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(The FASEB Journal. 2003;17:186-193.)
© 2003 FASEB

Vascular endothelial growth factor promotes proliferation of cortical neuron precursors by regulating E2F expression

YONGHUA ZHU, KUNLIN JIN, XIAO OU MAO and DAVID A. GREENBERG1

Buck Institute for Age Research, Novato, California, USA

1Correspondence: Buck Institute for Age Research, 8001 Redwood Boulevard, Novato, CA 94945, USA. E-mail: dgreenberg{at}buckinstitute.org


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Neurogenesis, or the production of new neurons, is regulated by physiological and pathological processes including aging, stress, and brain injury. Many mitogenic and trophic factors that regulate proliferation of nonneuronal cells are also involved in neurogenesis. These include vascular endothelial cell growth factor (VEGF), which stimulates the incorporation of bromodeoxyuridine (BrdU) into neuronal precursor cells in vitro and in the adult rat brain in vivo. Using BrdU labeling as an index of cell proliferation, we found that the in vitro neuroproliferative effect of VEGF was associated with up-regulation of E2F family transcription factors, cyclin D1, cyclin E, and cdc25. VEGF also increased nuclear expression of E2F1, E2F2, and E2F3, consistent with regulation of the G1/S phase transition of the cell cycle. The proliferative effect of VEGF was inhibited by the extracellular signal-regulated kinase kinase (MEK) inhibitor PD98059, the phospholipase C (PLC) inhibitor U73122, the protein kinase C (PKC) inhibitor GF102390X, and the phosphatidylinositol 3-kinase (PI3K) inhibitor wortmannin, indicating involvement of multiple signaling pathways. These findings help to provide a molecular basis for some of the recently identified neuronal effects of VEGF. Zhu, Y., Jin, K., Mao, X. O., Greenberg D. A. Vascular endothelial growth factor promotes proliferation of cortical neuron precursors by regulating E2F expression.


Key Words: neurogenesis • VEGF • transcription factors


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
NEUROGENESIS RELIES on the precisely controlled proliferation of neuronal precursor cells. It is subject to physiological regulation by glucocorticoids, sex hormones, growth factors, excitatory neurotransmission, learning, and stress, and it can be modified pharmacologically (1) . Neurogenesis in the adult brain is also induced by certain pathological events, such as mechanical injury (2) , seizures (3) , neuronal apoptosis (4) , and global (5) and focal (6) cerebral ischemia, and it may contribute to brain recovery and repair in these conditions.

Several mitogenic and trophic factors have been implicated in the processes of neurogenesis, including basic fibroblast growth factor (FGF-2; 7 ) and epidermal growth factor (EGF; ref 8 ). Vascular endothelial growth factor (VEGF), a 36–45 kDa dimeric glycoprotein with a major role in angiogenesis, promotes endothelial cell survival, proliferation, migration, and tube formation (9) . VEGF acts as a proinflammatory cytokine by increasing endothelial permeability and inducing the expression of adhesion molecules that bind leukocytes to endothelial cells (10 , 11) . VEGF also has direct neurotrophic and neuroprotective actions (12 13 14 15 16 17) , and recent evidence points to a role for VEGF as a directly (18) or indirectly (19) acting neurogenesis factor.

VEGF interacts with two high affinity tyrosine kinase receptors, the Fms-like tyrosine kinase Flt-1 and the kinase insert domain-containing receptor KDR/Flk-1, which are expressed on endothelial cells (20) . VEGF receptors are also found on nonvascular cells, including neurons, where they are thought to regulate retinal neural development (21 , 22) and cerebral responses to ischemia (23 , 24) .

The E2F family of transcription factors regulates cellular proliferation and differentiation. E2F1 is a primary target of cytokine receptor signals and a key regulator of the cell cycle machinery (25) . Together with its heterodimeric partner DP1, E2F1 promotes progression of the cell cycle beyond the G1 restriction point by binding to promoter regions of genes encoding factors essential for DNA synthesis and cell cycle control and inducing their transcription before and during S phase (26) . E2F activity is regulated by the retinoblastoma protein (Rb), a tumor suppressor gene. Binding of Rb and related proteins to E2Fs inhibits their ability to activate transcription and, in some cases, converts E2Fs from activators to repressors of transcription (27) .

