Department of Pharmacology, University College London, London WC1E 6BT, United Kingdom
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ABSTRACT |
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Shah, M. and
D. G. Haylett.
Ca2+ Channels Involved in the Generation of the Slow
Afterhyperpolarization in Cultured Rat Hippocampal Pyramidal
Neurons.
J. Neurophysiol. 83: 2554-2561, 2000.
The advantages of using isolated cells have led us to develop
short-term cultures of hippocampal pyramidal cells, which retain many
of the properties of cells in acute preparations and in particular the
ability to generate afterhyperpolarizations after a train of action
potentials. Using perforated-patch recordings, both medium and slow
afterhyperpolarization currents (mIAHP and
sIAHP, respectively) could be obtained from
pyramidal cells that were cultured for 8-15 days. The
sIAHP demonstrated the kinetics and pharmacologic characteristics reported for pyramidal cells in slices.
In addition to confirming the insensitivity to 100 nM apamin and 1 mM
TEA, we have shown that the sIAHP is also
insensitive to 100 nM charybdotoxin but is inhibited by 100 µM
D-tubocurarine. Concentrations of nifedipine (10 µM) and
nimodipine (3 µM) that maximally inhibit L-type calcium channels
reduced the sIAHP by 30 and 50%,
respectively. However, higher concentrations of nimodipine (10 µM)
abolished the sIAHP, which can be partially
explained by an effect on action potentials. Both nifedipine and
nimodipine at maximal concentrations were found to reduce the HVA
calcium current in freshly dissociated neurons to the same extent. The N-type calcium channel inhibitor, -conotoxin GVIA (100 nM),
irreversibly inhibited the sIAHP by 37%.
Together,
-conotoxin (100 nM) and nifedipine (10 µM) inhibited the
sIAHP by 70%. 10 µM ryanodine also
reduced the sIAHP by 30%, suggesting a role
for calcium-induced calcium release. It is concluded that activation of
the sIAHP in cultured hippocampal pyramidal
cells is mediated by a rise in intracellular calcium involving multiple
pathways and not just entry via L-type calcium channels.
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INTRODUCTION |
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In hippocampal pyramidal cells, a train of action
potentials is followed by a multicomponent afterhyperpolarization
(AHP), comprising a fast AHP (fAHP), a medium AHP (mAHP), and a slow AHP (sAHP) (Storm 1987, 1989
; see Sah
1996
for a review). The fAHP occurs immediately after an action
potential, lasts 1-10 ms, and is primarily a result of activation of
the large conductance calcium-activated potassium ion channels (BK
channels) (Sah 1996
) that are involved in the
repolarization of action potentials (Lancaster and Nicoll
1987
; Yoshida et al. 1991
). The mAHP has a fast
onset (<10 ms), can last up to several hundred milliseconds, and has a
decay time constant of ~39 ms. It appears to result from the activation of a number of different ion channels (Alger et al. 1994
; Storm 1987
, 1989
), including an
apamin-sensitive small conductance calcium-activated potassium ion
channel (SK channel) (Stocker et al. 1999
). In contrast
the sAHP has slow kinetics and is most easily detected after a train of
action potentials (for review see Storm 1990
). The
current underlying the sAHP (sIAHP)
shows a distinct rising phase, peaks between 400-700 ms after a train of action potentials, and decays with a time constant of ~1.5 s at
30°C (Lancaster and Adams 1986
). The sAHP is
insensitive to the bee venom toxin apamin (100 nM), 1 mM
tetraethyammonium (TEA), and 1 mM 4-aminopyridine (4AP)
(Lancaster and Nicoll 1987
; see Storm
1990
). It has been shown that the potassium ion channels giving
rise to the sAHP are calcium-activated (Lancaster and Adams 1986
) and have an estimated conductance of 10 pS
(Valiante et al. 1998
). Taken together, these findings
suggest that apamin-insensitive SK channels underlie the sAHP. Of the
three subtypes of cloned SK channels (SK1-SK3) (Kohler et al.
1996
), only SK1 channels (expressed in Xenopus
oocytes) are insensitive to apamin and thus may potentially underlie
the sIAHP (Ishii et al.
1997
; Kohler et al. 1996
; Vergara et al.
