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Originally published In Press as doi:10.1074/jbc.M202927200 on May 28, 2002

J. Biol. Chem., Vol. 277, Issue 32, 28861-28869, August 9, 2002
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Basic Fibroblast Growth Factor Evokes a Rapid Glutamate Release through Activation of the MAPK Pathway in Cultured Cortical Neurons*

Tadahiro NumakawaDagger §, Daisaku YokomakuDagger , Kazuyuki Kiyosue, Naoki AdachiDagger , Tomoya MatsumotoDagger , Yumiko NumakawaDagger , Takahisa Taguchi, Hiroshi HatanakaDagger , and Masashi YamadaDagger

From the Dagger  Division of Protein Biosynthesis, Institute for Protein Research, Osaka University, Suita, Osaka 565-0871, Japan, and the  Neuronics R. G. Division for Human Life Technology National Institute of Advanced Industrial Science and Technology (AIST) Midorigaoka, Ikeda, Osaka 563-8577, Japan

Received for publication, March 26, 2002

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

We examined the possibility that basic fibroblast growth factor (bFGF) is involved in synaptic transmissions. We found that bFGF rapidly induced the release of glutamate and an increase in the intracellular Ca2+ concentration through voltage-dependent Ca2+ channels in cultured cerebral cortical neurons. bFGF also evoked a significant influx of Na+. Tetanustoxin inhibited the bFGF-induced glutamate release, revealing that bFGF triggered exocytosis. The mitogen-activated protein kinase (MAPK) pathway was required for these acute effects of bFGF. We also found that pretreatment with bFGF significantly enhanced high K+-elicited glutamate release also in a MAPK activation-dependent manner. Therefore, we propose that bFGF exerts promoting effects on excitatory neuronal transmission via activation of the MAPK pathway.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Neuronal transmissions require mechanisms that modulate the balanced interaction of multiple factors, which control functional maintenance and plasticity in the central nervous system. Many factors including growth factors and the excitatory amino acid glutamate control neuronal functions. In particular, brain-derived neurotrophic factor (BDNF,1 one of the neurotrophins) plays a fundamental role in neuronal transmission and plasticity (1-7). We also reported that rapid glutamate release was induced by BDNF through the glutamate transporter in cultured cerebellar and cortical neurons (8, 9). However, very little work has been done concerning the roles of other neurotrophic factors in the acute modulation of central nervous system and peripheral nervous system functions (10, 11).

Initially, bFGF was identified as an angiogenic mitogen, but it is now recognized as a neurotrophic factor. For example, it has been found that bFGF exerts neurotrophic activities in cortical and hippocampal neurons (12, 13). However, the expression of bFGF in the central nervous system increases during postnatal development (14), implying that bFGF also has important roles in synaptic maturation. Indeed, bFGF modulates the efficacy of hippocampal synaptic transmission. bFGF enhances the generation of long term potentiation in hippocampal neurons (15, 16). These findings suggest that bFGF is involved in glutamatergic transmission, because it is widely accepted that the release of glutamate (presynaptic), as well as the NMDA and the AMPA receptor (postsynaptic), is essential for long term potentiation (17, 18). Several reports have indicated that these ionotropic glutamate receptor activities were regulated by bFGF. bFGF regulates the intracellular Ca2+ concentration after activation of the NMDA receptor (19). Long term treatment with bFGF decreases the NMDA receptor-dependent increase in the Ca2+ concentration, and the Ca2+ attenuation is required for the neuroprotective effects in excitotoxic cell death (20, 21). The expression of the glutamate receptor protein is also changed by bFGF. bFGF increased the AMPA receptor subunit GluR1 protein but did not alter levels of GluR2/3, GluR4, or the NMDA subunit NR1 (22). The expression of NMDA receptor protein and calbindin D28 (Ca2+-binding protein) was down-regulated by bFGF (23). These results raise the possibility that chronic bFGF treatment regulates not only neuronal survival but also postsynaptic plasticity in glutamatergic neurons. However, it is not known what kind of acute effects bFGF exerts on glutamatergic transmission and how bFGF stimulates neuronal transmission.

In the present study, we found that bFGF induced glutamate release in cultured cortical neurons. It was revealed that bFGF triggers the release through the exocytotic system in an extracellular Ca2+- and MAPK activation-dependent manner. We propose that not only neurotrophins but also bFGF exerts an acute effect on the excitability of glutamatergic neurons.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Cell Preparation-- Primary dissociated cortical cultures were prepared from the cerebral cortex of 2- or 3-day-old rats (SLC, Sizuoka, Japan) as reported previously (24, 25). Briefly, cells were gently dissociated with a plastic pipette after digestion with papain (90 units/ml, Worthington) at 37 °C. The dissociated cells were plated at a final density of 4 or 5 × 105/cm2 on polyethyleneimine-coated 12- and 24-well plates (4- and 2-cm2 surface area/well, respectively; Corning). The culture medium consisted of 5% precolostrum newborn calf serum, 5% heated-inactivated horse serum, 1% rat serum, and 89% of a 1:1 mixture of Dulbecco's modified Eagle's medium and Ham's F-12 medium containing 15 mM HEPES buffer, pH 7.4, 30 nM Na2SeO3, and 1.9 mg/ml NaHCO3. After maintenance for 6-8 days, the analysis was performed. Hippocampal and cerebellar cells were prepared from 2- to 3-day-old hippocampus and 5-day-old cerebellum, respectively. The procedures for these cultures were the same as for the cortical culture. These cultures were maintained for 9-12 days.