To date, six members of the E2F family, E2F1 through E2F6, have been cloned (28) and can be classified into three subgroups based on their structure, affinity for Rb, expression pattern, and function. The first group consists of E2F1, E2F2, and E2F3, which are involved in the regulation of cell proliferation and apoptosis. At physiological levels, these E2Fs associate exclusively with Rb, and ectopic expression of each is sufficient to induce quiescent cells to enter S phase. Conversely, conditional knockout of all three of these factors renders mouse embryonic fibroblasts incapable of entering S phase (29) . In postmitotic neurons, knockout of E2F1 confers resistance to staurosporine-induced apoptosis, whereas overexpression of E2F1 is associated with spontaneous apoptotic cell death (30) . E2F4–5 are poor transcriptional activators and are unable to induce quiescent cells to enter S phase; E2F6 lacks the sequences required for transcriptional activation or Rb family binding but can inhibit the transcription of E2F-responsive genes (28) .

In the present study, we characterized the in vitro mitogenic effect of VEGF on neuronal precursor cells from embryonic murine cerebral cortex and found that it involves induction of E2F1, E2F2, E2F3, and other cell cycle-related genes.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell culture
Cell cultures derived from CD1 mouse cortex were established as described previously (31 , 32) . Cerebral cortex from CD1 mice at gestational day 14 was dissected, treated with trypsin for 3 min at 37°C, and dissociated by trituration. Cell suspensions were plated at 3.5 x 105 cells/cm2 on 24-well plastic tissue culture dishes coated with poly-D-lysine in defined medium (Neurobasal/B27; Life Technologies, Inc., Rockville, MD), 2 mM glutamine, 25 U/mL penicillin, and 25 µg/mL streptomycin. Cultures were maintained in a humidified, 5% CO2 incubator at 37°C for 3 days before treatment.

VEGF treatment and bromodeoxyuridine uptake
Cultures were treated with VEGF165 (10–100 ng/mL; R&D Systems Inc., Minneapolis, MN) for up to 72 h. Bromodeoxyuridine (BrdU; 50 µg/mL; Sigma, St. Louis, MO) was added to cultures for the final 3 h of VEGF treatment, as described previously (33) , and BrdU incorporation was measured using a commercially available enzyme-linked immunsorbent assay (ELISA) kit (Roche, Indianapolis, IN) (34) .

Cell proliferation assays
To measure cell proliferation, cultures were assayed for the production of formazan by the reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), an index of the number of viable cells in culture, as described previously (35) . Cells were incubated with 5 mg/mL of MTT at 37°C for 2 h. The medium was removed, and cells were solubilized with dimethylsufoxide and transferred to 96-well plates. The formazan reduction product was detected by measuring absorbance at 570 nm in a Cytofluor Series 4000 multi-well plate-reader (PerSeptive Biosystems, Framingham, MA).

Immunocytochemistry
Cells were fixed with 70% ethanol and incubated with a mouse monoclonal (Roche; 1:200) or sheep polyclonal (Biodesign, Saco, ME; 1:200) antibody to BrdU, followed by rhodamine-labeled anti-mouse or anti-sheep immunoglobulin (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA; 1:200). Cells were stained with DAPI (Vector Laboratories, Burlingame, CA), mounted with mounting medium, and viewed by Nikon E800 epifluorescence microscopy. BrdU-labeled and DAPI-labeled cells were counted in 10 fields of each well at x40 magnification. Colocalization of BrdU with other markers was assessed by double-label immunocytohemistry using mouse monoclonal antiproliferating cell nuclear antigen (PCNA; Santa Cruz Biotechnology, Santa Cruz, CA; 1:1000) or rat monoclonal antiembryonic nerve cell adhesion molecule (ENCAM; BD Transduction Laboratories, San Diego, CA; 1:100), and staining was visualized with a FITC-labeled secondary antibody (Jackson Immuno-Research Laboratories, Inc.; 1:200).

