Cerebral Cortex Advance Access originally published online on June 1, 2005
Cerebral Cortex 2006 16(2):237-246; doi:10.1093/cercor/bhi101
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Activity-dependent ATP-waves in the Mouse Neocortex are Independent from Astrocytic Calcium Waves
1 Cellular Neuroscience, Max-Delbrück-Center for Molecular Medicine, Robert-Rössle-Str. 10, 13125 Berlin, Germany and 3 Institute of Genetics, Division of Molecular Genetics, University of Bonn, 53117 Bonn, Germany Eschenallee 3, 14050 Berlin, Germany, 2 Current address: Department of Psychiatry, Charité University Campus Benjamin Franklin, Berlin, Germany
Address correspondence to Helmut Kettenmann, Cellular Neuroscience, Max-Delbrück-Center for Molecular Medicine, Robert-Rössle-Str. 10, 13125 Berlin, Germany. Email: kettenmann{at}mdc-berlin.de.
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Abstract |
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 TopNotes Abstract IntroductionMaterial and MethodsResultsDiscussionSupplementary MaterialReferences |
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In the corpus callosum, astrocytic calcium waves propagate via a mechanism involving ATP-release but not gap junctional coupling. In the present study, we report for the neocortex that calcium wave propagation depends on functional astrocytic gap junctions but is still accompanied by ATP-release. In acute slices obtained from the neocortex of mice deficient for astrocytic expression of connexin43, the calcium wave did not propagate. In contrast, in the corpus callosum and hippocampus of these mice, the wave propagated as in control animals. In addition to calcium wave propagation in astrocytes, ATP-release was recorded as a calcium signal from ‘sniffer cells’, a cell line expressing high-affinity purinergic receptors placed on the surface of the slice. The astrocyte calcium wave in the neocortex was accompanied by calcium signals in the ‘sniffer cell’ population. In the connexin43-deficient mice we recorded calcium signals from sniffer cells also in the absence of an astrocytic calcium wave. Our findings indicate that astrocytes propagate calcium signals by two separate mechanisms depending on the brain region and that ATP release can propagate within the neocortex independent from calcium waves.
Key Words: astrocytes • ATP release • calcium wave • cortex • gap junction • slice
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Introduction |
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TopNotesAbstractIntroductionMaterial and MethodsResultsDiscussionSupplementary MaterialReferences |
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Astrocytes exhibit a form of long distance signaling, termed calcium (Ca2+) waves, which is the propagation of Ca2+ transients within a population of astrocytes. These Ca2+ waves have been described as occuring in primary cultures (Cornell-Bell et al., 1990
), cultured slices (Dani et al., 1992), isolated retina (Newman and Zahs, 1997; Newman, 2001) and acute brain slices (Schipke et al., 2002). In all preparations, astrocytic Ca2+ waves spread at a uniform speed of 10–20 µm/s, which is orders of magnitude slower than propagation of neuronal signals. Two different concepts have been proposed for the mechanism of the Ca2+ wave propagation among cultured astrocytes: (i) the intra- and intercellular diffusion of second messengers via gap junctions between highly coupled astrocytes with subsequent Ca2+ release from intracellular stores (Giaume and Venance, 1998; Venance et al., 1998); and (ii) the release of ATP from astrocytes into the extracellular space followed by activation of purinergic receptors on neighboring cells, which, in turn, leads to elevation of [Ca2+]i (Guthrie et al., 1999; Wang et al., 2000; Arcuino et al., 2002). Connexin expression, the molecular substrate for gap junctions, influences purinergic receptor expression. Deletion of connexin43 (Cx43), the major gap junction protein of astrocytes, potentiated the response of purinergic receptors triggered by ATP or UTP; the reduction in gap junctional communication in spinal cord astrocytes cultured from neonatal Cx43 deficient mice is accompanied by a functional switch in the expression of P2Y nucleotide receptor subtypes (Scemes et al., 2000).
