Serum and fibroblast growth factor stimulate quiescent astrocytes to re-enter the cell cycle

Brain Research, 439 (1988) 281-288 Elsevier

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BRE 13223

Serum and fibroblast growth factor stimulate quiescent astrocytes to re-enter the cell cycle Douglas A. Kniss and Richard W. Burry Department of Anatomy and Neuroscience Research Laboratory, The Ohio State University Co',lege of Medicine, Columbus, OH 43210 (U.S.A.) (Accepted 14 July 1987) Key words: Astrocyte; Cell cycle; Fibroblast growth factor; In vitro

An in vitro model was used to study the cytokinetics of astroglial cells derived from neonatal rat cerebellum. Confluent monolayers of astrocytes (85% astroglial as assessed by GFAP immunoreactivity) were subcultured at ilowcell density and after 2-3 days growth were rendered quiescent by shifting them to low serum medium (0.25%) for several days. Cells could be stimulated to re-enter the proliferative compartment by challenging them with high concentrations of fetal bovine serum (5-10% FBS) or fibroblast growth factor (FGF). FGF added alone at a concentration of 25 ng/ml caused quiescent astrocytes to re-enter the cell cycle nearly as effectively as 5-10% serum. Moreover, when FGF (25 ng/mi) was combined with 0.5% serum there was a potentiation of the mitogenic effect seen with FGF alone. This synchronization scheme is an important tool for continuing studies of the growth factor and hormonal requirements for as~roglial cell proliferation and differentiation.

INTRODUCTION The proliferation and differentiation of astroglial cells are fundamental events during the ontogeny of the vertebrate central nervous system (CNS). In addition, the re-entry of quiescent astrocytes into active proliferation is a general sequela seen subsequent to neural trauma initiated by: (1) physical damage 2"3' 17,18, (2) neurotoxic agents such as kainic acid 27 or (3) cerebrovascular infarction 8. Astroglial hyperplasia and hypertrophy following CNS injury are contributing factors in the formation of a gliotic scar at the lesion site which may be an impediment to regenerative efforts by d a m a g e d central neurons 1'9. A t the present time, little inforrnation is available concerning the signals which regulate astroglial cell cycle activity. Cell culture experiments suggest that multiple growth factors and hormones are required for optimal astrocyte proliferation in serum-free medium4,20,25.

Astrocytes proliferate in vitro in an asynchronous manner; that is, they are distributed more or less randomly throughout the cell cycle. Thus, it is difficult to determine the site of action within the cell cycle of various agents that affect glial proliferation. In the present report, experiments show that astrocytes can be synchronized with respect to cell cycle position. Astrocytes remain in a non-cycling but viable state and can be stimulated to re-enter the cell cycle by addition of 5 - 1 0 % serum or fibroblast growth factor (FGF) 4.~.25. This model may prove useful for analyzing the factors which cause quiescent populations of as troglial cells to resume active proliferation. MATERIALS AND METHODS Astroglial cell cultures Primary cultures of astroglial cells were prepared from 2-day-oid rats. Cerebellar tissue was removed and cleared of meninges using aseptic instruments.

Correspondence: D.A. Kniss. Present address: Laboratory of Developmental Neurobiology, Building 36, Room 2A21, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892, U.S.A. 0006-8993/88/$03.50 © 1988 Elsevier Science Publishers B.V. (Biomedical Division)

