(Received for publication, November 8, 1995; and in revised form, January 26, 1996)
From the
Nerve growth factor and basic fibroblast growth factor bind to
and activate receptor tyrosine kinases, causing sequential signaling
via the p21/extracellular signal-regulated
kinase pathway. The necessity and sufficiency of this signaling pathway
in transducing neuronal differentiation have been tested in the PC12
cell model. Although necessary for morphological changes, the
sufficiency of p21
-mediated signaling in these
events has come into question. We report that growth factor induction
of voltage-gated calcium channels, a hallmark of physiological
differentiation, also requires p21
-mediated
signaling, but cannot be driven by p21
activation alone. Thus, constitutive expression of the
dominant negative N17
mutant blocks growth
factor-induced increases in
-conotoxin GVIA-sensitive,
nimodipine-sensitive, and
-conotoxin GVIA/nimodipine-resistant
calcium currents, but it does not block sodium current induction.
However, manipulations that produce sustained activation of the
p21
signaling pathway and the neurite extension
characteristic of morphological differentiation fail to increase
calcium channel current densities. These results indicate the existence
of distinct signaling requirements for morphological and physiological
differentiation and further emphasize the importance of
p21
-independent signaling pathways in growth
factor-mediated neuronal development.
Key features of the developing neuronal phenotype include
process extension and ion channel expression, whereby cells establish
the synaptic contacts and the electrophysiological repertoire necessary
for intercellular signaling. Coordination of morphological and
physiological development essential to the proper wiring of the nervous
system is controlled in part by growth factors such as NGF ()and bFGF. An excellent model system for studying NGF- and
bFGF-induced neuronal differentiation is the well characterized PC12
pheochromocytoma cell line. In response to NGF or bFGF, PC12 cells
carry out a developmental program representative of a maturing neuron,
in which they cease cell division, extend neurites, and become
electrically excitable(1, 2, 3, 4) .
The acquisition of electrical excitability is due to an increase in the
number of voltage-gated sodium and calcium
channels(5, 6, 7, 8, 9, 10, 11, 12, 13) .
Although the bFGF- and NGF-activated signaling pathways responsible for
morphological differentiation are beginning to be understood, little is
known about the growth factor-activated pathways that govern
physiological differentiation. Indeed, a key question remains as to
whether or not the pathways identified with morphological changes are
necessary and sufficient for physiological differentiation,
particularly ion channel expression.
The signal cascade responsible
for growth factor-induced morphological differentiation in PC12 cells
is the same ubiquitous pathway activated by a number of mitogenic and
differentiating growth factors. Thus, NGF and bFGF bind to and activate
receptor tyrosine kinases, which in turn cause the sequential
activation of the membrane-associated GTP-binding protein
p21, followed by B-Raf kinase, Mek-1 kinase, and
the extracellular signal-regulated kinases ERK1 and
ERK2(14, 15, 16, 17, 18, 19, 20, 21) .
In PC12 cells, sustained activation of the
p21
/ERK pathway by NGF and bFGF is suggested to
be necessary for cessation of cell division and commitment to
morphological
differentiation(21, 22, 23, 24) ;
however, the sufficiency of this pathway in driving morphological
differentiation is now in question(25) . In contrast, EGF
stimulation of proliferation, rather than differentiation, is thought
to result from more transient activation of the same
p21
/ERK pathway by the EGF receptor tyrosine
kinase(22, 26, 27) . Only when the EGF
receptor is overexpressed in PC12 cells does EGF stimulation result in
morphological differentiation that is accompanied by the sustained
activation of the p21
/ERK pathway(23) .
Even though p21
/ERK stimulation is required in
both proliferative and differentiating scenarios, it seems inadequate
to explain all aspects of the mature neuronal phenotype. For instance,
NGF- and bFGF-induced expression of sodium channels, essential for
action potential initiation, is independent of
p21
(28, 29) . And although NGF-
and bFGF-induced calcium channel expression shifts PC12 cells to a more
-CoTX-sensitive (i.e. mature neuron-like) secretory
phenotype(12, 30) , the necessity or sufficiency of
p21
/ERK in this process remains unresolved.