So far, there have been only two reports on Ca2+ wave propagation in situ: the isolated retina and a white matter tract of acutely isolated brain slices (Newman and Zahs, 1999; Schipke et al., 2002). In the retina, Ca2+ wave propagation among astrocytes depends on gap junctional communication, while propagation between astrocytes and Müller cells involved purinergic signaling. In the corpus callosum of acutely isolated brain slices a glial Ca2+ wave was sensitive to purinergic receptor blockers but not to gap junction blockers (Schipke et al., 2002). It also involved Ca2+ release from cytoplasmic stores.
In this study, we investigated the mechanisms of astrocytic Ca2+ wave propagation in gray matter in acute brain slices of the frontal neocortex and in the CA1 region of the hippocampus. In the cortex, we determined the interplay between gap junctional coupling and ATP-release during wave propagation using connexin-deficient mice and a novel approach to record Ca2+ signals due to ATP-release from the slice surface
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Material and Methods |
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TopNotesAbstractIntroductionMaterial and MethodsResultsDiscussionSupplementary MaterialReferences |
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Animals, Preparation of Brain Slices, Stimulation and Calcium Recordings
All experiments were performed according to the guidelines of the German animal protection law. Both conditional Cx43(fl/fl):GFAP-cre mice and general Cx30 deficient mice [Cx30(–/–)] had a genetic background of 93% C57BL/6NCrlBR and 7% 129P2/OlaHsd. Genotyping of mice lacking Cx43 in astrocytes was done according to the instructions of Theis et al. (2003) and for Cx30 according to Teubner et al. (2003). Animals of these two strains were cross-bred and used as indicated in the text. For experiments, 10- to 14-day-old mice [either transgenic Cx30(–/–)/Cx43(fl/fl):GFAP-cre mice (Theis et al., 2003), transgenic GFAP/EGFP (Nolte et al., 2001) mice or out bred NMRI mice] were used. Slice preparation, staining with Fluo-4-AM and image recording was performed exactly as described in Peters et al. (2003). Electrical stimulation was accomplished with a conventional glass electrode filled with bath solution. The pipettes had a tip opening of 
20 µm. The tip of the pipette was placed on top of the slice, with the pipette only gently touching the upper cell layer. After the positioning of the pipette the slice was allowed to recover from mechanical stress for at least 2 min. Prior to the induction of the wave, the slice was superfused with Ca2+-free solution for 5 min, and then the bath perfusion was stopped. The wave was elicited by applying voltage pulses at 4 V, single stimulus duration 1.5 ms, 10 Hz stimulation stimulation frequency for 4 s. The pulses were generated by a patch clamp amplifier (EPC 9/2, HEKA Elektronik, Lambrecht, Germany) and isolated using a stimulus isolator (NeuroLog, NL 800, Digitimer Ltd, Welwyn Garden City, Hertfordshire, UK). This stimulation paradigm was chosen as it induced solid responses, but shorter stimulation, even only a few pulses, in some cases was also effective. The Ca2+ wave could be repetitively triggered within the same area. Time between stimulations was at least 5 min and slices were superfused with Ca2+ containing solution during recovery times.
To highlight and display newly responding cells during wave propagation, we subtracted from each image the pixel values of the previous image. To show all responding cells, the images showing the wave were averaged and the background was subtracted. Electrical stimulation was accomplished as described in Schipke et al. (2002).
Sniffer Cell Essay
The mouse glioma cell line GL 261 (Saic-Frederick, Inc., Frederick, MD) was stably transfected with the red fluorescent protein dsRedII (BD Bioscience, Heidelberg, Germany). Cells were grown to confluence in DMEM medium containing 10% fetal calf serum, and were trypsinated immediately before the experiment and diluted to a density of 1000 cells/µl. Cells were included in the Fluo-4-AM staining solution at a density of 200 cells/µl in a four-well plate, 500 µl total staining solution. Slices and sniffer cells were stained/incubated for 75–90 min at room temperature.
