。这些连接一起形成神经回路，它们是 大脑功能的结构基础。尽管它们很重要，但突触之间的联系 由于现有工具的匮乏，细胞类型在很大程度上是无法确定的。在这 项目，我们将开发一套基于基因组学的工具--统称为Synapse-Seq-- 使我们能够量化神经元投射、突触后密度和突触连接 活体中的单个细胞。首先，我们将使用AAV来介绍一种分子工具，它可以交易一个 条形码信使核糖核酸报告到感染细胞的突触前隔间，结合 单细胞测序和空间转录(Slide-seq)联合匹配细胞体位置 投射目标。接下来，我们将使用一个突触后定位系统，也是由AAV提供的， 结合突触体准备和原位测序来量化脊柱密度 跨越数千个单细胞。最后，我们将结合我们的突触前和突触后标记 具有新的原位测序读数以测量体内突触连接的系统。 总之，这些技术的成功开发将为神经科学家提供 一套强大的工具，用于常规测量活体神经连接的基本方面。

SUMMARY The brain is composed of thousands of highly specialized cell types that form very specific synaptic connections with each other. Together, these connections form neural circuits that are the structural basis of brain function. Despite their importance, synaptic connections amongst cell types are largely unascertained, because of the dearth of existing tools to do so. In this project, we will develop a suite of genomics-based tools--collectively termed Synapse-seq--that allow us to quantify neuronal projections, postsynaptic densities, and synaptic connections in individual cells in vivo. First, we will use AAV to introduce a molecular tool that trafficks a barcoded mRNA reporter to the presynaptic compartments of infected cells, combined with single-cell sequencing and spatial transcriptomics (Slide-seq) to jointly match cell body positions to projection targets. Next, we will use a postsynaptic targeting system, also delivered by AAV, combined with synaptosome preparations and in situ sequencing to quantify spine densities across thousands of single cells. Finally, we will combine our pre- and postsynaptic labeling systems with novel in situ sequencing readouts to measure synaptic connectivity in vivo. Together, the successful development of these technologies will provide neuroscientists with a suite of powerful tools to routinely measure fundamental aspects of neural connectivity in vivo.