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Astrocyte Ca²⁺ waves trigger responses in microglial cells in brain slices ¹

Max-Delbrück Center for Molecular Medicine, Cellular Neuroscience, D-13092 Berlin; and
^* Max Planck Institute of Experimental Medicine, Neurogenetics, D-37075 Göttingen, Germany

²Correspondence: Max Delbrück Center for Molecular Medicine, Robert-Rössle-Strasse 10, D-13092 Berlin, Germany. E-mail: hketten{at}mdc-berlin.de

SPECIFIC AIMS

Astrocytes, the largest glial population in the brain, can communicate over long distances (namely, via waves of intracellular Ca²⁺ elevation); this form of communication has been established in primary cultures, slice cultures, and the isolated retina. We have found conditions to trigger such Ca²⁺ waves in acute slices of brain tissue and have shown that the wave leads to a global purinergic response in glia, including an activation of microglial cells.

PRINCIPAL FINDINGS

1. In acute brain slices, a Ca²⁺ wave can be triggered in corpus callosum glial cells
We loaded 250–300 µm-thick coronal slices from 5- to 8-day-old mice with the Ca²⁺-sensitive dye Fluo-4-AM (10 µM) and placed a glass stimulation pipette with a tip opening of 15 µm onto the surface of the slice in the corpus callosum. As a white matter tract, the corpus callosum contains no neuronal cell bodies, and therefore all Fluo-4-labeled somata were from glia. To trigger a response, we stimulated electrically (4 s at 10 Hz) or chemically by ejection of ATP (0.1 mM) from a patch pipette. In response to either stimulation, a rapid Ca²⁺ increase was observed in cells close to the stimulation pipette. With a delay of several seconds, Ca²⁺ increased in cells more distant to the stimulation pipette. The Ca²⁺ signal propagated to the border of our observation area, indicating that the wave extended over a distance of >0.5 mm. The Ca²⁺ response of a given cell lasted for 1 min except for cells located in the close vicinity of the stimulation pipette. Cellular depolarization was not sufficient for wave propagation: as a control, local ejection of elevated [K⁺] (100 mM) from a patch pipette induced only a transient Ca²⁺ elevation restricted to the site of application. The wave was not confined to the corpus callosum; cells lining the ventricular wall, most likely ependymal cells, and cells in the adjacent cortical layers also responded with an increase in Ca²⁺. The wave spread with a similar velocity within the cortical layers as in the corpus callosum (Fig. 1 ).

View larger version (112K): [in this window] [in a new window]   Figure 1. Propagation of Ca2+ waves in an acute brain slice. A series of fluorescence images just before (control) and at defined times (as indicated) after electrical stimulation illustrate the spread of the Ca2+ signal within a slice (A). The position of the stimulation electrode is marked by the asterisk. B) Outline of the anatomical structures of the fluorescence images. Transient changes in Ca2+ are displayed in panel C. In area 1, close to the stimulation pipette, the increase in fluorescence (F/Fo) occurred right after stimulation (indicated by vertical line). At more distant areas the delay amounted to several seconds. Note that cells lining the ventricular wall respond with an intense signal.

2. The Ca²⁺ wave depends on Ca²⁺ release from internal calcium stores
To test for the source of Ca²⁺, we compared responses in Ca²⁺-containing and Ca²⁺-free solutions. In Ca²⁺-free solution, the wave spread even further and recruited more cells than in normal buffer. We therefore performed most of our experiments in Ca²⁺-free solution. However, complete depletion of internal Ca²⁺ stores abolished the Ca²⁺ wave as shown by the application of Thapsigargin, a blocker of Ca²⁺ uptake into the endoplasmic stores. The first application of ATP in the presence of Thapsigargin triggered a Ca²⁺ increase; a second application no longer led to a significant increase. Still in the presence of Thapsigargin, subsequent electrical stimulation did not elicit a Ca²⁺ wave, but the signal was restricted to the close proximity of the stimulation pipette (n=3).

3. The Ca²⁺ wave is propagating by an extracellular signaling molecule
In cultured astrocytes, Ca²⁺ signals spread either due to propagation via gap junctions or through release of ATP and consecutive activation of purinergic receptors. In our experiments, the wave propagated with a velocity of 13.9 ± 1.8 µm/s (n=26). The spread of the Ca²⁺ wave was strongly influenced by the bath perfusion. With the superfusion turned off, the wave traveled in almost concentric circles around the stimulation pipette. Turning on the superfusion led to wave propagation predominantly in the direction of the bath flow, indicating that a diffusible substance is part of the signaling process.

4. The Ca²⁺ wave depends on metabotropic purinergic receptor activity
To test for the involvement of purinergic receptors, we compared propagation of the Ca²⁺ wave in the presence and absence of an antagonist for purinergic receptors, Reactive Blue 2. When slices were incubated for 2 min with Reactive Blue 2 (30 µM), stimulation resulted only in a local Ca²⁺ increase in the close vicinity of the stimulation pipette as compared with the control (n=5). A similar result was obtained with suramin, another antagonist (n=5). To exclude the involvement of glutamatergic receptor activation, we carried out stimulations in the presence of 50 µM MCPG ((+)--methyl-4-carboxy-phenylglycine) (n=6) and 50 µM CNQX (6-cyano-7-nitroquinoxaline-2,3-dione) (n=4) in different sets of experiments. The wave was not altered in the presence of these blockers.

To exclude that electrical activity of axons is involved in the propagation of the wave, we incubated slices with 1 µM tetrodotoxin and 100 µM Cd²⁺, thus blocking the generation of action potentials and synaptic release, respectively. The Ca²⁺ wave was not affected, indicating that the wave spread independently of neuronal activity (n=4). To test for wave propagation via gap junctions, slices were incubated for 2 min with the gap junction blocker octanol (500 µM) (n=3); wave propagation was not affected.

