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ATP mediates rapid microglial response to local brain injury in vivo

Abstract

Parenchymal microglia are the principal immune cells of the brain. Time-lapse two-photon imaging of GFP-labeled microglia demonstrates that the fine termini of microglial processes are highly dynamic in the intact mouse cortex. Upon traumatic brain injury, microglial processes rapidly and autonomously converge on the site of injury without cell body movement, establishing a potential barrier between the healthy and injured tissue. This rapid chemotactic response can be mimicked by local injection of ATP and can be inhibited by the ATP-hydrolyzing enzyme apyrase or by blockers of G protein–coupled purinergic receptors and connexin channels, which are highly expressed in astrocytes. The baseline motility of microglial processes is also reduced significantly in the presence of apyrase and connexin channel inhibitors. Thus, extracellular ATP regulates microglial branch dynamics in the intact brain, and its release from the damaged tissue and surrounding astrocytes mediates a rapid microglial response towards injury.

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Figure 1: Transcranial two-photon imaging shows rapid dynamics of fine microglial processes.
Figure 2: Microglial processes move rapidly towards the site of injury induced either by the two-photon laser, or mechanically with a glass electrode.
Figure 3: Extracellular ATP and activation of purinergic G protein–coupled receptors are necessary for rapid chemotactic microglial response.
Figure 4: ATP-induced ATP release is essential for rapid microglial response.
Figure 5: The microglial response towards the laser ablation is inhibited after blocking connexin hemichannels, the conduits of ATP.

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References

  1. Thomas, W.E. Brain macrophages: evaluation of microglia and their functions. Brain Res. Brain Res. Rev. 17, 61–74 (1992).

    Article  CAS  Google Scholar 

  2. Hickey, W.F. Basic principles of immunological surveillance of the normal central nervous system. Glia 36, 118–124 (2001).

    Article  CAS  Google Scholar 

  3. Gonzalez-Scarano, F. & Baltuch, G. Microglia as mediators of inflammatory and degenerative diseases. Annu. Rev. Neurosci. 22, 219–240 (1999).

    Article  CAS  Google Scholar 

  4. Aschner, M., Allen, J.W., Kimelberg, H.K., LoPachin, R.M. & Streit, W.J. Glial cells in neurotoxicity development. Annu. Rev. Pharmacol. Toxicol. 39, 151–173 (1999).

    Article  CAS  Google Scholar 

  5. Kreutzberg, G.W. Microglia: a sensor for pathological events in the CNS. Trends Neurosci. 19, 312–318 (1996).

    Article  CAS  Google Scholar 

  6. Smith, S.J., Cooper, M.W . & Waxman, A. Laser microscopy of subcellular structure in living neocortex: can one see dendritic spines twitch? in The Biology of Memory, XXIII Symposium Medicum Hoechst (eds. Squire, L.R., Lindenlaub, E.) 49–71 (Schattauer Verlag, Stuttgart, 1990).

    Google Scholar 

  7. Brockhaus, J., Moller, T. & Kettenmann, H. Phagocytozing ameboid microglial cells studied in a mouse corpus callosum slice preparation. Glia 16, 81–90 (1996).

    Article  CAS  Google Scholar 

  8. Czapiga, M. & Colton, C.A. Function of microglia in organotypic slice cultures. J. Neurosci. Res. 56, 644–651 (1999).

    Article  CAS  Google Scholar 

  9. Petersen, M.A. & Dailey, M.E. Diverse microglial motility behaviors during clearance of dead cells in hippocampal slices. Glia 46, 195–206 (2004).

    Article  Google Scholar 

  10. Inoue, K. Microglial activation by purines and pyrimidines. Glia 40, 156–163 (2002).

    Article  Google Scholar 

  11. Honda, S. et al. Extracellular ATP or ADP induce chemotaxis of cultured microglia through Gi/o-coupled P2Y receptors. J. Neurosci. 21, 1975–1982 (2001).

    Article  CAS  Google Scholar 

  12. Nolte, C., Moller, T., Walter, T. & Kettenmann, H. Complement 5a controls motility of murine microglial cells in vitro via activation of an inhibitory G protein and the rearrangement of the actin cytoskeleton. Neuroscience 73, 1091–1107 (1996).

    Article  CAS  Google Scholar 

  13. von Zahn, J., Moller, T., Kettenmann, H. & Nolte, C. Microglial phagocytosis is modulated by pro- and anti-inflammatory cytokines. Neuroreport 8, 3851–3856 (1997).