Western blot analysis
Cells were washed twice with PBS, and whole-cell extracts were prepared by adding 10 vol of 1x sample buffer (2% SDS, 100 mM DTT, 60 mM Tris-pH6.8, and 10% glycerol) and boiling for 5 min. Nuclear extracts were prepared as described previously (36) ; 30 µg of protein was analyzed by 10–20% gradient SDS-PAGE and transferred to Immuno-Blot PVDF membranes (Bio-Rad Laboratories, Inc., Hercules, CA). Membranes were probed with primary antibody overnight, and the signal was detected with Boehringer-Mannheim chemiluminescence blotting kits. Protein bands were quantified using a ChemiImage System (Alpha Innotech Corporation, San Leandro, CA). Rabbit polyclonal anti-E2F1 (1:500), E2F2 (1:500), E2F3 (1:500), E2F4 (1:500), E2F5 (1:500), PCNA (1:2000), Flt-1 (1:250), and Flk-1 (1:250) antibodies were from Santa Cruz Biotechnology.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
VEGF promotes cell proliferation in cortical cultures
Cerebral cortical cells from E14 mouse embryos were cultured in defined medium as described previously (33) . Cultures contained primarily immature cells of neuronal lineage, as found previously (37) , with 91 ± 2% of cells staining for the neuron-specific RNA-binding protein Hu (38) , and 7 ± 2% staining for the basic helix-loop-helix neurogenic differentiation factor NeuroD (39) . In contrast, only 2 ± 2% were immunoreactive for the astroglial marker GFAP.

We used nuclear incorporation of BrdU into newly synthesized DNA as an index of cells in S phase of the mitotic cycle (40) . When cultures were treated with 50 ng/mL of VEGF for 24, 48, or 72 h and labeled with BrdU for the last 3 h, the number of BrdU-labeled cells was maximally (~20%) increased at 48 h (Fig. 1 ). Therefore, this duration of exposure to VEGF was used in subsequent experiments.



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  		Figure 1. VEGF stimulates BrdU incorporation in cortical cultures. Cultures enriched in neuronal precursors were treated with 50 ng/mL of VEGF for different times (unfilled bars) or with different concentrations of VEGF for 48 h (filled bars). BrdU (50 µg/mL) was added for the last 3 h of VEGF exposure, and BrdU incorporation was measured by ELISA as described in Materials and Methods. Data are means ± SE (n=3). *P < 0.05 relative to 0 h or 0 ng/mL (ANOVA and post hoc t tests).

VEGF at 25, 50, and 100 ng/mL increased BrdU labeling, with the maximal effect observed at 50–100 ng/mL. To confirm that BrdU incorporation was associated with cell proliferation, we stained cultures with antbodies against both BrdU and the cell-proliferation marker PCNA, which helps to regulate the fidelity of DNA replication (41) . Immunocytochemistry showed nuclear colocalization of PCNA and BrdU immunoreactivity (Fig. 2 ). To ascertain whether cells that incorporated BrdU in the presence of VEGF were of neuronal lineage, cultures were labeled with antibodies against BrdU and the immature neuronal marker ENCAM. These cultures showed nuclear BrdU and cytoplasmic ENCAM colocalized to the same cells.



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  		Figure 2. BrdU labeling colocalizes with the cell proliferation marker PCNA and the immature neuronal marker ENCAM. Cultures were treated with 50 ng/mL of VEGF for 48 h and with 50 µg/mL of BrdU for the last 3 h. They were then stained with antibodies against BrdU and either PCNA or ENCAM, followed by rhodamine (for BrdU, red)- or FITC (for PCNA and ENCAM, green)-labeled secondary antibodies, as described in Materials and Methods. Images labeled Merge show nuclear colocalization of PCNA and BrdU (left), or localization of ENCAM and BrdU to cytoplasm and nucleus, respectively (right) of the same cells. Data are representative fields from 3 experiments/column.

