Megaplexed Neuronal Visualization Using Combinatorial Labeling and Iterative Staining

项目摘要 神经元是在细胞环路内组织起来的，这些环路引发了所有的大脑活动。解剖学上的理解 对于弄清楚大脑是如何处理信息的，神经元结构是至关重要的。通常， 神经元的可视化依赖于用不同颜色的荧光蛋白标记来区分 然而，这种方法的一个主要限制是可用的颜色多样性较低。 因为神经元可以沿着复杂的路径长距离投射，所以许多重叠的细胞 当使用小的调色板时，彼此无法区分。我们提出了一种去耦合的技术 标记的多样性不受染料颜色的限制。我们建议用一种多肽组合来标记每个细胞 而不是单一的染料和进行迭代免疫荧光染色，这可以重复使用颜色。vbl.使用 这项技术，我们将创建100多万个可识别的标签，这些标签将用于绘制 脑组织中的细胞数量前所未有。 我们将使用标准的质粒法和转染法对细胞系进行概念验证研究。 技术(目标1)。接下来，我们将采用两种病毒策略对神经元进行基因标记(目标2)。我们会 在复制缺陷的Sindbis载体中编码多肽组合，可以迅速感染神经元和 诱导高水平的多肽表达，并工程腺相关病毒(AAV)来传递标记 因为AAV可以系统地和选择性地针对神经元亚型进行注射。总而言之，这些工具 将被用来绘制三维的整个病毒注射体积图(目标3)。预计将会有 根据这一提议开发的技术将对细胞标记和大脑连接产生巨大影响。

Project Summary Neurons are organized within cellular circuits which give rise to all brain activity. An anatomical understanding of neuronal architecture is crucial to figuring out how the brain processes information. Commonly, the visualization of neurons has relied on labeling with different colored fluorescent proteins in order to distinguish one from another, however a major limitation of this approach is the low diversity of colors that are available. Because neurons can project over long distance and along complex paths, many overlapping cells will be indistinguishable from one another when a small color palette is used. We propose technology which decouples labelling diversity from the limitation of dye colors. We propose labeling each cell with a combination of peptides rather than a single dye and performing iterative immunofluorescence staining which can re-use colors. Using this technology, we will create over one million discernable labels which will be used to map the location of an unprecedented number of cells within brain tissue. We will perform proof of concept studies on cell lines using standard plasmid preparation and transfection techniques (Aim 1). Next we will pursue two viral strategies to genetically label neurons (Aim 2). We will encode peptide combinations within replication-deficient Sindbis vectors which can infect neurons rapidly and induce high levels of peptide expression and also engineer adeno associated viruses (AAV) to deliver labels because AAV can be administered systemically and selectively target neuronal subtypes. Together, these tools will be used to map, in three dimensions, an entire viral injection volume (Aim 3). It is expected that the technology developed from this proposal will have a drastic impact on cellular labelling and brain connectomics.