Neuroscience Vol. 42, No. 2, pp. 561-568, 1991 Printed in Great Britain
0306-4522/91 $3.00 + 0.00 Pergamon Press plc © 1991 IBRO
EFFECTS OF BASIC FIBROBLAST GROWTH FACTOR ON THE DEVELOPMENT OF GABAERGIC N E U R O N S IN CULTURE J. C. DELOULME,* C. GENSBURGER,* S. SARHAN,~ N. SEILER~ and M. SENSENBRENNER*~ *Centre de Neurochimie du CNRS and INSERM U44, 5 rue Blaise Pascal, 67084 Strasbourg Cedex, France tMerrell Dow Research Institute, Strasbourg Center, 16 rue d'Ankara, 67084 Strasbourg Cedex, France Abstract--Six-day-old neuronal cultures derived from 14-day-old embryonic rat cerebral hemispheres were highly enriched in GABAergic neurons, as was demonstrated by immunocytochemistry using an anti-glutamate decarboxylase antiserum. They contained about 64% glutamate decarboxylase-positive neurons. About 8% of these neurons proliferated, as shown by a combination of glutamate decarboxylase immunocytochemistry and [3H]thymidine incorporation into cell nuclei. The proliferative activity of GABAergic precursor cells and changes in the cellular concentrations of the non-essential amino acids, including GABA under the effect of basic fibroblast growth factor were studied. When basic fibroblast growth factor was added to the cultures 4 h after seeding, the proliferation of the GABAergic neurons was stimulated about threefold. Under this culture condition, the concentration per cell of all amino acids increased, except those of GABA and fl-alanine. When basic fibroblast growth factor was added to cultures only on day four, the proliferation of the neuronal cells was no more enhanced. Under this condition of treatment, the concentrations of all non-essential amino acids, including those of GABA and fl-alanine were enhanced. Under both basic fibroblast growth factor treatments the concentration of GABA per GABAergic cell was increased. In contrast, the specific activity of glutamate decarboxylase was not stimulated under these conditions. We hypothesize that under the effect of basic fibroblast growth factor the capabilities of the cells to store GABA are improved.
Recent studies have shown that fibroblast growth factors (FGFs) have neurotrophic activity for neurons of the peripheral and central nervous system. Both acidic F G F ( a F G F ) and basic F G F ( b F G F ) support the survival and enhance the outgrowth of neurites of rat neurons from various brain regions, 6'11'17'18'32'33 and stimulate the proliferation of neuroblasts from cerebral hemispheres of 13-day-old rat embryos. 7,9 Basic F G F , but not a F G F , promotes the survival of chick ciliary ganglion neurons. 3° Little is known about the effect of F G F s on the expression o f neurotransmitter-related properties in cultured neurons. We have found that b F G F stimulates choline acetyltransferase activity in cultured chick ciliary ganglion n e u r o n s ) ° M o r e recently, Grothe et al. l° observed that b F G F promoted cholinergic development of rat septal neurons in culture. Other authors have mentioned that b F G F stimulated the uptake of G A B A and dopamine into G A B A e r g i c and dopaminergic neurons, respectively? Finally, intracerebroventricular injections of b F G F increased :~To whom correspondence should be addressed. Abbreviations: aFGF, acidic FGF; bFGF, basic FGF;
DMEM, Dulbecco's modified Eagle's medium; EDTA, ethylenediaminetetra-acetate; FCS, fetal calf serum; FGF(s), fibroblast growth factor(s); GABAr, GABA transaminase; GAD, glutamate decarboxylase; [3H]TdR, [methyl-3H]thymidine; HPLC, high-performance liquid chromatography; PBS, phosphate-buffered saline. 561
choline acetyltransferase activity in adult rats with partial septohippocampal lesions. 3 In this paper we have investigated the effect of b F G F on the in vitro development of neurons in dissociated cerebral hemisphere cultures from fetal rats under two conditions: (i) addition of b F G F to the culture medium 4 h after culture initiation; i.e. at a time when the neurons proliferate; (ii) exposure of neurons to b F G F only after four days of incubation; i.e. at a time when cell proliferation had essentially ceased. EXPERIMENTAL PROCEDURES
Neuronal cell cultures
Primary cultures of neuronal cells from cerebral hemispheres of 14-day-old Wistar rat embryos were prepared as previously described. 8 Briefly, cerebral hemispheres were dissected and cleaned of their meningeal membranes. The hemispheres were mechanically dissociated either with a 20-ml syringe by repeated passages through a needle of 1 mm i.d. or by filtration through a nylon sieve (mesh 48 #m). The cells were collected in Dulbecco's modified Eagle's H 16 medium (DMEM; Gibco) containing 20% fetal calf serum (FCS; Gibco) and antibiotics. The dissociated cells were cultured either in 35- or 60-mm Falcon Petri dishes precoated with poly-L-lysine (5 x 105 cells/ml). After 2 h, the cultures were switched to a serum-free, chemically defined medium: DMEM plus 5/zg/ml bovine insulin (Sigma, 1-5500), 50 #g/ml human transferrin (Sigma, T-2252), 20 nM progesterone (Sigma, P-0130), 100 # M putrescine (Sigma, P-7505), 30 nM sodium selenite (Merck) and antibiotics. Cultures
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were re-fed at four days with fresh medium and were incubated at 37°C in a 5% CO2-containing humidified atmosphere. Cultures were grown for a maximum of six days. Cultures were treated once, either at 4 h or four days after culture initiation with bFGF (5 ng/ml). The half-life of bFGF is about 20 h and after 48 h the amount of factor in the culture medium is low, so that no more biological activity is detected against astrocytes (Labourdette and Taupenot, unpublished observations). Therefore, under both conditions of treatment the exposure time to bFGF of the cultures was the same. Basic FGF was prepared in our laboratory from bovine brain by using heparin-Sepharose affinity chromatography, as was described previously. 