Supporting InformationGarré et al. 10.1073/pnas.1013793107SI Materials and MethodsMaterials. Culture medium and serum were from Gibco BRL.FGF-1 and other reagents were from SIGMA unless otherwisespecified.

Cell Cultures. Astrocytes from spinal cords of 1-d-old rat pups wereprepared and cultured as described with minor modifications(1). Cells were plated (at 2.0 × 104 cells/cm2) on 12-mm glasscoverslips or on 60-mm plastic culture dishes and kept in a main-tenance medium composed of DMEM supplemented with 10%FBS, Hepes (3.6 g/L), NaHCO3 (1.2 g/L), penicillin (100 UI/mL),and streptomycin (100 μg/mL). Cultures were grown at 37 °C ina humidified atmosphere of 5% CO2 and 95% O2. Astrocytes inconfluent monolayers after 15–20 d in culture were >98% pure asdetermined by GFAP immunoreactivity, and there were no cellspositive for OX42, a microglial marker.

Drugs and Solutions. Solution composition: Tyrode’s: NaCl (140mM), KCl (4 mM), CaCl2 (1.8 mM), MgCl2 (1 mM), Hepes (5mM), glucose (5mM), pyruvate (2mM), (pH=7.4); Hank’s: NaCl(137 mM); KCl (5 mM); CaCl2 (1.8 mM); MgCl2 (0.95 mM);KH2PO4 (0.4 mM); MgSO4 (0.4 mM); NaHCO3 (4 mM); NaH2-PO4 (0.3mM); glucose (5mM), pH= 7.40. Lucifer yellow used fordye uptake experiments was prepared from a 5% stock solution in150 mM LiCl and applied to cells at 500 μM final concentration.The ethidium bromide used for dye uptake experiments was pre-pared from a 1-mM stock solution and applied to cells at 1–10 μMfinal concentration. Octanol was diluted in ethanol (5%, vol/vol)or prepared directly in recording solution to reach a final con-centration of 0.4–1 mM. Carbenoxolone (CBX) and oxidized ATP(oATP) were dissolved in recording solution at a final concentra-tion of 0.2 mM. Brilliant blue G (BBG, 5 μM) and reactive blue(RB2, 50 μM) were dissolved in stock solutions of 5 mM and30mM, respectively, and the final concentration was applied to thecells in recording solution, or a small volume was added to theculture dish to reach the desired final concentration. Apyrasewas dissolved in PBS at 2 U/mL and diluted to the final concen-tration of 2 mU/mL. ATP was prepared as a 0.5-mM solutionin recording medium, and the pH was adjusted to 7.4 before re-cording. Benzoyl-ATP (Bz-ATP) was prepared in a 10-mM stocksolution and applied at the final concentration of 10 μM. The bathvolume was 2 mL.FGF-1 was prepared as a 10 μg/mL stock solution in 5,000 U/

mL heparin at least 1 h before each experiment. Final concen-trations were 10 ng/mL for FGF-1 and 5 U/mL for heparin.Confluent astrocyte cultures were washed three times in Ty-rode’s solution. Then, the maintenance medium (see Cell Cul-tures) was replaced by DMEM with 2% horse serum. Underthese conditions, cells were incubated with FGF-1 for differenttime periods at 37 °C in a humidified atmosphere of 5% CO2 and95% O2. Dye uptake (evaluated as fluorescence intensity in ar-bitrary units, AU) of astrocytes treated with heparin alone for allexposure times used did not differ from that by cells maintainedin basal conditions, i.e., incubated in DMEM with 2% horseserum (P > 0.05).

Lucifer Yellow and Ethidium Permeability Assays. Dye uptake wasmeasured in cultures under control conditions and after treat-ment with FGF-1 for various times. FGF-1 solution was washedout before recording. Then, the gap junction (GJ) permeablefluorescent tracers, lucifer yellow (LY2−, molecular weight of thefluorescent ion, 457) or ethidium as the bromide (Etd+, MW of