Growth factor-induced PC12 differentiation is an applicable model
for synaptic maturation in the developing nervous system, and
therefore, it is important to specify the signals responsible for
driving both the morphological and physiological aspects of this
process. To understand the coordination and divergence of these
processes, we are examining the relationships between the growth
factor-activated pathways governing neurite outgrowth as well as
expression of calcium and sodium channels. We report here that NGF or
bFGF induction of calcium channels, particularly those sensitive to
-CoTX, requires p21
as a signal
transducer. However, activation of p21
(and by
implication, ERKs) alone is not sufficient to explain growth
factor-induced functional expression of calcium channels. Thus, in the
context of neuronal differentiation, there appears to be a continuum of
signaling requirements for growth factor-induced morphological and
physiological responses. On one hand, p21
/ERK
signaling is required and may or may not be sufficient for
morphological differentiation, while sodium channel expression is
independent of p21
. Between these two different
requirements for p21
is calcium channel
induction, which we show to be dependent on p21
,
but for which p21
is not sufficient. Therefore,
although activation of the p21
/ERK pathway is
critical to morphological neuronal differentiation, it is clear that
other signals are required for complete development of the mature
physiological phenotype.
Nimodipine (Research
Biochemicals Inc., Natick, MA) was prepared fresh daily as a 5 mM stock solution in ethanol and diluted in bath solution immediately
prior to use. -CoTX (Bachem Bioscience, Philadelphia) was
prepared as a 0.5 mM stock solution in distilled water, and
aliquots were stored at -30 °C. A fresh aliquot was diluted
in the bath solution for each experiment. Drugs were pressure-applied
from blunt-tipped, fire-polished micropipettes positioned
5-10 mm from the cell.
Figure 1:
Calcium channel current-voltage
relationships are identical among wild-type PC12 cells and PC12 cells
constitutively expressing either the neomycin resistance marker gene
(Z-1) or the neomycin resistance gene and the dominant negative
N17mutant(17-2). A whole cell patch clamp was
used to record calcium channel currents with external 10 mM barium as the charge carrier and with external 135 mM tetraethylammonium and internal 150 mM CsCl to suppress
potassium currents. Cells were held at -90 mV, and 50-ms command
steps to the indicated voltages were given sequentially at 5-s
intervals. Current records were filtered at 5 kHz and leak-subtracted
with a standard P/N procedure. Peak inward current amplitudes are
plotted against command step voltages.
In addition, each of the cell lines exhibits the same three
pharmacologically distinct currents, a -CoTX-sensitive component,
a DHP-sensitive component, and a component that is resistant to these
compounds. Serial application of the calcium channel antagonists
-CoTX and DHP (nimodipine) (Fig. 2) was used to measure
the relative amount of each current component(12) . As an
indirect measure of channel density for each current, whole cell
current amplitudes for each component have been normalized to membrane
capacitance and are expressed as current densities. Again, all of the
cell lines are remarkably similar in the relative densities of each of
the three component currents in the absence of growth factor treatment (Fig. 3; see Fig. 3of (12) for wild-type data).
Inactivation time constants for the individual current components are
also similar (see below). Therefore, in terms of the densities and
types or properties of calcium channels expressed in the absence of
growth factors, there are no differences between wild-type PC12 cells
and PC12 cells expressing the neomycin resistance marker gene or the
marker gene and the dominant negative N17
mutant.
Figure 2:
Wild-type, Z-1, and 17-2 PC12 cells
similarly express at least three pharmacologically distinct calcium
channel currents. Pharmacologically distinct calcium channel currents
were distinguished by sequential application of 5 µM -CoTX and 5 µM nimodipine. The remaining
current was defined as
-CoTX/nimodipine-resistant current. To
facilitate comparison of records, all currents are scaled to similar
size (note scale bar). Single exponential fits to current decays
(Simplex optimization algorithm with user defined start parameters)
were used to obtain inactivation time constants (
). Growth factor
treatment did not affect current kinetics in any of these three cell
lines, although there were increases in the relative densities of all
current components in wild-type and Z-1 cells (see Fig. 3).
Recording procedures were identical to those described for Fig. 1, except that current records were refiltered at 2 kHz for
display.
