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Updated: Feb 26, 2026
Simultaneous Imaging of Microglial Dynamics and Neuronal Activity in Awake Mice
Published on: August 23, 2022
A new approach for ratiometric in vivo calcium imaging of microglia
Bianca Brawek1, Yajie Liang1, Daria Savitska1
1Institute of Physiology II, University of Tübingen, 72074, Tübingen, Germany.
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.
Insights
In vivo calcium (Ca2+) imaging in microglia is now possible using a novel viral vector. This technique reveals that steady-state Ca2+ levels in microglia are low but increase upon activation, serving as a key indicator.
Area of Science:
- Neuroscience
- Immunology
- Cell Biology
Background:
- Microglia, the brain's immune cells, exhibit diverse functions like phagocytosis and cytokine release.
- In vitro studies indicate calcium (Ca2+) dependency for microglial effector functions.
- In vivo studies of microglial Ca2+ signaling are limited due to technical challenges in labeling.
Purpose of the Study:
- To develop a novel method for in vivo Ca2+ imaging in microglia.
- To investigate steady-state and evoked Ca2+ signaling in microglia within their native environment.
- To establish Ca2+ levels as a potential marker for microglial activation.
Main Methods:
- Development of a microglia-specific viral vector regulated by microRNA-9.
- Expression of the genetically-encoded Ca2+ sensor Twitch-2B in microglia.
- In vivo imaging of Ca2+ signals in microglia using the developed system.
Main Results:
- Successful in vivo expression of Twitch-2B in microglia via the novel viral vector.
- Monitoring of spontaneous and evoked microglial Ca2+ signals in vivo.
- Demonstration of low, homogeneous steady-state intracellular Ca2+ levels in intact, in vivo microglia.
- Significant increase in Ca2+ levels post-acute slice preparation and cell culturing, correlating with CD68 and IL-1β upregulation.
Conclusions:
- The novel viral vector enables unprecedented in vivo Ca2+ imaging of microglia.
- Steady-state intracellular Ca2+ levels serve as a sensitive indicator of microglial activation.
- Microglial Ca2+ levels are highly responsive to environmental changes, particularly ex vivo conditions.