Immunocytochemistry-- Cells were double stained with anti-MAP2 (rabbit IgG; a gift from Dr. H. Murofushi, The University of Tokyo) and anti-c-Fos antibodies (rabbit IgG; Santa Cruz Biotechnology, Inc., Santa Cruz, CA). First, cultured cells were fixed in 4% paraformaldehyde containing 0.05% Triton X-100 at room temperature (25 °C) for 20 min and then incubated overnight with anti-MAP2 (1:5000). Secondary antibodies were applied at room temperature for 1 h. To visualize the staining, a Vectastain ABC kit (Vector Laboratories) together with 0.02% (w/v) 3,3'-diaminobenzidine·4HCl (DAB) dissolved in 0.05 M Tris-HCl buffer, pH 7.6, containing 0.01% (v/v) H2O2 was used. Next, cells were incubated with the anti-c-Fos (1:1000) antibody and visualized using DAB together with 0.1% (w/v) (NH4)Ni(SO4)2.

Detection of Amino Acid Neurotransmitters-- The amounts of amino acids released from the cultured neurons were measured as described previously (25). Briefly, the amounts released into the assay buffer (modified HEPES-buffered Krebs Ringer solution: KRH containing 130 mM NaCl, 5 mM KCl, 1.2 mM NaH2PO4, 1.8 mM CaCl2, 10 mM glucose, 1% bovine serum albumin, and 25 mM HEPES, pH 7.4) were measured by HPLC (Shimazu, Kyoto, Japan) with a fluorescence detector (Shimazu). The high K+ (HK+) solution consisted of 85 mM NaCl, 50 mM KCl, 1.2 mM NaH2PO4, 1.8 mM CaCl2, 10 mM glucose, 1% bovine serum albumin, and 25 mM HEPES, pH 7.4. The Ca2+-free solution was prepared by omitting the CaCl2. Sample fractions were collected every 1 min into tubes on ice by the batch method. They were then filtered with 0.22-µm membranes to remove cell debris. Samples were treated with o-phthalaldehyde and 2-mercaptoethanol for 5 min at 12 °C before being injected into the HPLC system and analyzed using a fluorescence monitor (excitation wavelength, 340 nm; emission wavelength, 445 nm). bFGF and BDNF dissolved in phosphate-buffered saline containing bovine serum albumin (BSA, 1 mg/ml) were added to cultured neurons by bath application. PD98059 (Calbiochem) and U0126 (Promega, Madison, WI), MAPK pathway inhibitors, were applied for 40 min at 50 and 10 µM, respectively. LY294002 (Calbiochem), a PI 3-kinase inhibitor, was applied for 30 min at 30 µM. Nifedipine (Research Biochemicals International, Natick, MA) and tetrodotoxin (TTX, as latoxan, Valence, France) were applied at 10 and 0.5 µM, respectively. Thapsigargin (1 µM) (Research Biochemicals) was added 30 min prior to the application of bFGF. Tetanustoxin (10 nM, List Biological Laboratories, Inc., Campbell, CA) was applied to the culture for 8-12 h before bFGF. L-trans-2,4-PDC (t-PDC) (Research Biochemicals) and DL-threo-beta -benzyloxyaspartate (DL-TBOA) (a gift from Suntory Institute for Bioorganic Research, Osaka, Japan) were applied at 10 µM. The glutamate release experiments were performed at least four times with independent cultures to confirm reproducibility. Representative data are shown in the figures.

Ca2+, Na+, and FM1-43 Imaging-- Cells were cultured on polyethyleneimine-coated glasses (Matsunami, Osaka, Japan) attached to flexiperm (In Vitro, Kalkberg, Germany). After a wash, cells were incubated for 1 h at 37 °C with 10 µM Fluo-3-AM (Molecular Probes, Eugene, OR) diluted in KRH. The dye intensity was monitored using a confocal laser microscope (RCM 8000: Nikon, Tokyo, Japan). Cells were irradiated with an excitation blue light beam (488 nm) produced by an argon ion laser. The emitted fluorescence was guided through a ×40 water-immersion objective to a pinhole diaphragm at 520 nm using a diachronic mirror. The intensity of emission was scanned at a dwell time of 1/30 s with a monitor video enhancer and images were obtained every 4 s. The image data were stored in a RCM work station and analyzed. For FM1-43 imaging, the indicator dye (2 µM, Molecular Probes) was loaded into cultured neurons by incubating with HK+ loading solution (KRH containing 50 mM KCl) for 10 min. After several washes, cells were illuminated with an argon ion laser (488 nM). The emission was observed at 520 nm using a diachronic mirror. To perform Na+ imaging, SBFI-AM (10 µM, Molecular Probes), a cell-permeable Na+ indicator, was used. The procedure for the experiment with SBFI-AM was the same as that with Fluo-3-AM. Cultured neurons were irradiated with an excitation ultraviolet beam (351 nm) produced by an argon ion laser, and the fluorescence emitted was guided through the objective to a pinhole diaphragm at 520 nm. Fluorescent dye intensity was normalized to the base line before application of the growth factors. It was confirmed that these dye fluorescence intensities were stable with minimum bleach for more than 5 min in vehicle solution. To confirm reproducibility, all imaging experiments were performed at least four times with independent cultures. Representative data are shown in the figures.