282 The tissue was rinsed in Buffer 114followed by modified Ham's F12 medium 15 supplemented with 6 g/l o-glucose, 0.5% bovine serum albumin (Miles), 50 b~ghnl gentamicin sulfate (GIBCO, Grand Island, NY), and 10% fetal bovine serum (FBS; Hyclone, Logan, UT). Th.~ tF~sue was mechanically dissociated without enzyme digestion. Single cell suspensions were plated into Falcon 75-cm 2 tissue culture flasks at 2-5 x 105 cells/cm2 with F12 + 10% FBS. Cultures were fed initially at day 2 in vitro and then twice weekly and maintained at 37 °C in 5% CO2/95% air. Primary cultures reached confluent density after 9-12 days in vitro and were subcultured for subsequent experiments by a modification of the method of McCarthy and de Vellis 19. Flasks were manually agitated for several rain to disperse residual neuronal material and oligodendrocytes. Cultures were then rinsed with Ca 2÷-, Mg2+-free Hank's balanced salt solution (CMF-HBSS). Trypsin-EDTA (GIBCO) was added to each flask for 20 min at 37 °C. Trypsinization was terminated by addition of an equal volume of serum-supplemented F12 medium. Cells were pelleted at 100 g for 5 min, then resuspended in F12 + 10% FBS, and counted in a hemocytometer. Cells were seeded into Falcon 24-well tissue culture plates at 1 x l0 s cells/well with F12 + 10% FBS and grown to confluence. For immunostaining and autoradiography, cells w~re seeded at 5 x 104 cells per well into 24-well plates containing 12-mm glass coverslips. Alternatively, for some experiments, cells were seeded into Miles Lab Tek 8-chamber glass slides at 2.5 x 104 cells/chamber. Confluent monolayers of astroglial cells were rinsed several times with serum-free F12 medium and then shifted to F12 + 0.25% FBS for 2-4 days at which time they were used for experiments.

DNA synthesis assay Proliferative activity was assessed by the ability of astrocytes to enter the S phase of the cell cycle and initiate DNA synthesis. Confluent monolayer cultures of astroglial cells were treated with test substances and then radiolabelad with 5 /zCi/ml of [3H]thymidine (TdR) (spec. act. 25 Ci/mmol, Amersham, Arlington Heights, IL) for 24 h. To measure [3H]TdR incorporation into DNA, cells were rinsed three times with CMF-HBSS, and then trypsinized

from wells and transferred" to chilled tubes. Trichloroacetic acid (TCA) was added to a final concentration of 10% and precivitation was carried out on ice for 15 min. Samples were collected on Whatman GF/C glass fiber filters under vacuum. Filters were washed with 5% TCA and cold 95% ethanol, and air dried. Filters were then counted in a liquid scintillation spectrometer (Beckman LS7800, 65% efficiency) using Formula 963 aqueous counting cocktail (New England Nuclear, Boston, MA).

[3H] Thymidine autoradiography Cells were seeded onto 12-mm coverslips or 8chamber Lab Tek slides and radiolabeled with 5 #Ci/mi of [3H]TdR for 24 h. Cultures were fixed with acid-alcohol and first processed for immunoperoxidase cytochemistry (see below). Next air-dried cultures were dipped in llford L4 emulsion (Polysciences, Warrington~ PA) and stored in the dark at 4 °C for 1-2 weeks6. Autoradiograms were developed with Kodak D-19, fixed with Kodak Rapid Fixer, washed in tap water and stained with 0.5% Toluidine blue in 1% sodium borate. To quantitate the labeling indices of autoradiograms, at least 500 cells were counted per sample under a light microscope using a final magnification of 400x.

Irnmunocytochemistry Cells were fixed with acid-alcohol for 15 min, rinsed with PBS, and treated for 10 min with 20% normal sheep serum. Cultures were then incubated with rabbit anti-bovine glial fibrillary acidic protein (1:100, GFAP) (a gift from Dr. L. Eng, Stanford University, Paid Alto, CA) or rabbit anti-chicken GFAP (Dr. B. Granger, Cal Tech, Pasadena, CA) for 30 min. Coverslips were then rinsed with PBS + 0.1% BSA and incubated with sheep anti-rabbit IgG conjugated to f, uorescein isothiocyanate (1:100, SAR-FITC, Cappel, Malvern, PA). Finally, cells were counterstained with propidium iodide (4/zg/ml in PBS) to visualize nuclei and mounted in phosphate-buffered saline (PBS):glycerol (1:1) 13. Immunofluorescence preparations were examined under UV illumination (Leitz) and photographed using TRI X film. To quantitate the percentage of GFAP ÷ astrocytes in the cultures, the number of GFAP-immunoreactive cells was divided by the total number

283 of cells counted. At least 500 cells were counted per slide.

