calcium waves between astrocytes from cx43 knockout mice

Calcium Waves Between Astrocytes From Cx43 Knockout Mice

ELIANA SCEMES1,3,*, ROLF DERMIETZEL2, and DAVID C. SPRAY31Deptartment of Physiology, Bioscience Institute, University of Sao Paulo, Sao Paulo, Brazil

2Department of Anatomy, University of Bochum, Bochum, Germany

3Department of Neuroscience, Albert Einstein College of Medicine, Bronx, New York

AbstractGap junctions are regarded as the primary pathway underlying propagation of Ca2+ waves betweenastrocytes, although signaling through extracellular space may also contribute. Results obtained fromastrocytes cultured from sibling Cx43 knockout (KO) and wild-type (WT) mice in six litters showedthat Ca2+ waves propagated more slowly in Cx43 KO than in WT astrocytes; however, because thisdifference in velocity was only seen in conditions where cell confluence was higher in WT than KOastrocytes, it is attributable to differences in plating density. By contrast, density-independentdifferences were observed in the amplitudes of the Ca2+ responses (15% smaller in KO astrocytes)and efficacy of spread (to 14% fewer cells in KO astrocytes). Blockade of purinergic receptors withsuramin reduced the velocities of the waves by 40% in WT and KO astrocytes and reduced theamplitudes by 20% and 6%, respectively. In the presence of heptanol, Ca2+ waves spread to only30% of the cells, with a 70% reduced velocity and 30% reduced amplitude. It is concluded that thepropagation of Ca2+ waves between astrocytes from Cx43 KO mice is not so greatly affected asexpected by deletion of the major gap junction protein between these cells. The residual 5% couplingcontributed by the additional connexins (Cx40, Cx45, and Cx46) expressed in KO astrocytes stillsuffices to provide a more substantial portion of Ca2+ wave propagation than does signaling throughextracellular purinergic pathways. These studies demonstrate that, even with severely reducedjunctional conductance, Cx43 KO astrocytes are capable of performing long-range Ca2+ wavesignaling, perhaps preserving one mechanism critical to neural function.

Keywordsgap junctions; intercellular communication; connexins; ATP receptors; suramin; heptanol

INTRODUCTIONThe participation of astrocytes in brain function as an integrated and a coordinated syncytiumis believed to rely on the presence of intercellular gap junction channels, which can provide apathway through which signaling molecules, such as cAMP, Ca2+ and IP3 spread between cells(see Saez et al., 1993). Gap junctions are constituted by two hemichannels each formed of sixconnexin subunits (Bennett et al., 1991). Of the 14 connexins so far identified in mammals,connexin43 (Cx43) is believed to be the major protein constituent of gap junctions in astrocytes(Dermietzel et al., 1989, 1991; Giaume et al., 1991; Nagy et al., 1992), although recent studiesindicate a minor contribution of other connexins (Cx40, Cx45, and Cx46) to junctionalcommunication in these cells (Dermietzel, 1996; Spray, 1996; Spray et al., 1998).

*Correspondence to: Dr. Eliana Scemes, Department of Neuroscience, 712 Kennedy Center, Albert Einstein College of Medicine, 1410Pelham Parkway South, Bronx, NY, 10461. E-mail: [email protected] .Contract grant sponsor: NIH; Contract grant numbers: NS07512 and NS34931.

NIH Public AccessAuthor ManuscriptGlia. Author manuscript; available in PMC 2007 March 2.

Published in final edited form as:Glia. 1998 September ; 24(1): 65–73.

