摘要：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(2+)-dependent, but our knowledge about in vivo Ca(2+) signalling in microglia is rudimentary. This is mostly due to technical reasons, as microglia largely resisted all attempts of in vivo labelling with Ca(2+) 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(2+) sensor Twitch-2B in microglia. The Twitch-2B-assisted in vivo imaging enables recording of spontaneous and evoked microglial Ca(2+) signals and allows for the first time to monitor the steady state intracellular Ca(2+) levels in microglia. Intact in vivo microglia show very homogenous and low steady state intracellular Ca(2+) 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(2+) level as a versatile microglial activation marker, which is highly sensitive to the cell's environment.
