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A new approach for ratiometric in vivo calcium imaging of microglia.

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-1?. These data identify the steady state intracellular Ca²⁺ level as a versatile microglial activation marker, which is highly sensitive to the cell's environment.

SUBMITTER: Brawek B

PROVIDER: S-EPMC5519759 | biostudies-literature | 2017 Jul

REPOSITORIES: biostudies-literature

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A new approach for ratiometric in vivo calcium imaging of microglia.

Brawek Bianca B Liang Yajie Y Savitska Daria D Li Kaizhen K Fomin-Thunemann Natalie N Kovalchuk Yury Y Zirdum Elizabeta E Jakobsson Johan J Garaschuk Olga O

Scientific reports 20170720 1

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<sup>2+</sup>-dependent, but our knowledge about in vivo Ca<sup>2+</sup> signalling in microglia is rudimentary. This is mostly due to technical reasons, as microglia largely ...[more]

PMID: 28729628

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Project description:Potassium is the most abundant intracellular metal in the body, playing vital roles in regulating intracellular fluid volume, nutrient transport, and cell-to-cell communication through nerve and muscle contraction. On the other hand, aberrant alterations in K+ homeostasis contribute to a diverse array of diseases spanning cardiovascular and neurological disorders to diabetes to kidney disease to cancer. There is an unmet need for studies of K+ physiology and pathology owing to the large differences in intracellular versus extracellular K+ concentrations ([K+]intra = 150 mM, [K+]extra = 3-5 mM). With a relative dearth of methods to reliably measure dynamic changes in intracellular K+ in biological specimens that meet the dual challenges of low affinity and high selectivity for K+, particularly over Na+, currently available fluorescent K+ sensors are largely optimized with high-affinity receptors that are more amenable for extracellular K+ detection. We report the design, synthesis, and biological evaluation of Ratiometric Potassium Sensor 1 (RPS-1), a dual-fluorophore sensor that enables ratiometric fluorescence imaging of intracellular potassium in living systems. RPS-1 links a potassium-responsive fluorescent sensor fragment (PS525) with a low-affinity, high-selectivity crown ether receptor for K+ to a potassium-insensitive reference fluorophore (Coumarin 343) as an internal calibration standard through ester bonds. Upon intracellular delivery, esterase-directed cleavage splits these two dyes into separate fragments to enable ratiometric detection of K+. RPS-1 responds to K+ in aqueous buffer with high selectivity over competing metal ions and is sensitive to potassium ions at steady-state intracellular levels and can respond to decreases or increases from that basal set point. Moreover, RPS-1 was applied for comparative screening of K+ pools across a panel of different cancer cell lines, revealing elevations in basal intracellular K+ in metastatic breast cancer cell lines vs. normal breast cells. This work provides a unique chemical tool for the study of intracellular potassium dynamics and a starting point for the design of other ratiometric fluorescent sensors based on two-fluorophore approaches that do not rely on FRET or related energy transfer designs.

Dataset Information

A new approach for ratiometric in vivo calcium imaging of microglia.

Publications

A new approach for ratiometric in vivo calcium imaging of microglia.

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