Combined immunocytochemistry and autoradiography Glial cells grown on 12-mm coverslips or Lab Tek slides were labeled for 24 h with 5/~Ci/ml [3H]TdR after which they were rinsed with PBS and fixed with acid-alcohol. Cultures were then treated sequentially with 20% normal sheep serum (10 min), rabbit anti-bovine G F A P (1:500, 30 min), sheep anti-rabbit IgG (1:300, 30 rain), and rabbit peroxidase-antiperoxidase complex (1:500, 30 min). The peroxidase reaction product was developed with 3,3'-diaminobenzidine tetrahydrochloride (0.5 mg/ml, Sigma, St. Louis, MO) and 0.015% H20 2 for 10 min. The cells were then rinsed with PBS, dehydrated in ethanol and air dried. Immunoperoxidase preparations were then processed for autoradiography as described above.

Polypeptide growth factors Purified bovine brain and pituitary FGF l° was a generous gift from Dr. Denis Gospodarowicz (University of California, San Francisco). The growth factor was added to astrogliai cell cultures in the presence of 0.5% bovine serum albumin (BSA). Partially purified human platelet-derived growth factor (PDGF) was a gift from Dr. W.J. Pledger (Vanderbilt University, Nashville, TN) and was purified as described by Heldin et al. 12. P D G F was added to cells at 5 units/ml in serum-free medium supplemented with 0.5% BSA. Epidermal growth factor (EGF) was a gift from Dr. S. Cohen (Vanderbilt University, Nashville, TN) and was purified from male mouse submaxillary gland 26. E G F was added (100 ng/ml) to cells in serum-free medium supplemented with 0.5% BSA. RESULTS

Fig. 1. Phase-contrast photomicrograph of a non-neuronal cerebellar culture (7 days in vitro). Arrows indicate mitotic cells. x99. t

not survive and within 7-10 a~,,~ in vitro the culture is comprised almost entirely of no,n-neuronal cells as assessed by phase contrast microscopy and the lack of neurofilament and tetanus toxin immunoreactive elements (Fig. 1). Secondary astrocyte cultures were prepared from confluent monolaycrs for use in experiments. Cells were trypsinized and replated at lower density (0.25-1.0 x 105 cells per well) in 24-well plates. Immunocytochemical staining of secondary cultures showed that they were 85% GFAP-immunoreactive, a marker for astroglial cells (Fig. 2, ref. 24). Quanti-

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Characterization of astroglial cells When dispersed rat cerebellar cells are plated at high density (2-5 x 105 cells per cm 2) in serum-supplemented medium, a monolayer of glial cells forms after about 7 days in vitro upon which small diameter neurons attach and begin to form neurites. Under conditions using the standard 3 mM KCI in this culture medium formulated by •,~,~,,,.l ..o~.,~..15the neurons do

Fig. 2. Immunofluorescence photomicrograph of a secondary culture of cerebellar astroglia 24 h after subculture in F12 + 10% FBS. Cells were fixed and stained with an'~ioGFAPa_n_tisera followed by FITC-conjugated sheep anti-rabbit IgG. Cultures were then counterstained with propidium iodide to visualize nuclei (arrows). x243.