VEGF-induced neural proliferation is mediated by multiple signaling pathways
The VEGF receptors Flt-1 and Flk-1 are both expressed on neurons (24) , but only Flk-1 has been implicated in neurogenesis (18 , 19) . To establish which VEGF receptors were involved in VEGF-induced neuroproliferation in the present study, we used a specific Flk-1 antagonist (SU1498) and the specific Flt-1 ligand, placenta growth factor (PlGF). SU1498 is a small-molecule synthetic inhibitor of the catalytic function of the Flk-1 tyrosine kinase (42) . PlGF is a 149-amino acid protein with 53% identity at the amino acid level to the platelet-derived growth factor-like region of human VEGF (43) . PlGF binds with high affinity to Flt-1, but not to Flk-1, and potentiates the action of low concentrations of VEGF in vitro (44) .

We treated cortical cultures with VEGF or PlGF and measured MTT absorbance as an index of cell proliferation (45) , as employed in prior studies of growth factor-induced neurogenesis in cell culture (37 , 46) . MTT absorbance in the presence of VEGF was significantly (~20%) higher than in untreated cells, whereas PlGF at concentrations as high as 200 ng/mL had no effect on MTT absorbance (Fig. 3 ). We also treated cortical cultures with 100 ng/mL of VEGF in the presence of 5 µM SU1498. SU1498 abolished the ability of VEGF to increase MTT absorbance in our cortical cultures, and a similar result was observed when 500 nM oxindole I, another Flk-1 antagonist (47) , was used. A decrease in the effect of VEGF on MTT absorbance could result not only from a decrease in cell proliferation but also from an increase in cell death. However, SU1598 does not cause cell death (measured using DNA damage assays) in our cultures at concentrations <=20 µM (not shown). These data suggest that Flk-1, but not Flt-1, mediates VEGF-induced proliferation of neuronal precursors in our cortical cultures.



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  		Figure 3. Effects of VEGF receptor ligands and protein kinase inhibitors on VEGF-induced cell proliferation in cortical cultures. Cultures were treated with VEGF (50 ng/mL) or PlGF (200 ng/mL) for 48 h, or with VEGF (50 ng/mL) for 48 h and the indicated inhibitors for 49 h beginning 1 h before the addition of VEGF. Inhibitors used were the Flk-1 inhibitor SU1498 (SU, 5 µM), the Flk-I inhibitor oxindole I (O, 500 nM), the PLC inhibitor U73122 (U, 1 µM), the p38 MAPK inhibitor SB203580 (SB, 10 µM), the PKC inhibitor GF102390X (GF, 1 µM), the PI3K wortmannin (W, 2 µM), and the MEK inhibitor PD98059 (PD, 20 µM). Cell proliferation was measured by MTT absorbance, as described in Materials and Methods, and expressed as percentage of absorbance in untreated cultures (Con). Data are means ± SE (n=3). Filled bars, P < 0.05 relative to VEGF (ANOVA and post hoc t tests).

Binding of growth factors to receptor tyrosine kinases like Flk-1 leads to the activation of intracellular signaling cascades. Several signaling mechanisms have been implicated in the effects of VEGF, including pathways that involve phospholipase C (PLC), p38 mitogen-activated protein kinase (MAPK), protein kinase C (PKC), phosphatidylinositol 3-kinase (PI3K), and MEK extracellular signal-regulated kinase (ERK) (reviewed in ref 48 ). To investigate which of these signaling pathways might be implicated in the neuroproliferative effects of VEGF, we used a series of protein kinase inhibitors to inactivate PLC, p38 MAPK, PKC, PI3K, or MEK. We expected that if a particular kinase were critical for the effects of VEGF on neuronal proliferation, inhibiting its activity should reverse these effects. As shown in Fig. 3 , treating cultures with the PLC inhibitor U73122 (1 µM), the PKC inhibitor GF102390X (1 µM), the PI3K inhibitor wortmannin (2 µM), or the MEK inhibitor PD98059 (20 µM) 1 h before the onset of VEGF administration blocked the effect of VEGF on cell proliferation, whereas the p38 MAPK inhibitor SB203580 (10 µM) had no effect. Although PD98059 can cause neuronal death under some conditions, it does not do so in normoxic cultures at concentrations <=50 µM (49) . These results indicate that VEGF-induced proliferation of neuronal precursors is mediated through downstream signal transduction pathways that employ PLC, PKC, PI3K, and MEK and that each of these pathways must operate for the effect of VEGF to occur.

