Department of Pharmacology and University Centre for Neuroscience, University of Alberta, Edmonton, Alberta T6G 2H7, Canada
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ABSTRACT |
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Lei, Saobo,
William F. Dryden, and
Peter A. Smith.
Nerve Growth Factor Regulates Sodium But Not Potassium Channel
Currents in Sympathetic B Neurons of Adult Bullfrogs.
J. Neurophysiol. 86: 641-650, 2001.
The TTX-sensitive
and -resistant components of the voltage-gated
Na+ current (TTX-s
INa and TTX-r
INa) are increased within 2 wk of cutting the axons of B-cells in bullfrog paravertebral sympathetic ganglia (BFSG). Axotomy also increases the noninactivating,
voltage-activated K+ current (M current
IM), whereas delayed rectifier
K+ current (IK)
is reduced. We found that similar effects were produced when BFSG B
cells were dissociated from adult bullfrogs and maintained in a defined-medium, neuron-enriched, low-density, serum-free culture.
Thus the density of TTX-s INa, TTX-r
INa, and
IM were transiently increased, whereas
IK density was decreased. Reduction in
voltage-sensitive, Ca2+-dependent
K+ current (IC)
was attributed to previously documented decreases in
Ca2+ channel current
(ICa). To test whether axotomy- or
culture-induced changes in ion channel function reflect loss of
retrograde influence of nerve growth factor (NGF), we examined the
effect of murine -NGF on TTX-s INa,
TTX-r INa,
IK, and
IM. Culture of neurons for 15 days in
the presence of NGF (200 ng/ml), more than doubled total
INa density but did not enhance
neurite outgrowth. The TTX-r INa
density was increased about threefold and the TTX-s
INa density increased 2.4-fold. NGF
did not affect the activation or inactivation kinetics of the total
Na+ conductance. Effects of NGF were blocked by
the transcription inhibitors, cordycepin (20 µM) and actinomycin D
(0.01 µg/ml). IK and
IM were unaffected by NGF, and
although IC was enhanced, this likely
reflected the known effect of NGF on
ICa in BFSG neurons. Na+ channel synthesis and/or expression in
adult sympathetic neurons is therefore subject to selective
regulation by NGF. Despite this, the increase in
INa and
IM as well as the decrease in
IK seen in BFSG neurons in culture or
after axotomy cannot readily be explained in terms of alterations in
the availability of target-derived NGF.
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INTRODUCTION |
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Damage to the axon
of a peripheral or a central neuron promotes characteristic changes in
the morphological, electrophysiological, and biochemical properties of
the surviving cell body (Gordon 1983; Titmus and
Faber 1990
). We have used the B cells of bullfrog sympathetic
ganglia (BFSG) to understand the relationship between axotomy-induced
changes in specific ion channels and changes in action potential (AP)
shape and regeneration (Gordon et al. 1987
; Jassar et al. 1993
, 1994
; Kelly et al. 1986
,
1989
; Petrov et al. 2001
; P. S. Pennefather and P. A. Smith, unpublished data).
In these cells, axotomy increases spike width and spike height but
decreases the peak amplitude and duration of the afterhyperpolarization (AHP) that follows the AP (Gordon et al. 1987;
Kelly et al. 1986
). These changes, which take about a
week to develop, result from decreases in Ca2+
channel current (ICa) and consequent
attenuation of Ca2+-dependent
K+ conductances (Jassar et al. 1993
,
1994
; Petrov et al. 2001
; P. S. Pennefather
and P. A. Smith, unpublished results). Attenuation of
voltage-sensitive Ca2+-dependent
K+ current (IC)
may be secondary to the decrease in
ICa (Jassar et al.
1994
). The delayed rectifier K+ current
(IK) is also reduced by axotomy,
whereas the noninactivating K+ current (M current
IM) and the tetrodotoxin-sensitive
(TTX-s) and TTX-resistant (TTX-r) components of the
Na+ current
(INa) are increased (Jassar et
al. 1993
, 1994
).
ICa is also reduced in vivo following
injection of nerve growth factor (NGF) antibodies under the skin of the
thighs (Lei et al. 1997). This area of the skin contains
the mucous glands that are targets for most B-cell axons (Horn
et al. 1988
). These results are consistent with the hypothesis
that the retrograde influence of NGF is responsible for maintaining the
properties of Ca2+ channels in adult BFSG neurons
and that the response of Ca2+ channels to axotomy
reflects interruption of this retrograde signal. Further corroborative
evidence for this idea was recently obtained by reversibly
disconnecting BFSG B cells from their targets by means of an injection
of 6-hydroxydopamine. Degeneration of peripheral sympathetic terminals
was associated with attenuation of cell body
ICa, and only after recovery and
reestablishment of target contact did cell body
ICa recover (Petrov et al.
