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ATP released from astrocytes mediates glial calcium waves - PubMed

  • ️Fri Jan 01 1999

ATP released from astrocytes mediates glial calcium waves

P B Guthrie et al. J Neurosci. 1999.

Abstract

Calcium waves represent a widespread form of intercellular communication. Although they have been thought for a long time to require gap junctions, we recently demonstrated that mouse cortical astrocytes use an extracellular messenger for calcium wave propagation. The present experiments identify ATP as a major extracellular messenger in this system. Medium collected from astrocyte cultures during (but not before) calcium wave stimulation contains ATP. The excitatory effects of medium samples and of ATP are blocked by purinergic receptor antagonists and by pretreatment with apyrase; these same purinergic receptor antagonists block propagation of electrically evoked calcium waves. ATP, applied at the concentration measured in medium samples, evokes responses that are qualitatively and quantitatively similar to those evoked by those medium samples. These data implicate ATP as an important transmitter between CNS astrocytes.

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Figures

Fig. 1.
Fig. 1.

Communication between noncontacting islands of astrocytes. Stimulation of an astrocyte island (approximately six contiguous cells) results in a calcium wave passing throughout the stimulated island and in activation of astrocytes in the noncontacting islands (arrows in left and right panels). Left, A phase-contrast image of four noncontacting islands of astrocytes. One astrocyte in thetop island was then electrically stimulated.Middle, right,F/F0 ratio images that show progression of the calcium wave throughout the field at the times indicated. Scale bar, 50 μm.

Fig. 2.
Fig. 2.

Human neutrophils respond to an extracellular message released during glial calcium waves. Left,Neutrophils were loaded with the calcium indicator fura-2 and seeded onto a astrocyte monolayer. Right, After electrical stimulation of a calcium wave in the astrocytes, calcium levels in the majority (but not all) of the neutrophils increased. Because the astrocytes were loaded with fluo-3, they did not appear in these images. The astrocytic fluo-3 fluorescence was used, in other regions of the culture, to confirm electrical stimulation efficacy before addition of the neutrophils. Scale bar, 50 μm.

Fig. 3.
Fig. 3.

Collection of extracellular message during an evoked calcium wave. Left, A schematic representation of the experimental protocol. A patch pipet with a 3–5 μm opening was positioned 10 μm above the astrocyte surface. A calcium wave was electrically evoked using an extracellular stimulation electrode, placed several cells away from the collection pipet. As the wave, monitored in real time, passed under the collection pipet, negative pressure was applied to the pipet, collecting 0.1–0.2 μl of medium. The stage was then rapidly moved to bring a distant area of the culture into the microscope field. The collection pipet was then brought into proximity of the naive astrocytes in that field, and the collected medium was gently expelled onto those astrocytes (arrowin the pipet indicates the direction of flow). Right, The typical results of such an experiment. When the collected medium was gently applied to those astrocytes, a dramatic rise in calcium was observed (top). Control medium (collected before calcium wave stimulation) had no effect (bottom), demonstrating that the response to the stimulated-field medium was not caused by a pressure artifact. (As the medium was expelled from the pipet, the oil filling the collection system advanced to the pipet tip; this change in material filling the pipet tip accounts for the change in apparent fluorescence observed within the pipet tip.) The calcium wave during which the sample was collected is not shown. Scale bar, 50 μm.

Fig. 4.
Fig. 4.

Mechanical stimulation of a calcium wave using glass microbeads. A small glass bead (30–50 μm), dropped through the medium onto a culture surface, initiates a calcium wave at the point where the bead lands. When thousands of beads are dropped throughout a single culture, thousands of such waves are simultaneously initiated and subsequently propagated throughout the culture. In this example, a single bead landed in the center of the field (phase-contrast image in the left panel, acquired after the fluorescence images), evoking the calcium wave seen in the middle andright panels. Scale bar, 50 μm.

Fig. 5.
Fig. 5.

ATP dose–response curve and the comparison with experimentally collected extracellular message samples. ATP evokes calcium responses in astrocytes. The number of astrocytes responding to control medium containing known amounts of exogenous ATP was determined and plotted against ATP concentration (mean ± SEM; four separate cultures used for each data point). The filled circlerepresents the mean number of cells responding to the medium of three separate samples containing extracellular message. The concentration of ATP in each sample was measured using the luciferin/luciferase assay. This point fell remarkably close to the dose–response curve determined using medium containing exogenous ATP. Thus, ATP is present in extracellular message samples at concentrations sufficient to account for much of the biological activity measured in those samples.

Fig. 6.
Fig. 6.

Purinergic antagonists block the biological activity of collected samples of extracellular message.Left, Samples of extracellular message evoke calcium responses in a majority of the astrocytes in the microscope field. Right, Addition of an aliquot of the same sample in the presence of suramin (100 μ

m

final concentration) virtually eliminated the biological activity. (The images shown are integrated to display every astrocyte responding to the sample during the time course of the experiment and do not represent a single time point.) Scale bar, 100 μm.

Fig. 7.
Fig. 7.

Local ATP application initiates astrocytic calcium waves. ATP (10 μ

m

) was locally applied from a patch pipet (arrow) using a pressure pulse 20 msec in duration. Left, Astrocytes in the path of the applied ATP responded with an immediate calcium elevation.Middle, right, The resulting calcium wave is clearly seen. Scale bar, 100 μm.

Fig. 8.
Fig. 8.

Purinergic antagonists block the propagation of an electrically evoked calcium wave. Left, When a single astrocyte was electrically stimulated, the evoked calcium wave propagated through many of the astrocytes in the field, normally involving 40 ± 7 cells. Right, Addition ofPPADS (10 μ

m

) to the observation saline virtually eliminated propagation of the calcium wave; only 6 ± 1 astrocytes show criterion calcium responses. Similar results were obtained using suramin (100 μ

m

). The astrocytes participating in the presence of PPADS included the astrocyte(s) immediately adjacent to the stimulating electrode, which were likely to be stimulated directly. (The images shown are integrated to display every astrocyte responding to the sample during the time course of the experiment and do not represent a single time point.) Scale bar, 100 μm.

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