1998
). No specific blocker of the sAHP or SK1 channels has yet
been identified to allow a test of this possibility. The sAHP can
however be inhibited by neurotransmitters such as acetylcholine,
noradrenaline, and histamine and potentiated by adenosine (see
Storm 1990
).
Studies of the source of calcium required to activate the sAHP in
hippocampal cells have provided conflicting results. In hippocampal
pyramidal slices, the sAHP was only partially reduced by the L-type
calcium channel inhibitor nimodipine (Moyer et al. 1992;
Rascol et al. 1991
). This contrasts with findings in
pyramidal cells in organotypic slice cultures where the sAHP was almost completely blocked by the L-type channel inhibitor isradipine (Tanabe et al. 1998
). These studies suggest that the
source of calcium for the sAHP may differ between hippocampal
preparations. Similarly, there is evidence both for (Tanabe et
al. 1998
; Torres et al. 1996
) and against
(Lancaster and Zucker 1994
; Zhang et al.
1995
) a role for calcium-induced calcium release (CICR) from intracellular stores.
In this study we have sought to study the role of L- and N-type calcium
channels and CICR in the activation of the sAHP. To improve drug access
to neurons, we decided to use isolated pyramidal cells. Previous
studies have reported difficulties in recording the sAHP in cultured
hippocampal pyramidal cells (Alger et al. 1994) but we
describe an isolated cell preparation that makes this possible. A
potential criticism of using cultured cells is that the isolation
procedure and culture conditions may lead to changes in calcium and
potassium channel distributions and densities and possibly to changes
in channel regulation. It was thus important to explore the basic
properties and the pharmacology of the
sIAHP in these cultured cells. Using
this preparation, we have been able to study the effects on the
sIAHP of the L-type calcium ion channel inhibitors, nifedipine and nimodipine, the N-type calcium channel inhibitor,
-conotoxin GVIA, and ryanodine, an inhibitor of
CICR. Some of this work has been published previously in abstract form
(Shah and Haylett 1999
).
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METHODS |
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Hippocampal cell culture method
Four-day-old Sprague-Dawley rats were decapitated and the whole
brain was removed and placed in cold (4°C) Gey's Balanced Salt
Solution (GBSS) supplemented with 0.6% wt/vol glucose and 8 mM
MgCl2 as described by Allen et al.
(1993). The brain was hemisected and 500-µm-thick coronal
slices were obtained using a MacIlwain tissue chopper. The CA1 and CA3
regions were dissected out. These regions were incubated in
Ca2+- and Mg2+-free Hanks'
Balanced Salt Solution (HBSS) containing 0.125% wt/vol trypsin and 1 mM HEPES buffer (Sigma, pH 7.3) at 37°C for 1 h. After the 1 h enzyme
treatment, the slices were repeatedly washed with
Ca2+- and Mg2+-free HBSS
supplemented with 8 mM MgCl2, 1 mg/ml bovine serum albumin, and 10% heat-inactivated fetal calf serum (FCS). Trituration was carried out in this solution with three fire-polished sterilized Pasteur pipettes of decreasing bore diameters (1-0.2 mm) to release individual pyramidal cells. The cell suspension was centrifuged at
27 × g and the cells resuspended in neurobasal medium
supplemented with 2% B27 serum free supplement, 0.02 mg × ml
1 gentamicin, 0.5% wt/vol
L-glutamine, and 10% FCS. 200 µl of the cell suspension
was plated in 35-mm tissue culture dishes (Nunc) coated with
poly-D-lysine (molecular weight > 300,000). The cells were maintained in culture in incubators continuously gassed with 95%
O2-5% CO2 at 37°C. After
24 h in culture the cells were re-fed with the culture medium
without FCS and retained in this medium until use.
Measurement of the sIAHP
The pyramidal cell cultures were used between 8 and 15 days
after preparation. All studies were made at a temperature of ~28°C. The cell cultures were superfused in the culture dishes at 5 ml × min1 with a bathing solution composed of (in
mM) 130 NaCl, 3 KCl, 2.5 CaCl2, 1.2 MgCl2, 5 HEPES free acid, 10 glucose, and 26 NaHCO3 (pH maintained at 7.2 by continuously
gassing with 95% O2-5%
CO2). DNQX (6,7-dinitroquinoxaline-2,3-dione; 5 µM) was added to block excitation by endogenously released glutamate.