2001
). These results would be expected if
ICa declined as a consequence of the
loss of the retrograde influence of NGF. Its protracted recovery could
then be ascribed to the gradual restoration of the retrograde influence
of target-derived NGF.
When BFSG neurons are placed in defined-medium culture, the changes in
ICa resemble those initiated by
axotomy in vivo: the current is reduced and inactivation is increased.
Inclusion of NGF in the cultures reverses these changes (Lei et
al. 1997).
Three questions arise from these observations. First, does tissue
culture produce an increase TTX-s INa,
TTX-r INa and
IM in the same way as axotomy? Is
IC unchanged and
IK attenuated? If this is so, might
the culture system be a useful model for examining the role of
target-derived NGF or other growth factors in the maintenance of ion
channels in BFSG neurons? Second, is the NGF-induced increase in
ICa but one aspect of a general
improvement in the "health" of sympathetic neurons that were
previously cultured without appropriate neurotrophic support? If this
was the case, NGF might be expected to increase all types of
ionic currents in culture. Third, because
INa and
IM increase after axotomy when the
retrograde influence of NGF should be reduced (Jassar et al. 1993; Petrov et al. 2001
), might NGF exert a
tonic downregulation of INa and
IM in the cell bodies of adult BFSG B
neurons in vivo? If this was so, NGF might be expected to
inhibit rather than potentate INa and
IM in BFSG neurons in culture.
To test these possibilities, we examined the effect of NGF on the
properties of Na+ and K+
currents in BFSG neurons in defined-medium, serum-free,
neuron-enriched, low-density culture (Lei et al. 1997,
1998
). This system removes any influences of glial cells
(Assouline et al. 1987
), adhesion molecules, or
injury-induced inflammation that may be active in vivo. Although the
changes in currents seen in culture resembled those seen with axotomy,
addition of NGF did not restore IK to control levels and did not affect IM.
Thus changes in K+ currents seen in culture or
after axotomy must occur via NGF-independent mechanisms. Since NGF
potentiated rather than inhibited the TTX-s and TTX-r components of
INa, the axotomy-induced increase in
these current must also occur via an NGF-independent mechanism. Some of
these findings have been communicated to the Society for Neuroscience (Dryden et al. 1998
).
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METHODS |
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All experimental procedures were reviewed and approved by the University of Alberta Animal Welfare Committee. This committee maintains standards set forth by the Canadian Council for Animal Care.
Isolation and culture of BFSG neurons
Adult bullfrogs were killed by rapid decapitation and spinal
cord function terminated by pithing. The VIIIth, IXth, and Xth paravertebral sympathetic ganglia were removed from both sides of the
animal, and the cells were dissociated using trypsin and collagenase as
previously described (Selyanko et al. 1990). Neurons were used for acute recording within 2-10 h of plating or were maintained for
2 wk in serum-free, low-density, neuron-enriched, defined-medium culture (Lei et al. 1997
, 1998
). The
culture medium consisted of diluted L-15 medium (73%) supplemented
with 10 mM glucose, 1 mM CaCl2, 100 units/ml
penicillin, and 100 µg/ml streptomycin. The total yield of ganglion
cells from each frog was preplated into two or three 35-mm culture
dishes. After 1-2 h, most of the nonneuronal cells adhered to the
bottom of the dishes, and the nonadherent cells, which were primarily
neurons, were harvested, redistributed to 30 culture dishes (35 mm;
NUNCLON "Surface"; Nalge-Nunc International, catalog No. 150318)
and cultured in fresh medium (3 ml/dish) supplemented with 10 µM
cytosine arabinoside (Ara-C). Neurons were allowed to adhere directly
to the plastic bottom of the dishes. For the culture groups with NGF,
murine
-NGF was added to the culture medium to make a final
concentration of 200 ng/ml. The dishes were placed in a humidified
glass chamber and maintained at room temperature (22°C) for
15
days. The culture medium was changed daily.
Electrophysiology
Currents were recorded by whole cell patch-clamp methods using
an Axoclamp 2A amplifier in discontinuous, single-electrode voltage-clamp mode. Generally, external solutions were 250 mOsmol/kg, and internal solutions were 240 mOsmol/kg.
INa was evoked by a series of
depolarizing voltage commands from a holding potential of 80 mV
(except where otherwise stated). With low-resistance patch electrodes
(2-5 M
), it was possible to use high switching frequencies >30kHz
with high clamp gains (>8 to <30 mV/nA). The fidelity of the clamp
was confirmed by examining the voltage recording. Recordings from cells
where the voltage trace was slow to rise or distorted were discarded.
