Abstract
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.
Highlights
The brain is highly heterogeneous [1,2,3], and requires single-cell resolution techniques for its analysis
Micropipette acts as a short-term diffusion-restricted sample reservoir after drawing in small volumes of reagents from a multiwell plate. (b) The micropipette is automatically transferred to the tissue slice bath and brought into a fixed point in the user’s field of view. (c,d) By using a stage controller in conjunction with fluorescence imaging of dye outflow from the micropipette and phase-contrast imaging of cell soma, the user can rapidly bring cells into contact with the micropipette and electroporate them using an electrical pulse sequence we designed for reliable highefficiency transfection. (e) When transfection of a reagent is completed, the system automatically removes, cleans, and washes the micropipette before beginning the transfection cycle of a new reagent
The fundamental operation of the technique relies upon loading a micropipette with a transfection reagent, which may contain a mixture of multiple agents such as plasmids, in an ionic solution, and positioning the tip opening on or near the cell of interest before applying an electrical signal to electroporate the membrane of the targeted cell
Summary
The brain is highly heterogeneous [1,2,3], and requires single-cell resolution techniques for its analysis. Genetic manipulation, labeling, and tracking of single cells in brain tissues enable the analysis of neuronal circuits [4,5,6], cellular dynamics, and genetics in ways not possible using viral or other bulk methods [7]. While optical microscopy techniques have been able to take advantage of the accessibility of brain slice cultures to achieve sub-cellular resolution imaging [23,24,25], technologies for genetically modifying and labeling cells with single-cell accuracy have been limited in both their speed and scalability. A high-throughput, scalable, easy-to-use, and reliable singlecell genetic manipulation technique could open new frontiers in neuroscience
Sign-in/Register to view highlights & summary 
Published Version (
Free)
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call