Institute of Physiology II, University of Tübingen, 72074, Tübingen, Germany.
Laboratory of Molecular Neurogenetics, Department of Experimental Medical Science, Wallenberg Neuroscience Center and Lund Stem Cell Center, Lund University, Lund, 221 84, Sweden.
Sci Rep. 2017 Jul 20;7(1):6030. doi: 10.1038/s41598-017-05952-3.
Microglia, resident immune cells of the brain, react to the presence of pathogens/danger signals with a large repertoire of functional responses including morphological changes, proliferation, chemotaxis, production/release of cytokines, and phagocytosis. In vitro studies suggest that many of these effector functions are Ca-dependent, but our knowledge about in vivo Ca signalling in microglia is rudimentary. This is mostly due to technical reasons, as microglia largely resisted all attempts of in vivo labelling with Ca indicators. Here, we introduce a novel approach, utilizing a microglia-specific microRNA-9-regulated viral vector, enabling the expression of a genetically-encoded ratiometric Ca sensor Twitch-2B in microglia. The Twitch-2B-assisted in vivo imaging enables recording of spontaneous and evoked microglial Ca signals and allows for the first time to monitor the steady state intracellular Ca levels in microglia. Intact in vivo microglia show very homogenous and low steady state intracellular Ca levels. However, the levels increase significantly after acute slice preparation and cell culturing along with an increase in the expression of activation markers CD68 and IL-1β. These data identify the steady state intracellular Ca level as a versatile microglial activation marker, which is highly sensitive to the cell's environment.
小胶质细胞是大脑中常驻的免疫细胞，它们对病原体/危险信号的存在会产生一系列功能反应，包括形态变化、增殖、趋化、细胞因子的产生/释放和吞噬作用。体外研究表明，其中许多效应功能是依赖 Ca 的，但我们对小胶质细胞体内 Ca 信号的了解还很初步。这主要是由于技术原因，因为小胶质细胞在很大程度上抵制了所有用 Ca 指示剂进行体内标记的尝试。在这里，我们介绍了一种新方法，利用小胶质细胞特异性 microRNA-9 调控的病毒载体，使基因编码的比率 Ca 传感器 Twitch-2B 在小胶质细胞中表达。Twitch-2B 辅助的体内成像使自发和诱发的小胶质细胞 Ca 信号的记录成为可能，并首次允许监测小胶质细胞内的稳态细胞内 Ca 水平。完整的体内小胶质细胞显示出非常同质和低的稳态细胞内 Ca 水平。然而，在急性切片制备和细胞培养后，以及激活标记物 CD68 和 IL-1β 的表达增加后，这些水平显著增加。这些数据将稳态细胞内 Ca 水平确定为一种多功能的小胶质细胞激活标志物，对细胞的环境高度敏感。