Figure 3:
Increases in calcium channel current
densities due to chronic growth factor treatment of PC12 cells are
inhibited in cells(17-2) expressing the dominant negative
N17 mutant. Cumulative data are shown for
calcium channel current densities in PC12 cells constitutively
expressing either the neomycin resistance marker (Z-1) or the neomycin
resistance marker and the dominant negative N17
mutant(17-2). Cells were grown with (NGF or bFGF) or without
(untreated) growth factors for 5-8 days. Whole cell calcium
channel current recordings and pharmacological dissection of the
component currents with
-CoTX and nimodipine were performed as
described for Fig. 1and Fig. 2. Individual component
currents were normalized to cell membrane capacitance as described
under ``Experimental Procedures.'' Each column represents the
mean ± S.E. of current densities taken from 6 to 33 cells. *,
significant difference from the untreated condition assessed by
Student's t test at p < 0.05. Because not
every cell was tested with both
-CoTX and nimodipine, total
current density may not exactly equal the sum of the three
pharmacologically distinct current
components.
Unlike wild-type PC12 cells, however, 17-2 PC12 cells fail to
respond to growth factors with increased calcium channel current
densities (Fig. 3). Total current density in untreated 17-2
cells (23.4 ± 2.3 pA/pF, mean ± S.E., n =
30) does not significantly increase after 5-8 days of treatment
with NGF or bFGF (18.9 ± 1.2 pA/pF, n = 30; and
28.1 ± 3.8 pA/pF, n = 15, respectively).
Moreover, bFGF or NGF treatment does not change the densities of any of
the three component calcium channel currents in 17-2 PC12 cells. The
densities of the -CoTX-sensitive, DHP-sensitive, and
-CoTX/DHP-resistant components in untreated 17-2 cells are 8.1
± 1.2 pA/pF (n = 14), 9.2 ± 1.6 pA/pF (n = 9), and 7.8 ± 1.9 pA/pF (n = 9), respectively, compared with 8.2 ± 1.0 pA/pF (n = 13), 4.9 ± 0.6 pA/pF (n =
13), and 10.0 ± 1.7 pA/pF (n = 7) with NGF and
10.2 ± 1.7 pA/pF (n = 12), 7.5 ± 1.9
pA/pF (n = 6), and 12.4 ± 4 pA/pF (n = 6) with bFGF. At this point, we do not know the reason
for the small but statistically significant decrease in DHP-sensitive
current in 17-2 cells treated with NGF. NGF also fails to increase
total and
-CoTX-sensitive calcium channel current densities in a
second PC12 line(17-26) expressing N17
(data not shown).
As has been reported previously(18, 31) , we observed
that expression of N17
blocks stimulation of neurite
outgrowth by both bFGF and NGF.
We found that Z-1 cells, the 17-2
counterpart cell line expressing only the neomycin resistance marker,
respond to application of NGF or bFGF in the same manner as wild-type
PC12 cells, with increased functional calcium channel expression (Fig. 3). After 5-8 days of treatment with NGF or bFGF,
the total calcium channel current density in Z-1 cells increases
>3-fold, from 20.3 ± 2.2 pA/pF (mean ± S.E., n = 33) to 62.8 ± 3.3 pA/pF (n = 21)
and 70.3 ± 7.7 pA/pF (n = 33), respectively. The
-CoTX-sensitive current component increases from 6.1 ± 1.1
pA/pF (n = 16) to 32.8 ± 2.8 pA/pF (n = 11) and 28.8 ± 4.7 pA/pF (n = 6)
for NGF- and bFGF-treated Z-1 cells, respectively. Compared with
wild-type PC12 cells, Z-1 cells respond to growth factors with
relatively larger increases in the
-CoTX/nimodipine-resistant
current component. NGF and bFGF increase this component from 9.1
± 0.1 pA/pF (n = 8) to 30.4 ± 3.2 pA/pF (n = 7) and 38.8 ± 7.3 pA/pF (n = 6), respectively. Like wild-type cells, Z-1 cells also
show a small but statistically significant increase in the
nimodipine-sensitive current component. Thus, the growth factor-induced
increases for both total and
-CoTX-sensitive calcium channel
current densities in Z-1 PC12 cells are similar to those reported for
wild-type PC12
cells(10, 11, 12, 32) . Z-1 cells
also extend neurites in response to NGF or bFGF. These results and
those from the two cell lines expressing the dominant negative
N17
mutant and the neomycin resistance marker suggest
that expression of N17
specifically inhibits growth
factor-induced increases in calcium channel functional expression in
PC12 cells.