Dye-coupling Experiments
Astrocytes in slices from GFAP/EGFP or Cx43(fl/fl):GFAP-cre-positive mice were filled via the patch pipette during whole-cell recordings (20 min) (Kressin et al., 1995). Only cells with stable input resistance over the 20 min period were considered for data analysis. During recording, the membrane was continuously de- and hyperpolarized between –160 and +20 mV. Current signals were amplified (EPC 9/2, HEKA Elektronik), filtered (10 kHz), sampled (30 kHz), and monitored with TIDA software (HEKA Elektronik). 150 µm thick slices were stained without cryosectioning. From 300 µm thick slices cryosectioning (50 µm) and biocytin detection was performed as described by D'Ambrosio et al. (D'Ambrosio et al., 1998) with few modifications: Sections were incubated with the Elite ABC kit (Vector, Burlingame, CA) for 48 hrs. The DAB reaction was stopped after exactly 60 min, NiCl2 instead of NiNH4SO4 was used for intensification. Slices were embedded in Aqua Poly/Mount (Polysciences, Inc.). Images were taken with a digital camera (Axiocam, Zeiss) and appropriate software (Axiovison, Zeiss).
Solutions and Electrodes
The standard bath solution contained 134 mM NaCl, 2.5 mM KCl, 2 mM CaCl2, 1.3 mM MgCl2, 26 mM NaHCO3, 1.25 mM K2HPO4, 10 mM glucose. By continuously gassing the solutions with carbogen, the pH was adjusted to 7.4. To achieve Ca2+-free conditions, the slice was superfused with nominally Ca2+-free solution for 5 min prior to experiments.
All pipettes were fabricated from borosilicate capillaries (Hilgenberg, Malsfeld, Germany). Recording pipettes had resistances of 3–5 M
. The pipette solution for patch clamp experiments was composed of (in mM): 130 KCl, 2 MgCl2, 0.5 CaCl2, 5 EGTA, 2 Na2-ATP, 10 HEPES, 0.5% biocytin (Sigma), pH 7.3. Drugs were applied via changing the perfusate in the following concentrations (µM): ATP (100), carbenoxolone (100), glutamate (100), 
-methyl-4-carboxyphenylglycerine (MCPG) (50), RB-2 (30), suramin (100), 1-aminocyclopentane-trans-1,3-dicaroxylic acid (trans-ACPD) (50), tetrodotoxin (TTX) (1).
Statistical Analysis
We analyzed populations of responsive cells within concentric rings in 50 µm increments around the point of stimulation. The threshold for a response was defined at 
(F/F0) = 0.1. The number of cells responding to stimulation was counted for each slice both for control conditions and in the presence of the drug. Total numbers of slices and reacting cells under both conditions are given for each series of experiments. Histograms show percentages of overall reacting cells compared to control conditions to obtain better visual comparability. For comparing responses in Ca2+-free versus Ca2+-containing solution, we directly compared the populations of responsive cells in dependence of distance from the recording pipette. For studying the effect of drugs in Ca2+-free solution, we compared two consecutive control responses in Ca2+-free solution with a control response and a consecutive response in the presence of the drug also in Ca2+-free solution. Data were analyzed using the 
2-test (SPSS Inc., Chicago, IL). Significance was defined as P < 0.05.
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Results |
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TopNotesAbstractIntroductionMaterial and MethodsResultsDiscussionSupplementary MaterialReferences |
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Astrocytic Ca2+ Waves Propagate in the Neocortex after Blocking Neuronal Activity
We have previously demonstrated that astrocytic Ca2+ waves elicited by electrical stimulation in the corpus callosum are not inhibited by blocking neuronal activity (Schipke et al., 2002). Here, we used the same stimulation paradigms to elicit Ca2+ waves in astrocytes in gray matter of acute slices from the mouse frontal neocortex. A focal stimulation (4s, 10 Hz) in standard bath solution led to a prompt increase of intracellular [Ca2+]i in a large population of astrocytes throughout the entire observation area of 470 x 375 mm (recording frequency was 1 Hz). This fast astrocytic response is sensitive to TTX or Ca2+ removal from the bath, indicating that the astrocytes respond to neuronal activity. In the present study, we have not investigated this form of neuron–glia communication any further.
