Carlson Greg C, Coulter Douglas A
Translational Research Laboratory, University of Pennsylvania School of Medicine, Room 2226, 125 S 31st Street, Philadelphia, Pennsylvania 19104-3403, USA.
Nat Protoc. 2008;3(2):249-55. doi: 10.1038/nprot.2007.539.
In many brain areas, circuit connectivity is segregated into specific lamina or glomerula. Functional imaging in these anatomically discrete areas is particularly useful in characterizing circuit properties. Voltage-sensitive dye (VSD) imaging directly assays the spatiotemporal dynamics of neuronal activity, including the functional connectivity of the neurons involved. In spatially segregated structures, VSD imaging can define how physiology and connectivity interact, and can identify functional abnormalities in models of neurological and psychiatric disorders. In the following protocol, we describe the in vitro slice preparation, epifluorescence setup and analyses necessary for fast charge-coupled device (CCD)-based VSD imaging combined with simultaneous whole-cell patch recording. The addition of single-cell recordings validates imaging results, and can reveal the relationship between single-cell activity and the VSD-imaged population response; in synchronously activated neurons, this change in whole-cell recorded V(m) can accurately represent population V(m) changes driving the VSD responses. Thus, the combined VSD imaging and whole-cell patch approach provides experimental resolution spanning single-cell electrophysiology to complex local circuit responses.
在许多脑区，神经回路连接被分隔到特定的层或小球中。在这些解剖学上离散的区域进行功能成像，对于表征神经回路特性特别有用。电压敏感染料（VSD）成像直接检测神经元活动的时空动态，包括所涉及神经元的功能连接。在空间上分隔的结构中，VSD成像可以确定生理学和连接性如何相互作用，并可以识别神经和精神疾病模型中的功能异常。在以下方案中，我们描述了基于快速电荷耦合器件（CCD）的VSD成像结合同步全细胞膜片钳记录所需的体外切片制备、落射荧光设置和分析。单细胞记录的加入验证了成像结果，并可以揭示单细胞活动与VSD成像的群体反应之间的关系；在同步激活的神经元中，全细胞记录的V（m）的这种变化可以准确地代表驱动VSD反应的群体V（m）变化。因此，VSD成像和全细胞膜片钳相结合的方法提供了从单细胞电生理到复杂局部回路反应的实验分辨率。