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Among the several proposed functions of gap junctions, such as the dissipation and homeostasisof K+ ions (Kuffler and Nichols, 1966), control of cell proliferation (Naus et al., 1996),regulation of cell volume (Kimelberg and Kettenmann, 1990; Bender et al., 1993), and thepropagation of intercellular Ca2+ waves between astrocytes, the last has been considered themechanism by which cooperative cell activity is coordinated (Sanderson, 1996). Evidenceimplicating gap junctions in the spread of calcium waves has included studies on Cx43transfected C6 glioma cells in which expression of Cx43 protein resulted in the ability of thecells to exhibit dye coupling and to propagate Ca2+ waves (Zhu et al., 1991; Charles et al.,1992) and the demonstration that treatment of cells with compounds that reduce gap junctionconductance (halothane and octanol) inhibited intercellular wave propagation (Saez et al.,1989). Whereas it is well established that gap junctions participate in Ca2+ wave propagationin astrocytes and other cell types, an extracellular signaling component was initially shown tomediate Ca2+ signaling in mast cells (Osipchuk and Cahalan, 1992) and has been recentlyproposed to be necessary for the maximal propagation of astrocytic calcium waves in confluentcultures (Hassinger et al. 1996).

This paper describing the properties of mechanically induced calcium waves between Cx43knockout astrocytes shows that gap junction channels contribute substantially to thepropagation of calcium waves between these glial cells. The results revealed that the residual5% coupling contributed by Cx40, Cx45, and Cx46 between knockout astrocytes is sufficientto provide a substantial portion of calcium wave propagation, which spreads with similarvelocity, amplitude, and to a similar number of cells as do such waves in wild-type astrocytes.

MATERIAL AND METHODSCell Culture

Purified primary astrocyte cell cultures were obtained from whole brain tissue that wasdissected from neonatal mice (GJA1M1 strain, obtained from Jackson Laboratories, BarHarbor, ME). After trypsinization (0.1% trypsin at 37°C), the dissociated cells were plated inimaging dishes (Glass bottomed microwells, model 15, MakTek Co.) containing Dulbecco’sessential culture medium supplemented with 45% (vol/vol) Ham’s F12, 10% (vol/vol) fetalcalf serum, penicillin (50 μg/ml), streptomycin (50 μg/ml), and glutamine (2 mM), buffered topH 7.3 with 21 mM sodium bicarbonate. Cells were maintained in 5% CO2, 95% air atmosphereat 37°C, and 100% humidity. About 90% of the cells were immunopositive for glial acidicfibrillary protein (GFAP). Astrocyte cultures derived from brains of wild-type (WT) and Cx43knockout (KO) littermates were established in parallel. Confocal microscopy experiments wereperformed on low, medium, and high density cultures of astrocytes.

Confocal MicroscopyIntracellular calcium measurements—Astrocytes plated on glass-bottomed microwellswere loaded with Indo-1-AM (10 μM; Molecular Probes, Eugene, OR) at 37°C for 45 min,after which they were rinsed and used for confocal microscopy. Intracellular Ca2+ wasmeasured in loaded astrocytes bathed in a solution containing 140 mM NaCl, 4 mM KCl, 2mM CaCl2, and 5 mM HEPES (pH 7.3). The ratio of Indo-1 fluorescence intensity emitted attwo wavelengths (390–440 nm and >440 nm) was imaged using UV laser excitation at 351nm. Ratio images were continuously acquired at 0.5 or 1 Hz after background and shadingcorrection using a Nikon real time confocal microscope (RCM 8000) with UV large pinholeand Nikon 40× water immersion objective (N.A. 1.15; working distance 0.2 mm). Indo-1fluorescence ratio images were continuously acquired before and 1–2 min after the inductionof intercellular calcium waves (see below). The ratiometric images were saved on an opticalmagnetic disk recorder as the average of 16 or 32 frames and then played back formeasurements of changes in calcium level using Polygon-Star software (Nikon). The gray

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levels (number of pixels per area) within the regions of interest (circular spots with radii of6.41 μm, containing about 200 pixels) were averaged and then used for analysis.

Velocity, amplitude, and efficacy of calcium wave spread—Calcium waves betweencultures of Indo-1-AM loaded astrocytes were evoked by mechanical stimulation of one cellin the confocal field (171 × 128 μm; 21,888 μm2) using a glass pipette with a 1–2 μm outerdiameter. The velocities of calcium waves were calculated as the distance (μm) between thestimulated and the non-stimulated cells divided by the time interval (s) between the half-maximal calcium increases within the stimulated and responding cells. Half-maximal calciumincreases were obtained from sigmoidal curves fitted to the ascending phase of each Indo-1fluorescence ratio increase using Origin 3.01 software (see Fig. 1D).