the cation, 314) were applied at room temperature to culturesfor 5 min at 500 μM and 5 μM final concentrations, respectively.Astrocytes were rinsed three times at the end of the dye exposureto stop uptake and reduce background labeling and held in Ty-rode’s solution for the brief period before photomicrographswere taken. Coverslips with labeled cells were mounted in aperfusion chamber and visualized in a Nikon microscope (Opti-phot) equipped with epifluorescence illumination and appropri-ate filters for LY2− (excitation wavelength peak, 426 nm; emissionwavelength peak, 520 nm), and Etd+ (excitation wavelength peak,528 nm; emission wavelength peak, 598 nm). Fluorescence andphase-contrast images were captured with a DC290 Zoom KodakDigital Camera (exposure time = 0.7 s). Fluorescence was digi-tized in arbitrary units (AU) with image processing software(Adobe Photoshop 7.0). Regions of interest including single cellswere evaluated for overall fluorescence and background sub-tracted using areas where the cultures were thin or where therewere no cells. Dye uptake was expressed as the difference F − F0,where F represents the average fluorescence from 10–15 as-trocytes and F0 corresponds to the background fluorescenceevaluated in the same field in regions where the cells were verythin or nonconfluent. At least three fields were selected at ran-dom near the center of each coverslip, and every cell in each fieldwas measured.For time-lapse measurements fluorescence signals were ac-

quired using UltraVIEW software for image acquisition andanalysis (Perkin-Elmer Life Sciences). To measure permeability,single astrocytes that were not in contact with other cells wereinjected from a patch pipette in whole cell recording mode con-taining of 500 μM LY2−; −20 mV pulses were applied to improvedelivery of LY2− into the cell. After the pipette was detached fromthe cell and resealing observed, fluorescence images were takenevery 2 min (no difference was found when pictures were takenevery 1 or 5 min, indicating that bleaching was minimal) andpermeability was measured as the time constant of the decay influorescence. An initial fast decay component was observed,which we interpreted as incomplete resealing of the membrane,but after ∼2 min from the time of detaching the pipette, the decayrate slowed to a value that remained constant over the time ofmeasurement, i.e., decay was virtually linear over the small rangeobserved. The decay in fluorescence was interpreted as LY2−

release through an intact and resealed membrane, and its slopewas taken as a measure of permeability.For Etd+ uptake, control and treated cultures were washedwith

Tyrode’s solution, 5 μM EtdBr was added as indicated, and thecultures were mounted on the stage of a fluorescence microscope(Olympus BMX51). Images were captured by a digital camera(Orca-ER; Hamamatsu) and imaging system (Imaging Suite,version 5.5; Perkin-Elmer) every 30 s (exposure time = 0.7 s). Thefluorescence intensity (F − F0) was averaged for the same 8–10cells in each image and plotted in arbitrary units vs. time. Mi-croscope and camera settings remained the same in any series ofmeasurements in which comparisons were made. The mean slopeof the relationship F − F0 vs. time represents the rate of Etd+

uptake in control and in FGF-1–treated astrocytes. The 5-μMconcentration used to load the cells minimized phototoxicityand still provided satisfactory fluorescence intensities. Over thetime course of our experiments (<40 min) we saw no saturation ofuptake, i.e., no decrease in the rate. We found no difference influorescence intensity over time when images were acquired at 6-srather than 30-s intervals, indicating that there was no significantbleaching.

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ATPMeasurement.Levels of ATP were measured using a luciferin-luciferase kit (Molecular Probes). Cultured astrocytes (cellseeding density, 25,000 cells/cm2) were washed three times inTyrode’s solution and then maintained in 500 μL of Tyrode’ssolution for 30 min to allow for accumulation of ATP in theextracellular medium. A sample of 250 μL of the medium wascollected and centrifuged at 1,000 rpm before recording. A totalof 300 μL of a reaction mixture containing a final concentrationof 0.1 μg/mL of luciferase and 150 μg/mL of luciferin and theculture medium was added to the plate reader. Baseline [blank]measurements were carried out on Tyrode’s solution accordingto the same procedure. The photon flux was counted during 15min using a luminometer (LUMIstar; BMG Labtech). Thereader was calibrated between 0.01–10 μM from a standard so-lution of ATP (Molecular Probes); the relation between lumi-nescence and ATP concentration was linear in this range.

