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Simultaneous Imaging of Microglial Dynamics and Neuronal Activity in Awake Mice
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Calcium Imaging of Microglial Network Activity in Stroke
Petr Tvrdik1,2, Kathryn N Kearns3,4, Khadijeh A Sharifi3,4
1Department of Neurosurgery, School of Medicine, University of Virginia, Charlottesville, VA, USA. tvrdik@virginia.edu.
Methods in Molecular Biology (Clifton, N.J.)
|August 9, 2019
Summary
Researchers developed a novel mouse model for tracking calcium signals in microglia, crucial for brain inflammation research. This tool aids in studying microglial calcium dynamics in conditions like ischemic stroke.
Area of Science:
- Neuroscience
- Cell Biology
- Neuroinflammation
Background:
- Microglial activation is central to brain inflammation and is regulated by calcium signaling.
- Genetically encoded calcium indicators (GECI) are vital for calcium imaging but have faced challenges in microglia due to their unique properties.
- Previous methods limited effective in vivo calcium imaging in microglia.
Purpose of the Study:
- To present a novel reporter mouse line, PC::G5-tdT, for efficient genetic encoding of calcium indicators in microglia.
- To enable robust in vivo calcium imaging of microglial populations in the brain.
- To provide guidelines for utilizing this system in studying microglial calcium dynamics, particularly in the context of ischemic stroke.
Main Methods:
- Development of the PC::G5-tdT reporter mouse line utilizing the Polr2a locus.
- Crossbreeding PC::G5-tdT with the Iba1(Aif1)-IRES-Cre line to achieve microglia-specific expression of GCaMP5G and tdTomato.
- Application of the reporter system for in vivo calcium imaging in the ischemic cortex following middle cerebral artery occlusion (MCAO).
- Detailed protocols for genetic manipulation, surgical procedures, in vivo imaging, and data analysis.
Main Results:
- Successful generation of a mouse line enabling specific GECI expression (GCaMP5G) and red fluorescent protein labeling (tdTomato) in all brain microglia.
- Demonstration of the system's utility in monitoring intracellular calcium in large microglial populations in vivo.
- Validation of the approach in a mouse model of ischemic stroke, allowing detailed observation of microglial calcium dynamics in a pathological context.
Conclusions:
- The PC::G5-tdT reporter system overcomes previous limitations in microglial calcium imaging.
- This tool facilitates advanced in vivo studies of microglial function in neurological diseases like stroke.
- The provided guidelines support the widespread adoption and application of this technique in neuroinflammation research.
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