For recording INa, the solution in the
bath (external) contained (in mM) 97.5 NaCl, 20 TEA-Br, 4 MnCl2, and 2.5 Tris-Cl (pH 7.2), and the solution
inside the pipette (internal) contained (in mM) 103 CsCl, 9 NaCl, 5 TEA-Br, 2.5 Cs-HEPES, and 1 Cs-EGTA (pH 7.2) (Jones
1987
).
To study K+ currents, the internal solution
consisted of (in mM) 110 KCl, 10 NaCl, 2 MgCl2,
0.4 CaCl2, 4.4 EGTA, 5 HEPES, 10 D-glucose, 0.125 cyclic AMP, and 0.1 leupeptin (pH 7.2),
and total outward current was initially recorded in response to
depolarizing steps from a holding potential of 80 mV in an external
solution containing (in mM) 40 KCl, 2 CaCl2, 40 NMG-Cl, 2.5 Tris-Cl, 94 sucrose, and 10 D-glucose. After
replacing the preceding external solution with a solution containing
(in mM) 0.1 CdCl2, 40 KCl, 2 MgCl2, 40 NMG-Cl, 2.5 Tris-Cl, 94 sucrose, and 10 D-glucose, the voltage-sensitive,
Ca2+-activated K+
conductance (IC), which depends on the
influx of extracellular Ca2+ (Jassar et
al. 1994
), was blocked, and the remaining current was
attributable to IK (delayed
rectifier). IC was recorded from a
holding potential of
40 mV. Outward currents were activated in
response to depolarizing voltage commands and recorded in the absence
and presence of extracellular Cd2+.
IC was then derived by taking the
difference between the total outward current at each potential and
IK that persisted after Ca2+channels were blocked. 40 mM extracellular
K+ was used to limit the amplitude of
IC and
IK and thereby to facilitate voltage
control. For IC and
IK, leak subtraction was done by
applying 1/4 amplitude hyperpolarizing pulses, multiplying the
responses by four and addition.
IM was studied using an external
solution containing (in mM) 117 NaCl, 2 KCl, 2 MgCl2, 2 CaCl2, 5 HEPES/NaOH (pH 7.2), and 10 D-glucose and an internal
solution consisting of (in mM) 110 KCl, 10NaCl, 2 MgCl2, 0.4 CaCl2, 4.4 EGTA,
5 HEPES/KOH (pH 7.2), and 10 D-glucose
(Selyanko et al. 1990).
IM deactivation and activation were
studied following a series of hyperpolarizing steps from a holding
potential of
30 mV. At this voltage,
IK was inactivated (Adams et
al. 1982
) as was much of the N-type Ca2+
conductance (gCa,N) that contributes
to Ca2+ influx and hence to the activation of
IC. Leak currents were calculated by
linear regression of the current-voltage relationship between
110 and
80 mV. These voltages are more negative than the activation range for
IM, and leak current therefore
dominates (Adams et al. 1982
). Pure
IM at the voltages between
70 and
30 mV was obtained by subtracting the leak current (Jassar et
al. 1994
).
The petri dishes were superfused with external solution at a flow rate
of 2 ml/min. This allowed exchange of solutions within ~2 min. Input
capacitance (Cin) was calculated by
integrating the capacitative transient that accompanied a 10-mV
depolarizing command from 80 mV. Since membrane capacitance per unit
area is constant, current densities were expressed in terms of current per unit capacitance, i.e., nanoampere per picofarad or for smaller currents, picoampere per picofarad.
All data are presented as means ± SE, and Student's two-tailed t-test or ANOVA was used to assess statistical significance (P < 0.05). In graphs where no error bars are visible, the error bars are smaller than the symbols used to designate the data points. All chemicals were from Sigma, St. Louis, MO, except for NGF (2.5 s) and anti-NGF antibody, which were from Alomone Laboratories, Jerusalem, Israel, and Leibovitz's L-15 medium and penicillin-streptomycin antibiotics, which were from Gibco BRL.
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RESULTS |
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Increase of Na+ currents in culture and enhancement of this effect by NGF
Figure 1 illustrates typical
recordings of total INa from an
acutely dissociated cell (A), a cell maintained in defined
medium for 15 days (B), and a cell maintained in defined
medium supplemented with 200 ng/ml NGF for 15 days (C). Even
in the absence of NGF, total INa
increased after 15 days in culture. A further and pronounced increase
in current occurred when neurons were cultured in the presence of NGF
for 15 days. Currents were elicited by a series of 12-ms depolarizing
voltage commands from a holding potential of 80 mV in 10-mV steps.