The following experiments were therefore performed in the presence of TTX or in Ca2+-free solution. As described below in detail, it allowed us to isolate an astrocyte Ca2+ wave. When we inhibited synaptic transmission in nominally Ca2+-free buffer, the Ca2+ wave spread among astrocytes at a velocity of 14 ± 5 µm/s (68 cells, Fig. 1A,B, supplementary data) as found in the corpus callosum (Schipke et al., 2002). Incubation with TTX had a similar effect: the wave propagated with a velocity of 12 ± 5 µm/s (276 cells; Fig. 2A). The number of responsive astrocytes was not affected by the presence of TTX or in Ca2+-free buffer (Fig. 2B). Thus, we conclude that astrocytic Ca2+ waves, similar as observed in culture or corpus callosum slices, can be observed in the neocortex when neuronal activity is impaired.
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Ca2+ Waves in the Neocortex are Dependent on Gap Junctions
To study the mechanism of astrocytic Ca2+ wave propagation in the neocortex, we switched to nominally Ca2+-free bath solution 5 min prior to stimulation. To exclude run-down effects, we elicited two consecutive waves under Ca2+-free conditions, which served as control for statistical analysis. To quantify the propagation of the wave, we analyzed the number of Ca2+-responsive cells in relation to the distance from the stimulation pipette in 50 µm increments.
Blockers for metabotropic glutamate receptors (MCPG; six slices, out of 80 cells reacting under Ca2+-free control conditions 82.5% still reacted in presence of MCPG) and for purinergic receptors (suramin, 12 slices, out of 160 cells reacting under Ca2+-free control conditions 81.3% still reacted in presence of suramin) did not affect the population of astrocytes involved in propagating the wave (Fig. 3A). However, in the presence of carbenoxolone (six slices; out of 83 cells reacting under Ca2+-free control conditions, 44.6% still reacted in presence of carbenoxolone), a gap junction blocker, the number of reacting cells was markedly reduced (Fig. 3A) (significance P < 0.005, 2-test). Incubation with carbenoxolone affected the responses in cells more distant from the stimulation pipette (<100 µm) (Fig. 3A,B) and as a consequence, the Ca2+ wave did not propagate beyond 100 µm. We conclude, that in the neocortex gap junctional coupling between astrocytes is a prerequisite for the long-distance propagation of Ca2+ waves.
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Ca2+ Wave Propagation in the Neocortex Requires Cx43 Expression
To circumvent the known problems of unspecific side effects of carbenoxolone and other gap junction blockers, we studied the wave propagation in transgenic mice with a conditional deletion of Cx43 expression in astrocytes. The animals used, Cx43(fl/fl):GFAP-cre-positive mice, carry two floxed Cx43 alleles and express Cre-recombinase under control of elements from the human GFAP promoter in astrocytes (Zhuo et al., 2001). This leads to a knockout of Cx43 only in (GFAP-positive) astrocytes (Theis et al., 2003). In three out of ten experiments the animals were additionally Cx30 deficient, but we did not observe differences between the two phenotypes. Animals not carrying cre transgene (cre negative) served as control. In the Cx43(fl/fl):GFAP-cre-positive mice there was no significant difference in astrocytic Ca2+ signaling following neuronal activation compared to wildtype animals: a Ca2+ increase was observed in a large population of astrocytes within the first second after stimulation in normal Ca2+-containing solution also in cells distant from the stimulation pipette (Fig. 4A, control). In contrast, in Ca2+-free conditions, in slices from Cx43(fl/fl):GFAP-cre-positive mice only few cells within a radius of <100 µm around the stimulation pipette responded to the stimulus (Fig. 4A, lower panel,B; P < 0.001; 10 slices, out of 219 cells reacting under Ca2+-containing control conditions, 30.6% still reacted under Ca2+-free conditions), but no wave was elicited. The distant cells were still able to generate Ca2+ responses since we sometimes observed spontaneous Ca2+ transients in these cells (Fig. 4B, supplementary data). In slices from GFAP-cre-negative mice Ca2+ wave propagation is already reduced: a significant smaller population of astrocytes responded to stimulation in Ca2+-free bath solution in the region beyond 100 µm compared with wildtype animals (Fig. 4C) (P = 0.02; 18 slices, out of 376 cells reacting under Ca2+-containing control conditions, 60.9% still reacted under Ca2+-free conditions). This is probably due to a reduced expression from the floxed Cx43 alleles. We conclude that in the neocortex, functional gap junctions are a prerequisite for astrocytic communication via the propagation of Ca2+ waves.