Immunoblotting-- Immunoblotting was performed as described previously (26). Cells were lysed in SDS lysis buffer containing 1% SDS, 20 mM Tris-HCl (pH 7.4), 5 mM EDTA, pH 8.0, 10 mM NaF, 2 mM Na3VO4, 0.5 mM phenylarsine oxide, and 1 mM phenylmethylsulfonyl fluoride. The lysates were boiled for 3 min and then clarified by ultracentrifugation at 60,000 × g for 30 min at 8 °C. The protein concentration of the supernatants was determined using a BCA protein assay kit (Pierce), and then 10-µg aliquots of protein were resolved by electrophoresis on 10% SDS-polyacrylamide gels. Proteins were transferred onto polyvinylidene fluoride membranes (Millipore Corp.) in 0.1 M Tris base, 0.192 M glycine, and 20% methanol using a semidry electrophoretic transfer system. The membranes were blocked with 0.1% Tween 20/Tris-buffered saline (T-TBS) containing 5% nonfat dried milk at room temperature for 1 h. Membranes were then probed with antibodies in T-TBS containing 1% nonfat dried milk at room temperature for 1 h. To detect MAPK phosphorylation and protein, anti-phospho-MAPK (1:5000 dilution, New England Biolabs, Inc.) and anti-MAPK (1:1000, Santa Cruz Biotechnology, Inc.) antibodies were used, respectively. After three washes with T-TBS, the membranes were incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG secondary antibody (Jackson ImmunoReserch Laboratories, Inc.) diluted 1:1000 with T-TBS at room temperature for 1 h. They were then washed at least four times with T-TBS and visualized using the Immunostar (Wako) chemiluminescence system.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

bFGF-induced Glutamate Release in Cultured Cortical Neurons-- To determine the acute effect of bFGF on presynaptic function, we examined the bFGF-induced glutamate release in cultured cortical neurons. The time course of the release is shown in Fig. 1A. The release of glutamate was observed immediately after the application of bFGF at 100 ng/ml and was maintained for 1-2 min (panel a in Fig. 1A). Neurotrophin BDNF-induced release in a sister culture is also shown (panel b in Fig. 1A). The time course analysis indicated that the bFGF-induced release was similar to the BDNF-induced one in time lapse. HK+ (50 mM KCl)-induced depolarization also evoked the release of glutamate (panel c in Fig. 1A). Compared with the HK+-evoked release, the bFGF- and BDNF-induced releases tended to be sustained, while the former achieved a much higher level (4-10-fold the growth factor-induced increases). The dose dependence of the bFGF-induced effect was examined (Fig. 1B). The release of glutamate began at 3 ng/ml and reached a plateau at 10-100 ng/ml. The maximum release was about 250-350% of the basal level in a series of independent cultures. Vehicle solution (BSA, 2 ng/ml) did not have an effect. The maximum levels of bFGF- and BDNF-induced release were almost the same at 100 ng/ml. These results indicated that not only BDNF but also bFGF induces an acute and transient release of glutamate in cultured cortical neurons.


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  		Fig. 1.   bFGF rapidly induced glutamate release in cultured cortical neurons. Cultured cortical neurons were prepared from 2- to 3-day-old rats and cultured for 6-9 days. A, time course analysis of the acute effect induced by bFGF. Panel a, the bFGF-induced release was maintained for 1-2 min and then there was a return to the basal level. The neurons were treated with bFGF at 100 ng/ml by bath application. Panel b, BDNF-induced release. BDNF was applied to cultures at 100 ng/ml. Panel c, release of glutamate evoked by high K+ (HK+ assay buffer containing 50 mM KCl). B, bFGF-induced glutamate release was dose-dependent. The effect of BDNF (100 ng/ml) was also examined (gray bar). The amounts of glutamate released by bFGF (100 ng/ml) and BDNF (100 ng/ml) were similar in sister cultures. Vehicle (final 2 ng/ml BSA) had no effect. Before exposing the culture to the two growth factors (1 min), basal fractions (1 min) were collected. Data represent the mean ± S.D. (n = 6). Statistical analysis was performed with Student's t test. **, p < 0.01 versus basal. C, no GABA was released on stimulation with bFGF (100 ng/ml). In contrast, HK+ evoked the release of both glutamate and GABA. Data represent the mean ± S.D. (n = 6). Statistical analysis was performed using Student's t test. **, p < 0.01 versus basal.

Next, we examined whether bFGF stimulates the release of other amino acid neurotransmitters. We found that the effect of bFGF was restricted to glutamate, which is an excitatory neurotransmitter (Fig. 1C). GABA, an inhibitory neurotransmitter, was not induced. Releases of glycine and taurine were not induced by bFGF either (data not shown). HK+, however, evoked the release of not only glutamate but also GABA. These results indicated that bFGF specifically influenced an excitatory amino acid transmitter in cultured cortical neurons.