INa and the underlying conductance
(gNa) activated quickly and had almost
completely inactivated at the end of the voltage command in acute cells
and in cells cultured for 15 days with or without NGF. Under all three
conditions, INa began to activate near
20 mV, and the peak currents generally occurred at +10 mV. Figure
1D shows the current-voltage relationship for total
INa from 23 acutely dissociated cells,
20 cultured cells in the absence of NGF for 15 days, and 20 cultured
cells in the presence of NGF for 15 days. This again illustrates that
INa recorded after 15 days in the
absence of NGF was significantly greater than that in acutely isolated
cells and that treatment of the cells with NGF for 15 days produced a
further increase in INa. Differences
were apparent at all test potentials and maximum total INa at +10 mV recorded in the presence
of NGF was significantly greater than the current recorded in its
absence (P < 0.01).
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Figure 1E shows the time course of the increase in total
INa as cells were maintained in
culture. There was a clear increase in current after 1 day in culture.
INa reached a peak of four times the
amplitude recorded from acutely isolated cells within the first 2 days
of culture (P < 0.01). Afterward, the current declined
gradually, but it was still 2.3 times larger that of the acutely
dissociated cells on the 15th day of culture (P < 0.01; Fig. 1E, ). NGF promoted further increases in
INa, but its onset of action was quite
slow. Only after 6 days was there a significant difference between the
amplitude of INa in cells cultured in
the presence and absence of NGF. All currents subsequently recorded in
the presence of NGF were significantly larger than those recorded in
its absence. By the 15th day, the current in the cells cultured with
NGF was 2.4 times larger than that of the cells cultured in the absence
of NGF (P < 0.01; Fig. 1E,
).
We have previously observed an increase in the size of BFSG B neurons,
as measured from their Cin, as they
are maintained in culture with or without NGF (Lei et al.
1997). Figure 1F shows the time course of the
increase in Cin for the cells used in
the present study. Cin did not change
within the first day of culture (P > 0.05). This
contrasted with INa, which more than
doubled on the first day (Fig. 1E). On the second day of
culture, Cin increased by only 39% in
contrast to the fourfold increase in the current (Fig. 1E).
The increase in INa therefore precedes the increase in Cin. At day 15, Cin was roughly doubled but NGF failed
to promote any further increase in cell size (Fig. 1F).
The effects of NGF on Na+ channels were further
studied by analysis of changes in current density. Figure 1G
() shows that the initial, transient peak of
INa seen in the absence of NGF was
more conspicuous when current density was used as an indicator because
on the second day of culture, there is far greater increase in
INa than in
Cin. The
INa density therefore reached a
maximum at this time but then declined until after 9 days; there was no statistical difference between the current density of the cultured cells and that of acutely dissociated cells. Addition of NGF to the
culture medium did not change the initial enhancement of the current
density, but it prevented the current density from declining with time
after the initial peak (Fig. 1G,
). By 15 days, current density in NGF-treated cells was 2.5 times larger than in untreated cells.
Although we used neuron-enriched, Ara-C-treated cultures, there is
evidence that neurotrophic factors can be produced by PC12 cells
(Gill et al. 1998) and by sympathetic neurons
(Korsching 1993
). We therefore wondered whether the
initial transient increase in INa
density seen after 2 days in culture in the absence of exogenous NGF
(Fig. 1G) may result from the presence of endogenous NGF or
some other neurotrophin in our system. To test for the involvement of
NGF, serum containing NGF antibody was included in the culture medium.
The control group was treated with serum containing a control Ig-G
antibody raised against nonneuronal protein. Anti-NGF (0.5 µg/ml)
inhibited only 34% of the enhanced current density on the second day
of culture (P < 0.05; Table 1). The current density of the cells
cultured with anti-NGF for 2 days was therefore still 71% higher than
that of the acutely dissociated cells (P < 0.05). The
antibodies therefore exerted only a very weak effect on the initial,
transient enhancement of INa that was
seen when BFSG neurons were placed in culture.
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NGF increases both TTX-sensitive and -insensitive components of INa
INa in BFSG B neurons consists of
a larger TTX-s component and a much smaller TTX-r component
(Jassar et al. 1993; Jones 1987
). NGF
induces TTX-r INa in PC12 cells
(Rudy et al. 1982b
) and increases expression of
-SNS
mRNA that codes for a TTX-r Na+ channel in DRG
neurons (Black et al. 1997
; Dib-Hajj et al.
1998
). By contrast, NGF decreases mRNA for one type of TTX-s
INa (sodium channel III;
-III) in
sensory neurons (Black et al. 1997
). We therefore
examined the effects of NGF on both TTX-s
INa and TTX-r INa in BFSG neurons.
