Heredia Dante J, Hennig Grant W, Gould Thomas W
Department of Physiology and Cell Biology, School of Medicine, University of Nevada.
Department of Pharmacology, Larner College of Medicine, University of Vermont.
J Vis Exp. 2018 Oct 4(140):58347. doi: 10.3791/58347.
The electrical activity of cells in tissues can be monitored by electrophysiological techniques, but these are usually limited to the analysis of individual cells. Since an increase of intracellular calcium (Ca) in the cytosol often occurs because of the electrical activity, or in response to a myriad of other stimuli, this process can be monitored by the imaging of cells loaded with fluorescent calcium-sensitive dyes. However, it is difficult to image this response in an individual cell type within whole tissue because these dyes are taken up by all cell types within the tissue. In contrast, genetically encoded calcium indicators (GECIs) can be expressed by an individual cell type and fluoresce in response to an increase of intracellular Ca, thus permitting the imaging of Ca signaling in entire populations of individual cell types. Here, we apply the use of the GECIs GCaMP3/6 to the mouse neuromuscular junction, a tripartite synapse between motor neurons, skeletal muscle, and terminal/perisynaptic Schwann cells. We demonstrate the utility of this technique in classic ex vivo tissue preparations. Using an optical splitter, we perform dual-wavelength imaging of dynamic Ca signals and a static label of the neuromuscular junction (NMJ) in an approach that could be easily adapted to monitor two cell-specific GECI or genetically encoded voltage indicators (GEVI) simultaneously. Finally, we discuss the routines used to capture spatial maps of fluorescence intensity. Together, these optical, transgenic, and analytic techniques can be employed to study the biological activity of distinct cell subpopulations at the NMJ in a wide variety of contexts.
组织中细胞的电活动可通过电生理技术进行监测，但这些技术通常仅限于对单个细胞的分析。由于细胞溶质中细胞内钙（Ca）的增加通常是由于电活动，或对众多其他刺激的反应而发生的，因此这个过程可以通过对加载了荧光钙敏染料的细胞进行成像来监测。然而，很难在整个组织内的单个细胞类型中对这种反应进行成像，因为这些染料会被组织内的所有细胞类型摄取。相比之下，基因编码钙指示剂（GECIs）可以由单个细胞类型表达，并在细胞内Ca增加时发出荧光，从而允许对单个细胞类型的整个群体中的Ca信号进行成像。在这里，我们将GECIs GCaMP3/6应用于小鼠神经肌肉接头，即运动神经元、骨骼肌和终末/突触周围施万细胞之间的三方突触。我们展示了这项技术在经典离体组织制备中的实用性。使用分光器，我们对动态Ca信号和神经肌肉接头（NMJ）的静态标记进行双波长成像，这种方法可以很容易地适用于同时监测两种细胞特异性GECI或基因编码电压指示剂（GEVI）。最后，我们讨论了用于捕获荧光强度空间图的常规方法。总之，这些光学、转基因和分析技术可用于在各种情况下研究NMJ中不同细胞亚群的生物活性。