Dye Transfer. Functional coupling between confluent astrocytes inculture was evaluated in two ways, by iontophoretic injection ofEtdBr into single cells and by scrape loading. During measure-ments following dye injection, cells were kept inHanks’ solution inthe absence or presence of La3+ (0.2 mM), which blocks Cx HCs,to reduce possible dye leakage through the nonjunctional cellmembrane; no difference was noted due to presence of La3+ (2).Coverslips to which the cells adhered were placed in a perfusionchamber that was mounted on the stage of an invertedmicroscope(TE 200; Nikon). Glass micropipettes (6030, 30–70 MΩ; A-MSystems) filled with EtdBr (1 mM in 150 mM of KCl) were usedto microinject cells using depolarizing current pulses (10 nA in-tensity, 200 ms duration). The incidence of dye coupling wascalculated as the percentage of injected cells from which the dyetransferred to at least one adjacent cell within 1 min.Scrape loading was used to introduce the GJ permeant fluo-

rescent dye, Lucifer yellow (LY2−, ion MW 457.2) into cells. Thetechnique was performed according to ref. 3 with minor mod-ifications. Cultured astrocytes were washed in a Ca2+/Mg2+-freeTyrode’s solution two to three times and then immersed in thesame solution containing 2 mM of LY2−. A razor cut in the centerof the monolayer permitted LY2− loading of cells at the marginof the cut. After 1 min, cells were washed for 5 min with normalCa2+/Mg2+ solution. In some experiments, the blocking agentCBX (100 μM) or apyrase (2 mU/mL) was applied to culturesbefore and during loading and dye diffusion. Labeled cells werecounted after 8 min.

Immunofluorescence. For detection of Cx43 or Px1, astrocytes werefixed for 20 min at –20 °C with 70% ethanol, rinsed twice in PBS,and incubated for 30 min at room temperature (RT) in blockingsolution containing 1% BSA and 10% goat serum in PBS. As-trocytes were then incubated overnight at 4 °C with polyclonalrabbit anti-Cx43 (1:500 in blocking solution) or polyclonal chickenanti-Px1 C terminal (1:500). The cultures were rinsed three timesin PBS and incubated for 1 h at RT with secondary antibodies,Cy3-conjugated goat anti-rabbit IgGs (1:1,000), fluorescein-con-jugated rabbit anti-chicken (1:1,000), or rhodamine-conjugatedrabbit anti-chicken (1:1,000) as indicated. Images were capturedwith a DC290 Zoom Kodak digital camera attached to a fluores-cence inverted microscope (TE 200; Nikon) or with a Hamamatsudigital camera (Orca-ER) attached to a fluorescence invertedmicroscope (IX70; Olympus).

Western Blotting. Cells were suspended in 100 μL of a solutioncontaining phosphatase inhibitors (0.1 M sodium pyrophosphate,0.1 MNaF, and 0.5 mMNa3VO4) and a protease inhibitor (2 mMphenylmethylsulfonyl fluoride) and lysed by sonication on ice.Protein concentration was measured by Bradford assay (Bio-Rad). Proteins were resolved by electrophoresis on 8% SDS-

polyacrylamide SDS/PAGE vertical slab minigels (Hoefer Sci-entific Instruments) in a discontinuous buffer system at 20 mAand then electrotransferred onto nitrocellulose at 30 mA over-night. Membranes were incubated for 30 min at room tempera-ture with 5% fat-free dried milk in Tris-buffered saline (TBS), pH7.4, and then exposed overnight at 4 °C to rabbit anti-Cx43 or-Cx45 antibodies (1:1,500 and 1:500, respectively, in 5% fat-freemilk in TBS). Blots were then rinsed repeatedly in TBS and in-cubated for 1 h at room temperature in goat anti-rabbit IgGsconjugated to alkaline phosphatase (1:3,000 in 5% fat-free milk inTBS). Blots were rinsed in TBS and bands were visualized bymeans of an ultrasensitive chemiluminescence detection kit(Pierce). Aliquots of rat heart homogenate were used to identifythe electrophoretic mobility of Cx43 reactive bands whose mo-bilities are affected by phosphorylation. Densitometric analyses ofimmunoblots were performed with a scanning densitometer(UC630; Umax).

