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Primary Microglia Isolation from Mixed Glial Cell Cultures of Neonatal Rat Brain Tissue
Published on: August 15, 2012
Purinergic receptors on microglial cells: functional expression in acute brain slices and modulation of microglial
Clemens Boucsein1, Robert Zacharias, Katrin Färber
1Max Delbrück Center for Molecular Medicine, Cellular Neuroscience, Robert-Rössle-Strasse10, D-13092, Berlin, Germany.
Abstract:
Microglial cells are the pathologic sensors in the brain. ATP released from damaged cells is a candidate for signalling neural injury to microglia. Moreover, ATP is an extracellular messenger for propagating astrocyte activity in the form of Ca2+ waves. To test for the functional expression of purinoreceptors in microglial cells we employed the patch-clamp technique in acute slices of adult mouse brain. ATP triggered a nonselective cationic and a K+ current. Pharmacological screening with purinergic ligands indicated the presence of P2Y1 and P2Y2/4 receptors linked to the activation of a K+ current and P2X receptors, including P2X7, linked to the activation of a nonselective cationic current. These findings suggest that microglial cells in situ express different purinergic receptors with distinct sensitivity and functional coupling. To test for the involvement of purinoreceptors in microglial activation, we stimulated cultured microglial cells with lipopolysaccharide and measured the release of tumour necrosis factor alpha, interleukin-6, interleukin-12 and macrophage inflammatory protein 1alpha, induction of K+ outward currents and nitric oxide release. All these parameters were reduced in the presence of purinergic ligands, indicating that purinergic receptor activation attenuated indicators of microglial activation.
Insights
Microglia utilize purinergic receptors to detect neural injury signals like ATP. Activating these receptors reduces microglial inflammatory responses, offering a potential therapeutic target for brain inflammation.
Area of Science:
- Neuroscience
- Cellular Biology
- Immunology
Background:
- Microglial cells act as the brain's primary immune responders.
- Extracellular ATP signals neural damage and propagates astrocyte activity.
- Purinergic receptors on microglia are potential mediators of these responses.
Purpose of the Study:
- To investigate the functional expression of purinergic receptors on microglial cells in situ.
- To determine the role of purinergic receptor activation in modulating microglial activation.
Main Methods:
- Patch-clamp electrophysiology in acute adult mouse brain slices.
- Pharmacological characterization using purinergic receptor ligands.
- Stimulation of cultured microglial cells with lipopolysaccharide (LPS).
- Measurement of inflammatory markers (TNF-α, IL-6, IL-12, MIP-1α), ion currents, and nitric oxide.
Main Results:
- ATP activated nonselective cationic and K+ currents in microglia.
- Microglia express functional P2Y1, P2Y2/4, and P2X receptors (including P2X7).
- Purinergic receptor activation significantly attenuated LPS-induced microglial activation markers.
Conclusions:
- Microglial cells express diverse purinergic receptors with distinct signaling pathways.
- Purinergic receptor modulation offers a potential strategy to dampen neuroinflammation.