Total INa was recorded in the normal external solution, and TTX-r currents were recorded in 1 µM TTX. The TTX-s component of INa was then derived by subtraction. Peak TTX-s INa density recorded after 15 days in NGF was about 2.4 times greater than after 15 days in culture alone (P < 0.01; Fig. 2A). A slightly stronger effect was seen on TTX-r current which was increased about 3 fold (P < 0.01; Fig. 2B). Even though NGF preferentially enhanced TTX-r current, the contribution of this component to the total INa was little changed; 15.8% of the total current was TTX-insensitive in the absence of NGF compared with 17.9% in its presence.
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Culture of neurons with or without NGF did not change the activation kinetics of total gNa
To study the effects of establishing cultures and the effects of
NGF on gNa activation kinetics, the
absolute currents evoked by a series of voltage steps were converted to
conductances by the equation gNa = INa/(Vm ENa), where
Vm is the command potential and
ENa is the Na+
equilibrium potential (+60 mV for
[Na+]o = 97.5 mM and
[Na+]i = 9 mM).
Conductance values were normalized and fitted to a Boltzmann equation
of the form g/gmax = {1 + exp[
(Vm
Va1/2)/ka]}
1,
where g is the conductance;
gmax, the maximal conductance;
Vm, the command potential;
Va1/2, voltage at which half activation is
achieved; ka, slope factor of
activation curve. Table 2 summarizes the
results fitted from 23 acutely dissociated cells, 20 cells cultured
without NGF for 15 days, and 20 cells cultured with NGF for 15 days.
There were no statistically significant differences among the values of
Va1/2 for the acutely dissociated cells,
cells cultured with and without NGF for 15 days, suggesting the voltage dependence of activation for those cells was not changed. Although the
slope factor (ka) for acutely
dissociated cells was statistically larger than that of the cells
cultured without NGF for 15 days (0.01 < P < 0.05)
and that of the cells cultured with NGF for 15 days (P < 0.01), there was no significant difference between the
ka value of the NGF-treated cells and
that of the cells cultured alone (P > 0.05). These
differences however had little influence on the overall Boltzmann fit
to the pooled data as the activation curves for acutely isolated cells
and cultured cells with or without NGF were almost superimposed (Fig.
2C).
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Culture of neurons with or without NGF did not change gNa inactivation kinetics
The voltage dependence of steady-state inactivation
("h plot"; Fig. 2D) was
obtained by clamping the neurons to a series prepulse potentials (from
70 to +10 mV) for 20 ms to allow inactivation to develop prior to the
application of a 12-ms test pulse to +10 mV. Currents recorded in
response to the test pulse were normalized to the maximal current and
then fitted by Boltzmann equation
I/Imax = {1 + exp[(V
Vi1/2)/ki]}
1
(where I is current; Imax
is maximal current; V is prepulse potential; Vi1/2 is voltage for half-maximal
inactivation reached, and ki is slope
factor of inactivation curve). Values of
Vi1/2 and
ki for each cell were obtained by
curve fitting. The results are also summarized in Table 2. There were
no significant differences for the values of
Vi1/2 and
ki among the acutely dissociated
cells, cultured cells with and without NGF for 15 days
(P > 0.05 for all the groups), suggesting that the
voltage dependence of inactivation has not been changed by culture with
or without NGF.
Inactivation time constants during 12-ms depolarizing voltage pulses
were obtained by single-exponential fitting to the decay of the
current. Inactivation rate (reciprocals of the time constants) is
plotted against voltage in Fig. 2E. Between 10 and +40 mV there is no significant difference in the inactivation rate among the
acutely dissociated cells, cultured cells without NGF for 15 days, and
cultured cells with NGF for 15 days (P > 0.05).
Transcription dependence of the effect of NGF on Na+ channels
The transcriptional inhibitors actinomycin D and cordycepin were used to test whether NGF-induced increases in INa involved changes in gene expression.
Inclusion of cordycepin (20 µM) or actinomycin D (0.01 µg/ml) did not significantly change the control INa density recorded after 12-15 days in culture. The inhibitors did, however, completely block the augmentation of current density induced by NGF. The results, which are shown in Fig. 3A, suggest that the effects of NGF on Na+ channels are dependent on gene induction.
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Effects of NGF in vivo
The preceding results pose a problem. If a retrograde influence of
target-derived NGF is responsible for maintaining
INa, the current would be predicted to
decrease when cells lose target contact as they are placed in culture.
Instead the opposite happens, INa
density transiently increases as neurons are cultured (Fig. 1G) and similar changes are seen with axotomy in vivo
(Jassar et al. 1993). In an attempt to resolve this
apparent contradiction, we also examined the effect of NGF on intact
BFSG B cells in vivo.