Biotinylation of Cell Surface Proteins. To evaluate the abundanceand electrophoretic mobility of nonjunctional connexin hemi-channels in the surface membrane, surface proteins were bio-tinylated using a kit obtained from Pierce. Briefly, confluentastrocytes were rinsed three times at 4 °C withHanks’ solution, pH8.0, and incubated for 30 min at 4 °C with sulfo-NHS-SS-biotin(0.5 mg/mL, Pierce). Biotinylated cells were then washed threetimes at 4 °C in Hanks’ solution containing 15 mM glycine (pH 8)to quench the biotinylation reaction and lysed by sonication ina solution containing phosphatase and protease inhibitors as de-scribed above. Protein concentration was measured by Bradfordassay. Biotinylated proteins were precipitated with avidin (im-munopure immobilized avidin cross-linked to agarose, Neu-trAvidin; Pierce) for 1 h at 4 °C (3 μg of biotinylated protein permicroliter of avidin as recommended by Pierce). Precipitatedproteins were washed three times at 4 °C in Hanks’ solution, SDS0.1%, Nonidet P-40 1%, pH 7.2. Protein samples were eluted in0.1 M glycine-HCl at pH 2.8; 10 μL of Tris pH 7.4 was added toeach sample (70 μL total volume) to neutralize the pH. The tubescontaining a sample were held in boiling water for 3 min in 1 M ofTris buffer + 5% β-mercaptoethanol to cleave the biotin moietyfrom the labeled protein and then analyzed by immunoblottingwith primary anti-Cx43.

Small Interfering RNA Against Px1 (siRNA Px1) and Cx43 Knockout.Spinal astrocytes were treated with 1 μM of siRNA Px1 targetingmouse pannexin 1 mRNA (sequence no. 3 in ref. 4: GGUG-CUGGAGAACAUUAAAtt). The effectiveness of this siRNAwas demonstrated by others (4) and is confirmed in Fig. S3.Confluent cultures (90%) were transfected overnight with 10 μL/mL lipofectamine reagent (Invitrogen), transfection reagentswere removed, and cells incubated for 4–6 h in DMEM, 2% FBSmedium before treatment with FGF-1, ATP, or other agents. Asilencer negative sequence from Ambion was used as a control(4). Connexin 43 knockout (KO) mice (originally reported in ref.5) were obtained from Drs. D. C. Spray and E. Scemes (Domi-nick P. Purpura Department of Neuroscience, Albert EinsteinCollege of Medicine, Bronx, NY) and cultured astrocytes wereprepared and cultured as described (1, 6).

Statistics. Data are presented as mean ± SEM; N expresses thenumber of independent experiments in each of which multiplecells were measured. Means for each group of cells treated in thesame way were compared using a nonparametric test for con-tinuous and unpaired variables, the Mann-Whitney U test. Dif-ferences were considered significant at P < 0.05. Statistics wereperformed using Graph Pad Prism 4 (2003) software. Graphicswere prepared using Sigma Plot (2000).

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1. Cassina P, et al. (2005) Astrocyte activation by fibroblast growth factor-1 and motorneuron apoptosis: Implications for amyotrophic lateral sclerosis. J Neurochem 93:38–46.

2. Contreras JE, et al. (2002) Metabolic inhibition induces opening of unapposedconnexin 43 gap junction hemichannels and reduces gap junctional communication incortical astrocytes in culture. Proc Natl Acad Sci USA 99:495–500.

3. Giaume C, Marin P, Cordier J, Glowinski J, Premont J (1991) Adrenergic regulation ofintercellular communications between cultured striatal astrocytes from the mouse.Proc Natl Acad Sci USA 88:5577–5581.

4. Locovei S, Scemes E, Qiu F, Spray DC, Dahl G (2007) Pannexin1 is part of the poreforming unit of the P2X(7) receptor death complex. FEBS Lett 581:483–488.

5. Reaume AG, et al. (1995) Cardiac malformation in neonatal mice lacking connexin43.Science 267:1831–1834.

6. Scemes E, Suadicani SO, Spray DC (2000) Intercellular communication in spinal cordastrocytes: Fine tuning between gap junctions and P2 nucleotide receptors in calciumwave propagation. J Neurosci 20:1435–1445.