bFGF Induced an Increase in the Intracellular Ca2+ Concentration-- To elucidate the mechanism of the bFGF-induced glutamate release, we focused on the intracellular Ca2+ accumulation induced by bFGF, because the mobilization of intracellular Ca2+ is believed to be an important step in neurotransmitter release. bFGF induced acute increases in the intracellular Ca2+ concentration in cortical neurons (Fig. 2). A marked increase was elicited immediately and followed by continuous oscillations (panel a in Fig. 2B). Some 71-85% of cells in cultures responded to bFGF. The Ca2+ increase was observed in the cell body and neurites (Fig. 2A). A similar response was observed on application of BDNF (panel b in Fig. 2B). Approximately 73-82% of cultured cells were BDNF-responsive. Previously, we reported that BDNF induced the release of glutamate in an intracellular Ca2+-dependent manner and that the increase in Ca2+ was mediated by the IP3 receptor (which is known to exist on intracellular Ca2+ stores) rather than by an influx of extracellular Ca2+ (8, 9). Therefore, we next examined the influence of thapsigargin (which induces depletion of Ca2+ from intracellular stores at 10 µM) on the bFGF-stimulated accumulation of Ca2+. Interestingly, although the BDNF-induced Ca2+ increase was inhibited by thapsigargin, the bFGF-induced increase was not affected (panels c and d in Fig. 2B). Under extracellular Ca2+-free conditions, no Ca2+ increase was induced by bFGF, but it was still observed in response to BDNF (panels e and f in Fig. 2B). Cd2+ (voltage-dependent Ca2+ channel blocker, 100 µM) and TTX (Na+ channel blocker, 0.5 µM) also blocked the bFGF-induced Ca2+ increase (Fig. 2C), indicating that the increase occurred through voltage-dependent Ca2+ channels. On the other hand, Cd2+ and TTX did not inhibit the BDNF-stimulated Ca2+ increase (data not shown). Previously, we showed that the intracellular Ca2+ concentration was either steady or displayed spontaneous synchronized oscillations (9). We had identified the growth factor-triggered or spontaneous synchronized oscillations as a synaptic transmission mediated by glutamate. Here we confirmed that the bFGF-induced Ca2+ increases (the first Ca2+ transient) were not affected by glutamate receptor antagonists, APV (10 µM) or CNQX (10 µM), but the subsequent oscillatory Ca2+ activities (synchronized oscillations after the first Ca2+ transient) were abolished. Furthermore, the bFGF-induced increase in the number of c-Fos (one of the immediate early genes)-positive neurons was examined by immunohistochemistry with anti-c-Fos antibody. Both bFGF and BDNF potentiated the number of neurons showing expression of c-Fos (control BSA, 7.93 ± 3.13%; bFGF, 70.5 ± 5.03%; BDNF, 51.55 ± 5.09%, percentages (c-fos/MAP2) of double positive cells, treatments with these factors were performed for 20 min). The induction of c-Fos expression by both these factors was not changed by APV or CNQX, indicating that the induction is triggered directly by bFGF and not through secondary effects such as stimulation by the released glutamate. Therefore, we performed Ca2+ imaging using resting cortical cultures, because the first rapid Ca2+ increase induced by growth factors was easy to clarify.


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  		Fig. 2.   bFGF increased the intracellular Ca2+ concentration in cultured cortical neurons. A, images of Fluo-3-filled cells. The increase in Ca2+ induced by bFGF in a selected area of the DIV6 culture is shown. Changes in the intracellular Ca2+ concentration were examined by ratiometry. Panel a, monochrome; panel b, pseudocolored basal image; and panel c, 4 s after bFGF application. Bar = 50 µm. B, voltage-dependent Ca2+ channels were involved in the bFGF-induced intracellular Ca2+ increase. Panels a and b, both bFGF and BDNF increased the Ca2+ levels in the presence of 1.8 mM Ca2+. Panels c and d, in the presence of thapsigargin (1 µM). Panels e and f, under extracellular Ca2+-free conditions. The trace indicates three cells, which were selected in the same well of a plate. From 71 to 85% of cultured cells were responsive to bFGF. C, characterization of the bFGF-induced Ca2+ increase. Plots summarize data from 18 selected cells in the same culture. (F, Intensity of the growth factor-induced first Ca2+ transient; F0, Intensity of base line before exposure to the growth factors). In the presence of Cd2+ (100 µM) and TTX (0.5 µM), cells did not respond to bFGF. Thapsigargin did no affect the bFGF-induced Ca 2+ increase, whereas it abolished the BDNF-induced one. For testing the effects of drugs, six to nine series of Ca2+ imaging experiments were performed with separated cultures. These results were reproducible.

bFGF Induced Glutamate Release via an Exocytosis Mechanism-- To test whether the bFGF-induced glutamate release is controlled by an exocytotic system, the Ca2+ dependence of the release and the involvement of exocytosis were examined (Fig. 3). Under extracellular Ca2+-free conditions, bFGF was not able to induce the release of glutamate (Fig. 3A). Cd2+ also abolished the bFGF-induced release. In the presence of nifedipine, an L-type Ca2+ channel blocker, and TTX, which are expected to block Ca2+ influx through the voltage-dependent Ca2+ channels, the bFGF-induced release was abolished, suggesting that the bFGF-induced glutamate release was dependent on Ca2+ influx through the voltage-dependent Ca2+ channels.


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  		Fig. 3.   bFGF induced glutamate release through an extracellular Ca2+ and exocytosis. A, Cd2+ (100 µM), nifedipin (10 µM), and TTX (0.5 µM) abolished the bFGF-induced release. Data represent the mean ± S.D. (n = 6). **, p < 0.01 versus basal (Student's t test). B, bFGF induced glutamate release through an exocytotic system. bFGF still induced glutamate release in the presence of t-PDC (10 µM) or DL-TBOA (10 µM). TeTx (10 nM) treatment for 8 h completely blocked the bFGF-induced release. Data represent the mean ± S.D. (n = 4). **, p < 0.01 versus basal (Student's t test). C, hippocampal and cerebellar neurons responded to bFGF. Panel a, bFGF-induced Ca2+ increases in cultured hippocampal (HIP) and cerebellar (CBL) neurons were observed. The traces indicate three cells, which were selected in the same well of a plate. Panel b, bFGF-induced acute glutamate release was also confirmed in cultured hippocampal (HIP) and cerebellar (CBL) neurons. Data represent the mean ± S.D. (n = 6). **, p < 0.01 versus basal (Student's t test).