23 Incubation of the cultures was continued until day six. lmmunoc y t ochemis try At different times, cultures were rapidly rinsed three times with phosphate-buffered saline (PBS) and fixed with 4% paraformaldehyde for 15 min, then postfixed with 30% methanol for 5 min at room temperature. After washing with PBS, the cultures were incubated for 2 h at room temperature or overnight at 4°C with rabbit anti-glutamate decarboxylase (-GAD) antiserum (diluted l : 200) containing 10% normal sheep serum, or with sheep anti-GAD antiserum (diluted 1 : 500) containing 10% normal rabbit serum, respectively. After washing with PBS, the preparations were incubated with 1% sheep anti-rabbit IgG or 0.2% donkey anti-sheep IgG conjugated with peroxidase (Institut Pasteur, Paris) for I h. After three washings with PBS, the preparations were treated for 5 min with a mixture containing 0.05% 3,Y-diaminobenzidine tetrahydrochloride and 0.01% hydrogen peroxide in PBS, washed with PBS, mounted with Kaiser's glycerol gelatin (Merck) and examined under a light microscope. Controls were performed with the corresponding normal serum which was diluted to the same extent as the specific antisera. Rabbit anti-GAD and sheep anti-GAD antisera were generous gifts of Dr M. Maitre (Centre de Neurochimie du CNRS, Strasbourg, France) and Dr M. L. Tappaz (INSERM U 171, Lyon, France), respectively. The characteristics of the antisera used have previously been described. 2°'28 Autoradiography At 24 h after initiation, cultures were exposed for 24 h to medium containing 0.05/iCi/ml of [methyl-3H]thymidine ([3H]TdR) (25 Ci/mmol; CEA, France). The radioactive medium was removed, cultures were rinsed five times with DMEM and supplied with fresh medium. After four days, cultures were submitted to immunoperoxidase staining as described above, rimed with distilled water and air-dried. The preparations were then submitted to an autoradiographic procedure. ~4 The autoradiographs were developed after 10 days of exposure in the dark at 4°C. Determination of amino acids After careful washing of the cultures with PBS, the cells were harvested with a rubber policeman, and the pellet obtained from three dishes was homogenized by sonication (Kontes, Micro-ultrasonic cell disrupter) with 0.6 ml of 0.2 M perchloric acid. After 1 h standing at +2°C, the acidinsoluble material was precipitated by centrifugation. The supernatants were diluted with 9 vol of 0.05 M perchloric acid. Aliquots (200 ~ 1) of the diluted perchloric acid extracts were applied on a reversed-phase column (Beckman Ultrasphere I.P.). Separation of the (non-essential) amino acids was accomplished by isocratic elution (flow rate 1 ml/min at 20°C) with a buffer containing 630 ml 0.2 M phosphoric acid, 270 ml 0.2% sodium acetate (pH 4.5), 100 ml of methanol, and 2.59 g sodium dodecylsulphate (Merck). After reaction of the column effluent with the same volume of o-phthalaldehyde,2-mercaptoethanol reagent, fluorescence intensity was
continuously recorded at 455 nm (excitation at 345 nm) (for details of the method, see Ref. 26). Determination of catecholamines and serotonin The cells harvested per dish were suspended in 600/~1 0.2 M perchloric acid (containing 250 mg sodium bisulphite and 100 mg EDTA/1), sonicated (2 × 30 s) and centrifuged. The supernatant (200#1) was separated by HPLC (isocratic elution mode). Column: Beckman Ultrasphere I.P. (250 mm in length, 4.6mm i.d.). Eluent: 800ml 0.1 M potassium acetate (pH 4.25 containing 2.16 g n-octanesulphonate and 100 mg EDTA/I) + 200 ml methanol; flow rate 1 ml/min (30°C). Detector: Coulochem Model 5100 A (Environmental Sciences Ass. Bedford, MA, U.S.A.) with a dual analytical cell (Model 5011). The coulometric cell was used as screen electrode (at 0.08 V), the amperometric cell as detector electrode (at 0.35V; working potentials relative to the reference electrode). The instrument was set at a sensitivity to allow the determination of 0.1 pmol noradrenaline, dopamine and serotonin per injection volume (signal-to-noise ratio > 5). Determination of protein, RNA and DNA These were determined in the precipitate of the perchloric acid homogenate (see above). For the separation of RNA, DNA and proteins, a modified Schmidt-Thannhauser procedure was used which was adapted to small samples. RNA was determined spectrophotometrically at 260 nm, DNA by a version of Burton's diphenylamine reaction, and proteins by a modified Lowry method (for a detailed description of the procedure, see Ref. 27). Yeast RNA, 2-deoxyribose (Serva, Heidelberg, F.R.G.) and bovine serum albumin (Sigma) served as standards. Enzyme assays GAD was assayed in homogenates obtained from the cells of five 60-mm dishes using [1-~4C]D,L-glutamate (49 Ci/mol; New England Nuclear Corp., Boston, MA, U.S.A.) as substrate, by a known 14CO2 trapping method. 35 For GABA-transaminase (GABAx) determination, homogenates were prepared of cells from five dishes in 0.1 ml 10mM potassium phosphate buffer pH 6.9 [containing pyridoxalphosphate (100/zM), EDTA (1 raM), glycerol (1.3 M) and Triton X-100 (0.13 vol%)]. After centrifugation (I000 g; 15 min) the supernatants were used for enzyme assay according to Scott and Jakoby. 25 Succinic semialdehyde dehydrogenase (from guinea-pig kidney) was added in excess to the incubation mixtures. RESULTS
Growth o f rat embryonic cerebral hemisphere neuronal cells The general appearance of the cultures has previously been d e s c r i b e d ) D u r i n g the first week, the cultures originating from 14-day-old rat embryos consisted essentially of process-bearing cells (Fig, la). These cells have been identified as n e u r o n a l cells by i m m u n o s t a i n i n g against neurofilament proteins. 8 A d d i t i o n o f b F G F to the culture m e d i u m at 4 h stimulated the proliferation o f the n e u r o n a l precursor cells during the first five days, as was d e m o n s t r a t e d by [125I]iododeoxyuridine a n d tritiated thymidine i n c o r p o r a t i o n ) After exposure to b F G F for 48 h, m a n y small r o u n d cells were visible either dispersed or associated into clumps. After six days the cell density was increased c o m p a r e d to controls (Table 1) a n d m o s t cells h a d formed processes a n d a dense fibre network h a d developed (Fig. lb). W h e n b F G F
Basic FGF and GABAergic neuronal development
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Fig. 1. Phase-contrast micrographs of neuronal cells from 14-day-oldfetal rat cerebral hemispheres grown for six days. (a) Control culture. (b, c) Cultures treated with bFGF (5 ng/ml) at 4 h and four days, respectively. Scale bar = 50 #m. was added only at day four after culture initiation, no notable morphological changes occurred; i.e. the bFGF-treated cultures were not different from control cultures (Fig. lc). The overall cell density (Table 1) and the fibre network were very similar after six days in both culture conditions. Moreover, in the presence and the absence of b F G F only a few radiolabelled
cells were visible on the autoradiographs, indicating that the proliferation of the neuronal precursor cells was not stimulated under this condition of treatment (not shown). During the first week, in control as well as in bFGF-treated cultures, only about 2% of the cells exhibited astroglial morphologies and contained glial