284 tative immunocytochemistry was performed using the double-staining protocol described by Jones and Kniss 13. The growth fractions of total non-neuronal cells and GFAP + astrocytes in the secondary cultures were measured using [3H]TdR autoradiography. This procedure measures the total proportion of cells in the culture that is within the proliferative compartment of the cell cycle. Fig. 3A shows the growth fraction of the total non-neuronal cell population in the culture after 5 days of continuous [3H]TdR labeling. A constant increase in the percentage of cells entering the S phase is seen through the first 4 days of labeling with a plateau reached at day 5. These results show that by day 5, over 80% of the cells are actively cycling while less than 20% of the cells are in the quiesceut state. When sister cultures were stained with a n t i - G F A P t, antibodies followed by autoradiography, it was found that the pattern of astroglial cell cycle behavior (GFAP + cells) was nearly identical to the total nonneuronal cell population (Fig. 3B). Note t~at the curves in Fig. 3A and B are nearly superimposible

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suggesting that examining the proliferative kinetics of the total population of non-neuronal cells is highly reflective of the behavior of the astroglia.

Growth kinetics in synchronized astroglial cultures In the experiments described above, asynchronous cultures were used. That is, cells were distributed more or less randomly throughout all phases of the cell cycle. This type of approach, however, only measures cells that are withm the proliferative compartment. Any cells that are either in the quiescent state or are in a portion of the cell cycle that is insensitive to perturbation will be refractory to alteration of cell cycle activity by various treatment regimens. Thus, an attempt was made to synchronize the astroglial cells with respect to cell cycle position. This protocol has the net effect of placing all cells at the same temporal point in order that every cell in the population has an equal probability of responding to cell cycle perturbing agents. The first series of studies examined the ability of variou~culture conditions to arrest the proliferation of astrocytes without compromising viability. Fig. 4A shows that astrocytes shifted to 0.25% FBS (solid bars) and labeled for 24 h incorporated [3H]TdR into D N A at 13% of control levels (10% serum; open bars), These data were supported by a parallel study in which autoradiography was combined with G F A P immunostaining (Fig. 4B). It was found that, while complete omission of serum from the basal medium caused a slightly more effective growth-arrest, serum-free medium failed to sustain the cells in a viable state for long periods in vitro. Low serum (0.25% FBS) fulfilled both criteria for the synchronization protocol; it arrested the growth of cells and maintained high cell viability. The next experiment tested whether the cells could be rescued from growth-arrest. Astrocytes were grown to confluence in 10% FBS and then shifted to 0.25% FBS for several days at which time half of the cultures were shifted back to 10% serum. Control cultures received fresh 0.25% serum. Both sets of cultures were radiolabeled with [3H]TdR and fixed for autoradiography at intervals up to 54 h. After an initial lag period of about 12 h, the cells that were shifted to 10% serum resumed active cell cycling as assessed by entry into D N A synthesis, while cultures treated with 0.25% FBS failed to re-enter the cell

285 showed that astrocytes could be arrested by culture in low serum and then rescued after restimulation with a high concentration of serum. In a parallel experiment, synchronous cultures (0.25% FBS) were shifted to 5% serum and pulselabeled for 1 h with [3H]TdR at intervals up to 24 h. U p o n release from cell cycle arrest, the astrocytes began to synthesize D N A after a lag of about 12 h confirming the autoradiographic data (Fig. 6). These data are similar to the cell cycle-arrest occurring in Gl/G0 of the cell cycle as shown by Pledger et al. 2°'21 in 3T3 cells. A maximum rate of D N A synthesis occurred at aboat 18 h and rapidly declined by 24 h (Fig. 6). The rapid diminution in the rate of D N A synthesis reflected the traverse of the synchronized astrocyte population past the S phase and into the G2/M phase. Thus, it was found that shifting cells to low serum was an effective method of achieving

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Fig. 4. Effect of serum concentration on DNA synthesis in astroglial cells. Cultures were treated with various concentrations of FBS and labeled with [3H]TdR for 24 h followed by (A) liquid scintillation counting or (B) GFAP staining and autoradiography. Triplicate samples were assayed under each conditions. Bars repzesent S.E.M.

growth-arrest while maintaining cell viability. The cells in low serum metAum cou:d then be restimulated to resume cycling by shifting them to a higher serum concentration.