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‘Sniffer Cells’ Detect the Release of ATP during the Ca2+ Wave
To determine whether the astrocytic Ca2+ wave in the neocortex was accompanied by the release of ATP, we developed an assay to record ATP release with temporal and spatial resolution in slices. We used dsRedII transfected cells from the mouse glioma cell line GL 261 as ‘sniffer cells’ for ATP as these cells respond to 100 nM ATP in Ca2+-free solution (Fig. 5C, right). Astrocytes stained within the slice do not show a response to this low ATP-concentration (Fig. 5C, left). The glioma cells were seeded on top of the slice, stained with Fluo-4-AM together with the slice, and imaging was performed as described above. Since not all GL 261 cells were transfected with dsRedII only 60–70% of the cells could be identified by their red fluorescence.
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In the neocortex of wildtype mice, the astrocytic Ca2+ wave was accompanied by a propagating Ca2+ response in the ‘sniffer cells’ (Fig. 5A, supplementary data). In the presence of RB-2, a purinergic receptor blocker, the signal in the sniffer cells was completely abolished, whereas the astrocytic Ca2+ wave in the slice was still propagating (Fig. 5E). The propagating Ca2+ signal in the ‘sniffer cells’ was also recorded in slices from Cx30(–/–)/Cx43(fl/fl):GFAP-cre-positive mice after stimulation in the neocortex, both, in Ca2+-free conditions (Fig. 5B) and in standard bath solution containing 1 µM TTX (Fig. 5D). The sniffer cell Ca2+ activity propagated beyond the point of stimulation but no underlying Ca2+ wave was observed in the astrocytes within the slice (Fig. 5B,D). In the presence of TTX, the ATP wave, visualized via the Ca2+ increase in the sniffer cells, spread unaffected over the entire visual field of 400 µm with a speed of 12 µm/s, comparable to the Ca2+ wave in astrocytes (supplementary data). To exclude the general absence of purinergic signaling in the Cx-deficient mice, we tested for the presence of functional purinergic receptors in cortical astrocytes in Cx30(–/–)/Cx43(fl/fl):GFAP-cre-positive mice. The application of 100 µM ATP led to the typical transient Ca2+ response as described previously for cultured astrocytes (Walz et al., 1994
) (four slices, data not shown). To exclude that sniffer cells respond to glutamate released from astrocytes (Pasti et al., 2001), we triggered Ca2+ waves in the presence of the metabotropic glutamate receptor blocker MCPG. The wave was not altered in astrocytes, nor was the reaction of the sniffer cells (four slices; Fig. 5A). Moreover, the GL 261 cells did not respond to application of glutamate (100 µM, n = 1; 1 mM, n = 3) or trans-ACPD, an agonist for metabotropic glutamate receptors (50 µM, n = 2, data not shown). These experiments indicate that in the neocortex electrical stimulation triggers a propagating wave of Ca2+ transients in astrocytes and a concomitant propagating wave of ATP release, which is not involved in astrocytic Ca2+ wave propagation. The ATP released does not reach a concentration level high enough to trigger responses in astrocytes, but is high enough to trigger a response in the more sensitive sniffer cells. Our results clearly demonstrate that the two events are not functionally linked, but can propagate independently from each other. Astrocytes in the Neocortex are Highly Coupled