J. C. DELOULMEet al.
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Table I. Percentage of glutamate decarboxylase-positive neuronal ceils cultured in the presence of basic fibroblast growth factor Culture Control bFGF added at 4 h bFGF added at four days
Days in culture
No. cells/dish x 10-5
Percentage GAD + cells
6 6 6
9.9 +__0.8 32.5 + 2.5*** 11.0 __+0.4*
64 _ 8 53 + 3* 55 _ 8
Neuronal cells from embryonic rat cerebral hemispheres were grown as described in Experimental Procedures with or without bFGF (5 ng/ml). The proportion of immunoreactive cells was determined by counting the number of labelled cells on immunoperoxidase-stained cultures and comparing them to the number of all cells visible under phase-contrast Total cells and GAD-positive (GAD +) cells were scored by counting 50 fields on each preparation. Values are means + S.D. of four cultures from two independent experiments. Statistical analysis was performed by use of Student's t-test. *P < 0.05, ***P < 0.001 vs control. fibriilary acidic protein (not shown) in agreement with previous results. 9
Influence o f basic fibroblast growth factor on GABA neurons In control a n d b F G F - t r e a t e d cultures m a n y cells were i m m u n o s t a i n e d for G A D (Fig. 2), a m a r k e r for G A B A e r g i c neurons. 19 Intensely a n d more weakly i m m u n o s t a i n e d cells were observed in b o t h culture conditions. T h e i m m u n o r e a c t i v i t y was f o u n d over the cell bodies a n d essentially the proximal part of the processes.
On these i m m u n o s t a i n e d p r e p a r a t i o n s G A D positive cells were counted. The results expressed as percentage of G A D - p o s i t i v e cells per total cell population are summarized in Table 1. After six days, control cultures c o n t a i n e d slightly more t h a n 6 0 % o f G A D - p o s i t i v e cells. In cultures treated with b F G F at 4 h the absolute n u m b e r of G A D - p o s i t i v e cells per culture (9.46 × 105) was greater t h a n in control cultures (6.72 x 105). However, the percentage o f G A D - c o n t a i n i n g cells was reduced to a b o u t 50%. W h e n b F G F was added only at day four, the percentage o f G A D - p o s i t i v e cells was again only a b o u t 10% less t h a n in control cultures.
Fig. 2. GAD immunoreactivity in neuronal cells grown for six days under normal conditions without bFGF. (a) Most cells are immunostained for GAD (Scale bar = 50/~m). (b) Immunoreactivity is restricted to the perikaryon and the proximal part of the neurites (Scale bar = 25 #m).
Basic FGF and GABAergic neuronal development
565
L
I
Fig. 3. Combination of autoradiography after [3H]thymidine incorporation and immunoperoxidase staining with anti-GAD antibody of a six-day-old neuronal cell culture treated with bFGF (5 ng/ml) (Scale bar = 20/~m). Several GAD-positive neurons are also radiolabelled.
The examination of the autoradiographs which were prepared after immunoperoxidase staining indicated the presence of [3H]TdR-labelled GAD-positive cells in control and b F G F - t r e a t e d cultures (Fig. 3). The quantitative evaluation of the autoradiographs demonstrated (Table 2) that in cultures exposed to b F G F at 4 h the percentage of proliferating cells per total cell population had increased about three-fold in the presence of b F G F . The increment was about the same for GAD-positive cells and GAD-negative cells. The stimulation of proliferating GAD-positive cells expressed per total GAD-positive cells was also in the same range. The cellular concentrations of the non-essential (i.e. intracellularly synthesized) amino acids after six days in culture are shown in Table 3. When b F G F
was added to the culture medium 4 h after culture initiation, the concentrations of all amino acids increased, except those of G A B A and /~-alanine. The specific activity of G A D decreased under this condition (Table 3). If b F G F was added to the cell culture only on day four, i.e. at a time when cell proliferation had ceased, the concentration of all non-essential amino acids, including those of G A B A and fl-alanine increased. In this condition the specific activity of G A D was not significantly changed compared to controls. GABAT activity remained unchanged after both b F G F treatments (Table 3). Catecholamines and serotonin were not found in the neuronal cells at the limit of detection of about 0.3 pmol/dish under any culture condition described in this work.
Table 2. Proliferative activity of glutamate decarboxylase-positive and glutamate decarboxylase-negative neuronal cells cultured in the absence or presence of basic fibroblast growth factor Cell
Control culture
bFGF added at 4 h
Fold stimulation
Percentage of cells per total cell population GAD-/3H-TdR + GAD-/3H-TdR GAD+/3H-TdR + GAD+/3H-TdR -
6.5 ___1.9 32.5 ___2.4 4.5 __.0.3 55.7 ___0.9
GAD+/3H-TdR ÷
7.8 ___2.5
17.4 + 1.8 28.3 +__3.2 12.4 +__2.7 42.1 ___1.3
2.7 2.7
Percentage of cells per total GAD + cells 22.6 __+4.0
2.9
Neuronal cells from embryonic rat cerebral hemispheres were grown for four days and exposed to [3H]TdR as described in Experimental Procedures with or without bFGF (5 ng/ml). At least 1000 cells per preparation were counted on autoradiographs after immunoperoxidase staining. Values are means + S.D. of four cultures from two independent experiments. GAD - / +, GAD-negative/positive.