cycle in significant numbers (Fig. 5). Even after 54 h of constant labeling the cells maintained in 0.25% serum did not re-enter the S phase. The data clearly

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Fig. 5. Rescue of growth-arrested astrocytes after shifting them to 10% FBS. Cells were cultured for several days in 0.25% serum and then shifted to 10% FBS (Xs) or treated with fresh 0.25% FBS (circles). Both groups were radiolabeled with [3H]TdR and then fixed for autoradiography at intervals up to 54 h. Triplicate samples were assayed at each point and standard errors varied by 10%.

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Fig. 6. Kinetics of re-entry into DNA synthesis of astrocytes stimulated with 5% FBS. Growth-arrested astrocytes were stimulated to 5% serum and labeled for 1 h at intervals up to 24 h. DNA synthesis was measured by liquid scintillation counting. Triplicate samples were assayed at each time point.

286 FGF-induced resumption o f cell cycle activity in quiescent astrocytes Several polypeptide growth factors were tested for their ability to induce a resumption of cell cycle activity in low serum growth-arrested astrocytes. FGF isolated from bovine brain and pituitary was effective in stimulating quiescent astrocytes back into the cell cycle as assessed by their ability to re-enter the S phase and synthesize DNA (Table I). As shown in Table I, when G1/G0-arrested astrocytes were treated with FGF (25 ng/ml) in serum-free medium they resumed active D N A synthesis at a rate approximately equal to 3% FBS. Interestingly, when quiescent cells were exposed to FGF in the presence of 0.5% FBS (a roon-mitogenic concentration of serum) their rate of re-entry into DNA synthesis was nearly doubled compared to 5% FBS-treated cells. This represented approximately a 10-fold increase in nuclear labeling over unsupplemented control cultures and a nearly 2-fold increase over cells treated with 5% serum. Dose-response experiments with FGF demonstrated even more dramatically the apparent synergistic interaction between FGF and low concentrations of serum. When quiescent astrocytes were treated with FGF in serum-free medium half-maximal stimulation of DNA synthesis was seen at about 1-2 ng/ml (Fig. 7). When astrocytes were treated with various concentrations of FGF in the presence of 0.5% FBS, the half-maximal stimulation occurred at approximately the same concentration of FGF (1-2 ng/ml). However, maximal stimulation of DNA synthesis by FGF in the presence of low serum was

TABLE I Effect of serum and fibroblast growth factor on re-entry of quiescent astrocytes into the cell cycle

Quiescent cultures of astroglial cells were treated with test substances and labeled for 24 h with 5 gCi/ml [3H]TdR. Cultures were then fixed and processed for GFAP immunostaining followed by autoradiography. Labeling indices were calculated after counting at least 500 cells per coverslip (n = 2). Values in table represent mean + S.D. Treatment

[3H] TdRI G FA P +cells

Unsupplemented medium 0.5% Serum 5% Scram FGF (25 ng/ml) alone FGF (25 ng/ml) + 0.5% serum

5.1 +_0.21 11.2 ± 0.35 30.8 _+ 1.20 32.7 _ 2.10 57.6 _+7.40

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Fig. 7. Dose-response curves for FGF-stimulated astrocytes in serum-free and 0.5% serum medium. Cells were rendered quiescent by growth in 0.25% serum for several days. The solid line indicates quiescent cells that were challenged with the indicated concentrations of FGF and labeled for 24 h. The dashed line indicates quiescent cells shifted to 0.5% serum and then challenged with the indicated concentrations of FGF and labeled for 24 h. about twice the level seen in serum-free medium (Fig. 7). These results clearly indicate that F G F and a low concentration of serum can interact synergistically to stimulate quiescent astrocytes back into the cell cycle. Insulin, epidermal growth factor, and platelet-derived growth factor were unable to stimulate quiescent astrocytes to resume cell cycle activity in serumfree medium or medium supplemented with 0.5% FBS under the assay conditions used here (data not shown). DISCUSSION The factors that control the entry of quiescent astrocytes into cell division are important to the understanding of the glial response to traumatic injury. The study of the mechanisms that control astrocyte proliferation requires a system of enriched astrocytes that can be easily manipulated. The primary cell culture of cerebellar astrocytes offered these advantages. Initially: the proportion of proliferating cells in culture was examined by measuring maximal nuclear labeling after chronic exposure to [3H]thymidine. After labeling for 5 days, 84% of the GFAP ÷ cells were labeled indicating that about 16% of the cells did not enter the S phase of the cell cycle during this time. One interpretation of this result is that the astroglial