VEGF enhances E2F-1, E2F-2, and E2F-3 but not E2F-4 and E2F-5 expression
The G1/S checkpoint controls the passage of eukaryotic cells from G1 phase into S phase of the cell cycle. Many cell cycle kinases and transcription factors are pivotal in controlling this checkpoint. To investigate how VEGF regulates the cell cycle in cultures containing proliferating neuronal precursors, we measured levels of E2F transcription factors and cyclins by Western blotting in control and VEGF-treated cortical cultures. E2F transcription factors are expressed during neurogenesis in the developing mouse brain, with high levels in neuroproliferative areas such as the ventricular zone (50) . In hippocampus of adult rodents, E2F1 is transcriptionally induced by global cerebral ischemia (51) , which also stimulates neurogenesis in this region (5) , suggesting that E2F expression and neurogenesis may be linked.

E2F1, E2F2, and E2F3 were up-regulated by VEGF in a time-dependent manner (Fig. 4 ), while levels of E2F4 and E2F-5, which do not appear to be involved in proliferation control (52) , were unchanged (Fig. 5 ). These changes in E2F1–3 expression were accompanied by increases in the expression of 1) PCNA, which helps to regulate the fidelity of DNA replication (41) ; 2) cell division cycle (CDC) 25, a protein tyrosine phosphatase that interacts with PCNA at the G2/M transition (53) ; and 3) cyclins D1, E, and A, which are associated with progression through G1 phase and the G1/S transition (50 , 54) . Consistent with the effects of signaling inhibitors on VEGF-induced cell proliferation measured by MTT absorbance, VEGF-induced E2F1, E2F2, E2F3, PCNA, and CDC25 expression was attenuated by inhibitors of Flk-1, PLC, PKC, PI3K, and MEK but not by an inhibitor of p38 MAPK.



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  		Figure 4. Effects of VEGF and signaling inhibitors on E2F1, E2F2, E2F3, PCNA, and CDC25 protein expression. Cultures were incubated with 100 ng/mL of VEGF for 0–48 h or for 48 h in the presence of SU1498 (SU, 5 µM), U73122 (U, 1 µM), SB203580 (SB, 10 µM), GF102390X (GF, 1 µM), wortmannin (W, 2 µM), or PD98059 (PD, 20 µM). Protein samples (50 µg) were loaded on 10–12% gradient SDS-PAGE gels and transferred to PVDF membranes for Western analysis. VEGF produced a time-dependent increase in the expression of all 5 proteins, which was attenuated by all the inhibitors except SB203580. Data are representative blots from 3 experiments per panel.



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  		Figure 5. Effects of VEGF on cyclin A, E, and D1 and E2F4 and E2F5 expression. Cultures were incubated with 100 ng/mL of VEGF for 0–48 h, and 50 µg protein samples were loaded on 10–12% gradient SDS-PAGE gels and transferred to PVDF membranes for Western analysis. VEGF produced a time-dependent increase in the expression of cyclins A, E, and D1 but not E2F4 or E2F5. Data are representative blots from 3 experiments per panel.

VEGF enhances nuclear expression of E2F1–3
E2F transcription factors must be present in the nucleus to gain access to target DNA sequences (28) . Therefore, we evaluated whether induction of E2F expression by VEGF is associated with an increase in nuclear expression of E2Fs. Western blotting of nuclear extracts from cortical cultures treated with 50 ng/mL of VEGF for 0–48 h showed an increase in nuclear levels of those E2Fs that undergo VEGF-induced up-egulation–E2F1, E2F2, and E2F3 but not E2F4 (Fig. 6 ).