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Table 3. Effect of exposure of rat cerebral hemisphere neurons to basal fibroblast growth factor on the cellular concentration of the non-essential amino acids, and on glutamate decarboxylase and GABA transaminase activity
Control cells ~-Alanine fl-Alanine y-Aminobutyrate (GABA) Aspartate Glutamate Glutamine Glutathion Glycine Serine Threonine
Amino acid concentration (pmol/nmol deoxyribose) Treated cells bFGF added at 4 h bFGF added at four days
12_+ 2 0.6 + 0.2 41 + 2 12__+ 1 73 ± 6 79 ± 8 22+ 11 85 + 8 19+4 37 + 8
22± 7 (183)* 26___4 (217) 0.5 + 0.1 (83) 0.8 + 0.3 (133)** 42___ 1 (102) 59__+ 8 (144)** 19 + 6 (158)* 19 + 3 (158) 97_+ 15 (133)* 108 _+ 18 (148) 111 + 25 (141)* 104 ___12 (132) 53+24 (241)* 4 6 + 8 (209) 102 + 16 (120)* 110___ 9 (129) 27__+8 (142) 27+ 3 (142) 45 + 10 (122) 54 + 15 (146) GAD activity (nmol/h per nmol deoxyribose) 0.025 +_0.0001 0.012 + 0.001 (48)* 0.014 + 0.006 (56)* Activity (nmol/h per nmol deoxyribose) 0.77 + 0.15 0.80 _+ 0.11 0.81 +0.09
Neurons from embryonic rat cerebral hemispheres were exposed to bFGF either at 4 h after culture initiation or at day four. Cells were harvested on day six of cell culture. Mean values + S.D. (n = 4) (percentage of control in parentheses). *Statistically significant difference (P < 0.05) between control cells and cells exposed at 4 h to bFGF. **Statistically significant difference (P < 0.05) between cells exposed at 4 h and at four days to bFGF. Analysis of variance (single factor experiment). Winer B. J. (1971) Statistical Principles in Experimental Design. McGraw Hill, New York. DISCUSSION The GABAergic system has been demonstrated to differentiate relatively early during prenatal development. The first GABAergic cells have been observed on embryonic day 14 in the rat cerebral cortexJ 6'31 In the present study we found that of the cells deriving from 14-day-old cerebral hemispheres, about 64% were G A B A neurons after six days of incubation in serum-free medium. Others have reported the presence of 20-50% of GABAergic neurons in the cultured cell population deriving from cerebrum or hippocampus. L,2,24,34The higher percentage of G A B A neurons obtained under our culture conditions could be due to the fact that these cultures were prepared from brains of younger animals (14-day-old embryos vs 17-21-day-old embryos, or of newborn rats). Noradrenergic, dopaminergic and serotoninergic cells, if present at all, constitute only a very small fraction of the cell population as appears from the virtual absence of the biogenic amines in our cell cultures. This finding is in agreement with the small percentage of noradrenergic ~3 and serotoninergic 36 neurons in cortical structures of the rat embryo brain. The proportion of cholinergic neurons is also small, as has previously been demonstratedfl 9 We assume that in addition to GABAergic neurons, glutamatergic neurons constitute the major cell population under our culture conditions. Our cultures seem, therefore, well suited to study the effect of growth factors on the development of GABAergic neurons. As has been mentioned before, several in vitro and in vivo studies have shown that b F G F elicit trophic effects on cholinergic n e u r o n s 7 4'I°'2L3° A few studies have indicated that b F G F promotes the survival
and development of dopaminergic and GABAergic neurons in culture. 6''° Our report is the first to demonstrate an effect of b F G F on the proliferation of GABAergic neurons and on the cellular concentration of G A B A in cultured neuronal cells. Indeed, b F G F at a concentration of 5 ng/ml enhanced the proliferative activity of the GABAergic neurons, as determined by G A D immunocytochemistry and [3H]TdR autoradiography. After six days in culture, GAD-positive neurons showed [3H]TdR labelling of their nuclei. In the presence of b F G F the percentage of GAD-positive and [aH]TdR-labelled cells was increased by 15%, compared with cells grown in the absence of b F G F . In a previous work, 9 we have shown that b F G F stimulates the proliferation of rat neuronal precursor cells. The present results indicate that among this proliferating population, b F G F affects at least one defined neuronal population, namely the presumptive GABAergic neurons. In contrast, preliminary experiments performed in our laboratory, demonstrate that the few cholinergic neurons present in these cultures are not stimulated to proliferate under the influence of b F G F . Exposure of neuronal cells either at 4 h or on day four after culture initiation caused about the same enhancement of all non-essential amino acids, with the exception of G A B A and fl-alanine (Table 3). Free amino acid pools have previously been determined in primary cultures of neurons, 5'22 but the available data do not allow direct comparison with our data. F r o m the changes of amino acid patterns in fetal and newborn mouse brain, 15 it is apparent that brain development is not accompanied by a general increase of the concentration of the non-essential amino acids: e.g. aspartate and G A B A increase, glycine and serine
Basic FGF and GABAergic neuronal development decrease during development. Thus it is not possible to explain our observations as a general effect of b F G F on cell maturation. Since the proportion of G A B A e r g i c cells decreased by about 10% after exposure of the cells to b F G F , we have to assume that even in the case where cells were exposed to the growth factor at 4 h, a slight increase of G A B A occurred, which compensated for the decrease of G A B A e r g i c cells, so that the average G A B A concentration remained constant. The 40% increase of the G A B A concentration in cells exposed to b F G F at day four (Table 3) indicates a specific effect of the growth factor on G A B A e r g i c cells in the non-proliferating stage. The reason for this enhancement of the G A B A concentration is not known. The decrease of G A D activity excludes enhanced G A B A formation; from the fact that G A B A x activity was not affected by the treatment it seems unlikely that the rate of G A B A degradation was altered. An alternative possibility to explain our finding is the improvement of the storage capability of the cells for G A B A .
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CONCLUSION U n d e r our in vitro conditions the neuronal cultures derived from 14-day-old cerebral hemispheres are rich in GABAergic neurons and their proliferation is stimulated by b F G F ; b F G F has a general effect on the concentration of all non-essential amino acids, both at 4 h and four days after culture initiation, but a specific effect on the G A B A content, if non-proliferating neurons are exposed to the growth factor. These observations raise the question as to whether b F G F also exerts such trophic effects on GABAergic neurons in vivo.
Acknowledgements--We are grateful to Mr B. Kntdgen
for technical assistance. We thank Mrs C. ThomassinOrphanides for excellent secretarial assistance. This work was supported by a grant from the french "Association Franqaise contre les Myopathies". J. C. Deloulme is a recipient of a fellowship from the "Association Franqaise contre les Myopathies".
