242 H
from Kyowa Hakko Kogyo Ltd. N-Acetyl-S-farnesyl-
L
- cysteine AFC was from Calbiochem, U73122 was from
Biomol Research Lab., and wortmannin and forskolin were from Sigma. Other drugs were purchased from Wako
Chemicals.
3. Results
3.1. Effects of bFGF in relation to GABA and calbindin immunoreactivity
Serum deprivation from the medium of hippocampal primary cultures, 24 h after plating at an initial density of
4 2
5310 cells cm , resulted in a gradual but marked de-
crease in the total number of surviving neurons. As previously demonstrated, addition of bFGF in the serum-
free medium dramatically prevented the decrease in the number of hippocampal neurons. We performed immuno-
cytochemical staining with anti-GABA antibody Fig. 1A,B, and compared control and bFGF-treated cultures as
to the number of surviving GABA-positive and GABA- negative neurons. As shown in Fig. 1C triangles, while
the total number of neurons gradually decreased after serum deprivation, the number of GABA-positive neurons
remained constant. Moreover, addition of bFGF 5 ng ml markedly and significantly prevented the decrease in the
total number of surviving neurons, whereas the number of GABA-positive neurons in bFGF-treated cultures showed
no significant differences from that in control cultures Fig. 1C. These results suggest that, under the present ex-
perimental conditions, serum deprivation causes selective death of GABA-negative neurons that is reversed by
bFGF. bFGF has also been shown to act as a differentia- tion factor for calbindin-expressing neurons [33]. Accord-
ingly, we examined calbindin expression in high-density
4
cultures of hippocampal neurons initial density of 5310
2
cells cm . In control cultures at 4 DIV, calbindin-positive neurons represented a small population: only 2.460.37
in number of total neurons was positively stained with anti-calbindin antibody. Consistent with previous reports
Fig. 1. The effect of bFGF on the number of GABA-positive and
[6,33], treatment with bFGF 5 ng ml markedly and
GABA-negative hippocampal neurons. A GABA-immunocytochemistry
significantly increased the number of calbindin-positive
in control cultures at 4 DIV. Neurons immunoreactive for anti-GABA
neurons Fig. 2.
antibody are pointed by arrows. B GABA-immunocytochemistry in bFGF 5 ng ml-treated cultures at 4 DIV. C Time-dependent changes
21
in the number of GABA-positive triangles and total circles neurons.
3.2. Ca channels and the effects of bFGF
Open symbols, control cultures; closed symbols, bFGF 5 ng ml-treated cultures. bFGF inhibited the decrease in the number of total neurons,
We have previously demonstrated that an L-type VDCC
whereas it did not show significant effect on the number of GABA-
blocker nicardipine at a concentration of 5 mM suppresses
positive neurons. P,0.05; P,0.01 vs. control at each corresponding time point.
the promoting effect of bFGF on the formation of neurite branches [27]. Here we questioned if L-type VDCCs are
also involved in the actions of bFGF on neuronal survival and calbindin expression. Nicardipine 1–5 mM co-ap-
concentrations of nicardipine had no significant effect on plied with bFGF 5 ng ml counteracted the effect of
the increase in the number of calbindin-expressing neurons bFGF on neuronal survival in a concentration-dependent
by bFGF Fig. 3A, open bars. Consistent with previous manner Fig. 3A, hatched bars. In contrast, the same
findings, the increase in neuritic branches by bFGF 1
H . Katsuki et al. Brain Research 885 2000 240 –250
243
Fig. 3. The effect of nicardipine on the actions of bFGF. A bFGF 5 ng ml and nicardipine at indicated concentrations were simultaneously
applied to high-density hippocampal cell cultures at 1 DIV. At 4 DIV, the numbers of total neurons hatched bars and calbindin-positive neurons
open bars were determined. Nicardipine inhibited the effect of bFGF on survival of total neurons, but had no significant effect on the increase in
the number of calbindin-expressing neurons. P,0.01 vs. no drug
[[
treatment 0 mM nicardipine; P,0.01 vs. bFGF alone. B bFGF 1
ng ml and nicardipine 5 mM were simultaneously applied to low- density cell cultures at 2 DIV. At 2 DIV open bars and 4 DIV hatched
bars, the number of branch points along the longest neurite was counted. Nicardipine inhibited the increase in neuritic branches by bFGF. P,
[[
0.01 vs. control; P,0.01 vs. bFGF alone.
neuronal survival, enhancement of calbindin expression, or
Fig. 2. The effect of bFGF on the expression of calbindin D in
acceleration of neuritic branch formation data not shown.
