Abstract
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 Ca2+-dependent, but our knowledge about in vivo Ca2+ signalling in microglia is rudimentary. This is mostly due to technical reasons, as microglia largely resisted all attempts of in vivo labelling with Ca2+ indicators. Here, we introduce a novel approach, utilizing a microglia-specific microRNA-9-regulated viral vector, enabling the expression of a genetically-encoded ratiometric Ca2+ sensor Twitch-2B in microglia. The Twitch-2B-assisted in vivo imaging enables recording of spontaneous and evoked microglial Ca2+ signals and allows for the first time to monitor the steady state intracellular Ca2+ levels in microglia. Intact in vivo microglia show very homogenous and low steady state intracellular Ca2+ 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 Ca2+ level as a versatile microglial activation marker, which is highly sensitive to the cell’s environment.
Highlights
Microglia are resident immune cells of the central nervous system (CNS) classically thought to mediate the innate defence responses against pathogens as well as brain injury[1, 2]
Depending on the strength of the DAMP/pathogen-associated molecular pattern molecules (PAMPs) stimulus, microglial cells engage in different effector responses including cytoskeletal rearrangements, process extension, migration to the site of injury, enhanced phagocytosis as well as release of proinflammatory cytokines, nitric oxide (NO) and neurotrophic factors[2, 12]
In vitro data suggest that well known PAMPs, such as LPS, cause chronic elevations of [Ca2+]i and that these chronic elevations are required for effector responses of microglia such as release of NO or certain cytokines and chemokines[13, 14]
Summary
Microglia are resident immune cells of the central nervous system (CNS) classically thought to mediate the innate defence responses against pathogens as well as brain injury[1, 2]. Because retroviruses only transduce dividing cells, the virus had to be injected 2 days after stab wound injury, triggering microglial proliferation This approach enabled monitoring agonist-evoked microglial Ca2+ signals in brain slices and theoretically it can be applied in vivo. Ageing and amyloid accumulation dramatically increased the incidence of somatic Ca2+ transients in cortical microglia[22] Despite these encouraging results, electroporation technique cannot be widely used for analyses of microglial physiology because of several limitations: (i) it is very laborious since each cell has to be approached individually, (ii) it is not applicable in longitudinal experiments and (iii) it cannot be excluded that electroporation itself modifies the cell’s function. To enable reliable in vivo measurements of steady state intracellular Ca2+ level as well as Ca2+ transients we chose to express in microglia our novel ratiometric GECI Twitch-2B25 by means of a microRNA-9-regulated lentiviral vector[26]
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