A simple method of in vitro electroporation allows visualization, recording, and calcium imaging of local neuronal circuits.
A simple method of in vitro electroporation allows visualization, recording, and calcium imaging of local neuronal circuits.
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DOI：
10.1016/j.jneumeth.2010.05.017
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发表时间：
2010-08-15
影响因子：
3
通讯作者：
Urban, Nathaniel N.
中科院分区：
文献类型：
--
作者：
Hovis, Kenneth R.;Padmanabhan, Krishnan;Urban, Nathaniel N.
关键词：
自卡哈尔早期绘画以来，神经元结构的表征对于理解神经元功能至关重要。随着电生理技术的发展，提供了前所未有的访问这些细胞的生理，神经元功能的实验问题也变得更加容易处理。荧光示踪剂可以标记单个或群体神经元的解剖结构，为解剖学与生理学的联系打开了大门。然而，在实验上，目前的技术在体外大量标记的细胞往往会影响神经元的功能，探索结构-功能的问题，创造一个障碍。在这里，我们描述了一种新的技术，用于在细胞或细胞群内进行高度局部化的电穿孔，该技术能够在体外引入膜不可渗透的带电染料，包括葡聚糖缀合的荧光团、酰肼示踪剂和钙指示剂染料。我们证明，这种技术是高度通用的，允许标记的大或小面积的组织，允许在急性脑切片中确定的神经元回路的细胞形态和生理活动的调查。此外，这种方法允许随后的有针对性的全细胞补丁记录的基础上定义良好的连接，以及在一个快速的时间尺度上的目标电路的生理活动的评估。
Since Cajal's early drawings, the characterization of neuronal architecture has been paramount in understanding neuronal function. With the development of electrophysiological techniques that provide unprecedented access to the physiology of these cells, experimental questions of neuronal function have also become more tractable. Fluorescent tracers that can label the anatomy of individual or populations of neurons have opened the door to linking anatomy with physiology. Experimentally however, current techniques for bulk labeling of cells in vitro often affect neuronal function creating a barrier for exploring structure-function questions. Here we describe a new technique for highly localized electroporation within a cell or cell population that enables the introduction of membrane impermeable charged dyes including dextran-conjugated fluorophores, hydrazide tracers, and calcium indicator dyes in vitro. We demonstrate that this technique is highly versatile, allowing for labeling of large or small areas of tissue, allowing for the investigation of both cellular morphology and physiological activity in identified neuronal circuits in acute brain slices. Furthermore, this approach allows subsequent targeted whole-cell patch recording based on well-defined connectivity as well as assessment of physiological activity in targeted circuits on a fast time scale.
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