To further identify the mechanism behind the bFGF-induced release, we tested the effect of tetanustoxin (TeTx, which inhibits synaptic vesicle-dependent release). Pretreatment with TeTx for 8-12 h before the application of bFGF completely inhibited the release of glutamate. Furthermore, the influence of glutamate transporter inhibitors was examined. In the presence of t-PDC (10 µM) and DL-TBOA (10 µM), the bFGF-induced release was not affected (Fig. 3B), suggesting that it occurred through exocytosis. Furthermore, we examined the acute effects of bFGF on the release of glutamate and accumulation of intracellular Ca2+ in cultured hippocampal and cerebellar neurons. These cultured neurons also responded to bFGF (Fig. 3C), suggesting that bFGF exerted acute effects on central nervous system glutamatergic neurons. By contrast, cultured astroglial cells (95-99% pure judging from immunocytochemistry) exhibited no response to bFGF (glutamate release: basal, 24.5 ± 3.88; bFGF-induced, 26.6 ± 3.52 (×10-10 mol/well), n = 6). All these results suggested that the bFGF induced the release of excitatory transmitters through an extracellular Ca2+-dependent pathway in central nervous system neurons.

To further characterize the bFGF-induced release and the involvement of the exocytotic system, we performed an exocytosis assay with the style dye FM1-43 (Fig. 4, A and B). After depolarization-induced FM dye loading, we tested whether bFGF triggers a reduction in the fluorescent intensity of FM. As expected, HK+ and bFGF reduced the intensity (panels a and b in Fig. 4B). However, the destaining effect of BDNF tended to be weaker than that of bFGF (panels c and d in Fig. 4B). Vehicle (solution containing BSA) had no effect. Although HK+ also attenuated the intensity of fluorescence, the extent of the destaining by HK+ was less than expected. However, because the number of FM fluorescent buttons, which responded to HK+, seemed to be much larger than that which responded to bFGF, the HK+-evoked glutamate release was thought to be more extensive. The reduction in FM dye by bFGF meant that bFGF likely evoked the depolarization. Thus, we performed an Na+ imaging assay using a Na+ indicator SBFI-AM in cultured cortical neurons. Acute application of bFGF induced a significant influx of Na+, suggesting that bFGF acutely evoked cell depolarization (panels a and c in Fig. 4C). BDNF induced an accumulation of Na+ as well (panel c in Fig. 4C), although the extent of the influx was less than that caused by bFGF. Furthermore, we confirmed that HK+ also induced an influx of Na+, which was 2-3-fold greater than the bFGF-induced one.


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  		Fig. 4.   Acute effects of bFGF on destaining of FM1-43 fluorescent and Na+ influx. A, bFGF triggered a reduction in the fluorescence of the style dye, FM1-43. Panels a and b, basal; panel c, 1 min after bFGF application (panel a, monochrome; panels b and c, pseudocolor). B, time course analysis of bFGF-reduced FM intensity. Each trace indicates the loss of FM fluorescence (vesicle-like buttons) in a single cell. Panel a, bFGF application (n = 9, the number of vesicle-like buttons); panel b, HK+ (50 mM) (n = 6); panel c, BDNF (n = 6). All these results suggest that exocytosis is involved in the bFGF-induced release. Representative data are shown. Panel d, plots of the summarized data were obtained from 12 cells (four independent cultures). The loss was determined by comparing the stimulation intensity (100 s after stimulation) with basal intensity (4 s before stimulation). Vehicle (BSA) had no effect. C, Na+ imaging was performed using Na+ indicator SBFI-AM (100 µM). Panel a, bFGF-induced intracellular Na+ accumulation. Panel b, HK+-induced Na+ accumulation. The traces indicate data obtained from six independent neurons (F/F0). Panel c, plots of data from 12 selected cells in the same culture. (F and F0, intensity 100 s after stimulation and intensity of pretreatment base line, respectively). Four series of Na+ imaging experiments were performed with three separated cultures. These results were reproducible.

MAPK Signal Transduction Was Required for the bFGF-induced Glutamate Release-- What kind of intracellular signal pathways are involved in the bFGF-induced glutamate release? First, the effect of genistein, a broad tyrosine kinase inhibitor, on the bFGF-induced release was tested. bFGF did not induce glutamate release after treatment with genistein (1 µM) in cultured cortical neurons (data not shown). Next, we examined the effect of an inhibitor of the MAPK pathway on the bFGF-induced release. MEK (an upstream regulator of MAPK) was required for the survival-promoting effects of bFGF on cultured cortical and hippocampal neurons (27). U0126 and PD98059, inhibitors of MEK, completely blocked the bFGF-induced glutamate release (Fig. 5A). On the other hand, BDNF still induced the release in the presence of these MEK inhibitors as reported previously (8). Furthermore, to confirm that bFGF and BDNF stimulated the activation of MAPK in the cultured cortical neurons, we performed Western blotting using anti-phospho-MAPK antibody. bFGF and BDNF induced phosphorylation of MAPK, although the level of phosphorylation was somewhat lower with bFGF. U0126 and PD98059 markedly attenuated the bFGF-stimulated phosphorylation of MAPK, although the inhibitory effect of U0126 was stronger than that of PD98059. The BDNF-induced phosphorylation of MAPK was also completely blocked by U0126. These results indicated that bFGF and BDNF similarly activate MAPK in cultured cortical neurons. It was therefore revealed that activation of the MAPK pathway is involved in the bFGF-induced, but not BDNF-induced, release of glutamate.