-NGF (1 µg/g body wt) was therefore injected
under the skin of the thigh of adult bullfrogs. This location contains
the mucous glands that receive innervation from BFSG B cells
(Horn et al. 1988
; Jobling and Horn
1996
). NGF was injected into the right leg 1 wk before
examination of the properties of the ipsilateral ganglion cells. Figure
3B compares the
INa-V relationships for acutely isolated B cells with those from animals that have received NGF
injections. Although INa is generally
larger in the injected frogs, the difference fell just short of
attaining statistical significance. For example, at 0 mV, 0.1 < P < 0.05.
Effects of NGF on voltage-dependent Ca2+-activated K+-channel current (IC)
To examine whether NGF affects the properties of
IC, the current was elicited by 50-ms
pulses from a holding potential of 40 mV to minimize contamination by
IK because, at this voltage, IK is largely inactivated
(Adams et al. 1982
). To obtain adequate voltage control,
[K+]O, was raised to 40 mM (EK =
23 mV).
Figure 4, A and
B, illustrates IC traces
obtained by digitally subtracting the
Cd2+-resistant current from the total current for
a cell cultured without NGF for 15 days and a cell cultured with NGF
for 15 days, respectively. The current in the NGF-treated cell is much
greater. Figure 4C shows the current-voltage relationship of
IC from all cells studied.
IC began to activate when the command
potential was 30 mV and reached a maximum at +30 mV. At more positive
voltages, the current declined gradually because of the decreased
influx of Ca2+ via Ca2+
channels (Jassar et al. 1994
).
IC of the cells cultured with NGF was
significantly greater than that of the cells cultured without NGF for
15 days (the differences between the values of the cultured cells
without NGF and those of the cells cultured with NGF were statistically
significant for all voltages positive to
20 mV, P < 0.01). Figure 4D shows the time course of the change in
IC. In the absence of NGF, total
IC decreased slightly throughout the
15 days experimental period (Fig. 4D,
). Inclusion of NGF in the culture medium more than doubled the current seen in the absence
of NGF (Fig. 4D,
).
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Figure 4E shows the effect of culture with or without NGF on IC density. This decreased significantly after 3 days of culture because Cin increased at this time (Fig. 1F). Inclusion of NGF prevented the decrease in IC density.
NGF increases N- and L-type Ca2+ currents
(ICa,(N);
ICa,(L)) and attenuates the
inactivation of Ca2+ channels in BFSG B neurons
(Lei et al. 1997). Since the activation of
IC channels requires influx of
Ca2+ via voltage-gated Ca2+
channels, it is possible that effects of NGF on
IC reflect its action on
Ca2+ channels rather than on
IC channels per se. We therefore
plotted the means of the Ca2+ current from cells
cultured with NGF for 1-15 days (Ba2+ was used
as the charge carrier) (data from Lei et al. 1997
)
against the corresponding IC
amplitudes. IC amplitudes were
linearly proportional to their corresponding Ca2+
channel current amplitudes (Fig. 4F, P < 0.01 for a correlation coefficient of 0.829). These results are
consistent with the hypothesis that the effects of NGF on
IC are secondary to its effect on
Ca2+ channels.
Lack of effect of NGF on delayed-rectifier K+ current (IK)
IK was recorded from a holding
potential of 80 mV in the presence of 100 µM
Cd2+ to block
IC.
IK started to activate at
30 mV and
increased with increasing depolarization. During a relatively brief,
50-ms depolarization voltage command, the current did not inactivate.
Figure 5, A and B,
illustrates typical recordings of IK
from a cultured cell without NGF and a cultured cell with NGF for 15 days, respectively. The current amplitudes are quite similar. Figure
5C shows that IK density
(at +80 mV) decreased with time in culture and that this decrease was
not prevented by NGF. The observed IK
density 1.05 ± 0.06 nA/pF seen after 1 day in culture is similar
to the value seen in control, freshly dissociated neurons (B. S. Jassar and P. A. Smith, unpublished observations). Figure
5D shows that the IK-voltage relationship was not
changed by NGF.
|
Lack of effect of NGF on M current (IM)
Hyperpolarizing command pulses from a holding potential of 30 mV
promote gM deactivation. The
conductance then reactivates on repolarization to
30 mV (Adams
et al. 1982
). Figure 6,
A-C, illustrates representative current responses evoked
from an acutely isolated cell, a cell cultured without NGF for 15 days,
and a cultured cell with NGF for 15 days. Although there was an obvious increase in IM in cells that were
maintained in culture, the presence of NGF had no additional effect on
the amplitude of IM relaxations over
the 15-day experimental period. Figure 6D shows that the IM-voltage relationship from 15 days
cultured cells was not altered by NGF. Figure 6E shows the
time course for changes in IM at
30 mV for cells cultured with or without NGF. There is an initial transient increase followed by a sustained increase in
IM over the 15-day experimental
period. NGF neither prevented nor augmented these changes.