To determine the extent of coupling among astrocytes in the neocortex in comparison to the corpus callosum, where Ca2+ wave propagation is realized via ATP-release, we filled single astrocytes with biocytin, a gap junction-permeable dye. We identified astrocytes using slices from a transgenic animal in which EGFP is expressed under the GFAP promoter (Nolte et al., 2001). Biocytin was transferred to the astrocyte within 20 min by diffusion via the patch pipette. In the neocortex, after staining for biocytin we always observed a network of labeled astrocytes after a single cell was injected with (14 slices) (Fig. 6A, left panel). In 50 µm cryosections, the dye could clearly be recognized in up to 180 (mean 94) cells, spanning a region up to 600 µm (mean 390 µm) in diameter (Fig. 6B). In the corpus callosum, we either could not find the cell after injection (10 slices) or only one or two cells were labeled with biocytin (four slices) (Fig. 6A, right panel). We never observed a network of cells after biocytin injection although applying the same procedures as in the neocortex. Our results indicate that astrocytes in the neocortex are highly coupled, in contrast to astrocytes within the corpus callosum. To verify the absence of coupling in Cx30(–/–)Cx43(fl/fl):GFAP-cre-positive mice, we injected astrocytes that were identified by their passive membrane current pattern in the cortex of these animals (six slices). We never observed coupling to neighboring cells, only a network of fine processes belonging to a single cell (Fig. 6C). The somata of the injected cells were not detected as they are damaged or removed when removing the patch pipette.
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In the Gray Matter of the Hippocampus the Propagation of the Ca2+ Wave Depends on Purinergic Signaling
To exclude a general inability of the Cx30(–/–)/Cx43(fl/fl):GFAP-cre-positive mice to generate astrocytic Ca2+ waves, we checked for the presence of waves in the corpus callosum. Electrical stimulation elicited a glial Ca2+ wave (Fig. 7A) both in Ca2+-containing and Ca2+-free conditions, and the wave spread with the same velocity as described previously (Schipke et al., 2002). There was, however, one striking observation: while in control animals of the wildtype genotype, the wave propagated readily from the corpus callosum into the neocortex (Schipke et al., 2002), the propagation in Cx30(–/–)/Cx43(fl/fl):GFAP-cre-positive mice stopped at the border to the neocortex (Fig. 7A) but propagated within the corpus callosum (Fig. 7A,C; eight slices; out of 146 cells reacting under Ca2+-containing control conditions, 87.7% reacted under Ca2+-free conditions).
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To test if gap junctions are generally required for the propagation of astrocytic Ca2+ waves in gray matter, we analyzed the wave propagation in another gray matter region, the CA1 stratum radiatum of the hippocampus. In the CA1 region of Cx30(–/–)Cx43(fl/fl):GFAP-cre-positive mice, the wave still spread in Ca2+-free solution (Fig. 7B,C; three slices; out of 102 cells reacting under Ca2+-containing control conditions, 93.1% reacted under Ca2+-free conditions).
In the CA1 region of wildtype animals, in Ca2+-free solution, the wave propagated with a low speed of 9 ± 4 µm/s (345 cells) (Fig. 9). We tested the effect of a gap junction and a purinergic receptor blocker on the propagation of this slow Ca2+ wave. In contrast to the neocortex, carbenoxolone had no significant influence on the number of reacting cells (Fig. 8B,C; P = 0.847; eight slices; out of 118 cells reacting under Ca2+-free control conditions, 90.7% reacted in presence of carbenoxolone), but blockade of purinergic receptors with suramin partially blocked the propagation of the Ca2+ signal, the number of reacting cells was significantly reduced (Fig. 8C; P = 0.015; six slices; out of 118 cells reacting under Ca2+-free control conditions, 59.3% reacted in presence of suramin). Therefore, astrocytic signaling in the CA1 region of the hippocampus during Ca2+ wave propagation depends on the release of ATP and activation of purinergic receptors but not on functional coupling.