Amplitudes of calcium waves were considered to be the maximal increments in intracellularcalcium observed in responding cells, calculated for each cell as the value of Indo-1fluorescence ratio rise at the peak of the response divided by the basal fluorescence ratio valueacquired before the induction of the calcium wave.

The efficacy of calcium spread between glial cells is reported here as the proportion of cellsresponding with an intracellular calcium increase during the propagation of the wave in relationto the total number of cells within the field of view.

Contribution of Extracellular Signaling and Intercellular Communication to the Propagationof Calcium Waves

The contribution of ATP-mediated calcium waves between cultured astrocytes was evaluatedby exposing sibling cultures of WT and Cx43 KO astrocytes to 50 μM suramin (a purinergicP2 antagonist, see King et al., 1996; Bolego et al., 1997) and comparing the velocities,amplitudes, and efficacies of calcium spread with those of untreated cultures.

Heptanol, a potent gap junction channel blocker in astrocytes (Dermietzel et al., 1991), wasbath applied (3 mM final concentration) to astrocytes cultured from both WT and Cx43 KOsiblings in order to measure the effects of gap junction blockade on calcium wave spreadbetween these glial cells.

RESULTSProperties of Calcium Waves Between WT and Between Cx43 KO Astrocytes

The properties of calcium spread between cultured whole brain astrocytes derived from sixlitters of WT and Cx43 KO mice were analyzed in terms of velocity, amplitude, and efficacyof the spread.

When one cell in the confocal field was mechanically stimulated, there was a rapid increase inIndo-1 fluorescence ratio, indicating the rise in intracellular calcium levels. Within a fewseconds after stimulation of one cell, the majority of the cells present in the confocal fielddisplayed increases in intracellular calcium levels (see Fig. 1). The velocity with which thisphenomenon propagated from the stimulated cell to the neighbors was variable, ranging from2.1 to 53.5 μm/sec for WT astrocytes and from 0.1 to 47 μm/sec for Cx43 KO cells. Theamplitudes of intracellular calcium changes in the responding cells also varied, from 1.03- to3.34-fold from basal levels in WT and from 1.01- to 2.08-fold in Cx43 KO astrocytes. Thisvariability in velocities and amplitudes of the wave was not correlated with the distance ofresponding cells from the stimulated one, i.e., the velocity and amplitude of calcium wavespreading to the first order cells (located at about 20 μm from the stimulated cell) was as variableas the spread to the third order astrocytes (located at about 65 μm from the stimulus) (Fig. 2).

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These results indicate that, within the limits of the confocal field (171 × 121 μm), the spreadof calcium waves between WT and between Cx43 KO astrocytes was non-uniform.

The analysis of the overall data obtained from astrocytes of the six litters of WT and Cx43 KOmice revealed that the deletion of Cx43 gene by homologous recombination resulted indecreased velocity, amplitude, and efficacy of calcium wave spread (see last rows of Table 1).However, when the properties of calcium waves between individual paired sibling WT andCx43 KO astrocytes are compared (Table 1), a totally different picture emerges, indicating thatthe properties of calcium waves between astrocytes are only slightly altered by Cx43 deletion.

The velocity of calcium wave propagation calculated for all the six litters of WT astrocytes(18.80 ± 0.74 μm/sec) was significantly higher than between Cx43 KO astrocytes (12.1 ± 0.79μm/s). However, comparison of sibling littermates showed that the propagation velocities weresignificantly reduced in only three out of six litters (A–C but not D–F; Table 1) in Cx43 KOastrocytes. Because Cx43 KO astrocytes from litters A, B, and C were evaluated in lowerdensity than their WT siblings, the observed decreases in velocity may be due to this differencein cell culture density. This hypothesis is supported by the fact that in cultures of cells fromWT and KO littermates (litters D, E, and F), which were cultured at comparable degrees ofconfluence, calcium waves travelled between WT and between KO astrocytes with similarvelocities (Table 1).