It seems unlikely that the failure of growth factors to
increase calcium current densities in cells expressing N17 could be due to clonal selection of cells lacking the appropriate
growth factor-sensitive calcium channel types. As shown above, based on
threshold voltage for activation, voltage for maximal current, and
pharmacological sensitivities, composite whole cell calcium channel
currents are identical among untreated wild-type, 17-2, and Z-1 PC12
cells. Also, untreated cells from each of the lines express, in roughly
equal amounts, currents sensitive to either
-CoTX or nimodipine
and a current resistant to both drugs.
To kinetically describe
individual calcium channel current components, we have applied
-CoTX and then digitally subtracted the remainder current from
the initial current to obtain ``pure''
-CoTX-resistant
and
-CoTX-sensitive component currents. Inactivation time
constants were then determined for each component (Fig. 2). In
wild-type cells, inactivation kinetics of the
-CoTX-sensitive and
-CoTX-resistant currents observed for untreated cells (mean
= 122 and 913 ms, n = four cells) are
essentially unchanged by growth factor treatment (
= 153
and 950 ms, n = four cells), suggesting that there is
an increase in channel numbers, but no change in channel
types(12) . This result was confirmed for Z-1 and 17-2 cells,
and among all three lines, inactivation time constants for these two
current types are comparable both with and without growth factor
treatment. The inactivation time constants in untreated Z-1 and 17-2
cells for
-CoTX-sensitive (
= 125 and 135 ms, n = three to eight cells throughout) and
-CoTX-resistant (
= 890 and 879 ms) currents are
unaffected by either NGF (
= 150 and 167 ms for Z-1 and
17-2
-CoTX-sensitive currents;
= 914 and 811 ms for
-CoTX-resistant currents) or bFGF (
= 156 and 139 ms
and
= 799 and 907 ms). Thus, each cell type under all
treatment conditions has similar current components, and in each, the
-CoTX-sensitive component inactivates much faster than the
-CoTX-insensitive components. In this regard, our results are
consistent with other studies on wild-type PC12 cells (6, 10, 11, 32) . However, unlike
Garber et al.(6) , but in agreement with
others(10, 11, 32) , we did not observe the
presence of a low threshold or ``T''-type calcium channel
current under any condition.
Figure 4:
Increases in sodium channel current
densities due to chronic growth factor treatment of PC12 cells are not
inhibited by the dominant negative N17 mutant.
Cells were grown with (NGF or bFGF) or without (untreated) growth
factors for 5-8 days. At the top are shown individual sample
records for whole cell sodium channel currents in PC12 cells
constitutively expressing either the neomycin resistance marker (Z-1)
or the neomycin resistance marker and the dominant negative
N17
mutant(17-2). Cells were held at -90
mV, and command steps to the indicated voltages were given sequentially
at 1.5-s intervals. Current records were filtered at 5 kHz and
leak-subtracted with a standard P/N procedure. Cumulative data for
whole cell currents normalized to cell membrane area are shown in the
column graph at the bottom. Each column represents the mean ±
S.E. of current densities taken from 10 to 15 cells. *, significant
difference from the untreated condition assessed by Student's t test at p < 0.05.
First, cells
transfected with a constitutively active form of p21,
under the control of the inducible murine mammary tumor virus promoter
(GSRas1)(28) , were treated with 1 µM dexamethasone for 3 days. Whole cell calcium channel current
densities were then recorded from those cells with neurites longer than
two-cell diameters, i.e. cells that showed clear morphological
differentiation, and these densities were compared with those from
cells that were chronically treated with 0.01% dimethyl sulfoxide
vehicle alone. Expression of exogenous ras transcript occurs
within hours after start of dexamethasone treatment(28) , and
robust neurite outgrowth is observed within the first day of treatment.