287 cell cycle time is quite long and that even at 5 days more cells could be entering the S phase for the first time. This is unlikely because the labeling of cells had reached a plateau by 3 days incubation (Fig. 3B), and did not show a continuous increase. An alternative interpretation of the long time required to achieve maximal labeling is that a portion of the astrocyte population withdraws transiently from the active proliferative phase of the cell cycle and then re-enters at a later time. Support for such an interpretation comes from studies by Tomlinson et al. 29 in which they demonstrated that blastemal cells from regenerating urodele amphibian limbs withdra~ from the cell cycle and remain quiescent, but are capable of re-entry into the cell cycle. Both explanations of the apparent long cell cycle time are currently under study. A concerted effort was expended to synchronize the astrocytes with respect to cell cycle position. It was reasoned that this procedure would place all of the cells at the same temporal point and thus would allow questions to be asked concerning the site of action within the cell cycle of various agents. Several methods exist for arresting cells in a specific phase of the cell cycle including: density-dependent arrest in G1/G0 (ref. 16) specific metabolic inhibitors 11 and serum or amino acid deprivation 5"7. Once cells are arrested at the same temporal point, they can be released from the cell cycle blockade with growth factors or serum, or the chemical inhibitors can be removed, and all cells ~ill resume cycling in a synchronous manner. That astrocytes indeed can be arrested in G~/G0 and then restimulated to resume cycling actively in a synchronous manner was shown in studies of the kinetics of entry into the S phase after serum stimulation (see Figs. 5 and 6). Upon exposure to 5% serum of Gl/G0-arrested astrocytes, they entered rapidly into DNA synthesis (S phase) after a lag of about 12 h. This lag time has been shown by Pledger et al. 21"22 to reflect the time required for 3T3 cells to exit the Go state and traverse the G 1 t o the S period. The rapid increase in the rate of DNA synthesis in the present study is reflective of a wave of astrocytes entering the S phase in synchrony. Had the cells traversed the

G~/G0 to the S period in an asynchronous fashion, one would predict a more gradual rise in the rate of DNA synthesis. The rapid decline in the rate of DNA synthesis in the pulse-labeling experiment (see Fig. 6) also reflects a synchronous exit from the S phase by the astrocytes. This synchronization scheme will be useful for studying the specific signals which trigger quiescent (differentiated) astrocytes back into the cell cycle. Indeed, in the present study, this synchronization protocol was used to show that FGF, but not EGF, PDGF, or insulin, could cause astroglial cells to exit the quiescent state and re-enter the proliferating compartment. Interestingly, FGF added alone was about as effective as 5% serum in stimulating DNA synthesis in astrocytes. Moreover, when FGF was combined with 0.5% FBS (a non-mitogenic concentration of serum), the cells were restimulated at nearly twice the level of 5% serum. This apparent synergy between FGF and a serum component(s) is reminiscent of the multifactor requirements for optimal cell cycle progression in various fibroblast cell