The analysis of the overall data from the six littermates also indicated that the calcium wavestravelling through the Cx43 KO astrocytic network had a significantly larger amplitudeattenuation than the waves spreading through WT cells (Table 1). In responding WT astrocytes,intracellular calcium rose 1.81 ± 0.04 fold during the spread of calcium waves while a 1.54 ±0.03 fold increment of intracellular Ca2+ occurred in Cx43 KO astrocytes (Table 1). However,such attenuation in calcium wave amplitude was observed to be statistically significant in onlythree of six sibling littermates (litters B, C, and D but not A, E, and F in Table 1). In contrastto the velocities of calcium waves discussed above, density of cell cultures did not account forthe differences in the amplitude attenuation between litters. Such behavior may be related todifferences involving other steps responsible for the generation of intracellular calciumresponses.

The analysis of data from astrocytes from WT and Cx43 KO siblings surprisingly showed thatthe efficacy of calcium spread (measured as the proportion of responding cells relative to thenumber of cells present in the confocal field) was not statistically different in any of the sixlitters studied (Table 1). Although analysis performed combining data from all six littersrevealed a significant decrease in responding cells in KO cultures, the difference in efficacyof calcium spread between WT and between Cx43 KO astrocytes was not as high as expectedfor the deletion of gene expressing the major protein forming the astrocytic gap junctions. Incultures of WT astrocytes, 94% of the cells present in the confocal microscope field participatedin the propagation of calcium waves, while 80% of the cells from Cx43 KO mice displayedintracellular calcium changes after mechanical stimulation of one cell (Table 1).

Contribution of Extracellular Signaling and Intercellular Communication to the Propagationof Calcium Waves

It has been reported that extracellular signals contribute to the propagation of calcium wavesbetween astrocytes (Enkvist and McCarthy, 1992; Hassinger et al., 1996) and in some othercell types, an ATP-dependent and gap junction-independent signaling mechanism has beenidentified as an extracellular component for the process of intercellular calcium spread(Osipchuk and Cahalan, 1992; Schlosser et al., 1996; Cao et al., 1997).

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An extracellular, ATP-dependent, signaling component in the propagation of calcium wavesis here reported to be present in mouse astrocytes. Suramin, a purinergic receptor antagonist(King et al., 1996; Centemeri et al., 1997; Reetz et al., 1997), reduced both the velocity andthe amplitude but did not affect the efficacy of calcium wave spread between WT and Cx43KO astrocytes (Table 2). The addition of 50 μM suramin to cultures of WT astrocytes reducedthe velocity of calcium waves from 17.22 ± 1.25 μm/sec to 10.66 ± 0.86 μm/sec and reducedthe amplitude of the calcium response from 2.02 ± 0.06 fold to 1.61 ± 0.04 fold. In culturedCx43 KO astrocytes, the velocity of calcium waves was reduced from 15.56 ± 2.31 μm/sec to8.92 ± 0.91 μm/sec after blocking the purinergic receptors with suramin; under the samecircumstances, intracellular calcium increments were attenuated from 1.72 ± 0.05 to 1.66 ±0.10-fold. Blockade of this extracellular component, however, did not affect the efficacy ofcalcium wave spread; 98% of WT and 83% of Cx43 KO astrocytes present in the confocalfields participated in the propagation of the waves after the addition of suramin.

Heptanol, which has been shown to reversibly block astrocyte gap junction channels(Dermietzel et al., 1991), strongly affected the efficacy of calcium wave spread, reducing by75% the number of cells involved in calcium wave propagation between WT as well as betweenCx43 KO astrocytes (Table 2). The remaining active population of WT cells propagated thewaves at a velocity of 5.02 ± 1.2 μm/sec with an attenuated amplitude (1.30 ± 0.13-foldincrement in Ca2+ level), values much lower than those observed under control conditions(19.86 ± 1.54 μm/sec and 2.22 ± 0.05 fold, respectively) (Table 2, Fig. 3). In Cx43 KOastrocytes, the velocity of calcium waves between responding cells was reduced from 18.40 ±1.59 to 9.90 ± 2.52 μm/s and intracellular Ca2+ increments decreased from 1.77 ± 0.06- to 1.22± 0.09-fold in the presence of heptanol (Table 2. Fig. 3).