Patch electrodes with resistances of 4-10 M
were pulled from thin
borosilicate glass (Clark Electromedical Instruments; GC15OTF-15). The
tips of the pipettes were coated with Sylgard and fire polished. To obtain perforated patches, the electrodes were filled with a solution composed of 126 mM KMeSO4, 14 mM KCl, 10 mM
HEPES, 3 mM MgCl2, 2 mM
Na2ATP, 0.3 mM Na2GTP, and
1.2 mg × ml
1 amphotericin B (pH adjusted
to 7.25 with 1 M KOH).
Cells with a soma of 12-18 µm diameter and with at least one thick
process were assumed to be pyramidal (Fig. 1B). The
sIAHP was recorded under hybrid clamp
conditions using an Axoclamp 2A amplifier (Axon Instruments). A train
of 13 action potentials was evoked by passing 5-ms current pulses (at a
frequency of 76.5 Hz) under discontinuous current-clamp conditions and
the cell was then voltage-clamped at approximately 50 mV to record
the sIAHP (Axoclamp sampling rate
1.5-5kHz). The pulse protocol was provided by a Master-8 pulse
generator (Intracel). The sIAHP was evoked every 10 s. The current signals were filtered using the Axoclamp 2A low-pass filter at 0.3 kHz. For action potential recording, the voltage signals were filtered at 3 kHz. Signals were recorded on a
chart recorder and an oscilloscope. Signals were also digitized at 48 kHz (VR-10 digital data recorder; Instrutech) and recorded continuously
on a video recorder. sIAHPs were also
stored on a computer using pClamp6 (Axon Instruments) for later
analysis. The first 2 s of the
sIAHP were acquired at a sampling
frequency of 2 kHz and the remainder at a sampling frequency of 0.5 kHz. Action potentials were acquired separately at a sampling frequency of 20 kHz.
Drugs were applied by switching to a superfusion fluid containing the drug using a multiway tap. The flow of the superfusion solution was directed onto the patched cell.
Measurement of calcium ion currents
Because cultured hippocampal cells have extensive dendritic processes, it is difficult to voltage-clamp them adequately. Therefore calcium ion currents were studied using acutely dissociated cells under whole cell conditions. The pyramidal cells were isolated and plated as described above. Recordings were made from cells between 3.5 and 8 h after isolation.
Pyramidal cells could again be identified by their morphology (Fig.
1A). The cells were superfused with a solution composed of
the following (in mM): 115 NaCl, 2 KCl, 2 CaCl2,
0.5 MgCl2, 11 glucose, and 10 HEPES (pH adjusted
to 7.4 with 1 M NaOH). TEA (25 mM), TTX (0.3 µM), and DNQX (5 µM)
were added to block potassium currents, sodium currents, and AMPA and
kainate receptor activation, respectively. Patch-clamp recordings were
made with a List EPC-7 amplifier using 7-10 M pipettes filled with
solution composed of the following (in mM): 135 CsCl, 0.5 CaCl2, 2 MgCl2, 10 HEPES, 3 EGTA, 2 Na2ATP, and 0.3 Na2GTP (pH adjusted to 7.3 with NaOH). The
calcium ion current was generated using a procedure similar to that
described by Deak et al. (1998)
. The cells were voltage clamped at
80 mV. Using a ramp protocol, the cells were depolarized from
100 to +40 mV at a speed of 1400 mV × s
1 every 10 s. The leak current was
determined using Cd2+ (200 µM) to block calcium
currents. Because the maximum voltage error was calculated to be <5
mV, series resistance compensation was not carried out. The signals
were filtered at a frequency of 1 kHz (8 pole bessel filter) and
digitized at a sampling frequency of 3.33 kHz using pClamp6 software
(Axon Instruments).
Data analysis
Data were analyzed using pClamp6 software. The current traces
shown in the figures are the average of three records. The average of
the amplitude of three successive records of the
sIAHP in the presence of the drug was
expressed as a percentage of the amplitude of the average of six
successive records before application of the drug. The effects of drugs
on action potential duration were measured at a potential of 20 mV.