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DISCUSSION |
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There were three main findings from these experiments.
First, the electrophysiological changes seen when BFSG neurons are maintained in defined-medium culture in the absence of NGF resemble those produced by axotomy in vivo. Thus TTX-s
INa, TTX-r
INa, and
IM are increased, whereas
IK is decreased (Jassar et al.
1993, 1994
). Both axotomy and culture produced an increase in
INa that was not accompanied by
alterations in channel kinetics. Moreover, culture-induced changes in
the Ca2+-dependent K+
conductance, IC, are likely secondary
to previously documented changes in
ICa (Lei et al. 1997
).
Second, the effects of NGF were selective for certain types of
currents; TTX-s INa and TTX-r
INa were increased, whereas the K+ currents, IM
and IK, were unaffected. Again,
increases in IC likely reflected
NGF-induced increases in ICa
(Jassar et al. 1993; Lei et al. 1997
).
The overall implication is that NGF-induced increases in
INa and
ICa cannot be ascribed to a
generalized improvement in the "health" of cultured sympathetic
neurons that had previously been denied appropriate neurotrophic support.
Third, there was no evidence for down-regulation of channel function by
NGF. Thus increases in INa or
IM (Jassar et al. 1993, 1994
) that are seen in culture or that are induced by axotomy do not reflect removal of a tonic, retrograde inhibitory effect of NGF
on ion channel function. Because axotomy, culture, and NGF application
all increased INa, NGF-dependent and
-independent mechanisms must control expression of
Na+ channels on the cell bodies of BFSG neurons.
A broad range of concentrations (from 2 to 300 ng/ml) have been used to
maintain or examine responses of neuronal cultures to NGF
(Hilborn et al. 1998; Levine et al. 1995
;
Mandelzys et al. 1995
; Riccio et al.
1997
; Sjogreen et al. 2000
;
Tsui-Pierchala et al. 2000
). In one study, 2 ng/ml NGF
was used to maintain cultures prior to testing for physiological
responses with 200 ng/ml (Riccio et al. 1997
). The
concentration of NGF (200 ng/ml) that we used is thus toward the upper
end of the range used by other investigators. Despite this, it does not
promote a growth response yet serves to distinguish positive effects on
Na+ and Ca2+ channels
(Lei et al. 1997
) from a lack of effect on
K+ channels. It would be useful to know whether a
selective effect could be observed with lower concentrations of NGF.
Changes in INa
The increase in INa that followed
either the inclusion of NGF in cultures (Fig. 1) or the injection of
NGF under the skin of the thighs (Fig. 3B) is consistent
with the long-standing hypothesis that the availability of
target-derived NGF is necessary for the maintenance of
Na+ channel function in sympathetic neurons
(Rudy et al. 1982a; Toledo-Aral et al.
1995
). NGF may also fulfil this function in small, nociceptive sensory neurons (Fjell et al. 1999
).
If NGF is responsible for maintaining
INa, why does the current
increase after axotomy (Jassar et al. 1993)
or in NGF-free culture (Fig. 1) when access to target-derived NGF is
restricted or denied? The implication is that two opposing processes
control INa expression: one is NGF and
the other is an NGF-independent phenomenon that is associated with the
loss of an axon. One possibility is that axotomy, or the establishment
of NGF-free cultures, causes the accumulation in the soma of
Na+ channels destined for the axon. This idea is
supported by the observation that chronic constriction of sensory
nerves leads to accumulation of Na+ channels in
the plasma membrane at the site of injury (Novakovic et al.
1998
). Another possibility is that the increase in
gNa reflects a change in the rate of
insertion or degradation of channels that have already been
synthesized. Further information about the mechanism of action of NGF
may be obtained by examining the time course of effects of the
transcription inhibitors, cordycepin and actinomycin D. These
experiments remain to be done.
The initial transient increase in INa
seen in cultured BFSG neurons is too rapid to reflect a response to NGF
released from the cultured neurons themselves (Gill et al.
1998). The increase is apparent even after 1 day in culture
(Fig. 1E), whereas the response to exogenously applied NGF
takes at least 3 days to develop, and it is 6 days before the
difference attains statistical significance (Fig. 1E). If
the initial, transient increase in INa
reflects NGF-independent inappropriate insertion of
Na+ channels into the soma, it remains to be
explained why this effect is attenuated, albeit slightly, by NGF
antibodies (Table 1).