DISCUSSIONAstrocytes are strongly coupled to each other by gap junction channels (Dermietzel et al.,1989, 1991; Giaume et al., 1991; Nagy et al., 1992) that provide the intercellular pathway bywhich ions (Kuffler and Nicholls, 1966), metabolites (Tabernero et al., 1996; Giaume et al.,1997) and signaling molecules (e.g., IP3, cAMP, Ca2+: see Saez et al., 1993; Charles et al.,1991, 1992; Giaume and McCarthy, 1996) are spread throughout the astrocytic syncytium.With regard to the various functions that have been proposed for gap junctions betweenastrocytes, the spread of intercellular Ca2+ waves has been hypothesized to provide amechanism by which cooperative cell activity is coordinated (Sanderson, 1996). Becauseconditions that increase or decrease gap junction conductance or expression have been foundto increase or decrease the number of cells through which Ca2+ waves propagate (Enkvist andMcCarthy, 1992; Charles et al., 1992), it has been postulated that the number of open gapjunction channels dictates the rate and amount of second messenger diffusion, therebydetermining velocity, amplitude, and efficacy of the propagated Ca2+ waves.

The major gap junction protein expressed between astrocytes is connexin 43 (Cx43), asevidenced by immunocytochemistry, Northern and Western blot analyses, and byelectrophysiological recordings (Giaume et al., 1991; Dermietzel et al., 1991; Nagy et al.,1992). However, recent studies have indicated that these glia express minor amounts of othergap junction proteins, including Cx40, Cx45, and Cx46 (Dermietzel, 1996; Spray et al.,1998). Electrophysiological and dye coupling studies on astrocytes obtained from Cx43-null(Cx43 knockout) mice have demonstrated that these cells are coupled with respect to electricalcurrent and Lucifer Yellow diffusion (Spray, 1996); comparing levels of macroscopicconductance with amplitudes of single channel conductances, it has been calculated that, onaverage, cultured wild-type astrocytes express about 200 functional channels, whereas inastrocytes from Cx43 KO mice this functional channel number is about 12 (Spray et al.,1998).

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This paper reports the properties (velocity, amplitude, and efficacy) of mechanically inducedcalcium waves between astrocytes cultured from wild-type (WT) and Cx43 knockout (KO)mice. The relative contributions of extracellular signals (Enkvist and McCarthy, 1992;Hassinger et al., 1996) and of the other gap junction channels present in the Cx43 KO cells tothe propagation of calcium waves were evaluated in experiments where suramin and heptanolwere used to block purinergic P2 receptors and gap junction channels, respectively.

It is shown here that the propagation of calcium waves between astrocytes was diminished incultures from Cx43 KO mice, but the parameters of propagation (see Table 1) were not sogreatly affected as would be expected to result from deletion of the major gap junction proteinexpressed in this tissue. A recent study by Naus et al. (1997) reported a more substantial, thoughalso incomplete, deficit in Ca2+ wave propagation (efficacy in that study was reduced by 54%)in cultured astrocytes from Cx43 KO mice.