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20, Nanopoulos D., Belin M. F., Maitre M., Vincendon G. and Pujol J. F. (1982) Immunocytochemical evidence for the existence of GABAergic neurons in the nucleus raphe dorsalis. Possible existence of neurons containing serotonin and GABA. Brain Res. 232, 375-389. 21, Otto D,, Frotscher M. and Unsicker K. (1989) Basic fibroblast growth factor and nerve growth factor administered in gel foam rescue medial septal neurons after fimbria fornix transection. J. Neurosci. Res. 22, 83-91. 22. Patel A. J. and Hunt H. (1985) Concentration of free amino acids in primary cultures of neurons and astrocytes. J. Neurochem. 44, 1816-1821. 23, Pettmann B., Weibel M., Sensenbrenner M. and Labourdette G. (1985) Purification of two astroglial growth factors from bovine brain. Fedn Eur. biochem. Socs Lett. 189, 102-108. 24. Robain O., Barbin G., Ben-Ari Y., Rozenberg F. and Prochiantz A. (1987) GABAergic neurons of the hippocampus: development in homotopic grafts and in dissociated cell cultures. Neuroscience 23, 73~86. 25. Scott E. M. and Jakoby W. B. (1959) Soluble 7-aminobutyric-glutamic transaminase from Pseudomonasfluorescens. J. biol. Chem. 234, 932-936. 26. Seiler N. and Kn6dgen B. (1985) Determination of amino acids by separation of their ion pairs with dodecylsulphate. J. Chromatogr. 341, 11-21. 27. Seiler N. and Schmidt-Glenewinkel T. (1975) Regional distribution of putrescine, spermidine and spermine in relation to the distribution of RNA and DNA in the rat nervous system. ,L Neurochem. 24, 791-795. 28. Tappaz M. L., Wassef M., Oertel W. H., Paut L. and Pujol J. F. (1983) Light- and electron-microscopic immunocytochemistry of glutamic acid decarboxylase (GAD) in the basal hypothalamus: morphological evidence for neuroendocrine ~,-aminobutyrate (GABA). Neuroscience 9, 271-287. 29. Thomas W. E. (1985) Morphology of acetylcholinesterase-containing neurons in primary cultures of dissociated rat cerebral cortex. Brain Res. 361, 392-395. 30. Unsicker K., Reichert-Preibsch H., Schmidt R., Pettmann B., Labourdette G. and Sensenbrenner M. (1987) Astroglial and fibroblast growth factors have neuronotrophic functions for cultured peripheral and central nervous system neurons. Proc. natn. Acad. Sci. U.S.A. 84, 5459-5463. 31. Van Eden C. G., Mrzljak L., Voorn P. and Uylings H. B. M. (1989) Prenatal development of GABA-ergic neurons in the neocortex of the rat. J. comp. Neurol. 289, 213-227. 32. Walicke P. A. (1988) Basic and acidic fibroblast growth factors have trophic effects on neurons from multiple CNS regions~ J. Neurosci. 8, 2618-2627. 33. Walicke P., Cowan W. M., Ueno N., Baird A. and Guillemin R. (1986) Fibroblast growth factor promotes survival of dissociated hippocampal neurons and enhances neurite extension. Proc. natn. Acad. Sci. U.S.A. 83, 3012-3016. 34. White W. F., Snodgrass S. R. and Dichter M. (1980) Identification of GABA neurons in rat cortical cultures by GABA uptake autoradiography. Brain Res. 190, 139-152. 35. Yamaguchi T. and Matsumura Y. (1977) Purification of L-glutamate decarboxylase by affinity chromatography. Biochem. Biophys. Acta 481, 706-711. 36. Yamamoto M., Steinbusch H. W. M. and Jessell T. M. (1981) Differentiated properties of identified serotonin neurons in dissociated cultures of embryonic rat brain stem. J. Cell Biol. 91, 142-152. (Accepted 15 November 1990)
0306-4522/91 $3.00 + 0.00 Pergamon Press plc © 1991 IBRO
EFFECTS OF BASIC FIBROBLAST GROWTH FACTOR ON THE DEVELOPMENT OF GABAERGIC N E U R O N S IN CULTURE J. C. DELOULME,* C. GENSBURGER,* S. SARHAN,~ N. SEILER~ and M. SENSENBRENNER*~ *Centre de Neurochimie du CNRS and INSERM U44, 5 rue Blaise Pascal, 67084 Strasbourg Cedex, France tMerrell Dow Research Institute, Strasbourg Center, 16 rue d'Ankara, 67084 Strasbourg Cedex, France Abstract--Six-day-old neuronal cultures derived from 14-day-old embryonic rat cerebral hemispheres were highly enriched in GABAergic neurons, as was demonstrated by immunocytochemistry using an anti-glutamate decarboxylase antiserum. They contained about 64% glutamate decarboxylase-positive neurons. About 8% of these neurons proliferated, as shown by a combination of glutamate decarboxylase immunocytochemistry and [3H]thymidine incorporation into cell nuclei. The proliferative activity of GABAergic precursor cells and changes in the cellular concentrations of the non-essential amino acids, including GABA under the effect of basic fibroblast growth factor were studied. When basic fibroblast growth factor was added to the cultures 4 h after seeding, the proliferation of the GABAergic neurons was stimulated about threefold. Under this culture condition, the concentration per cell of all amino acids increased, except those of GABA and fl-alanine. When basic fibroblast growth factor was added to cultures only on day four, the proliferation of the neuronal cells was no more enhanced. Under this condition of treatment, the concentrations of all non-essential amino acids, including those of GABA and fl-alanine were enhanced. Under both basic fibroblast growth factor treatments the concentration of GABA per GABAergic cell was increased. In contrast, the specific activity of glutamate decarboxylase was not stimulated under these conditions. We hypothesize that under the effect of basic fibroblast growth factor the capabilities of the cells to store GABA are improved.
Recent studies have shown that fibroblast growth factors (FGFs) have neurotrophic activity for neurons of the peripheral and central nervous system. Both acidic F G F ( a F G F ) and basic F G F ( b F G F ) support the survival and enhance the outgrowth of neurites of rat neurons from various brain regions, 6'11'17'18'32'33 and stimulate the proliferation of neuroblasts from cerebral hemispheres of 13-day-old rat embryos. 7,9 Basic F G F , but not a F G F , promotes the survival of chick ciliary ganglion neurons. 3° Little is known about the effect of F G F s on the expression o f neurotransmitter-related properties in cultured neurons. We have found that b F G F stimulates choline acetyltransferase activity in cultured chick ciliary ganglion n e u r o n s ) ° M o r e recently, Grothe et al. l° observed that b F G F promoted cholinergic development of rat septal neurons in culture. Other authors have mentioned that b F G F stimulated the uptake of G A B A and dopamine into G A B A e r g i c and dopaminergic neurons, respectively? Finally, intracerebroventricular injections of b F G F increased :~To whom correspondence should be addressed. Abbreviations: aFGF, acidic FGF; bFGF, basic FGF;
DMEM, Dulbecco's modified Eagle's medium; EDTA, ethylenediaminetetra-acetate; FCS, fetal calf serum; FGF(s), fibroblast growth factor(s); GABAr, GABA transaminase; GAD, glutamate decarboxylase; [3H]TdR, [methyl-3H]thymidine; HPLC, high-performance liquid chromatography; PBS, phosphate-buffered saline. 561
choline acetyltransferase activity in adult rats with partial septohippocampal lesions. 3 In this paper we have investigated the effect of b F G F on the in vitro development of neurons in dissociated cerebral hemisphere cultures from fetal rats under two conditions: (i) addition of b F G F to the culture medium 4 h after culture initiation; i.e. at a time when the neurons proliferate; (ii) exposure of neurons to b F G F only after four days of incubation; i.e. at a time when cell proliferation had essentially ceased. EXPERIMENTAL PROCEDURES
Neuronal cell cultures
Primary cultures of neuronal cells from cerebral hemispheres of 14-day-old Wistar rat embryos were prepared as previously described. 8 Briefly, cerebral hemispheres were dissected and cleaned of their meningeal membranes. The hemispheres were mechanically dissociated either with a 20-ml syringe by repeated passages through a needle of 1 mm i.d. or by filtration through a nylon sieve (mesh 48 #m). The cells were collected in Dulbecco's modified Eagle's H 16 medium (DMEM; Gibco) containing 20% fetal calf serum (FCS; Gibco) and antibiotics. The dissociated cells were cultured either in 35- or 60-mm Falcon Petri dishes precoated with poly-L-lysine (5 x 105 cells/ml). After 2 h, the cultures were switched to a serum-free, chemically defined medium: DMEM plus 5/zg/ml bovine insulin (Sigma, 1-5500), 50 #g/ml human transferrin (Sigma, T-2252), 20 nM progesterone (Sigma, P-0130), 100 # M putrescine (Sigma, P-7505), 30 nM sodium selenite (Merck) and antibiotics. Cultures