However, the densities of the
-CoTX-, nimodipine-, and
-CoTX/nimodipine-resistant currents (5.6 ± 1.4 pA/pF, n = 17; 4.9 ± 1.4 pA/pF, n = 9;
and 6.8 ± 1.6 pA/pF, n = 9, respectively),
recorded from cells treated with dimethyl sulfoxide alone, were not
significantly affected by growth in 1 µM dexamethasone
(6.4 ± 0.8 pA/pF, n = 15; 3.9 ± 0.7
pA/pF, n = 5; and 3.6 ± 0.9 pA/pF, n = 5, respectively). This is reflected by the lack of change
in total calcium current density after dexamethasone treatment (Fig. 5). In wild-type PC12 cells, NGF application activates
endogenous p21
within minutes(27) . In one
experiment with wild-type PC12 cells, we found that after 24 h of NGF
treatment, mean total calcium current density was unchanged (19.2
± 3.4 pA/pF (n = 3) compared with 17.3 pA/pF (n = 9)), but after 48 h of treatment, current density
had increased to 36.2 ± 7.5 pA/pF (n = 7) (in
cells with at least one neurite of length greater than one-cell
diameter). Although this change in current density is still
statistically insignificant, it suggests that growth factor induction
of calcium channels is well underway within 48 h. Thus, it seems
reasonable to conclude that had induction of activated p21
been sufficient to induce calcium channels, then increased
current density would have been observed at 3 days after start of
dexamethasone treatment, especially as p21
is
overexpressed when driven by the murine mammary tumor virus promoter.
The failure of p21
induction to up-regulate calcium
channel density in GSRas1 cells could not be attributed to some
suppressive action of the dexamethasone treatment itself since in
GSRas1 cells treated for 6 days with NGF and dexamethasone, calcium
channel density increased to a level (72.5 ± 8.0 pA/pF, n = 8) comparable to that observed with NGF treatment alone.
Figure 5:
Induced expression of activated
p21 (GSRas1 + dexamethasone) or sustained
stimulation of endogenous p21
(v-Crk + EGF)
fails to increase calcium channel current densities in PC12 cells.
Cumulative data are shown for calcium channel current densities in two
different PC12 cell lines. One expresses activated p21
under the control of the murine mammary tumor virus promoter
(GSRas1), and the other expresses v-Crk, an oncogenic adaptor protein
that allows sustained p21
/ERK activation and
morphological differentiation in response to EGF receptor stimulation. A, 3-day application of 1 µM dexamethasone (Dex) to GSRas1 cells (recordings from 29 cells) produced
sustained activation of the p21
/ERK signaling
pathway(28) , but failed to increase calcium channel current
density relative to 0.01% dimethyl sulfoxide-treated vehicle controls (n = 38 cells). Exposure of these cells to NGF (100
ng/ml for 6 days) and dexamethasone produced normal increases in
calcium channel current density (n = eight cells),
indicating that dexamethasone itself does not suppress calcium channel
induction. B, 5-7-day application of EGF (25 ng/ml) to
v-Crk cells also did not significantly increase calcium current density (n = 18 cells) relative to water-treated vehicle
controls (n = 11 cells). However, these cells responded
to treatment with NGF (100 ng/ml for 6 days) with significant increases
in calcium channel current densities (n = five cells).
Whole cell calcium channel current recordings and normalization to
whole cell capacitance were performed as described for Fig. 1and 2. Each column represents the mean ± S.E. of
current densities. Activation of p21
by
dexamethasone (A) or EGF (B) produced no significant
differences in calcium current densities (Student's t test at p < 0.05).
We used a second approach to further rule out the possibility that
dexamethasone suppressed the induction of calcium channels, as has been
observed for some other NGF-inducible events(33) , and to see
if p21 stimulation alone was sufficient to induce calcium
channel up-regulation. Application of EGF to PC12 cells stably
transfected with v-Crk, an oncogenic adapter protein similar in
function to the p21
guanine nucleotide exchange
factor-docking protein Grb2, results in sustained activation of
endogenous p21
and the downstream mitogen-activated
protein kinase pathway(34, 35, 36) . As a
result, in cells expressing v-Crk, EGF induces morphological
differentiation. PC12 cells expressing v-Crk were grown in the presence
of EGF for 5-7 days, sufficient time to produce robust neurite
outgrowth. In cells with at least one neurite greater than two-cell
diameters in length, there was a small but statistically insignificant
increase in calcium channel current density compared with
v-Crk-expressing cells grown in the absence of EGF (Fig. 5). As
a positive control, these cells were treated with NGF (for 6 days), and
they responded with a significant increase in calcium channel current
density (64.0 ± 20 pA/pF, n = 5). Therefore,
this experiment and the experiment with expression of activated
p21
suggest that p21
signaling alone, even
if it is sustained, is insufficient to increase calcium channel current
density in PC12 cells.