28k
hippocampal neurons. A Calbindin-immunocytochemistry in control
The effective blockade by nicardipine of bFGF actions
21
cultures at 4 DIV. B Calbindin-immunocytochemistry in bFGF 5
implies that Ca influx through L-type VDCCs is in-
ng ml-treated cultures at 4 DIV. Neurons immunoreactive for anti-
volved in the effect of bFGF, especially on neuronal
calbindin D antibody are pointed by arrows. C Time-dependent
28k
survival and neurite branch formation. Therefore, we
changes in the number of calbindin-positive neurons. Open symbols, control cultures; closed symbols, bFGF 5 ng ml-treated cultures.
performed next set of experiments to verify if the elevated
21
Treatment with bFGF resulted in a marked increase in the number of
cytosolic Ca is required for the actions of bFGF. Cells
calbindin-expressing neurons. P,0.01 vs. control at each corre-
were treated
with 1,2-bis2-aminophenoxy-ethane-
sponding time point.
N ,N,N9,N9-tetraacetic acid tetrakisacetoxymethyl ester
BAPTA AM; 1–10 mM, a membrane-permeable deriva-
21
ng ml was markedly inhibited by concurrent application tive of a potent Ca
chelator BAPTA, which is expected
21
of 5 mM nicardipine Fig. 3B. We also used v-conotoxin to diminish increases in cytoplasmic free Ca
. A previous GVIA, a selective N-type VDCC blocker [37], to examine
study by others [16] have demonstrated that treatment with if N-type VDCC activity is involved in the actions of
BAPTA AM at 5 mM effectively prevents PC12 cell death
21
bFGF. v-Conotoxin GVIA 1 mM had no significant caused by disruption of intracellular Ca
homeostasis. We influences on the effects of bFGF, i.e. promotion of
found that co-application of BAPTA AM with bFGF
244 H
attenuated the effects of bFGF on neuronal survival Fig. 4A and neurite branch formation Fig. 4B. Interestingly,
BAPTA AM was also effective in attenuating the increase in the number of calbindin-expressing cells by bFGF Fig.
4A.
21
We next questioned whether Ca influx via L-type
VDCCs is sufficient for the promotion of neuronal survival and formation of neurite branches. To activate L-type
VDCCs, we utilized culture medium containing a high
1
level of K 24.2 mM instead of 4.2 mM in normal
medium, and Bay K 8644 5 mM, an L-type VDCC agonist. Bay K8644 at 1 mM has been shown to mimic the
1
effect of high K depolarization in olfactory neurons [5].
1
Treatment with high K medium alone, or in combination
with Bay K8644, had no significant effect on neuronal
1
survival Fig. 5A, hatched bars. The effect of high K and
Bay K8644 was also examined on the number of calbin-
Fig. 5. The effect of VDCC activation on neuronal survival, calbindin expression and neurite branching. A Bay K8644 5 mM was simul-
1
taneously applied with medium containing 4.2 normal or 24.2 mM K to high-density hippocampal cell cultures at 1 DIV. At 4 DIV, the numbers
of total neurons hatched bars and calbindin-positive neurons open bars were determined. Neither of these treatments had significant effects in the
number of total neurons and calbindin-expressing neurons. B Bay K8644 5 mM was simultaneously applied with medium containing 4.2
1
or 24.2 mM K to low-density cell cultures at 2 DIV. At 2 DIV open
Fig. 4. The effect of BAPTA AM on the actions of bFGF. A bFGF 5 bars and 4 DIV hatched bars, the number of branch points along the
ng ml and BAPTA AM at indicated concentrations were simultaneously longest neurite was counted. Treatment with Bay K8644 plus high K
applied to high-density hippocampal cell cultures at 1 DIV. At 4 DIV, the resulted in a significant increase in the number of neuritic branches.
