Brain Under Surveillance: The Microglia Patrol

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Science  15 Jul 2005:
Vol. 309, Issue 5733, pp. 392-393
DOI: 10.1126/science.1114852

Biophysicists and biologists have long worked together to develop tools to analyze the limits of the living world—molecules and cells at one end and whole organisms at the other. In contrast, important intermediate levels of organization, namely the organs and tissues, had received little attention until very recently. For instance, we understand very little about what controls the density, shape, and size of organs, or how cells direct their movements or communicate with each other within tissues. This has been due in part to technical limitations. The environment that cells encounter within tissues can include multiple three-dimensional chemotactic gradients and numerous physical constraints imposed by interactions with the extracellular matrix and with other cells. Such a complex milieu is virtually impossible to reconstitute in vitro.

The advent of two-photon microscopy (1) and its use on tissues of living animals is rapidly advancing our understanding of cell behavior and fate within tissues (2, 3). Based on the simultaneous absorption of two photons, the technique allows greater imaging depth and minimal phototoxicity compared to conventional fluorescence microscopy. Whereas other imaging techniques require surgical dissection that can damage tissue, two-photon microscopy allows direct imaging of cells in the undisturbed physiological environment of an intact organ. Two recent studies by Nimmerjahn et al. (4) and Davalos et al. (5) used this powerful imaging approach to examine the activity of microglia, the most abundant immune cell in the brain, in live mice. Microglia comprise ~10% of the cells in the central nervous system. Under pathological conditions such as neurodegenerative disease, stroke, and tumor invasion, these cells become activated, surround damaged and dead cells, and clear cellular debris from the area, much like phagocytic macrophages of the immune system. In healthy mammalian brain tissue, microglia display characteristically elongated cell bodies with spine-like processes that often branch perpendicularly. Until Nimmerjahn and Davalos applied two-photon microscropy to a live and healthy mammalian brain, it was generally thought that microglia are essentially quiescent cells—dormant and nonmotile. But a static state is hardly what was observed.

The technique allowed the Nimmerjahn and Davalos groups to transcranially visualize microglia in live animals. Information was recorded from up to 200 μm below the brain's surface through a surgically thinned section of skull. Both groups generated transgenic mice whose microglia were fluorescently labeled. The easily detectable cells were observed for several hours in the brains of anesthesized mice. Whereas microglial cell bodies and main branches were stable for hours, their evenly distributed and highly ramified processes were remarkably motile, continuously and randomly undergoing cycles of formation, extension, and withdrawal on time scales of minutes (1.5 μm/min). These processes also displayed motile (4 μm/min), filopodia-like protrusions that typically formed bulbous tips with an average lifetime of 4 min. Although the function of these tips remains unclear, it is possible that they constitute specialized phagocytosis domains that clear accumulated metabolic products and deteriorated tissue. This high “resting” motility may serve a housekeeping function, enabling microglia to effectively sample and assess the status of the local surroundings and control their microenvironment. The restructuring activity of microglial processes is in sharp contrast to the apparent stability of dendritic processes of surrounding neurons. Microglial processes and protrusions were also observed to directly contact astrocytes, neuronal cell bodies, and blood vessels, suggesting that in healthy brain tissue, microglia communicate with other cortical cells to coordinately monitor the general health of the brain.

Both groups also performed laser-induced injury of individual capillaries in the brains of the transgenic mice. Within a few minutes, time-lapse imaging revealed rapid, targeted movements of nearby microglial processes toward the injured site (see the figure). The average velocity of microglial extensions radially impinging on the target site was similar to extension rates during the resting state. Within 30 min after laser ablation, processes of nearby microglia reached the damaged site and appeared to accumulate and fuse together, forming a spherical containment around the damaged area and establishing a potential barrier between healthy and injured tissue (5). Microglia responded to mechanical injury in a similar way. The shielding of injured sites suggests a neuroprotective role for microglia. Furthermore, the early formation of spherical inclusions within the microglial processes suggests immediate phagocytic engulfment and removal of damaged tissue or leaked blood components. Together, these findings confirm the idea that microglia represent the first line of defense against invading pathogens or other types of brain tissue injury.

Microglia patrol the brain and shield it from injury.

Microglia continually extend (green) and retract (yellow) processes, surveying their immediate environment within the brain. The processes move rapidly toward a site of injury, such as a damaged blood vessel in the brain, in response to the localized release of a chemoattractant (gradient of orange) from the injured sited. Once at the target site, the processes form a barrier to protect healthy tissue.


To identify the molecular signals that mediate this targeted microglial response, Davalos et al. (5) made use of the observation that microglial migration can be induced in cell culture with nucleotides that signal through P2Y receptors expressed at the cell surface. They demonstrate that localized application of ATP to the mouse brain (through either a craniotomy or a small electrode; neither invasive technique itself elicited a response from microglia), which mimics nucleotide release from injured tissue, attracted microglial processes, similar to the microglial response to injury. Apyrase, an ATPase (adenosine triphosphatase) that hydrolyzes ATP and ADP, substantially reduced both the baseline motility of microglial processes as well as their response to laser-induced tissue injury. Furthermore, they showed that activation of P2Y receptors on microglia in the surrounding tissue is necessary for the rapid microglial response toward the injured site. Previous studies showed that extracellular ATP can induce ATP release from astrocytes. ATP also mediates communication between astrocytes and between astrocytes and microglia. This ATP-induced ATP release was essential for attracting microglial processes. Indeed, when the authors applied apyrase and then a nonhydrolyzable ATP analog from a microelectrode, they observed no such rapid microglial response. Applying connexin channel inhibitors, which inhibit ATP release from astrocytes, also blocked the microglial response toward the laser ablation site. Resting motility of microglial processes in the intact brain also seems to be modulated by the same ATP signaling mechanisms that mediate injury-induced responses, because apyrase and connexin channel inhibitors nearly abolished microglial baseline dynamics.

These two elegant studies provide direct evidence for the highly dynamic nature of microglia, indicating that the brain is under constant immune surveillance by these cells. In the adult mammalian brain, there is generally little movement of cellular processes, except perhaps for those associated with synaptic plasticity that underlie learning and memory. Microglia are apparently never at physical rest either.

Although the development of two-photon microscopy opens new perspectives for the analysis of intact organs, some major technical issues remain. Improvements in the resolution, depth penetration, image acquisition speed, and photon detector sensitivity of the microscopes will enhance our ability to follow intracellular signaling events and cellular traffic in living tissues. Likewise, the generation of mice expressing fluorescent proteins under the control of different cell type-specific promoters or inducible promoters should allow the study of multiple cell functions within intact organs using two-photon microscopy. Accurate methods to quantify image information are also needed. Despite these obstacles, the development of intact organ imaging should continue to have a major impact in biology over the coming years.


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