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  		Figure 6. Increased nuclear expression of E2F1–3 in VEGF-treated cortical cultures. Cultures were treated with 100 ng/mL of VEGF for 0–48 h. Nuclear extracts (40 µg protein) were loaded on 12% SDS-PAGE gels and transferred to PVDF membranes for Western analysis. VEGF increased nuclear expression of E2F1, E2F2 and E2F3 but not E2F4. Data are representative blots from 3 experiments per panel.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The major finding of this study is that the angiogenesis factor VEGF stimulates the proliferation of neuronal precursor cells cultured from murine cerebral cortex via multiple signal transduction pathways and cell-cycle proteins. In addition to the role of neurogenesis in embryonic development, neurogenesis has been shown to persist in the adult brain, where it may be involved in cell replacement after injury (1) . Several growth factors have been implicated in adult neurogenesis (55) , and growth factors are likely mediators of neurogenesis after brain injury, such as that due to cerebral ischemia. In ischemia, candidate mediators include those factors that are known to be induced in the ischemic brain and that have mitogenic effects on nonneuronal cells, such as endothelial cells. Given these considerations, and the fact that some evidence points to an intimate association between angiogenesis and neurogenesis in at least some sites in the adult brain (56) , VEGF would appear to be a strong candidate as an injury-induced neurogenesis factor. Two recent reports are consistent with this hypothesis (18 , 19) .

To demonstrate neurogenesis, at least two criteria must be satisfied:first, precursor cells must be shown to proliferate, and second, the progeny that result must be shown to differentiate toward a mature neuronal phenotype. Because individual methods to demonstrate cell proliferation may have pitfalls (57) , we used BrdU labeling (which identifies cells in S phase of the cell cycle), expression of PCNA (which is induced during S phase and regulates the fidelity of DNA replication), and MTT absorbance (which reflects the increased metabolic activity associated with increases in cell number) to measure neurogenesis. To demonstrate that cells in our cultures that were induced to proliferate by VEGF were of neuronal lineage, we examined the expression of a neuronal differentiation marker, ENCAM. Together, the results of these investigations point to neurogenesis as the cytoproliferative process induced by VEGF in our cultures.

Several growth factors have the capacity to stimulate neurogenesis in vitro or in vivo, including EGF (58) , FGF-2 (7 , 59) , brain-derived neurotrophic factor (BDNF) (60 61 62 63) , heparin-binding epidermal growth factor (37) , and stem cell factor (46) . Each of these factors interacts with different receptor tyrosine kinases and activates distinct but overlapping sets of signal transduction pathways. In endothelial cultures, for example, VEGF, EGF, and FGF-2 all stimulate cell proliferation, but whereas binding of VEGF to Flk-1 activates ERK via MEK, PLC{gamma}, and PKC{varepsilon}, neither PLC{gamma} nor PKC{varepsilon} is involved in the activation of ERK by EGF or FGF-2 (64) . The PI3K/Akt pathway also has an important role in VEGF-induced cell-cycle progression in endothelial cells (65) and contributes to the neurotrophic effects of several growth factors, including BDNF (66) . In previous studies, we found that the ability of VEGF to prevent the death of HN33 (mouse hippocampal neuronxneuroblastoma) cells after serum withdrawal was associated with activation of the PI3K/Akt pathway (15) and that protection of these cells from hypoxia by VEGF was blocked by the PI3K inhibitors wortmannin and LY294002 (16) . Our observation that PLC, PKC, PI3K, and MEK are all required for VEGF-induced proliferation of neuronal precursors in our cultures is, therefore, consistent with previous findings regarding signaling pathways employed by VEGF and by other neurotrophic factors.

In many cells, including neural precursors, transition from G1 to S phase of the cell cycle is regulated by the retinoblastoma gene Rb and by E2F transcription factors (50) . In G1, binding of hypophosphorylated Rb to E2F/dimerization partner heterodimers represses transcription of E2F-responsive genes. Entry into S phase occurs when complexation with cyclins activates cyclin-dependent kinases (Cdks); Cdks phosphorylate Rb, which dissociates from E2F/DP dimers and permits transcriptional activation to proceed. VEGF-stimulated proliferation of endothelial cells appears to involve this pathway, because VEGF increases endothelial cyclin D1 expression, Cdk4 kinase activity, Rb phosphorylation, and E2F promoter activity (67 68 69) . Because the proliferative effect of VEGF in our cortical cultures was accompanied by increased expression of several cyclins and E2Fs, our results suggest that VEGF may induce neurogenesis through a similar mechanism.


   ACKNOWLEDGMENTS
 
This work was supported by grant NS-37695 from the National Institutes of Health.

Received for publication July 18, 2002. Accepted for publication October 15, 2002.


   REFERENCES
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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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