The effects of calcium channel blockers on the time course of the
sIAHP were measured by fitting the
sIAHP empirically to the equation
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When analyzing the effects of drugs on the calcium ion current, the average peak current of the last two records in the presence of the drug (2-min bath application) was expressed as a percentage of the average peak current of two successive records obtained both before application of the drug and after its washout. All records were leak subtracted.
Materials
All solutions and chemicals were obtained from Sigma except for
the following: GBSS, Ca2+,
Mg2+-free HBSS, neurobasal medium, B27 serum-free
supplement, gentamicin, and FCS were obtained from Gibco; bovine serum
albumin was obtained from ICN Pharmaceuticals;
KMeSO4 was purchased from Pfaltz and Bauer; and
-conotoxin GVIA was obtained from Peptide Institute and Alamone Laboratories.
Stock solutions of most drugs were made in water and stored at 20°C
until required. Stock solutions of nifedipine, nimodipine, and
ryanodine were made in dimethyl sulfoxide (DMSO) and stored at
20°C. These were diluted to the appropriate concentrations in the
external bathing solution. The maximum concentration of DMSO applied
(0.1%) had no effect on the sIAHP.
Suitable precautions were taken to ensure that nifedipine, nimodipine,
and ryanodine were protected from exposure to light.
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RESULTS |
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Both freshly dissociated and cultured hippocampal pyramidal cells
were identified by their morphology (Fig.
1). The soma had a width of 12-18 µM
and at least one apical dendrite emerging from it. The cultured
pyramidal cells were easily visualized as glial cell growth was
minimized by the use of the B27 serum free supplement (Brewer et
al. 1993). Recordings were made only from neurons that had
resting membrane potentials of at least
50 mV. Conventional whole
cell recording was observed to cause significant rundown of the
sIAHP, which quite often disappeared
within 1-2 min of establishing whole cell conditions. This was much
less marked when perforated patches were tested. Stable recordings of
the sIAHP could be obtained for up to
60 min.
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General properties of the sIAHP
Approximately 50-60% of the cells exhibited a
sIAHP with an amplitude >20pA. The
sAHP recorded under current-clamp conditions in these cells (Fig.
2A) had similar kinetics to
the sIAHP recorded using the hybrid
clamp (Fig. 2B). In some cells exhibiting a
sIAHP, a
mIAHP was also observed (Fig.
3A). In a few cells (~20%),
a mIAHP was observed without any
sIAHP. The amplitude of the
sIAHP increased at more positive
potentials (Fig. 2B) as expected for a selective change in
potassium permeability. To record the
sIAHP, cells were routinely held at
50 mV. The amplitude of the sAHP increased with the number of action
potentials (Fig. 2, C and D) as has been reported
in slices (Lancaster and Adams 1986
). However, the
time-to-peak and the decay time constant of the
sIAHP did not change with the action
potential number. The amplitude of the
sIAHP was near maximal with a train of
13 or more action potentials (Fig. 2D). Based on this
observation, a train of 13 action potentials was used to generate the
sIAHP in subsequent experiments.
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The effect of drugs on the sIAHP was
investigated in cells exhibiting a
sIAHP >50pA. In these cells, the mean
amplitude of the sIAHP was 173 ± 15 (SE) pA (n = 78). The
sIAHP peaked ~600 ms after the last
action potential and had a decay time constant of 1.30 ± 0.06 s (n = 32), which is similar to the value
reported for the sIAHP (1.5 s) in
hippocampal slices (see Storm 1990).
Pharmacological characterization of the sIAHP
The sIAHP could be abolished by a
2-min bath application of 200 µM Cd2+
(n = 4; Fig. 3B) and is therefore dependent
on calcium entry. The sIAHP was
observed to be insensitive to 100 nM apamin (inhibition = 8.9 ± 7.6%, n = 6; Fig. 3A) and 1 mM TEA (2.0 ± 6.4%, n = 6; Fig. 3C)
as previously reported (Lancaster and Adams 1986
;
Lancaster and Nicoll 1987
). Although TEA appeared to
have no effect on the sIAHP, the width
of the action potentials increased from 1.27 ± 0.18 to 2.03 ± 0.46 ms (P = 0.057). Apamin did not have any effect
on the action potentials (P > 0.05). In cells that had both a mIAHP and a
sIAHP, 2-min bath applications of
either 100 nM apamin (Fig. 3A) or 200 µM
Cd2+ abolished the mAHP suggesting that
apamin-sensitive SK channels mediate the mAHP in these cultured neurons
(see also Stocker et al. 1999
). The effects of
Cd2+ and TEA were reversible within 5 min of
washout. Although there was little change in the amplitude of the
sIAHP for 15 min after washout, the
effect of apamin on the mIAHP was
irreversible within this time period.