numbers of total neurons hatched bars and calbindin-positive neurons P,0.05 vs. control. C The effects of drugs on neurite branch
open bars were determined. Increases in total and calbindin-expressing formation stimulated by direct activation of VDCCs. Nicardipine nicar.;
neurons by bFGF were significantly attenuated by BAPTA AM. P, 5 mM, U73122 U; 100 nM or forskolin fors.; 100 mM was applied to
[[ 1
0.01 vs. no drug treatment 0 mM BAPTA AM; P,0.01 vs. bFGF
low-density cell cultures at 2 DIV, simultaneously with high K 24.2
alone. B bFGF 1 ng ml and BAPTA AM 1 mM were simultaneouly mM plus Bay K8644 5 mM. At 2 DIV open bars and 4 DIV hatched
applied to low-density cell cultures at 2 DIV. At 2 DIV open bars and 4 bars, the number of branch points along the longest neurite was counted.
1
DIV hatched bars, the number of branch points along the longest neurite Only nicardipine was effective in blocking the effect of high K plus Bay
[[ 1
was counted. BAPTA AM blocked the effect of bFGF. P,0.01 vs. K8644. P,0.01 vs. control cont.;
P,0.01 vs. high K plus Bay
[[
control; P,0.01 vs. bFGF alone.
K8644 alone.
H . Katsuki et al. Brain Research 885 2000 240 –250
245
1
din-expressing neurons. High K treatment marginally
increased the number of calbindin-expressing neurons, but its effect was not altered by co-application of Bay K8644
Fig. 5A, open bars. On the other hand, Bay K8644 or
1
high K alone induced a modest increase in the number of
branches of the longest neurite, and the combination of these treatments significantly increased the number of
neurite branches Fig. 5B. Increase in neurite branching
1
by high K plus Bay K8644 was almost entirely blocked
by co-application of 5 mM nicardipine Fig. 5C, indicat- ing that the activation of L-type VDCCs is crucial for this
21
effect. Taken together, these results suggest that Ca influx via L-type VDCCs is sufficient for the acceleration
of neurite branching but not for the promotion of neuronal survival or the increase in the number of calbindin-ex-
pressing cells.
3.3. Signaling mechanisms in the effects of bFGF We next examined potential involvement of various
intracellular signal transduction pathways in the effects of bFGF. U73122, a broad spectrum inhibitor of PLC, was
used for possible counteraction against the effect of bFGF. U73122 effectively inhibits PLC activity at concentrations
of 0.1–1 mM [30]. When U73122 1 mM was tested on the survival of hippocampal neurons in high density
cultures, the effect of bFGF 5 ng ml was not blocked by this agent Fig. 6A, hatched bars. In the same cultures,
Fig. 6. The effect of U73122 on the actions of bFGF. A bFGF 5
increase in calbindin-expressing neurons by bFGF 5 ng
ng ml and U73122 1 mM were simultaneously applied to high-density
ml was not inhibited by U73122 1 mM either. Rather,
hippocampal cell cultures at 1 DIV. At 4 DIV, the numbers of total neurons hatched bars and calbindin-positive neurons open bars were
the increase in the number of calbindin-expressing neurons
determined. Increase in total surviving neurons was not affected by
was significantly potentiated Fig. 6A, open bars. In
U73122, whereas the increase in calbindin-expressing cells was sig-
contrast, promoting effect of bFGF 1 ng ml on neurite
[[
nificantly augmented by U73122. P,0.01 vs. control; P,0.01 vs.
branch formation was markedly attenuated when U73122
bFGF alone. B bFGF 1 ng ml and U73122 100 nM were simul-
100 nM was applied concurrently with bFGF Fig. 6B,
taneously applied to low-density cell cultures at 2 DIV. At 2 DIV open bars and 4 DIV hatched bars, the number of branch points along the
while U73122 alone had no significant influences on the
longest neurite was counted. U73122 significantly attenuated the effect of
number of branches of the longest neurite. The same
[[
bFGF. P,0.01 vs. control; P,0.01 vs. bFGF alone.
concentration 100 nM of U73122 did not inhibit the formation of neurite branches stimulated by co-application
1
of high K 24.2 mM and 5 mM Bay K8644 Fig. 5C.
Several lines of evidence suggest that protein kinase C We also tested possible counteraction by wortmannin
plays a role in receptor tyrosine kinase signaling [4]. 100 nM and AFC 20 mM of the effect of bFGF.