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  		Fig. 5.   MAPK pathway was involved in the bFGF-induced glutamate release. A, U0126 (10 µM) and PD98059 (50 µM), inhibitors of MEK, completely inhibited the bFGF-induced glutamate release. BDNF still induced the release even in the presence of these MEK inhibitors. Data represent the mean ± S.D. (n = 6); **, p < 0.01 versus basal. Student's t test. B, bFGF- and BDNF-stimulated activation of MAPK in cultured cerebral cortical neurons. Cultured cortical neurons pretreated without (-) or with 10 µM U0126 (U) or 50 µM PD98059 (PD) for 40 min were then incubated with 2 µg/ml BSA (con.), 2 µg/ml BSA plus 100 ng/ml bFGF (+bFGF), or BDNF (+BDNF) for 5 min and lysed. The lysates were immunoblotted with anti-phospho-MAPK antibody (upper panel) or anti-MAPK antibody (lower panel). The arrows on the right indicate the positions of MAPK. Molecular weights are shown on the left of the immunoblots.

bFGF Potentiated HK+-induced Glutamate Release through the Activation of MAP Kinase Pathway-- Here, we found that bFGF activates MAPK and that the activation is essential for the bFGF-induced glutamate release. As shown in Fig. 6A, interestingly, the phosphorylation level of MAPK gradually increased for at least 20 min after the application of bFGF. The phosphorylation of MAPK began to be observed at 3 min after bFGF exposure. The intensity of phosphorylation then increased, reaching a plateau at 20-40 min. Because the bFGF-induced glutamate release occurred within 1-2 min, the weak but significant MAPK phosphorylation at the early phase (within 3 min) may lead to the glutamate release. On the other hand, there is the possibility that MAPK activation at a later phase (20-40 min after bFGF stimulation) is also involved in the modulation of synaptic transmission. Thus, in addition to the acute effects of bFGF, we examined the effect of pretreatment with bFGF on the HK+-evoked release. Time course analysis showed that treatment with bFGF for 20-60 min enhanced the HK+-evoked release (Fig. 6, B and C), and vehicle (BSA solution) had no effect (Fig. 6B), suggesting that pretreatment with bFGF potentiated the activity-dependent release. The effects of inhibitors of the MAPK pathway were tested next. Both U0126 and PD98059 (data not shown) completely abolished the bFGF-enhanced release. There are several reports that PI 3-kinase is also activated by bFGF (28, 29). However, a PI 3-kinase inhibitor (LY294002, 30 µM) had no effect on the HK+-evoked release with or without bFGF treatment (Fig. 6C), excluding the involvement of PI 3-kinase-mediated intracellular signaling systems. These results indicated that the later phase phosphorylation of MAPK is required for the bFGF-potentiated glutamate release.


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  		Fig. 6.   bFGF enhanced HK+-evoked glutamate release in cultured cortical neurons. A, Time course analysis of the bFGF-stimulated activation of MAPK in cultured cortical neurons. Cultured cortical neurons were incubated with 100 ng/ml bFGF for 0, 3, 5, 20, and 40 min and lysed. The lysates were immunoblotted with anti-phospho-MAPK antibody (upper panel) or anti-MAPK antibody (lower panel). The positions of MAPK are indicated by the arrows on the right. Molecular weights are shown on the left of the immunoblots. B, time course analysis of the effect of treatment with bFGF on HK+-evoked glutamate release. Panel a, bFGF (100 ng/ml) was applied to cortical neurons for 0, 5, 20, 40, and 60 min, and then basal and HK+-evoked release was examined. Pretreatment with bFGF for 20, 40, and 60 min potentiated the HK+-evoked glutamate. Basal release was not changed by bFGF pretreatment. Vehicle treatment (BSA (2 µg/ml)-added assay solution, for 20 min) had no effect on the HK+-evoked release. Before HK+ stimulation (1 min), basal fractions (1 min) were collected. Data represent the mean ± S.D. (n = 6). **, p < 0.01 versus basal. Student's t test. C, U0126 (10 µM), an inhibitor of MEK, completely inhibited the bFGF-potentiated effect on the HK+-evoked release. PI 3-kinase inhibitor, LY294002 (30 µM), did not affect the HK+-evoked release. Data represent the mean ± S.D. (n = 6). Statistical analysis was performed using Student's t test. **, p < 0.01 versus basal.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Growth factors play important roles in the regulation of synaptic transmission in central nervous system neurons. Reports have indicated that BDNF and other neurotrophins trigger and modulate synaptic transmission in the acute phase. However, as concerns the effects of bFGF, very little was known. Here, we report an acute effect of bFGF on neurotransmitter release and the underlying intracellular signaling. We found that bFGF induced a rapid and transient glutamate release in cultured cortical neurons. Sequential experiments revealed that exocytosis, which requires an influx of Ca2+ and the activation of the MAPK pathway, is involved in the bFGF-induced release. Furthermore, we found that pretreatment with bFGF significantly enhanced the high K+-elicited glutamate release in a MAPK activation-dependent manner. These results suggest that bFGF acutely influences excitability in central nervous system neurons through activation of the MAPK pathway.