Two possible mechanisms could account for the observed results: the safety factor for Ca2+

wave propagation may be so high that the residual junctional permeability present in Cx43 KOastrocytes is sufficient to fulfill this function, and/or an extracellular signaling mechanism (aswas first demonstrated in mast cells: Osipchuk and Cahalan, 1992, and now shown inastrocytes: Hassinger et al., 1996) may substantially contribute to Ca2+ wave propagation inastrocytes. Because gap junction blockade by heptanol had a much greater impact on theproperties of calcium wave spread between Cx43 KO astrocytes than did blockade of purinergicreceptors by suramin (see Table 2), it is strongly suggested that the residual junctional channelspresent in the Cx43 KO astrocytes provide the necessary and sufficient requirement for thepropagation of Ca2+ waves between astrocytes. Thus, although an extracellular route forCa2+ wave spread has been demonstrated by the finding that Ca2+ waves can leap smallboundaries in cultures (Enkvist and McCarthy, 1992; Hassinger et al., 1996), this extracellularroute for intercellular communication appears to be less critical under the experimentalconditions described here than does the direct route of signal transfer through gap junctionchannels. This is consistent with the view that phospholipase C (PLC) activity, IP3 formation,intracellular Ca2+ stores and gap junctional communication comprise the critical steps for theinitiation and propagation of these Ca2+ waves in cultured astrocytes (Venace et al., 1997).

The currently accepted mechanistic model for propagation of mechanically inducedintercellular Ca2+ waves is based on diffusion of IP3 through gap junctions (see Sanderson,1996). In this model, mechanical stimulation generates intracellular IP3 through the activationof PLC, which then diffuses across cell boundaries, leading to the release of Ca2+ fromintracellular stores via IP3 receptors (IP3R); although IP3 concentration would decline as afunction of distance from the source cell, regenerative responses at concentrations abovethreshold for Ca2+ mobilization are explained by local amplification through feedback ofCa2+ concentration on IP3 release (Sanderson, 1995; Sneyd et al., 1995). Thus, the velocity,amplitude and number of cells recruited into the Ca2+ wave would all be dependent on theconcentration of IP3 liberated from intracellular stores by the initial stimulus.

It is shown here that astrocytes cultured from both wild-type mouse and Cx43 KO siblingsexhibited Ca2+ waves that propagated at about 12 μm/s with little attenuation and to almost allthroughout the field of view. Although conduction velocity, amplitude, and number ofresponding cells indicated decreased intercellular communication in the Cx43 KO astrocytes,studies presented here have been limited to relatively small cell populations. More substantialdifferences would be expected to occur at the fringes of the area, where propagation fails,examination of which will require low power microscope objectives with high numericalaperture and high UV transparency.

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Gap junctions in other systems are permeable to both Ca2+ and IP3 (Saez et al., 1989; Christet al., 1992; Sanderson, 1995; Charles et al., 1991, 1992), and the permeability of junctions inCx43 KO astrocytes to an anion of similar size (Lucifer Yellow) suggests that the residualchannels permit IP3 diffusion. Whether Ca2+ or other second messenger molecules furthercontribute to this phenomenon is unresolved, although the relatively short range of diffusionexpected for Ca2+ ions within cytoplasm (diffusion distance of 0.08 μm, compared with 13μm for IP3; see Kasai and Petersen, 1994) would be expected to result in such rapid attenuationof the signal as to render it useless unless continuously amplified through Ca2+-inducedCa2+ release.

Cx43 KO mice die at birth due to cardiac hyperplasia obstructing the right ventricular outflowtract; however, their brains are grossly normal in appearance and exhibit no profound differencefrom WT animals in cortical lamination (Dermietzel, 1996). These observations suggest eitherthat gap junctions between astrocytes play no role in brain development or organization or thatthe residually expressed connexins are sufficient for this task. The studies presented heredemonstrate that, even with severely reduced junctional conductance (Spray et al., 1998), Cx43KO astrocytes are capable of performing long-range Ca2+ wave signaling, perhaps preservingone mechanism critical to neural function.

Acknowledgements

The authors are grateful to Ms. Delia Vieira for culturing astrocytes and to Dr. Yang Gao for the GFAP stainingmentioned in the Material and Methods section. This work was supported by NIH-NS07512 and NS34931 to DCS.