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were re-fed at four days with fresh medium and were incubated at 37°C in a 5% CO2-containing humidified atmosphere. Cultures were grown for a maximum of six days. Cultures were treated once, either at 4 h or four days after culture initiation with bFGF (5 ng/ml). The half-life of bFGF is about 20 h and after 48 h the amount of factor in the culture medium is low, so that no more biological activity is detected against astrocytes (Labourdette and Taupenot, unpublished observations). Therefore, under both conditions of treatment the exposure time to bFGF of the cultures was the same. Basic FGF was prepared in our laboratory from bovine brain by using heparin-Sepharose affinity chromatography, as was described previously. 23 Incubation of the cultures was continued until day six. lmmunoc y t ochemis try At different times, cultures were rapidly rinsed three times with phosphate-buffered saline (PBS) and fixed with 4% paraformaldehyde for 15 min, then postfixed with 30% methanol for 5 min at room temperature. After washing with PBS, the cultures were incubated for 2 h at room temperature or overnight at 4°C with rabbit anti-glutamate decarboxylase (-GAD) antiserum (diluted l : 200) containing 10% normal sheep serum, or with sheep anti-GAD antiserum (diluted 1 : 500) containing 10% normal rabbit serum, respectively. After washing with PBS, the preparations were incubated with 1% sheep anti-rabbit IgG or 0.2% donkey anti-sheep IgG conjugated with peroxidase (Institut Pasteur, Paris) for I h. After three washings with PBS, the preparations were treated for 5 min with a mixture containing 0.05% 3,Y-diaminobenzidine tetrahydrochloride and 0.01% hydrogen peroxide in PBS, washed with PBS, mounted with Kaiser's glycerol gelatin (Merck) and examined under a light microscope. Controls were performed with the corresponding normal serum which was diluted to the same extent as the specific antisera. Rabbit anti-GAD and sheep anti-GAD antisera were generous gifts of Dr M. Maitre (Centre de Neurochimie du CNRS, Strasbourg, France) and Dr M. L. Tappaz (INSERM U 171, Lyon, France), respectively. The characteristics of the antisera used have previously been described. 2°'28 Autoradiography At 24 h after initiation, cultures were exposed for 24 h to medium containing 0.05/iCi/ml of [methyl-3H]thymidine ([3H]TdR) (25 Ci/mmol; CEA, France). The radioactive medium was removed, cultures were rinsed five times with DMEM and supplied with fresh medium. After four days, cultures were submitted to immunoperoxidase staining as described above, rimed with distilled water and air-dried. The preparations were then submitted to an autoradiographic procedure. ~4 The autoradiographs were developed after 10 days of exposure in the dark at 4°C. Determination of amino acids After careful washing of the cultures with PBS, the cells were harvested with a rubber policeman, and the pellet obtained from three dishes was homogenized by sonication (Kontes, Micro-ultrasonic cell disrupter) with 0.6 ml of 0.2 M perchloric acid. After 1 h standing at +2°C, the acidinsoluble material was precipitated by centrifugation. The supernatants were diluted with 9 vol of 0.05 M perchloric acid. Aliquots (200 ~ 1) of the diluted perchloric acid extracts were applied on a reversed-phase column (Beckman Ultrasphere I.P.). Separation of the (non-essential) amino acids was accomplished by isocratic elution (flow rate 1 ml/min at 20°C) with a buffer containing 630 ml 0.2 M phosphoric acid, 270 ml 0.2% sodium acetate (pH 4.5), 100 ml of methanol, and 2.59 g sodium dodecylsulphate (Merck). After reaction of the column effluent with the same volume of o-phthalaldehyde,2-mercaptoethanol reagent, fluorescence intensity was
continuously recorded at 455 nm (excitation at 345 nm) (for details of the method, see Ref. 26). Determination of catecholamines and serotonin The cells harvested per dish were suspended in 600/~1 0.2 M perchloric acid (containing 250 mg sodium bisulphite and 100 mg EDTA/1), sonicated (2 × 30 s) and centrifuged. The supernatant (200#1) was separated by HPLC (isocratic elution mode). Column: Beckman Ultrasphere I.P. (250 mm in length, 4.6mm i.d.). Eluent: 800ml 0.1 M potassium acetate (pH 4.25 containing 2.16 g n-octanesulphonate and 100 mg EDTA/I) + 200 ml methanol; flow rate 1 ml/min (30°C). Detector: Coulochem Model 5100 A (Environmental Sciences Ass. Bedford, MA, U.S.A.) with a dual analytical cell (Model 5011). The coulometric cell was used as screen electrode (at 0.08 V), the amperometric cell as detector electrode (at 0.35V; working potentials relative to the reference electrode). The instrument was set at a sensitivity to allow the determination of 0.1 pmol noradrenaline, dopamine and serotonin per injection volume (signal-to-noise ratio > 5). Determination of protein, RNA and DNA These were determined in the precipitate of the perchloric acid homogenate (see above). For the separation of RNA, DNA and proteins, a modified Schmidt-Thannhauser procedure was used which was adapted to small samples. RNA was determined spectrophotometrically at 260 nm, DNA by a version of Burton's diphenylamine reaction, and proteins by a modified Lowry method (for a detailed description of the procedure, see Ref. 27). Yeast RNA, 2-deoxyribose (Serva, Heidelberg, F.R.G.) and bovine serum albumin (Sigma) served as standards. Enzyme assays GAD was assayed in homogenates obtained from the cells of five 60-mm dishes using [1-~4C]D,L-glutamate (49 Ci/mol; New England Nuclear Corp., Boston, MA, U.S.A.) as substrate, by a known 14CO2 trapping method. 35 For GABA-transaminase (GABAx) determination, homogenates were prepared of cells from five dishes in 0.1 ml 10mM potassium phosphate buffer pH 6.9 [containing pyridoxalphosphate (100/zM), EDTA (1 raM), glycerol (1.3 M) and Triton X-100 (0.13 vol%)]. After centrifugation (I000 g; 15 min) the supernatants were used for enzyme assay according to Scott and Jakoby. 25 Succinic semialdehyde dehydrogenase (from guinea-pig kidney) was added in excess to the incubation mixtures. RESULTS
Growth o f rat embryonic cerebral hemisphere neuronal cells The general appearance of the cultures has previously been d e s c r i b e d ) D u r i n g the first week, the cultures originating from 14-day-old rat embryos consisted essentially of process-bearing cells (Fig, la). These cells have been identified as n e u r o n a l cells by i m m u n o s t a i n i n g against neurofilament proteins. 8 A d d i t i o n o f b F G F to the culture m e d i u m at 4 h stimulated the proliferation o f the n e u r o n a l precursor cells during the first five days, as was d e m o n s t r a t e d by [125I]iododeoxyuridine a n d tritiated thymidine i n c o r p o r a t i o n ) After exposure to b F G F for 48 h, m a n y small r o u n d cells were visible either dispersed or associated into clumps. After six days the cell density was increased c o m p a r e d to controls (Table 1) a n d m o s t cells h a d formed processes a n d a dense fibre network h a d developed (Fig. lb). W h e n b F G F
Basic FGF and GABAergic neuronal development
563
Fig. 1. Phase-contrast micrographs of neuronal cells from 14-day-oldfetal rat cerebral hemispheres grown for six days. (a) Control culture. (b, c) Cultures treated with bFGF (5 ng/ml) at 4 h and four days, respectively. Scale bar = 50 #m. was added only at day four after culture initiation, no notable morphological changes occurred; i.e. the bFGF-treated cultures were not different from control cultures (Fig. lc). The overall cell density (Table 1) and the fibre network were very similar after six days in both culture conditions. Moreover, in the presence and the absence of b F G F only a few radiolabelled
cells were visible on the autoradiographs, indicating that the proliferation of the neuronal precursor cells was not stimulated under this condition of treatment (not shown). During the first week, in control as well as in bFGF-treated cultures, only about 2% of the cells exhibited astroglial morphologies and contained glial