Our results with transcription inhibitors suggest that induction of
gene expression in response to NGF was involved in the regulation of
Na+ channels. We do not yet know whether the gene
or genes that were induced encode Na+ channel
proteins per se or whether they encode subsidiary proteins that are
required for normal Na+ channel function. In the
light of studies on PC12 cells; however, we favor a direct effect on
Na+ channel genes. In that system, NGF induces
brain type II/IIA Na+ channel mRNA (Fanger
et al. 1993, 1995a
,b
) as well as the expression of peripheral
nerve type I (PN1) mRNA (D'Arcangelo et al. 1993
; Toledo-Aral et al. 1997
). These both code for TTX-s
channels. Although the types of TTX-s Na+ channel
genes that may be induced by NGF in adult BFSG neurons remain to be
determined, the
-subunits of type III Na+
channels are unlikely to be involved these are downregulated by NGF in
adult rat sensory neurons (Black et al. 1997
).
The finding that TTX-r INa was
potentiated by NGF (Fig. 2B) goes along with the observation
that -SNS mRNA is upregulated (Black et al. 1997
) and
TTX-r channels in axotomized sensory neurons are "rescued" by NGF
(Dib-Hajj et al. 1998
). In BFSG, however, this may
reflect an action of NGF on some other mRNA that codes for a TTX-r
channel as
-SNS is apparently not expressed in sympathetic neurons
(Akopian et al. 1996
). These conclusions are in accord with the results from PC12 cells where NGF increases functional Na+ channels and induces TTX-r currents
(Rudy et al. 1982b
).
General lack of effect of NGF on K+ channels
IM in BFSG increases in culture
or after axotomy (Jassar et al. 1994), but since it is
insensitive to NGF (Fig. 6), this increase is likely to reflect an
NGF-independent mechanism, i.e., NGF neither maintains nor
tonically inhibits the current. The mechanisms underlying gM regulation therefore require
further investigation. IK was not
affected by NGF (Fig. 5), and of the three K+
channel types examined, only voltage-dependent,
Ca2+-activated K+ channels
(IC) appeared to be affected by NGF
(Fig. 4). Because IC channels are
dependent on Ca2+ influx for their activation
(Lancaster and Pennefather 1987
; Pennefather et
al. 1985
), the observed enhancement of
IC could reflect the documented effect
of NGF on voltage-gated Ca2+ channels (Lei
et al. 1997
). This is possible because the
IC amplitude correlated well with that
of the Ca2+ channel currents in NGF-treated cells
(Fig. 4F). Despite this correlation, we cannot discount a
possible additional direct effect of NGF on
IC channels per se. Such an effect has
been seen in NIH3T3 and C3H10T1/2 cell lines where NGF increases the
expression of Ca2+-activated
K+ channels (Huang and Rane 1994
).
Selectivity of action of NGF
In this and our previous study (Lei et al. 1997),
we showed that treatment of cultured BFSG neurons with NGF selectively
increases INa and
ICa, whereas
IK and
IM are unaffected. Because its effects are selective, we have argued that NGF-induced alterations in ion
channel function cannot be ascribed to a general improvement in the
health of cultured neurons on exposure to neurotrophin. This idea is
supported by the observation that injection of NGF into the target
field of BFSG B neurons increases INa
in vivo (Fig. 3B). Moreover, the selective effects of NGF on
Na+ and not on K+ channels
in culture is not part of a generalized "growth response", as under
the conditions employed, NGF does not increase
Cin (Fig. 1F). It also does
not stimulate neurite outgrowth (Lei et al. 1997
).
Similar neurotrophin modulation of ion channels without the initiation
of a growth response has been described in cultured basal forebrain
neurons (Levine et al. 1995
). These findings therefore reinforce the emerging concept that the complement of ion channel types
seen in a given neuronal type relate specifically to the trophic agents
that are at that neuron's disposal. It is possible that each ion
channel type is maintained by one or more specific target-derived
trophic substance(s). A particularly clear example of this idea has
been provided in sensory neurons. Here it was shown that TTX-r
INa was regulated by NGF but not by
brain-derived neurotrophic factor (BDNF), whereas
GABAA channels were regulated by BDNF and not by
NGF (Oyelese et al. 1997
).
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ACKNOWLEDGMENTS |
---|
We thank P. Stemkowski and C. Ford for help with the in vivo experiments.
This research was supported by the Heart and Stroke Foundation of Canada and the Canadian Institutes of Health Research. S. Lei received a studentship stipend from the Alberta Paraplegic/Rick Hansen Man-in-Motion Foundation.
Present address of S. Lei: Laboratory of Cellular and Molecular Neurophysiology, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892-4495.
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FOOTNOTES |
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Address for reprint requests: P. A. Smith, Dept. of Pharmacology, 9.75 Medical Sciences Building, University of Alberta, Edmonton, Alberta T6G 2H7, Canada (E-mail: peter.a.smith{at}ualberta.ca).
Received 22 February 2001; accepted in final form 23 April 2001.
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REFERENCES |
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