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Fig. 1.Analysis of properties of calcium waves spreading between cultured astrocytes. A,B: A three-dimensional representation of the images of Indo-1-AM loaded astrocytes acquired with aNikon RCM-8000 confocal microscope. The positions (in μm) and the temporal changes influorescence ratio of loaded astrocytes are shown before (A) and after (B) applying amechanical stimulus (at time 14 sec) to one cell (located at position 0 μm within the confocalfield). C: The time course of Indo-1 fluorescence ratio changes observed in the stimulated (cellA) and responding cells (cells B,D,E,H, and I) that are shown in part (B). D: Sigmoidal curveswith equation in inset fitted to the ascending portions of Indo-1 fluorescence ratio changes andthe half maximal (EC50) values calculated using Origin 3.0 software. The time interval (tE-

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tA) between the two half-maximal (EC50) responses of the stimulated (cell A) and a respondingcell (cell E) was used to calculate the velocity of calcium wave propagation between the twocells.

SCEMES et al.


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Fig. 2.Propagation of calcium waves between cultured wild-type (A,B) and between Cx43 knockout(C,D) astrocytes. Indo-1 fluorescence ratio (A,C) and intracellular calcium amplitude changes(B,D) recorded from cells located at about 20 μm (squares and diamond symbols) and at 65μm (up and down triangles) away from a mechanically stimulated cell (black circle). Note thatin both cultures of WT and KO astrocytes, the two cells located at about 20 μm or the ones at65 μm apart from the stimulated cell display different intracellular calcium level changes aswell as different latencies of responses, indicating that calcium waves propagate from the pointof stimulation without uniform velocities and amplitudes over distance.

SCEMES et al.


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Fig. 3.Heptanol blockade of calcium waves spreading between WT and between Cx43 KO astrocytes.Three-dimensional representation of images acquired in the confocal microscope from Indo-1-AM loaded WT (A,B) and KO (C,D) astrocytes in the absence (A,C) and in the presence (B,D)of 3 mM heptanol. In both WT and KO astrocytes the spread of calcium waves shown to occurover a distance of about 100 μm from the stimulated cell (located at position 0 μm) wasprevented by heptanol.

SCEMES et al.


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SCEMES et al. TA

BLE

1Pr

oper

ties o

f cal

cium

wav

es b

etw

een

astro

cyte

sa

Litt

ers

Vel

ocity

(μm

/sec

)Pr

obab

ility

Am

plitu

de (f

old)

Prob

abili

tyE

ffica

cy (n

umbe

r of

cells

)N

AW

TH20

.02

± 1.

220.

0326

1.52

± 0

.04

0.59

50.

98 ±

0.0

298

KO

M16

.50

± 1.

221.

48 ±

0.0

41.

00 ±

0.0

062

BW

TM15

.10

± 1.

220.

0192

1.67

± 0

.01

0.00

610.

95 ±

0.0

329

KO

L10

.80

± 1.

551.

38 ±

0.0

50.

75 ±

0.1

024

CW

TM18

.10

± 1.

130.

0001

2.16

± 0

.06

0.00

070.

91 ±

0.0

789

KO

L10

.60

± 1.

161.

74 ±

0.0

80.

89 ±

0.0

535

DW

TH19

.86

± 1.

540.

9236

2.22

± 0

.05

0.00

010.

92 ±

0.0

829

KO

H20

.08

± 1.

751.

87 ±

0.0

61.

00 ±

0.0

024

EW

TM11

.75

± 1.

180.

6417

1.61

± 0

.05

0.46

251.

00 ±

0.0

014

KO

M15

.60

± 3.

711.

54 ±

0.0

90.

53 ±

0.2

811

FW

TM14

.37

± 1.

870.

3500

1.54

± 0

.10

0.68

471.

00 ±

0.0

015

KO

M15

.52

± 3.

051.

60 ±

0.1

10.

88 ±

0.0

114

A–F

WT

18.8

0 ±

0.74

0.00

011.

81 ±

0.0

40.

