Astrocytes generate Na+-mediated metabolic waves - PubMed (original) (raw)
Astrocytes generate Na+-mediated metabolic waves
Yann Bernardinelli et al. Proc Natl Acad Sci U S A. 2004.
Abstract
Glutamate-evoked Na+ increase in astrocytes has been identified as a signal coupling synaptic activity to glucose consumption. Astrocytes participate in multicellular signaling by transmitting intercellular Ca2+ waves. Here we show that intercellular Na+ waves are also evoked by activation of single cultured cortical mouse astrocytes in parallel with Ca2+ waves; however, there are spatial and temporal differences. Indeed, maneuvers that inhibit Ca2+ waves also inhibit Na+ waves; however, inhibition of the Na+/glutamate cotransporters or enzymatic degradation of extracellular glutamate selectively inhibit the Na+ wave. Thus, glutamate released by a Ca2+ wave-dependent mechanism is taken up by the Na+/glutamate cotransporters, resulting in a regenerative propagation of cytosolic Na+ increases. The Na+ wave gives rise to a spatially correlated increase in glucose uptake, which is prevented by glutamate transporter inhibition. Therefore, astrocytes appear to function as a network for concerted neurometabolic coupling through the generation of intercellular Na+ and metabolic waves.
Figures
Fig. 1.
Astrocytes generate intercellular waves. (A) Intensity-modulated-display images of SBFI excitation ratio recorded in cultured astrocytes showing the
elevation over time after single-cell mechanical stimulation. The top image shows the pipette tip position over the cell. The false colors represent SBFI ratio values corresponding to
ranging from dark green for resting values to red for elevated values. (Scale bar, 200 μm.) (B) Single cell SBFI ratio values plotted against time. The corresponding cells (white regions, Right) were randomly selected along a line (dotted line) starting from the stimulated cell 1. The red arrows represent the onset of response in each cell defined as the timepoint preceding a signal increase >2 SD over baseline. The graph shows that the
increase occurred sequentially in a cell-by-cell manner along the line. (C)Na+ increase for regions selected at a radial distance of 180 μm (dotted circle, Right). The arrows shows that at an equal given distance,
increase is synchronous. (D)
increase for cells selected 210 μm from the stimulated spot (dotted circle, Right). The red arrows indicate that, at this distance,
increases are no longer synchronous (n = 6 experiments).
Fig. 2.
Coexistence and characteristics of Na+ and Ca2+ waves. (A) Simultaneous Na+ and Ca2+ imaging showed that a Ca2+ wave is evoked by electrical stimulation and parallels the observed Na+ wave. (Upper) Intensity-modulated display image series of SBFI ratio (Na+ wave). (Lower) Fluorescence image series of Fluo-4 recorded simultaneously (Ca2+ wave). An initial reference image was subtracted from this image series to represent only the Ca2+ elevation over time after stimulation. (Scale bar, 200 μm.) (B) Fluo-4 fluorescence and SBFI ratio were plotted against time for four selected cells (circles in A Upper Right). (D) The speed of the Na+ wave was 40% slower than that of the Ca2+ wave. Data are means ± SEM (*, P < 0.05; n = 9 experiments).
Fig. 3.
Mechanism of Ca2+ and Na+ wave propagation. Results from Na+ (Upper) or Ca2+ (Lower) waves triggered by a first electrical stimulation as a control (white bars) and a second consecutive stimulation (black bars) are shown. Data are presented as percentage of the control wave speed. The waves triggered by two consecutive stimulations were not significantly different (2nd stim) (n = 9 experiments). Blocking gap junctions with 500 μM octanol (n = 8 experiments) or 20 μM carbenoxolone (CBX, n = 6 experiments) had a modest, if any, effect on the Ca2+ wave speed, but a more pronounced effect on the Na+ wave. Both Ca2+ and Na+ waves were inhibited up to 70% by the purinergic receptor antagonist suramin (100 μM, n = 6 experiments). Chelating intracellular Ca2+ by treatment with 50 μM BAPTA-AM massively inhibited both waves (n = 6 experiments). Application of the glutamate transporter inhibitor TBOA (500 μM) resulted in a strong inhibition of the Na+ wave. By contrast, the Ca2+ wave was not influenced by the presence of TBOA (n = 7 experiments). Na+ waves were ≈50% reduced by application of either glutamate oxidase (GLOD; 1 unit/ml) in the extracellular medium (n = 6 experiments) or glutamate decarboxylase (GAD; 40 units/ml) (n = 6 experiments). Ca2+ waves were not influenced by either GLOD or GAD. Data are means ± SEM (*, P < 0.05; **, P < 0.01; ***, P < 0.001). n.s., Not significant.
Fig. 4.
Na+ waves enhance cellular glucose uptake. (A) 2-NBD-glucose uptake (dotted line, filled diamonds) expressed as percentage of the 2-NBD-glucose fluorescence intensity measured in cells >200 μm away from the electrode tip (equivalent to basal 2-NBD-glucose uptake). 2-NBD-glucose intensity was plotted against distance from the electrically stimulated spot in regions depicted in the image (C). The amplitude of (in mM), obtained in separate experiments (solid line, open diamonds), shows the high degree of correspondence of the spatial spreading of Na+ wave and the enhanced glucose uptake. Resting
values were 6.6 ± 0.7 mM (n = 36 cells, four experiments). (B) 2-NBD-glucose uptake measured in the absence (dotted line, filled diamonds) or the presence of 500 μM TBOA (solid line, open diamonds) as described in A. Data are means ± SEM (n = 4 experiments each). The linear slopes of fluorescent signal in the range of 48-165 μM were -1.9 ± 0.7 μM-1 for control and -0.08 ± 0.06 μM-1 for TBOA (P < 0.05). (C) Transmitted light image of cells showing the electrode tip position and an overlay of the regions selected for analysis. (D and E) Images of 2-NBD-glucose fluorescence after electrical stimulation. The two images shown were acquired on the same cells, first in the presence of TBOA (E) and then in control solution after washout of the drug (D). (Scale bar, 200 μm.)
Fig. 5.
Model for Na+ and metabolic wave transmission mechanism. elevation is followed by the release of ATP and the release of glutamate in the extracellular space. Extracellular ATP then binds to purinoceptors on neighboring astrocytes, which induce a
response and participate in the propagation of the Ca2+ signals in addition to inositol 1,4,5-triphosphate (IP3) (17). In parallel, the released glutamate is taken up by Na+/glutamate cotransporters, resulting in
increases. Na+ has also the ability to diffuse through gap junctions and participate in the Na+ wave extension. The
elevation is sufficient to enhance Na+/K+-ATPase activity and, therefore, glucose consumption in astrocytes implicated by the wave.
References
- Robinson, M. B. & Dowd, L. A. (1997) Adv. Pharmacol. 37, 69-115. - PubMed
- Magistretti, P. J., Pellerin, L., Rothman, D. L. & Shulman, R. G. (1999) Science 283, 496-497. - PubMed
- Voutsinos-Porche, B., Bonvento, G., Tanaka, K., Steiner, P., Welker, E., Chatton, J. Y., Magistretti, P. J. & Pellerin, L. (2003) Neuron 37, 275-286. - PubMed
- Nedergaard, M., Takano, T. & Hansen, A. J. (2002) Nat. Rev. Neurosci. 3, 748-755. - PubMed
- Chatton, J.-Y., Marquet, P. & Magistretti, P. J. (2000) Eur. J. Neurosci. 12, 3843-3853. - PubMed
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