The present study indicated that bFGF induced the release of glutamate, but not GABA, in cultured cortical neurons. In contrast, GABA as well as glutamate were released by HK+-induced depolarization, suggesting that bFGF specifically acts on glutamatergic neurons to trigger glutamate release. In cultured hippocampal neurons, bFGF modulated GABA-mediated neuronal transmissions (30). However, in our system, there was no change in the amount of GABA released by bFGF. bFGF and BDNF triggered continuous synchronized Ca2+ oscillations in cultured cortical neurons (Fig. 2B). We previously reported that synchronized spontaneous Ca2+ oscillations were potentiated by acute application of BDNF and that BDNF-induced glutamate release was essential for these potentiated oscillations (9). We had confirmed that cortical cells displaying oscillations were glutaminase (a marker for glutamatergic neurons)-positive by immunocytochemistry, suggesting that glutamate, but not GABA-mediated, transmissions were enhanced by these growth factors. Thus, we concluded that bFGF plays a dominant role in stimulating glutamatergic neurons in cortical cultures.

The increase in the intracellular Ca2+ concentration seems to be stimulated through different mechanisms among growth factors. Phospholipase C (PLC), which produces IP3, is activated by growth factors, and IP3 triggers release of Ca2+ from intracellular storage sites (31). Neurotrophins are known to increase the intracellular Ca2+ concentration through the release of Ca2+ from intracellular stores (32-34). In addition, Drosophila TRP proteins and some mammalian homologs (TRPC1-7 proteins) are thought to mediate capacitative Ca2+ (35, 36) in a membrane potential-independent manner. Interestingly, Li et al. (37) reported that TRPC3 mediates Ca2+ and Na+ entry downstream of a pathway, including TrkB (BDNF-specific receptor), PLC-gamma , and IP3 in pontine neurons. We previously showed that BDNF increases the Ca2+ concentration in a PLC-gamma /IP3-dependent manner and that this increase is necessary for the BDNF-induced glutamate release in cultured cortical and cerebellar neurons (8, 9). On the other hand, pharmacological experiments revealed that the Ca2+ influx induced by bFGF was via voltage-dependent Ca2+ channels and that bFGF-induced glutamate release was dependent on the Ca2+ influx. In contrast, BDNF still induced both the release of glutamate and an increase in Ca2+ under conditions in which the influx of extracellular Ca2+ was blocked, implying that BDNF and bFGF stimulate Ca2+ increases through different mechanisms. These results raise the possibility that bFGF- and BDNF-induced glutamate releases are mediated through different mechanisms. To test this possibility, we examined the contribution of exocytosis to the bFGF-induced release, because a reverse transport system (through a glutamate transporter) was involved in the BDNF-induced release in developing cortical neurons (9). Pretreatment with TeTx completely inhibited the bFGF-induced release (Fig. 3), while the transporter inhibitors had no effect. Furthermore, we confirmed that bFGF reduced the FM1-43 intensity, while BDNF had a weaker effect than bFGF (Fig. 4, A and B). Furthermore, Na+ imaging experiments suggested that bFGF evoked significant cell depolarization in comparison with BDNF. All these results imply that exocytosis was the dominant system in the bFGF-induced glutamate release. The question then arises whether the BDNF-induced effect in our system is mediated through conventional synapses or whether there is spillover of glutamate via synapses or non-exocytotic release, as suggested for some transmitters (38-40). Katsumori et al. (41) has reported that depolarization-evoked release was occurred via reverse transport in hippocampal slices in neonates rather than via exocytosis as in adults. Therefore, in addition to exocytosis, a reverse transport system may be involved in the BDNF-induced glutamate release at this cortical stage. At a more mature stage, BDNF may exert an acute effect via exocytosis.

The glutamate release was induced by bFGF in a dose-dependent manner (Fig. 1), suggesting that bFGF receptor-stimulated intracellular signaling is required. What kind of intracellular signaling is required for the bFGF-induced glutamate release? It is possible that a MAPK pathway is involved. This pathway includes Ras (Raf kinase), MEK (MAP kinase kinase), S6 kinase (p90 ribosomal S6 kinase), and MAPK. For the survival-enhancing effect of bFGF on cultured cortical and hippocampal neurons, MEK was required (27). Recently, Yang et al. (11) indicated that glial cell line-derived neurotrophic factor acutely modulated the excitability in midbrain dopaminergic neurons through MAPK-dependent A-type K+ channel inhibition. Therefore, we examined the contribution of the MAPK pathway to the bFGF-induced glutamate release. MEK inhibitors completely inhibited the release (Fig. 5). On the other hand, the BDNF-induced release was not blocked. Both bFGF and BDNF stimulated activation of MAPK in the cultured cortical neurons. These results indicate that the MAPK pathway is involved in the bFGF-induced, but not BDNF-induced, glutamate release even though bFGF and BDNF similarly activate MAPK in cultured cortical neurons (Fig. 6).