0001

0.94

± 0

.03

216

KO

12.1

± 0

.79

1.54

± 0

.03

0.80

± 0

.05

146

a Vel

ociti

es, a

mpl

itude

s and

eff

icac

ies o

f cal

cium

wav

e sp

read

bet

wee

n ce

lls w

as o

btai

ned

from

six

sibl

ing

pairs

of w

ild ty

pe (W

T) a

nd C

x43

knoc

kout

(KO

) (lit

ters

A–F

). Th

e pr

obab

ility

val

ues

obta

ined

from

one

way

ana

lysi

s of v

aria

nce

used

to c

ompa

re th

e re

spon

ses o

f WT

and

KO

cel

ls o

f eac

h lit

ters

are

show

n on

ly fo

r the

vel

ociti

es a

nd a

mpl

itude

s of c

alci

um w

aves

. The

upp

erca

se H

(hig

h), M

(med

ium

), an

d L

(low

) ref

er to

cel

l cul

ture

den

sitie

s. Th

e bo

ttom

row

s sho

w th

e ca

lciu

m w

ave

prop

erty

val

ues c

alcu

late

d fo

r dat

a co

mbi

ned

for a

ll si

x lit

term

ate

WT

and

KO

ast

rocy

tes.

N c

orre

spon

ds to

the

num

ber o

f cel

ls fr

om w

hich

the

velo

city

and

am

plitu

de d

ata

(mea

n ±

stan

dard

err

or) w

ere

obta

ined

.


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SCEMES et al. TA

BLE

2Ef

fect

s of s

uram

in a

nd h

epta

nol o

n th

e pr

oper

ties o

f cal

cium

wav

es b

etw

een

astro

cyte

sa

Litt

ers

Vel

ocity

(μm

/sec

)Pr

obab

ility

Am

plitu

de (f

olds

)Pr

obab

ility

Effi

cacy

(num

ber

ofce

lls)

N

D,E

,F{W

Tc

WTs

KOc

KOs

17.2

2 ±

1.25

0.00

012.

02 ±

0.0

60.

0001

0.94

± 0

.06

4310

.66

± 0.

861.

61 ±

0.0

40.

86 ±

0.0

710

315

.56

± 2.

31

0.00

62

1.72

± 0

.05

0.04

6

0.83

± 0

.10

258.

92 ±

0.9

11.

66 ±

0.1

00.

92 ±

0.0

389

D,F

{WTc

WTh

KOc

KOh

19.8

6 ±

1.54

0.00

012.

22 ±

0.0

50.

0001

0.94

± 0

.067

295.

02 ±

1.2

01.

30 ±

0.1

30.

25 ±

0.1

015

18.4

0 ±

1.59

0.01

9

1.77

± 0

.06

0.00

01

0.95

± 0

.03

389.

90 ±

2.5

21.

22 ±

0.0

90.

38 ±

0.2

111

a Vel

ociti

es, a

mpl

itude

s, an

d ef

ficac

ies o

f cal

cium

wav

es sp

read

bet

wee

n ce

lls w

ere

mea

sure

d fr

om si

blin

gs in

thre

e lit

ters

(D, E

, F) o

f wild

type

(WT)

and

Cx4

3 kn

ocko

ut (K

O) m

ice.

The

cel

ls fr

omlit

term

ates

D–F

are

the

sam

e as

show

n in

Tab

le 1

. The

pro

babi

lity

valu

es o

btai

ned

from

one

way

ana

lysi

s of v

aria

nce

used

to c

ompa

re th

e ef

fect

s of t

he tw

o tre

atm

ents

are

show

n on

ly fo

r the

vel

ociti

esan

d am

plitu

des o

f cal

cium

wav

es. T

he lo

wer

case

c, s

, and

h c

orre

spon

d to

exp

erim

ents

per

form

ed u

nder

con

trol c

ondi

tions

and

afte

r sur

amin

and

hep

tano

l tre

atm

ents

, res

pect

ivel

y. N

cor

resp

onds

to th

e nu

mbe

r of c

ells

from

whi

ch th

e ve

loci

ty a

nd a

mpl

itude

dat

a (m

ean

± st

anda

rd e

rror

) wer

e ob

tain

ed.


calcium waves between astrocytes from cx43 knockout mice

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