J. C. DELOULMEet al.
564
Table I. Percentage of glutamate decarboxylase-positive neuronal ceils cultured in the presence of basic fibroblast growth factor Culture Control bFGF added at 4 h bFGF added at four days
Days in culture
No. cells/dish x 10-5
Percentage GAD + cells
6 6 6
9.9 +__0.8 32.5 + 2.5*** 11.0 __+0.4*
64 _ 8 53 + 3* 55 _ 8
Neuronal cells from embryonic rat cerebral hemispheres were grown as described in Experimental Procedures with or without bFGF (5 ng/ml). The proportion of immunoreactive cells was determined by counting the number of labelled cells on immunoperoxidase-stained cultures and comparing them to the number of all cells visible under phase-contrast Total cells and GAD-positive (GAD +) cells were scored by counting 50 fields on each preparation. Values are means + S.D. of four cultures from two independent experiments. Statistical analysis was performed by use of Student's t-test. *P < 0.05, ***P < 0.001 vs control. fibriilary acidic protein (not shown) in agreement with previous results. 9
Influence o f basic fibroblast growth factor on GABA neurons In control a n d b F G F - t r e a t e d cultures m a n y cells were i m m u n o s t a i n e d for G A D (Fig. 2), a m a r k e r for G A B A e r g i c neurons. 19 Intensely a n d more weakly i m m u n o s t a i n e d cells were observed in b o t h culture conditions. T h e i m m u n o r e a c t i v i t y was f o u n d over the cell bodies a n d essentially the proximal part of the processes.
On these i m m u n o s t a i n e d p r e p a r a t i o n s G A D positive cells were counted. The results expressed as percentage of G A D - p o s i t i v e cells per total cell population are summarized in Table 1. After six days, control cultures c o n t a i n e d slightly more t h a n 6 0 % o f G A D - p o s i t i v e cells. In cultures treated with b F G F at 4 h the absolute n u m b e r of G A D - p o s i t i v e cells per culture (9.46 × 105) was greater t h a n in control cultures (6.72 x 105). However, the percentage o f G A D - c o n t a i n i n g cells was reduced to a b o u t 50%. W h e n b F G F was added only at day four, the percentage o f G A D - p o s i t i v e cells was again only a b o u t 10% less t h a n in control cultures.
Fig. 2. GAD immunoreactivity in neuronal cells grown for six days under normal conditions without bFGF. (a) Most cells are immunostained for GAD (Scale bar = 50/~m). (b) Immunoreactivity is restricted to the perikaryon and the proximal part of the neurites (Scale bar = 25 #m).
Basic FGF and GABAergic neuronal development
565
L
I
Fig. 3. Combination of autoradiography after [3H]thymidine incorporation and immunoperoxidase staining with anti-GAD antibody of a six-day-old neuronal cell culture treated with bFGF (5 ng/ml) (Scale bar = 20/~m). Several GAD-positive neurons are also radiolabelled.
The examination of the autoradiographs which were prepared after immunoperoxidase staining indicated the presence of [3H]TdR-labelled GAD-positive cells in control and b F G F - t r e a t e d cultures (Fig. 3). The quantitative evaluation of the autoradiographs demonstrated (Table 2) that in cultures exposed to b F G F at 4 h the percentage of proliferating cells per total cell population had increased about three-fold in the presence of b F G F . The increment was about the same for GAD-positive cells and GAD-negative cells. The stimulation of proliferating GAD-positive cells expressed per total GAD-positive cells was also in the same range. The cellular concentrations of the non-essential (i.e. intracellularly synthesized) amino acids after six days in culture are shown in Table 3. When b F G F
was added to the culture medium 4 h after culture initiation, the concentrations of all amino acids increased, except those of G A B A and /~-alanine. The specific activity of G A D decreased under this condition (Table 3). If b F G F was added to the cell culture only on day four, i.e. at a time when cell proliferation had ceased, the concentration of all non-essential amino acids, including those of G A B A and fl-alanine increased. In this condition the specific activity of G A D was not significantly changed compared to controls. GABAT activity remained unchanged after both b F G F treatments (Table 3). Catecholamines and serotonin were not found in the neuronal cells at the limit of detection of about 0.3 pmol/dish under any culture condition described in this work.
Table 2. Proliferative activity of glutamate decarboxylase-positive and glutamate decarboxylase-negative neuronal cells cultured in the absence or presence of basic fibroblast growth factor Cell
Control culture
bFGF added at 4 h
Fold stimulation
Percentage of cells per total cell population GAD-/3H-TdR + GAD-/3H-TdR GAD+/3H-TdR + GAD+/3H-TdR -
6.5 ___1.9 32.5 ___2.4 4.5 __.0.3 55.7 ___0.9
GAD+/3H-TdR ÷
7.8 ___2.5
17.4 + 1.8 28.3 +__3.2 12.4 +__2.7 42.1 ___1.3
2.7 2.7
Percentage of cells per total GAD + cells 22.6 __+4.0
2.9
Neuronal cells from embryonic rat cerebral hemispheres were grown for four days and exposed to [3H]TdR as described in Experimental Procedures with or without bFGF (5 ng/ml). At least 1000 cells per preparation were counted on autoradiographs after immunoperoxidase staining. Values are means + S.D. of four cultures from two independent experiments. GAD - / +, GAD-negative/positive.