Accordingly, we examined the effects of agents conven- Wortmannin is a potent and selective inhibitor of PI
tionally used for manipulation of protein kinase C ac- 3-kinase [42], which is reported to inhibit the survival-
tivities. Co-application of 30–100 nM calphostin C, a promoting effect of insulin-like growth factor on cerebellar
potent inhibitor of protein kinase C with an IC of 50 nM
50
granule neurons at 10–100 nM, and almost completely [17], had no significant influences on the effect of bFGF on
block PI 3-kinase activity in the same preparation at 100 neuronal survival, calbindin expression and neurite branch
nM [7]. AFC inhibits Ras-mediated signal transduction formation data not shown. On the other hand, application
through the inhibition of carboxyl methylation of the of 1 mM phorbol-12-myristate-13-acetate PMA, a potent
C-terminal cysteine residue of Ras proteins [34], and is activator of protein kinase C, largely attenuated the effect
previously demonstrated to inhibit the enhancement by of 5 ng ml bFGF on neuronal survival Fig. 8A. Accele-
21
bFGF of VDCC-mediated Ca responses in hippocampal
ration of neurite branching and stimulation of calbindin neurons at a concentration of 20 mM [15]. However,
expression by bFGF were not significantly inhibited by neither AFC Fig. 7 nor wortmannin not shown at-
co-application of PMA Fig. 8A,B. tenuated the effect of bFGF on neuronal survival, calbindin
Intracellular cyclic AMP-mediated events, including expression and neurite branching.
those via activation of protein kinase A, may either mimic
246 H
Fig. 7. The effect of AFC on the actions of bFGF. A bFGF 5 ng ml Fig. 8. The effect of PMA on the actions of bFGF. A bFGF 5 ng ml
and AFC 20 mM were simultaneously applied to high-density hip- and PMA 1 mM were simultaneously applied to high-density hippocam-
pocampal cell cultures at 1 DIV. At 4 DIV, the numbers of total neurons pal cell cultures at 1 DIV. At 4 DIV, the numbers of total neurons
hatched bars and calbindin-positive neurons open bars were deter- hatched bars and calbindin-positive neurons open bars were deter-
[[
mined. P,0.05; P,0.01 vs. control. B bFGF 1 ng ml and AFC mined. P,0.01 vs. control;
P,0.01 vs. bFGF alone. PMA blocked 20 mM were simultaneouly applied to low-density cell cultures at 2
the effect of bFGF on total neuron number, whereas it did not show DIV. At 2 DIV open bars and 4 DIV hatched bars, the number of
significant effect on calbindin expression. B bFGF 1 ng ml and PMA branch points along the longest neurite was counted. P,0.01 vs.
1 mM were simultaneouly applied to low-density cell cultures at 2 DIV. control. AFC had no significant influences on the effects of bFGF.
At 2 DIV open bars and 4 DIV hatched bars, the number of branch points along the longest neurite was counted. P,0.01 vs. control. PMA
had no significant influence on the effect of bFGF on neurite branch
or inhibit the effects of growth factor receptor activation
formation.
[35,41]. Therefore, we tested the effect of an adenylyl cyclase activator, forskolin, either alone or on the effect of
bFGF. Our previous study has demonstrated that forskolin give insight into the mechanisms involved in multiple
at 50 mM markedly attenuates the effect of bFGF on cellular consequences such as promotion of survival,
VDCC responses [15]. Forskolin 100 mM alone had no enhancement of calbindin expression and acceleration of
significant influences on neuronal survival, calbindin ex- neuritic branching. Our results indicate differential in-
pression or neurite branch formation. Application of volvement of VDCC activities in different bFGF actions.
forskolin, however, blocked the effect of bFGF on neuro- Three kinds of bFGF actions also differ from each other
nal survival and neurite branch formation Fig. 9. In with respect to the intracellular signaling pathways in-
contrast, forskolin 100 mM did not attenuate the effect of volved.
1
high K 24.2 mM plus Bay K8644 5 mM on neurite
branch formation Fig. 5C. The increase in calbindin- 4.1. Multiple biological effects of bFGF
expressing cells by bFGF was not affected by co-applica- tion of forskolin Fig. 9A, open bars.
Examination with anti-GABA antibody revealed that serum deprivation caused selective decrease of GABA-
negative neurons in high cell density cultures, since the
4. Discussion number of GABA-positive neurons did not decline during