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Discussion |
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TopNotesAbstractIntroductionMaterial and MethodsResultsDiscussionSupplementary MaterialReferences |
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Ca2+ Waves in Gray and White Matter
Here we demonstrate that Ca2+ waves could be evoked in acute brain slices of gray matter in a large population of astrocytes. In contrast to the corpus callosum, where, under standard conditions, the electrically induced wave spreads with a velocity (Schipke et al., 2002) comparable to that in cultured astrocytes (Giaume and Venance, 1998), in the neocortex the slow astrocytic Ca2+ wave could only be unmasked after blocking neuronal signal transmission. The presence of astrocytic Ca2+ waves in the neocortex is supported by a recent study in which we elicited cortical spreading depression and observed a concomitant Ca2+ wave in astrocytes (Peters et al., 2003). The astrocyte wave slowed to 15 µm/s when it propagated beyond the area of spreading depression. This speed seems to be the general propagation velocity of the intrinsic astrocytic wave as also observed in culture (Venance et al., 1998) and in white matter (Schipke et al., 2002) (Fig. 9). In the CA1 region of the hippocampus, we determined a slightly slower speed, 9 ± 4 µm/s (345 cells), but it still lies within the same order of magnitude (Fig. 9).
Ca2+ Wave Propagation in the Neocortex Depends on Functional Gap Junctions
For the propagation of Ca2+ waves in cultured astrocytes, two mechanisms have been described: ATP release combined with purinergic signaling (Guthrie et al., 1999) and the propagation of a signal, most likely IP3, via gap junctions (Giaume and Venance, 1998). Studies in acutely isolated tissue of the corpus callosum (Schipke et al., 2002) demonstrate that astrocytic Ca2+ waves propagated by ATP release and purinergic signaling. We now provide evidence that among astrocytes in cortical slices the waves propagate with a mechanism involving gap junctions: the astrocytic Ca2+ wave in the neocortex cannot be elicited in the presence of a gap junction blocker or in transgenic mice deficient for the major astrocytic gap junction proteins Cx43 and Cx30, and is not affected by purinergic receptor blockers. In young mice Cx43 is the dominant gap junctional protein in astrocytes, while later in development (after postnatal day 15) astrocytes express in addition Cx30 (Kunzelmann et al., 1999; Nagy et al., 1999). Since we recorded from slices of animals from postnatal day 10 to day 14, we did not observe a difference between Cx43-deficient and Cx43/Cx30-deficient mice. In contrast to findings from cultured astrocytes, deletion of astrocytic connexins does not involve changes in the propagation mechanism in our hands, although we cannot exclude a change in purinergic receptor subtypes (Scemes et al., 2000). Even if there was an increased sensitivity to purinergic agonists in the Cx-deficient mice, this does not seem to be sufficient to rescue the astrocytic Ca2+ wave propagation.
Distinct Mechanisms of Ca2+ Wave Propagation
The mechanism of Ca2+ wave propagation via gap junctions is not common to all astrocytes in gray matter. For example, coupling was described for hippocampal astrocytes (Theis et al., 2003; Wallraff et al., 2004) to a comparable extent as observed here for cortical astrocytes. However, in the CA1 region of the hippocampus the wave was not sensitive to gap junction blockade but to blockade of purinergic signaling. Additionally the wave propagation was not altered in slices from Cx30(–/–)/Cx43(fl/fl):GFAP-cre-positive mice. Thus, the extent of coupling between cells does not determine the mechanism for the propagation of an astrocytic Ca2+ wave.
We conclude that communication between populations of astrocytes can differ among brain regions. This adds another facet to the emerging picture that astrocytes comprise a functionally heterogeneous cell population as previously shown for the expression of receptors and transporters (Israel et al., 2003; Matthias et al., 2003).