5. The wave spreads within astrocytes and nonastrocytic macroglial cells
To identify cells that participate in the wave, we used transgenic mice in which astrocytes are labeled by the enhanced green fluorescent protein (EGFP) that is expressed under control of the GFAP promoter. We have used Calcium Orange as a Ca²⁺ sensor, which allowed for imaging of EGFP fluorescence and intracellular Ca²⁺ simultaneously. The wave propagated in EGFP-positive as well as in EGFP-negative cells. To further characterize the responding cells, we studied the membrane current pattern with the patch-clamp technique applied to cells that were part of the Ca²⁺ wave. We recorded two different current patterns in response to voltage steps in de- and hyperpolarizing direction: cells with a linear current-voltage curve exhibited passive membrane currents with no sign of voltage dependency and without the tail currents typical for oligodendrocytes; these cells thus are most likely immature astrocytes. A second population of cells was characterized by a lack of inward currents, a prominent delayed rectifying current, and small sodium inward currents. This current pattern is characteristic for glial precursor cells. Thus, the wave propagates within astrocytes and glial precursor cells.

6. The glial Ca²⁺ wave triggers activation of membrane currents in microglia
Microglial cells represent the major immunocompetent element of the central nervous system (CNS) and react with a complex and graded response to any type of CNS injury. To test whether microglial cells participate in the Ca²⁺ wave, we recorded electrophysiological signals from identified microglia while eliciting a Ca²⁺ wave because double labeling with the Ca²⁺ sensor and the microglia-specific dye tomato lectin (lectin from lycopersicon esculentum, coupled to Texas Red) showed that microglia did not take up the Ca²⁺ dye. We approached the labeled cells with the patch pipette and recorded membrane currents while eliciting a Ca²⁺ wave by electrical stimulation 50–100 µm away from the patch pipette. Five of 13 microglia investigated showed the transient induction of an outwardly rectifying current when the Ca²⁺ wave passed the cell. This current corresponds to the currents observed in cultured microglia after stimulation with ATP. Thus, microglial cells sense the activation of other glial cells and participate in this global glial activity (Fig. 2 ).

View larger version (89K): [in this window] [in a new window]   Figure 2. Microglial cells sense and respond to the calcium wave. A) Illustration of the spread of a Ca2+ wave elicited by electrical stimulation. Membrane currents were recorded from a microglial cell. The position of the recording and stimulation pipette is indicated in the first image. The bar denotes 20 µm; the time after stimulation is indicated in each image recorded before stimulation (control). B) Transient changes in Ca2+ from 5 cells (indicated in the last image in panel A) are displayed in combination with the membrane currents recorded from the microglial cell. The membrane of the microglial cell was clamped at -20 mV and repetitively clamped to a series of de- and hyperpolarizing values. The currents measured at different clamping potentials allowed us to construct current-to-voltage curves at 2 s intervals. It is evident that with a delay to stimulation, outward currents increased in amplitude. C) Current voltage curve of the stimulation-induced current is displayed showing features of an outward rectifying K+ channel.

CONCLUSIONS

Our experiments demonstrate that a global glial response can be activated in brain tissue and can spread hundreds of micrometers from its site of origin. This response is not restricted to astrocytes, but spreads to microglia, glial progenitor cells, and cells lining the ventricular wall, most likely ependymal cells. Our data indicate that, like in the retina, the propagation of the Ca²⁺ wave is mediated by release of ATP and subsequent activation of purinergic receptors and involves Ca²⁺ release from cytoplasmic stores. Astrocytic Ca²⁺ waves and concomitant ATP release could communicate injury to uninjured areas and thereby activate microglial purinergic receptors. This could lead to the release of cytokines since stimulation of microglial purinergic receptors in culture leads to the release of tumor necrosis factor and modulation of interleukin 1ß (IL-1ß) release. Cytokine release from microglia can then influence the Ca²⁺ wave. Increased ATP release from astrocytes after interferon treatment has been shown, and it is known that IL-1ß influences Ca²⁺ wave propagation in cultures of human fetal astrocytes. ATP and cytokines might therefore be key molecules to communicate a local brain insult and to regulate the tissue response (Fig. 3 ). All types of glial cells, astrocytes, oligodendrocytes and their progenitors, and microglial cells express various types of purinoreceptors, and this receptor family can be viewed as the backbone of a common glial communication system.

View larger version (22K): [in this window] [in a new window]   Figure 3. The glial communication network. Electrical stimulation or ATP ejection from a micropipette triggers a cytosolic Ca2+ increase in astrocytes in the immediate surrounding of the pipette. The Ca2+ increase spreads like a wave to neighboring astrocytes. Since the activity of purinergic receptors is essential and we have evidence for the release of a substance, we assume that ATP is the extracellular messenger. Besides astrocytes, glial precursor cells and cells lining the ventricular wall (most likely ependymal cells) respond with a cytosolic Ca2+ increase. Furthermore, purinergic receptors on microglia are activated when the Ca2+ wave passes by (as substantiated using the patch-clamp technique). Thus, the entire glial population senses and/or participates in this long-distance signaling event and purinergic receptors are the key elements. It is conceivable that the nonastrocytic glial cells also respond with the release of substances. For microglial cells, cytokines are potential candidates since ATP can trigger cytokine release in culture.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.01-0514fje; to cite this article, use FASEB J. (December 28, 2001) 10.1096/fj.01-0514fje

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