    Article  CAS  Google Scholar 

  14. Stence, N., Waite, M. & Dailey, M.E. Dynamics of microglial activation: a confocal time-lapse analysis in hippocampal slices. Glia 33, 256–266 (2001).

    Article  CAS  Google Scholar 

  15. Koshinaga, M. et al. Rapid and widespread microglial activation induced by traumatic brain injury in rat brain slices. J. Neurotrauma 17, 185–192 (2000).

    Article  CAS  Google Scholar 

  16. Jung, S. et al. Analysis of fractalkine receptor CX(3)CR1 function by targeted deletion and green fluorescent protein reporter gene insertion. Mol. Cell. Biol. 20, 4106–4114 (2000).

    Article  CAS  Google Scholar 

  17. Grutzendler, J., Kasthuri, N. & Gan, W.B. Long-term dendritic spine stability in the adult cortex. Nature 420, 812–816 (2002).

    Article  CAS  Google Scholar 

  18. Galbraith, J.A. & Terasaki, M. Controlled damage in thick specimens by multiphoton excitation. Mol. Biol. Cell 14, 1808–1817 (2003).

    Article  CAS  Google Scholar 

  19. Dubyak, G.R. & el-Moatassim, C. Signal transduction via P2-purinergic receptors for extracellular ATP and other nucleotides. Am. J. Physiol. 265, C577–C606 (1993).

    Article  CAS  Google Scholar 

  20. Zimmermann, H. & Braun, N. Extracellular metabolism of nucleotides in the nervous system. J. Auton. Pharmacol. 16, 397–400 (1996).

    Article  CAS  Google Scholar 

  21. Illes, P. & Alexandre Ribeiro, J. Molecular physiology of P2 receptors in the central nervous system. Eur. J. Pharmacol. 483, 5–17 (2004).

    Article  CAS  Google Scholar 

  22. Cotrina, M.L. et al. Connexins regulate calcium signaling by controlling ATP release. Proc. Natl. Acad. Sci. USA 95, 15735–15740 (1998).

    Article  CAS  Google Scholar 

  23. Guthrie, P.B. et al. ATP released from astrocytes mediates glial calcium waves. J. Neurosci. 19, 520–528 (1999).

    Article  CAS  Google Scholar 

  24. Cotrina, M.L., Lin, J.H., Lopez-Garcia, J.C., Naus, C.C. & Nedergaard, M. ATP-mediated glia signaling. J. Neurosci. 20, 2835–2844 (2000).

    Article  CAS  Google Scholar 

  25. Anderson, C.M., Bergher, J.P. & Swanson, R.A. ATP-induced ATP release from astrocytes. J. Neurochem. 88, 246–256 (2004).

    Article  CAS  Google Scholar 

  26. Schipke, C.G., Boucsein, C., Ohlemeyer, C., Kirchhoff, F. & Kettenmann, H. Astrocyte Ca2+ waves trigger responses in microglial cells in brain slices. FASEB J. 16, 255–257 (2002).

    Article  CAS  Google Scholar 

  27. Verderio, C. & Matteoli, M. ATP mediates calcium signaling between astrocytes and microglial cells: modulation by IFN-gamma. J. Immunol. 166, 6383–6391 (2001).

    Article  CAS  Google Scholar 

  28. Wang, Z., Haydon, P.G. & Yeung, E.S. Direct observation of calcium-independent intercellular ATP signaling in astrocytes. Anal. Chem. 72, 2001–2007 (2000).

    Article  CAS  Google Scholar 

  29. Beyer, E.C. & Steinberg, T.H. Evidence that the gap junction protein connexin-43 is the ATP-induced pore of mouse macrophages. J. Biol. Chem. 266, 7971–7974 (1991).

    CAS  Google Scholar 

  30. Stout, C.E., Costantin, J.L., Naus, C.C. & Charles, A.C. Intercellular calcium signaling in astrocytes via ATP release through connexin hemichannels. J. Biol. Chem. 277, 10482–10488 (2002).

    Article  CAS  Google Scholar 

  31. Braet, K., Vandamme, W., Martin, P.E., Evans, W.H. & Leybaert, L. Photoliberating inositol-1,4,5-trisphosphate triggers ATP release that is blocked by the connexin mimetic peptide gap 26. Cell Calcium 33, 37–48 (2003).

    Article  CAS  Google Scholar 

  32. Coco, S. et al. Storage and release of ATP from astrocytes in culture. J. Biol. Chem. 278, 1354–1362 (2003).

    Article  CAS  Google Scholar 

  33. Rouach, N. et al. Gap junctions and connexin expression in the normal and pathological central nervous system. Biol. Cell. 94, 457–475 (2002).