Sniffer Cells Record ATP Release
Using the luciferin–luciferase bioluminescence assay Wang et al. (2000) recorded simultaneous ATP release during a Ca2+ wave in cultured astrocytes. Using the same assay in the isolated retina, it was found that ATP release propagated somewhat faster than the Ca2+ waves (Newman, 2001). We have tried to apply the luciferin–luciferase bioluminescence assay to freshly isolated slices, but failed due to high background activity. We, therefore, used a novel assay to detect the release of ATP from slices, the ‘sniffer cells’. The glioma cell line GL 261 expresses highly sensitive purinergic receptors and these cells were seeded onto the surface of the slice during staining with Fluo-4-AM. The observed Ca2+ signal in these cells is due to release of ATP from the slice since it could be blocked by the purinergic receptor blocker Reactive Blue-2 (Ralevic and Burnstock, 1998) and was mimicked by ATP.
We exclude relevant ATP release from sniffer cells since there the signal was not influenced by sniffer cell density on the slice surface; Ca2+ waves and responses in sniffer cells were also observed in slices with only very few (3–5) sniffer cells present. Also, formation of gap junctions between the GL 261 cells and astrocytes seems rather impossible as the sniffer cells sometimes move during the experiment (5–10 µm during the recording time of 140 s).
Based on diffusion models, ATP released from a point source, e.g. from damaged cells at the stimulation site, would diffuse over a distance of up to 110 ± 30 µm (Crank, 1975; Hazel and Sidell, 1987). Indeed, in some cases we observed a clear stimulus artifact ranging up to 100 µm from the point of stimulation without a traveling wave, both for astrocytic Ca2+ waves and for ATP release monitored via Ca2+ responses in the sniffer cells, but the maximal distance observed for the spreading of the ATP wave of up to 400 µm argues against a single point release model. It is not entirely clear how the electrical stimulus triggers the initial Ca2+ signal, but as we were able to elicit a wave at least four times at the same stimulation site, we assume that cells are not destroyed or damaged by our stimulation paradigm. We found that both a propagating wave of ATP and a propagating glial Ca2+ wave can be elicited by electrical stimulation, but the two events can occur independently. In the neocortex of Cx-deficient mice we recorded Ca2+ signals in the sniffer cell population that were spreading in a wave-like manner from the point of stimulation while Ca2+ signals in the astrocytes were absent. This is compatible to previous studies demonstrating that ATP release is Ca2+-independent and therefore the ATP wave can propagate independently of the Ca2+ wave (Wang et al., 2000). It has been shown that ATP release from astrocytes can even be triggered by ATP itself (Anderson et al., 2004). Since the ATP wave was observed both in the presence of TTX and in Ca2+-free solution, we exclude a release of ATP through hemichannels since it only occurs in low Ca2+ (Ye et al., 2003). Thus we conclude that (i) the ATP wave travels independently from an astrocytic Ca2+ wave in the neocortex; and (ii) ATP release does not occur via hemichannels in the neocortex and in the corpus callosum.
In parallel with the propagation of astrocytic Ca2+ waves, glutamate concentrations can also increase in the extracellular space (Innocenti et al., 2000). We exclude that the signal in the sniffer cells is due to glutamate receptor activation since (i) the sniffer cells express no functional glutamate receptors; (ii) the signal was not inhibited by employing metabotropic glutamate receptor antagonists; and (iii) the mechanisms leading to the release of glutamate is Ca2+-sensitive (Wang et al., 2000). The ability of astrocytes in gray matter to propagate information by two distinct pathways, the astrocytic Ca2+ wave and propagating ATP release, supports their role as elements of cellular communication and as source for neuromodulatory substances.
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Supplementary Material |
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TopNotesAbstractIntroductionMaterial and MethodsResultsDiscussionSupplementary MaterialReferences |
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Supplementary material can be found at: http://cercor.oupjournals.org/
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TopNotesAbstractIntroductionMaterial and MethodsResultsDiscussionSupplementary MaterialReferences |
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* These authors contributed equally.
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Acknowledgments |
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The work was supported by grants of the German Research Association (SFB 515 and 400) and Funds of the Chemical Industry to K.W.
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References |
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TopNotesAbstractIntroductionMaterial and MethodsResultsDiscussionSupplementary MaterialReferences |
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