Apamin-sensitive SK channels are sensitive to the plant alkaloid,
D-tubocurarine (Cook and Haylett 1985;
Kohler et al. 1996
) and AHPs in various neurons can also
be blocked by tubocurarine (IC50 < 100 µM)
(Bourque and Brown 1987
; Dun et al.
1986
). In chromaffin cells, which also express apamin-sensitive
SK channels, the IC50 for tubocurarine is 20 µM
(Park 1994
). In cultured hippocampal pyramidal cells,
tubocurarine at a concentration of 50 µM had little effect on the
sIAHP (an increase in amplitude of
15.4 ± 18.5%, n = 4). Increasing the
concentration to 100 µM reduced the amplitude of the
sIAHP by 25.9 ± 8.3%
(n = 6; Fig. 3D), which was completely
reversed within a 5-min washout period. The shape of the
sIAHP was unaffected by tubocurarine.
It was observed that both 50 and 100 µM tubocurarine completely
abolished the mIAHP in cells that had
both the mIAHP and the
sIAHP, providing further evidence that
apamin-sensitive SK channels underlie the mAHP in these cultured
neurons (Stocker et al. 1999
).
Charybdotoxin is a potent blocker of both large conductance (BK) and
intermediate conductance (IK) calcium-activated potassium ion channels
(McManus 1991). Because magnocellular neurons of the rat
supraoptic nucleus have been demonstrated to have a
charybdotoxin-sensitive sAHP (Greffrath et al. 1998
), we
decided to test the effects of charybdotoxin on the
sIAHP in cultured hippocampal
pyramidal cells. A 2-min application of charybdotoxin (100 nM)
significantly increased the amplitude of the
sIAHP by 21.7 ± 5.4%
(n = 7; P < 0.01; Fig. 3E)
and also significantly (P < 0.01) increased the action
potential width from 1.30 ± 0.27 ms (under control conditions) to
1.56 ± 0.29 ms (Fig. 3F). This broadening of the
action potentials can be explained by block of BK channels which are
involved in action potential repolarization in hippocampal pyramidal
cells (Lancaster and Nicoll 1987
; Yoshida et al.
1991
). The increase in amplitude of the
sIAHP may be a result of increased
influx of calcium through voltage-gated calcium channels.
The sIAHP was sensitive to
neurotransmitters, as previously reported (see Storm
1990). Muscarine (3 µM; n = 4) and
noradrenaline (1 µM; n = 3) abolished the
sIAHP (Fig.
4, A and B). The
effects of both reversed in <5 min. Both agonists also caused a
reduction of the outward holding current (Fig. 4C), an
effect which has also been demonstrated in previous studies (see
Storm 1990
). Neither agonist modified the action
potentials.
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Calcium channel inhibitors
The L-type calcium channel inhibitor, nifedipine (1 µM),
inhibited the sIAHP amplitude by
29.1 ± 4.0% (n = 10). This effect was maximal
within 2 min of bath application and was also reversible within 5 min
of washout. Increasing the concentration of nifedipine to 10 µM did
not inhibit the sIAHP any further
(28.3 ± 5.1%, n = 14; Fig.
5A), suggesting that L-type
channels had been maximally inhibited at both concentrations.
Nifedipine had no effect on the time-to-peak or decay time constant of
the sIAHP (control value for
time-to-peak = 0.85 ± 0.09 s and
3 = 0.68 ± 0.06 s; in the presence
of nifedipine time-to-peak = 0.83 ± 0.10 s and
3 = 0.66 ± 0.05 s;
n = 14; P > 0.05; Fig. 5A).
The possibility that the degree of block by nifedipine depends on the
number of action potentials was also tested. Nifedipine produced almost the same degree of block when applied to cells in which the
sIAHP was induced using either 8 or 20 action potentials [27.2 ± 3.3%, (n = 3) and
29.4 ± 4.5%, (n = 5) respectively]. Therefore
in this test the effect of nifedipine on the
sIAHP is independent of the number of
action potentials.