    Article  CAS  Google Scholar 

  34. Srinivas, M. & Spray, D.C. Closure of gap junction channels by arylaminobenzoates. Mol. Pharmacol. 63, 1389–1397 (2003).

    Article  CAS  Google Scholar 

  35. Hansson, E. & Ronnback, L. Glial neuronal signaling in the central nervous system. FASEB J. 17, 341–348 (2003).

    Article  CAS  Google Scholar 

  36. Fields, R.D. & Stevens-Graham, B. New insights into neuron-glia communication. Science 298, 556–562 (2002).

    Article  CAS  Google Scholar 

  37. Ciccarelli, R. et al. Involvement of astrocytes in purine-mediated reparative processes in the brain. Int. J. Dev. Neurosci. 19, 395–414 (2001).

    Article  CAS  Google Scholar 

  38. Wang, X. et al. P2X7 receptor inhibition improves recovery after spinal cord injury. Nat. Med. 10, 821–827 (2004).

    Article  CAS  Google Scholar 

  39. Nakki, R., Nickolenko, J., Chang, J., Sagar, S.M. & Sharp, F.R. Haloperidol prevents ketamine- and phencyclidine-induced HSP70 protein expression but not microglial activation. Exp. Neurol. 137, 234–241 (1996).

    Article  CAS  Google Scholar 

  40. Binder, D.K., Papadopoulos, M.C., Haggie, P.M. & Verkman, A.S. In vivo measurement of brain extracellular space diffusion by cortical surface photobleaching. J. Neurosci. 24, 8049–8056 (2004).

    Article  CAS  Google Scholar 

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Acknowledgements

We thank H. Suk-Woo, G. Shakhar, Y-C. Chen and R. Uglesich for offering useful comments and help with experiments. This work is supported by grants from the National Institute of Health and the Dana Foundation to M. L. D and W-B. G.

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Correspondence to Wen-Biao Gan.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Video 1

Baseline dynamics of fine microglial processes. Microglia in the cerebral cortex display a highly branched morphology with each cell soma decorated by long processes with fine termini. Timelapse imaging of microglia in the intact mouse brain reveals rapid extension, retraction, shape and volume changes of fine processes over intervals of seconds to minutes, while microglial cell bodies and main branches remain morphologically stable over hours. (MOV 147 kb)

Supplementary Video 2

Rapid microglial response after laser-induced ablation. This movie shows the dynamics of microglial processes before and immediately after a small laser ablation is induced ~40 µm from the pial surface. Within the first minutes post-ablation, the tips of the processes of the cells immediately surrounding the ablation appear bulbous and slightly enlarged. In the next few minutes, these cells extend their processes toward the damaged site where they appear to fuse together and form a spherical containment around it. During this period, the same cells also retract those processes that previously lied in directions opposite to the site of injury. Most of the cellular content of each of the immediate neighbors is directed towards the damaged site within the first 1-3 hours, whereas the cell bodies remain at approximately the same location for at least 10 hours. Cells located further away also respond in a directional way, sending their processes towards the ablation but without ever reaching the already contained injury site. (MOV 1031 kb)

Supplementary Video 3

Rapid microglial response after dual laser ablation. Immediately after the first ablation, the surrounding cells begin extending their processes toward the injury site, and retracting those on opposite sides. When the second ablation occurs 20 minutes after the first one, the previously retracting processes of the cell lying in between the two ablations started to extend toward the second site of damage. Although the cell had committed a number of its processes to respond to the first ablation, the same cell was still able to detect the second ablation and immediately assign its remaining processes on the opposite side to the new ablation. (MOV 537 kb)

Supplementary Video 4

Rapid microglial response after a small-scale mechanical injury, induced with a glass electrode through a small craniotomy. Using a micromanipulator, we inserted the glass electrode inside the brain and performed lateral movements around an area of 50 µm in diameter. We observed that similar to the laser-induced response, microglial processes assumed the bulbous morphology at their termini and rapidly moved into the damaged tissue. (MOV 143 kb)

Supplementary Video 5

Local injection of 10 mM ATP in an ACSF solution containing 3% rhodamine-dextran (to make the electrode visible) induces rapid microglial response toward the tip of the glass electrode in a way strikingly similar to that towards the laser-induced injury. (MOV 413 kb)

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Davalos, D., Grutzendler, J., Yang, G. et al. ATP mediates rapid microglial response to local brain injury in vivo. Nat Neurosci 8, 752–758 (2005). https://doi.org/10.1038/nn1472

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