Fig. S1. FGF-1 increases LY2− and Etd+ uptake by spinal astrocytes in culture. Control astrocytes (Top) were relatively impermeable. A total of 500 μM of LY2−

and 5 μM of Etd+ were applied for 5 min to cultures in control conditions or after 2- or 7-h FGF-1 treatment (Middle and Bottom), and the cells were rapidlywashed and photographed. LY2− and Etd+ were taken up by cells treated for 2 h or 7 h with FGF-1 (10 ng /mL). The labeling was heterogeneous and colocalizedto a moderate degree. After a 7-h treatment, cytoplasm was more retracted to a perinuclear position leaving processes between cells (black arrows in thephase image). LY2− and Etd+ puncta may be cellular domains with higher density of dye binding sites. Some puncta do not label with both LY2− and Etd+; seewhite arrows.

Fig. S2. The FGF-1–induced Etd+ uptake in spinal astrocytes requires kinase activity of the FGF-1 receptor. (A and B) Uptake was measured in spinal astrocytesunder control conditions (open circles) or after 2-h (A) or 7-h (B) application of FGF-1 (filled circles). Pretreatment with PD170374 (1 μM, PD), a blocker of thekinase activity of activated FGF-1 receptor, applied 45 min before the start of and during FGF-1 treatment completely blocked Etd+ uptake in both A and B(stars). PD alone did not alter control uptake (A, red plus signs). (C) Quantitation. *P < 0.05, ***P < 0.005 FGF-1 vs. control (n = 3 experiments).

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Fig. S3. Control and FGF-1–treated spinal astrocytes express Px1. (A) Px1 detected with an anti–C-terminal antibody in rat spinal astrocytes under controlconditions was largely in the perinuclear cytoplasm or Golgi. Nuclei were labeled with Hoechst 33258 stain. (B) siRNA against Px1 virtually abolished im-munolabeling. (C and D) After 2- or 7-h treatment with 10 ng/mL FGF-1 (typical of n = 4) Px1 immunoreactivity was prominent at the cell periphery. (E) Westernblot detection of Px1 in astrocyte homogenates from cultures under control conditions and 24 h after transfection with siRNA Px1. A single band with a mobilityof ∼50 kDa was prominent in control astrocytes and greatly reduced in astrocytes 24 h after transfection with siRNA (to 20% of control, n = 3, P < 0.01).

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Fig. S4. FGF-1–treated spinal astrocytes release ATP, and exogenous ATP mimics the FGF-1–induced astrocyte permeabilization. (A) Determined by a luciferin-luciferase assay, levels of ATP in the medium of spinal astrocyte cultures were increased after treatment with FGF-1 for 2 h or 7 h (**P < 0.01 vs. control, 2 h vs.7 h, P > 0.05, n = 6). (B) Normalized rate of Etd+ uptake under control conditions, after a 7-h FGF-1 treatment and after a 7-h FGF-1 treatment with APY (2 mU/mL), BBG (5 μM), oATP (200 μM), or reactive blue 2, a P2Y blocker, (RB2, 50 μM). Blockers were applied 15 min before the onset and during the FGF-1 treatmentand during uptake measurement. APY, BBG, and oATP partially blocked uptake. RB2 did not have a significant effect. Thus, FGF-1 may have some effectindependent of ATP release. (C and D) Etd+ uptake was evaluated in cells treated for 2 h (C) or 7 h (D) with 500 μM of ATP. To compensate for ATP hydrolysis byecto-ATPases, ATP was applied at 0, 2, and 4 h during a 7-h treatment. Effects on uptake by CBX (200 μM), BBG (5 μM), RB2 (50 μM), or octanol (Oct, 1 mM)applied 15 min before and during uptake measurement were determined. (C) After 2 h of ATP, uptake was markedly reduced by CBX and BBG but not by RB2or Oct, consistent with mediation by P2X7Rs and Px1 HCs. (D) After 7 h of ATP, CBX block was the greatest although incomplete; BBG and Oct blockedsomewhat less (but not significantly so) and RB2 did not have a significant effect. (E and F) Effect of a single application of 10 μM BzATP (an activator of P2X7Rsand blocker of P2YRs) on Etd+ uptake 2 h (E) and 7 h (F) later with blocker treatment as for ATP. (E) After 2 h of BzTP, CBX and BBG markedly reduced uptake;Oct had no effect, consistent with Px1 mediation. (F) After 7 h of BzATP, CBX produced nearly complete block; BBG and Oct each produced partial block,consistent with uptake through both Px1 and Cx HCs (four experiments, 7–15 cells in each experiment). *P < 0.05, **P < 0.01 vs. control; #P < 0.05, ##P < 0.01 vs.FGF-1, ATP, or BzATP alone.