Our results show that NGF or bFGF induction of a 3-fold
increase in total calcium channel current density in PC12 cells is
dependent on signaling via p21. Growth factor treatment
induces p21
-dependent differential increases in each of
three pharmacologically distinct calcium current components. There is a
5-fold increase in the
-CoTX-sensitive current, a 1.5-fold
increase in the DHP-sensitive current, and a 4-5-fold increase in
the
-CoTX/DHP-resistant current, yet all of these increases are
blocked in two PC12 cell lines expressing dominant negative N17
(17-2 and 17-26). Constitutive expression of N17
does not appear to affect the types or properties of calcium
channels expressed in PC12 cells either in the absence or presence of
NGF or bFGF treatment. Also, our observation, in agreement with Fanger et al.(29) , that increases in sodium channel density
are unaffected in N17
-expressing cells suggests that the
dominant negative mutant block of calcium channel induction is
specific, i.e. other signaling events driven by NGF and bFGF
receptors remain intact in cells expressing dominant negative
p21
. It could be argued that functional expression of
calcium channels is dependent on their insertion into actively growing
membrane and that N17
inhibition of calcium channels is
secondary to its inhibition of neurite outgrowth. However, this is
unlikely since NGF induction of calcium channels is unaffected by
growing cells in suspension, a condition that prevents neurite
extension(11) .
Although we have shown that p21 is necessary for induction of calcium channels by growth factors,
it appears that p21
signaling alone, even if it is
sustained, is not sufficient to mediate this action. Induction of
oncogenic p21
with dexamethasone and EGF stimulation of
PC12 cells expressing the oncogenic adaptor protein v-Crk both produce
sustained activation of the p21
/ERK pathway as well as
neurite extension(28, 36) . However, we do not observe
calcium channel induction in either case. These results and those
described above suggest that the functional expression of calcium
channels in response to NGF and bFGF requires activation of the
p21
signaling pathway, but that other
p21
-independent signaling events are also necessary.
The identity of the p21-independent pathways required
for calcium channel induction may lie in those
p21
-independent pathways implicated in morphological
differentiation of PC12 cells. Trk receptor activation and subsequent
autophosphorylation on critical tyrosines is required for the
association and activation of phospholipase C-
,
phosphatidylinositol 3-kinase, and
SHC(37, 38, 39) , and phospholipase C-
and SHC appear to be necessary for growth factor-induced morphological
differentiation, while phosphatidylinositol 3-kinase is
not(38, 39) . In addition, non-receptor tyrosine
kinases such as c-Src, c-Yes, and Fyn may be activated by growth
factors that promote neuronal
differentiation(25, 40) . Infection of PC12 cells with
v-src seems to be sufficient for induction of calcium
channels(41) , but not sodium channels(28) , but this
does not preclude the necessity of Src family members for sodium
channel induction. Subsequent studies using mutant Trk
receptors(39) , mutant platelet-derived growth factor receptors
expressed in PC12 cells(25) , and antisense application (42) may elucidate whether or not phospholipase C-
, SHC,
phosphatidylinositol 3-kinase, or one of the members of the Src
non-receptor tyrosine kinase family mediates calcium and sodium channel
induction.
p21/ERK-mediated signaling appears to be
necessary for growth factor-induced morphological differentiation, but
different experimental paradigms have produced conflicting results
regarding the sufficiency of p21
/ERK signaling for
neurite outgrowth(24, 25) . Similarly, we have shown
that p21
signaling alone is insufficient to produce an
important component of physiological differentiation, functional
expression of a sympathetic neuron-like calcium channel phenotype.
Thus, the observations that process extension, calcium channel
induction, and sodium channel induction (43) depend to varying
degrees on p21
lend credence to the idea that distinct
signal pathways mediate different aspects of neuronal differentiation.
Furthermore, it suggests that sustained activation of the
p21
/ERK pathway may not explain all aspects of neuronal
differentiation produced by NGF or bFGF. The ability of cells to
independently regulate sodium channel induction, calcium channel
induction, and neurite outgrowth suggests that process extension can
occur in the absence of events dependent on electrical activity.
Additional molecules in the extracellular matrix, such as L1 and
neuronal cell adhesion molecule, may activate the outgrowth of axons (44) , which, upon reaching their target neurons, are exposed
to cytokines and growth factors that cause increased expression of
sodium and calcium channels. The magnitude and timing of these events
could then be the critical determinants of synaptic efficacy and
ultimately survival.