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.2004 Apr;2(4):E96.
doi: 10.1371/journal.pbio.0020096. Epub 2004 Apr 13.

Calcium dynamics of cortical astrocytic networks in vivo

Affiliations

Calcium dynamics of cortical astrocytic networks in vivo

Hajime Hirase et al. PLoS Biol.2004 Apr.

Abstract

Large and long-lasting cytosolic calcium surges in astrocytes have been described in cultured cells and acute slice preparations. The mechanisms that give rise to these calcium events have been extensively studied in vitro. However, their existence and functions in the intact brain are unknown. We have topically applied Fluo-4 AM on the cerebral cortex of anesthetized rats, and imaged cytosolic calcium fluctuation in astrocyte populations of superficial cortical layers in vivo, using two-photon laser scanning microscopy. Spontaneous [Ca(2+)](i) events in individual astrocytes were similar to those observed in vitro. Coordination of [Ca(2+)](i) events among astrocytes was indicated by the broad cross-correlograms. Increased neuronal discharge was associated with increased astrocytic [Ca(2+)](i) activity in individual cells and a robust coordination of [Ca(2+)](i) signals in neighboring astrocytes. These findings indicate potential neuron-glia communication in the intact brain.

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Conflict of interest statement

The authors have declared that no conflicts of interest exist.

Figures

Figure 1
Figure 1. In Vivo Loading and Imaging of Astrocytes Using Fluo-4 AM
(A) Acute slice prepared 1 h after dye loading. Scale bar, 200 μm. (B) Higher magnification reveals cells with typical astrocyte morphology. Scale bar, 20 μm. (C) Average bulk fluorescence as a function of the depth from the pial surface. (D) Schematic drawing of the experimental arrangement. Abbreviations: EKG, electrocardiogram. PMT, photomultiplier. LFP, glass micropipe for local field potential and multiple unit recording. The same pipette was used to deliver bicuculline. (E) Image taken 50–150 μm below pial surface in vivo. Flattened xyz stack. (F) Fluo-4 AM loaded cells (left) were stained for S100B immunoreactivity (right), and the images were merged (center). See Video S3 for large-scale staining. Scale bar, 20 μm.
Figure 2
Figure 2. Time-Lapse Imaging of Astrocytes In Vivo
Four astrocytes, from which fluorometric Ca2+ imaging (0.5 Hz) was made, are outlined. A blood vessel, outlined by the astrocyte end feet, runs diagonally across the viewed area. White arrows show the end foot connected to the imaged astrocyte.
Figure 3
Figure 3. Frequency Domain Analysis of Population Dynamics of Fluorescence in Astrocytes in Control State and during Bicuculline-Induced Neuronal Hyperactivity
Insets show local field potentials in a control animal and regular spiking in a bicuculline treated mouse (scale bar: 2.0 s, 500 μV). Asterisks show significant differences (p < 0.05) between groups at various frequencies.
Figure 4
Figure 4. Spatio-Temporal Dynamics of Astrocyte Ca2+ Activity
(A) Definition of nearby (less than 50 μm) and distant (greater than 50 μm) cell pairs. (B) Fluorescence changes in two nearby astrocytes. (C) Cross-correlogram of fluorescent intensity. (D) Mean cross-correlation ofΔF/F0 in all nearby (thick line) and distant (thin line) cell pairs in control condition (left) and in the presence of bicuculline (right). Note large increase ofΔF/F0 correlation in nearby cell pairs in the bicuculline condition (error bar: standard error of the mean). (E) Relationship between distance of the two cells and the magnitude of correlation at zero timelag. Note lack of a reliable relationship in the control condition (left). Note also the significant negative correlation between the distance and correlatedΔF/F0 changes in cell pairs in the bicuculline-treated cortex (right).
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