lines21,22.28. Work by de Vellis and colleagues2°'2s and Bottenstein 4 has elegantly shown that chemically defined media formulated for astrocytes require a multiplicity of hormones and growth factors for optimal growth in vitro. The present protocol for selectively arresting astrocytes in GI/G 0 of the cell cycle and then releasing them synchronously will provide a means to test in a mechanistic way the role of various hormones and growth factors in astroglial cell proliferation and differentiation. ACKNOWLEDGEMENTS The authors wish to thank Diane M. Hayes for technical assistance, and Linda Hoover and Brenda Judge for photographic assistance. This work was supported in part by a Grant-in-Aid of Research from Sigma Xi (D.A.K.), an Alumni Graduate Research Award from The Ohio State University (D.A.K.), a Presidential Fellowship (D.A.K.), and a U.S. Public Health Services grant from the National Institutes of Health NINCDS NS-19961 (R.W.B.).

288 REFERENCES 1 Barrett, C.P., Donati, E.J. and Guth, L., Differences between adult and neonatal rats in their astrogliai response to spinal injury, Exp. Neurol., 84 (1984) 374-385. 2 Bignami, A. and Dahl, D., The astroglial response to stabbing. !mmunofluorescence studies with antibodies to astrocyte specific protein (GFA) in mammalian and submammalian vertebrates, Neuropath. Appl. Neurobiol., 2 (1976) 99-110. 3 Billingsley, M.L., Straw, J.A. and Mandel, H.G., Glial DNA synthesis and cell proliferation in the lesioned frontal cortex of the rat, Brain Research, 247 (1982) 325-344. 4 Bottenstein, J.1S., Growth and differentiation of neural ceils in defined medium. In J.E. Bottenstein and G. Sato (Eds.), Cell Cu!ture in the Neurosciences, Plenum, 1985, pp. 3-43. 5 Brooks, R.F., Regulation of the fibroblast cell cycle by serum, Nature (London), 260 (1976) 248-250. 6 Burry, R.W. and Lasher, R.S., Electron microscopic autoradiography of the uptake of [3H]GABA in dispersed cell cultures of rat cerebellums. II. The development of GABAergic synapses, Brain Research, 151 (1978) 19-29. 7 Campisi, J., Morreo, G. and Pardee, A.B., Kinetics of G1 transit following brief starvation for serum factors, Exp. Cell Res., 152 (1984) 459-466. 8 DuBois, M., Bowman, P.D. and Goldstein, G.W., Cell proliferation after ischemic infarction in gerbil brain, Brain Research, 347 (1985) 245-252. 9 Frank, E., Adaptive and maladaptive regeneration in the spinal cord. In J.G. Nicholls (Ed.), Repair and Regeneration in the N~,rvous System, Springer, New York, 1982, pp. 243-254. 10 Gospodarowicz, D., Cheng, J., Lui, G.-M., Baird, A. and Bnhlen, P,, Isolation of bran fibroblast growth factor by heparin-sepharose affinity chromatography: identity with pituitary fibroblast growth factor, Proc. Natl. Acad. Sci. U.S.A., 81 (198a) 6963-6967. 11 Hamlin, J.L. and Pardee, A.B., Control of DNA synthesis in tissue culture cells, In Vitro, 14 (1978) 119-127. 12 Heldin, C.-H., Westermark, B. and Wasteson, A., Platelet-derived growth factor: purification and partial characterization, Proc. Natl. Acad. Sci. U.S.A., 76 (1979) 3722-3726. 13 Jones, K.H. and Kniss, D.A., Propidium iodide as a nuclear counterstain for immunofluorescence studies on cells in culture, J. Histochem. Cytochem,, 35 (1987) 123-125. 14 Kniss, D.A. and Burry, R.W., Glucocorticoid hormones inhibit DNA synthesis in glial cells cultured in chemically defined medium, Exp. CellRes., 161 (1985) 29-40. 15 Lasher, R.S., The uptake of [3H]GABA and differentiation of steUate neurons in cultures of dissociated postnatai rat cerebellum, Brain Research, 69 (1974) 235-254. 16 Lieberman, M.A. and Glaser, L., Density-dependent regu-

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