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Updated: Jun 27, 2026
Two-photon Imaging of Microglial Processes' Attraction Toward ATP or Serotonin in Acute Brain Slices
Published on: January 31, 2019
Microglial cell migration stimulated by ATP and C5a involve distinct molecular mechanisms: quantification of
1Department of Pharmacology, University of Washington, 1959 NE Pacific St., Seattle, WA 98195, USA.
Abstract:
Microglial cells, the macrophages of the brain, play an essential role in the propagation of neuroinflammation. Increased microglial cell migration in response to specific chemoattractants has been documented, but less is known about the differences between these stimuli and the signal transduction pathways that mediate their effects. Current methods to measure cell migration are often labor-intensive and rely on the manual counting of cell number, so more efficient and objective methods are needed. Here we present an improved and higher-throughput Boyden chamber technique that measures microglial cell migration by using DRAQ5, a nuclear dye that emits in the near-infrared. Out of a panel of chemoattractants tested, we found that ATP and C5a potently stimulate the migration of mouse primary microglial cells. The stimulatory effects of ATP and C5a displayed significant additivity, suggesting that each chemoattractant stimulated migration through independent molecular mechanisms. Accordingly, we found key differences in these responses: ATP stimulated a combination of both chemokinesis and chemotaxis, and this response was mediated by the ROCK signaling pathway; whereas C5a stimulated only chemotaxis and this response was mediated by the Rac1 signaling pathway. Finally, we found that functional PI3-kinase is only required for random basal microglial cell migration. Thus, our results show that distinct nonoverlapping signal transduction pathways control different modes of microglial cell migration and suggest that the targeting of these distinct molecular mechanisms should modulate different aspects of neuroinflammation propagation.
Insights
This study reveals distinct molecular pathways controlling microglial cell migration, crucial for understanding neuroinflammation. ATP and C5a activate separate signaling routes, offering targeted therapeutic strategies for brain inflammation.
Area of Science:
- Neuroscience
- Immunology
- Cell Biology
Background:
- Microglial cells are key players in brain neuroinflammation.
- Understanding microglial migration pathways is crucial for neuroinflammation research.
- Existing cell migration assays are often labor-intensive and subjective.
Purpose of the Study:
- To develop a higher-throughput method for measuring microglial cell migration.
- To investigate and compare the signaling pathways activated by different chemoattractants.
- To elucidate the distinct mechanisms underlying microglial migration modes.
Main Methods:
- An improved, higher-throughput Boyden chamber assay utilizing DRAQ5 nuclear dye.
- Testing a panel of chemoattractants on primary mouse microglial cells.
- Analyzing signal transduction pathways including ROCK, Rac1, and PI3-kinase.
Main Results:
- ATP and C5a were identified as potent chemoattractants for microglial cells.
- ATP-induced migration involved chemokinesis and chemotaxis via the ROCK pathway.
- C5a-induced migration involved chemotaxis mediated by the Rac1 pathway.
- PI3-kinase was essential only for random basal migration.
Conclusions:
- Distinct, non-overlapping signaling pathways control different modes of microglial migration.
- ATP and C5a utilize independent molecular mechanisms to stimulate microglial migration.
- Targeting these specific pathways could modulate neuroinflammation propagation.