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Fig. S5. Intercellular transfer of Etd+ injected into spinal astrocytes under control conditions and after FGF-1 treatment; transfer is less after FGF-1 treatment.(A) The dye spread to as many as three cells in the control and possibly one after a 7-h FGF-1 treatment (which activated the astrocytes). Phase contrast imagesare shown above and the corresponding fluorescence images below. (B) The incidence of dye coupling (defined as the percentage of injected cells from whichdye spread to at least one neighboring cell) was decreased at 2-h and 7-h FGF-1 treatment (four experiments for each duration; in each experiment 10–15 cellsmicroinjected within 15 min of the end of treatment). *P < 0.05 vs. control. Coupling after 2 h and 7 h of FGF-1 treatment did not differ significantly.

Fig. S6. FGF-1 reduces intercellular dye transfer via gap junctions in spinal astrocytes, an action reduced by APY. Transfer measured by scrape loading. (A)Loading and intercellular transfer of LY2− in control medium, in control medium after application of 0.1 mM CBX 15 min before loading, after 7-h treatmentwith FGF-1 (7 h FGF-1), and after 7 h of FGF-1 treatment with apyrase (APY) for 15 min before and during FGF-1 treatment. (B) Percentage of cells containingdye relative to control in experiments as shown in A. *P < 0.05 vs. control, ***P < 0.005 vs. control, ##P < 0.01 vs. FGF-1 alone, n = 4. Both CBX and FGF-1reduced dye transfer. APY partially protected dye transfer during FGF-1 treatment.

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Fig. S7. Cx43 distribution determined by indirect immunofluorescence and by Western analysis of total and biotinylated cell surface proteins. (A) Cx43distribution in spinal astrocytes under control conditions (Left) and after 7 h of FGF-1 (Right). Both punctate and diffuse staining appeared somewhat reducedby FGF-1. White arrows: probable gap junctions. Green arrows: probable internalized junctions. Red arrows: processes indicating activation of astrocytes. (B)Western blot of Cx43 on the cell surface isolated by biotinylation (Biot) and in total cell homogenates (total) under control conditions and after a 7-h FGF-1treatment (Cx43, n = 4). Both surface and total Cx43 were reduced by FGF-1 with no marked change in phosphorylation as indicated by mobility of the severalbands. Heart homogenate (Left) was used as a standard for phosphorylation. P2 and P3 denote phosphorylated forms and NP, the unphosphorylated form.

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Fig. S8. FGF-1 does not permeabilize cortical astrocytes, unlike its effect on spinal astrocytes. (A and B) Etd+ uptake by cortical astrocytes (Cort) is not affectedby FGF-1 at 2 or 7 h of treatment. Faster basal uptake may be due to greater surface-to-volume ratio relative to the larger spinal astrocytes. (C and D) Incontrast uptake by spinal astrocytes (Sp) is increased by 2-h and 7-h FGF-1 treatment. (E and F) Summary data for cortical and spinal astrocytes from threeexperiments with comparable cultures of cortical and spinal astrocytes (∼20 DIV). Cultures of cortical and spinal cells were prepared from P1 pups from thesame mothers and examined at the same age. Uptake rate after FGF-1 is normalized to that by the respective untreated cortical or spinal cord astrocytes. *P <0.05, ***P < 0.001.

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Fig. S9. FGF-1 causes greater permeabilization of spinal than cortical astrocytes. Uptake of 5 μM Etd+ in response to 10 ng/mL FGF-1. First through thirdcolumns: Control, 2-h FGF-1 treatment, and 7-h FGF-1 treatment. (A) Cortical astrocytes and (B) spinal astrocytes in phase and fluorescence micrographs (Upperand Lower). Cortical astrocytes are smaller than spinal astrocytes and show greater basal uptake of Etd+ with little effect of FGF-1. Labeling was heteroge-neous. After 7 h of FGF-1, the phase images of both cortical and spinal astrocytes show retraction of cytoplasm around the cell bodies, leaving processesbetween cells a sign of activation.

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