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Table 3. Effect of exposure of rat cerebral hemisphere neurons to basal fibroblast growth factor on the cellular concentration of the non-essential amino acids, and on glutamate decarboxylase and GABA transaminase activity
Control cells ~-Alanine fl-Alanine y-Aminobutyrate (GABA) Aspartate Glutamate Glutamine Glutathion Glycine Serine Threonine
Amino acid concentration (pmol/nmol deoxyribose) Treated cells bFGF added at 4 h bFGF added at four days
12_+ 2 0.6 + 0.2 41 + 2 12__+ 1 73 ± 6 79 ± 8 22+ 11 85 + 8 19+4 37 + 8
22± 7 (183)* 26___4 (217) 0.5 + 0.1 (83) 0.8 + 0.3 (133)** 42___ 1 (102) 59__+ 8 (144)** 19 + 6 (158)* 19 + 3 (158) 97_+ 15 (133)* 108 _+ 18 (148) 111 + 25 (141)* 104 ___12 (132) 53+24 (241)* 4 6 + 8 (209) 102 + 16 (120)* 110___ 9 (129) 27__+8 (142) 27+ 3 (142) 45 + 10 (122) 54 + 15 (146) GAD activity (nmol/h per nmol deoxyribose) 0.025 +_0.0001 0.012 + 0.001 (48)* 0.014 + 0.006 (56)* Activity (nmol/h per nmol deoxyribose) 0.77 + 0.15 0.80 _+ 0.11 0.81 +0.09
Neurons from embryonic rat cerebral hemispheres were exposed to bFGF either at 4 h after culture initiation or at day four. Cells were harvested on day six of cell culture. Mean values + S.D. (n = 4) (percentage of control in parentheses). *Statistically significant difference (P < 0.05) between control cells and cells exposed at 4 h to bFGF. **Statistically significant difference (P < 0.05) between cells exposed at 4 h and at four days to bFGF. Analysis of variance (single factor experiment). Winer B. J. (1971) Statistical Principles in Experimental Design. McGraw Hill, New York. DISCUSSION The GABAergic system has been demonstrated to differentiate relatively early during prenatal development. The first GABAergic cells have been observed on embryonic day 14 in the rat cerebral cortexJ 6'31 In the present study we found that of the cells deriving from 14-day-old cerebral hemispheres, about 64% were G A B A neurons after six days of incubation in serum-free medium. Others have reported the presence of 20-50% of GABAergic neurons in the cultured cell population deriving from cerebrum or hippocampus. L,2,24,34The higher percentage of G A B A neurons obtained under our culture conditions could be due to the fact that these cultures were prepared from brains of younger animals (14-day-old embryos vs 17-21-day-old embryos, or of newborn rats). Noradrenergic, dopaminergic and serotoninergic cells, if present at all, constitute only a very small fraction of the cell population as appears from the virtual absence of the biogenic amines in our cell cultures. This finding is in agreement with the small percentage of noradrenergic ~3 and serotoninergic 36 neurons in cortical structures of the rat embryo brain. The proportion of cholinergic neurons is also small, as has previously been demonstratedfl 9 We assume that in addition to GABAergic neurons, glutamatergic neurons constitute the major cell population under our culture conditions. Our cultures seem, therefore, well suited to study the effect of growth factors on the development of GABAergic neurons. As has been mentioned before, several in vitro and in vivo studies have shown that b F G F elicit trophic effects on cholinergic n e u r o n s 7 4'I°'2L3° A few studies have indicated that b F G F promotes the survival
and development of dopaminergic and GABAergic neurons in culture. 6''° Our report is the first to demonstrate an effect of b F G F on the proliferation of GABAergic neurons and on the cellular concentration of G A B A in cultured neuronal cells. Indeed, b F G F at a concentration of 5 ng/ml enhanced the proliferative activity of the GABAergic neurons, as determined by G A D immunocytochemistry and [3H]TdR autoradiography. After six days in culture, GAD-positive neurons showed [3H]TdR labelling of their nuclei. In the presence of b F G F the percentage of GAD-positive and [aH]TdR-labelled cells was increased by 15%, compared with cells grown in the absence of b F G F . In a previous work, 9 we have shown that b F G F stimulates the proliferation of rat neuronal precursor cells. The present results indicate that among this proliferating population, b F G F affects at least one defined neuronal population, namely the presumptive GABAergic neurons. In contrast, preliminary experiments performed in our laboratory, demonstrate that the few cholinergic neurons present in these cultures are not stimulated to proliferate under the influence of b F G F . Exposure of neuronal cells either at 4 h or on day four after culture initiation caused about the same enhancement of all non-essential amino acids, with the exception of G A B A and fl-alanine (Table 3). Free amino acid pools have previously been determined in primary cultures of neurons, 5'22 but the available data do not allow direct comparison with our data. F r o m the changes of amino acid patterns in fetal and newborn mouse brain, 15 it is apparent that brain development is not accompanied by a general increase of the concentration of the non-essential amino acids: e.g. aspartate and G A B A increase, glycine and serine
Basic FGF and GABAergic neuronal development decrease during development. Thus it is not possible to explain our observations as a general effect of b F G F on cell maturation. Since the proportion of G A B A e r g i c cells decreased by about 10% after exposure of the cells to b F G F , we have to assume that even in the case where cells were exposed to the growth factor at 4 h, a slight increase of G A B A occurred, which compensated for the decrease of G A B A e r g i c cells, so that the average G A B A concentration remained constant. The 40% increase of the G A B A concentration in cells exposed to b F G F at day four (Table 3) indicates a specific effect of the growth factor on G A B A e r g i c cells in the non-proliferating stage. The reason for this enhancement of the G A B A concentration is not known. The decrease of G A D activity excludes enhanced G A B A formation; from the fact that G A B A x activity was not affected by the treatment it seems unlikely that the rate of G A B A degradation was altered. An alternative possibility to explain our finding is the improvement of the storage capability of the cells for G A B A .
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CONCLUSION U n d e r our in vitro conditions the neuronal cultures derived from 14-day-old cerebral hemispheres are rich in GABAergic neurons and their proliferation is stimulated by b F G F ; b F G F has a general effect on the concentration of all non-essential amino acids, both at 4 h and four days after culture initiation, but a specific effect on the G A B A content, if non-proliferating neurons are exposed to the growth factor. These observations raise the question as to whether b F G F also exerts such trophic effects on GABAergic neurons in vivo.
Acknowledgements--We are grateful to Mr B. Kntdgen
for technical assistance. We thank Mrs C. ThomassinOrphanides for excellent secretarial assistance. This work was supported by a grant from the french "Association Franqaise contre les Myopathies". J. C. Deloulme is a recipient of a fellowship from the "Association Franqaise contre les Myopathies".
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