There is a question of whether the MAPK pathway is upstream or downstream of the Ca2+ influx because the bFGF-dependent glutamate release was mediated by the Ca2+ increase. Pende et al. (42) reported that bFGF rapidly induces an extracellular Ca2+-independent phosphorylation of MAPK. On the other hand, Ca2+ also induces activation of the MAPK pathway (43). We tested the bFGF-induced Ca2+ increase in the presence of MEK inhibitors. PD98059 and U0126 partially blocked the increase (ratio values (bFGF-stimulated/basal): none, 262 ± 109%; PD98059, 137 ± 45.2%; U0126, 135 ± 49.9%; n = 12, n indicates the cell numbers selected). Furthermore, we observed the bFGF-stimulated phosphorylation of MAPK under extracellular Ca2+-free conditions (data not shown). These results indicate that the MAPK pathway is located upstream of bFGF-stimulated Ca2+ influx. Glial cell line-derived neurotrophic factor-modulated excitation of dopaminergic neurons is primarily mediated by MAPK-dependent A-type K+ channel inhibition, and this inhibition seemed to result in an increase in Ca2+ current (11). In the present study, K+ channel inhibition may be functioning as well as in the dopaminergic neurons. We cannot exclude the possibility that MAPK worked downstream of the bFGF-induced Ca2+ influx because MEK inhibitors significantly but did not completely inhibit the Ca2+ increase. There is adequate evidence that the increase in intracellular Ca2+ evoked by depolarization induces the activation of MAPK. For example, glutamate and potassium are effective in activating MAPK in hippocampal neurons (44-47). Therefore, both Ca2+-dependent and -independent MAPK activation may be required for the bFGF-induced glutamate release, although further investigation is needed to clarify the mechanisms by which Ca2+ and MAPK regulate each other.

Studies have suggested that BDNF potentiates activity-dependent synaptic transmission. For example, BDNF produces a long lasting (2-3 h) enhancement of field excitatory postsynaptic potential in the hippocampus that requires local protein synthesis (48). BDNF enhances glutamate release, and novel protein synthesis is required for the enhancement by BDNF in cortical neurons (49). We also reported that BDNF enhanced depolarization-evoked glutamate release in a PLC-gamma -dependent manner in cultured cortical neurons (50). However, the underlying signaling, which is essential for the activity-dependent synaptic modulation by bFGF, is not known. The time course experiment showed that the rapid bFGF-induced glutamate release finished within 1-2 min of the bFGF stimulation. However, the activation of MAPK was sustained for 20-40 min. Interestingly, the HK+-evoked glutamate release was significantly potentiated by bFGF pretreatment, and MAPK was essential for the potentiation. These studies indicate that different signal transductions are important in the late-phase modulation of synaptic efficacy by each growth factor.

Recent studies have suggested that bFGF is a multifunctional growth factor in neuronal cells (51). bFGF and FGFR1 (high affinity receptor for bFGF; Ref. 52) are present in the developing and adult brain (14). Astrocytes also synthesized and released bFGF (53). Here we showed a novel function of bFGF in central nervous system neurons at an acute phase. Thus, in developing and adult brain, bFGF, which is derived from neurons or astrocytes, may modulate glutamatergic transmission.

    ACKNOWLEDGEMENT

We thank the Regeneron Pharmaceutical Co. and Takeda Chemical Industries, Ltd. for generously donating the BDNF and bFGF, respectively.

    FOOTNOTES

§ Japan Society for the Promotion of Science Postdoctoral Fellow. To whom correspondence should be addressed: Division of Protein Biosynthesis, Inst. for Protein Research Osaka University, Suita, Osaka 565-0871, Japan. Tel.: 81-727-51-9524; Fax: 81-727-51-9628; E-mail: t-numakawa@aist.go.jp.

Published, JBC Papers in Press, May 28, 2002, DOI 10.1074/jbc.M202927200

This work was supported in part by grants-in-aid for Scientific Research from the Ministry of Education, Science, Sports, Culture and Technology of Japan and from CREST of the Japan Science and Technology Cooperation.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

    ABBREVIATIONS

The abbreviations used are: BDNF, brain-derived neurotrophic factor; bFGF, basic fibroblast growth factor; t-PDC, L-trans-pyrollidine-2,4-dicalboxylic acid; DL-TBOA, DL-threo-beta -benzyloxyaspartate; MAPK, mitogen-activated protein kinase; MAP, mitogen-activated protein; PLC-gamma , phospholipase C-gamma ; IP3, inositol 1,4,5-trisphosphate; PI 3-kinase, phosphatidylinositol 3-kinase; NMDA, N-methyl-D-aspartic acid; DAB, 3,3'-diaminobenzidine; HPLC, high performance liquid chromatography; BSA, bovine serum albumin; TTX, tetrodotoxin; SFBI-AM, 1,3-benzenedicarboxylic acid, 4,4'-[1,4,10-trioxa-7,13-diazacyclopentadecane-7,13-diylbis(5-methoxy-6,12-benzofurandiyl)]bistetrakis[(acetyloxy)methyl]ester; GABA, gamma -aminobutyric acid; TeTx, tetanustoxin; MEK, mitogen-activated protein kinase/extracellular signal-regulated kinase kinase; FM1-43, N-(3-triethylammoniumpropyl)-4-(4-(dibutylamino)styryl)pyridium dibromide; AMPA, alpha -amino-3-hydroxy-5-methylisoxazolepropionate; APV, amino-5-phosphonovaleric acid; CNQX, 6-cyano-7-nitroquinoxaline-2,3-dione.

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RESULTS
DISCUSSION
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