High-Throughput Single-Cell Manipulation in Brain Tissue

The complexity of neurons and neuronal circuits in brain tissue requires the genetic manipulation, labeling, and tracking of single cells. However, current methods for manipulating cells in brain tissue are limited to either bulk techniques, lacking single-cell accuracy, or manual methods that provide single-cell accuracy but at significantly lower throughputs and repeatability. Here, we demonstrate high-throughput, efficient, reliable, and combinatorial delivery of multiple genetic vectors and reagents into targeted cells within the same tissue sample with single-cell accuracy. Our system automatically loads nanoliter-scale volumes of reagents into a micropipette from multiwell plates, targets and transfects single cells in brain tissues using a robust electroporation technique, and finally preps the micropipette by automated cleaning for repeating the transfection cycle. We demonstrate multi-colored labeling of adjacent cells, both in organotypic and acute slices, and transfection of plasmids encoding different protein isoforms into neurons within the same brain tissue for analysis of their effects on linear dendritic spine density. Our platform could also be used to rapidly deliver, both ex vivo and in vivo, a variety of genetic vectors, including optogenetic and cell-type specific agents, as well as fast-acting reagents such as labeling dyes, calcium sensors, and voltage sensors to manipulate and track neuronal circuit activity at single-cell resolution.脑组织内的神经元（neurons）与神经元环路（neuronal circuits）结构复杂，亟需实现单细胞水平的遗传操控（genetic manipulation）、标记（labeling）与追踪（tracking）。然而，当前用于脑组织细胞操控的技术存在显著局限：要么为批量操作技术，无法实现单细胞精度；要么为手动方法，虽可达到单细胞精度，但通量（throughputs）与重复性（repeatability）均显著偏低。本研究实现了在同一份脑组织样本中，以单细胞精度对靶细胞进行高通量、高效、可靠且组合式的多种遗传载体（genetic vectors）与试剂（reagents）递送。本系统可自动从多孔板（multiwell plates）中吸取纳升级体积的试剂至微量移液管（micropipette），依托稳定的电穿孔（electroporation）技术对脑组织内的单细胞进行靶向转染，并通过自动清洁完成微量移液管的预处理，以重复开展转染循环（transfection cycle）。我们分别在器官型脑片（organotypic slices）与急性脑片（acute slices）中实现了相邻细胞的多色标记，并将编码不同蛋白质异构体（protein isoforms）的质粒转染至同一脑组织内的神经元中，以分析其对线性树突棘密度（dendritic spine density）的影响。该平台还可快速向离体与活体样本递送多种遗传载体，包括光遗传（optogenetic）试剂与细胞类型特异性试剂，以及荧光染料、钙传感器（calcium sensors）、电压传感器（voltage sensors）等快速作用试剂，从而实现单细胞分辨率（single-cell resolution）下的神经元环路活动操控与追踪。