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A 20-min bath application of -conotoxin GVIA (100 nM), which is a
selective blocker of N-type calcium channels (Tsien et al.
1988
), irreversibly reduced the
sIAHP by 35.8 ± 3.4%
(n = 8; Fig. 5B), suggesting calcium entry
via N-type calcium ion channels also plays a role in the activation of
the sIAHP. Because of the irreversibility of the effects of
-conotoxin, it was essential to
use only those cells that demonstrated stable
sIAHPs for at least 5 min before drug
application (as shown in Fig. 5C). It should also be noted
that after washout of
-conotoxin, the
sIAHPs remained at their reduced
amplitude for at least 15 min and occasionally up to 30 min (see Fig.
5C). Application of
-conotoxin had no effects on the
time-to-peak or decay time constant of the
sIAHP (control value for
time-to-peak = 0.82 ± 0.13 s, control
3 = 1.15 ± 0.12 s; after 20-min
application of
-conotoxin time-to-peak = 0.83 ± 0.09 s and
3 = 1.15 ± 0.18 s;
n = 8; P > 0.05; Fig. 5B).
The IC50 for
-conotoxin block of N-type
channels has been given as 60 pM (Wagner et al. 1988
).
Application of nifedipine (10 µM) and
-conotoxin (100 nM) together
reduced the sIAHP by 69.6 ± 2.6% (n = 4; Fig. 5B). Neither nifedipine
nor
-conotoxin had any effects on the duration of the action
potentials (Fig. 5, D and E). With coapplication
of nifedipine and
-conotoxin, the action potential width increased
from 0.98 ± 0.10 ms (under control conditions) to 1.70 ± 0.31 ms (P = 0.056).
The effects of a second L-type calcium channel antagonist, nimodipine,
were also studied. Nimodipine at concentrations up to 3 µM only
caused a partial inhibition of the
sIAHP (Fig.
6, A and B). The
maximal effects of nimodipine occurred within 2 min of bath application
and reversed within 5 min of washout. The block with 3 µM nimodipine
was not significantly different from that with 1 µM
(P > 0.05). The IC50 for
inhibition of L-type channels by nimodipine is ~50 nM in neurons
(Marchetti et al. 1995) so that it is likely that almost
maximal inhibition of L-type channels was achieved by nimodipine at the
concentrations used in this study (0.3-10 µM; see also results of
direct measurements of calcium currents). There was no effect of
nimodipine (at either 1 or 3 µM) on the time-to-peak or decay time
constant of the sIAHP (control value
for time-to-peak = 0.51 ± 0.01 s and
3 = 0.67 ± 0.15 s; in the presence
of nimodipine time-to-peak = 0.54 ± 0.03 s and
3 = 0.64 ± 0.11 s;
n = 9; P > 0.05; Fig. 6B),
nor was there any change in the action potential width.
|
Increasing the nimodipine concentration to 10 µM resulted in complete abolition of the sIAHP (Fig. 6C) within 2 min of application. This can at least partially be explained by the ability of 10 µM nimodipine to reduce the action potential amplitude during a train of 13 action potentials in a use-dependent fashion (Fig. 7). The threshold of firing of the action potentials was also raised (see Fig. 7, B and D) and the last current pulse during the train of 13 often failed to trigger an action potential (Fig. 7B). The use-dependent effect on the action potentials reversed during the 10-s intervals between trains. These findings suggest that the complete block of the sIAHP by nimodipine at high concentrations cannot be solely attributed to block of L-type calcium ion channels.
|
The effects of nimodipine on the sIAHP as well as the action potentials were reversible within 5 min at all concentrations.
The effects of inhibitors on calcium currents
The effects of both nifedipine and nimodipine on the calcium current were examined directly in freshly dissociated cells. Using the protocol described in METHODS to evoke the calcium current, a low-voltage-activated (LVA) and a high-voltage-activated (HVA) calcium current were observed in all cells. It should be noted that rundown of the calcium current was quite common and therefore the inhibition of drugs was calculated as a percentage of the average of control and recovery currents. Data were only collected from cells in which the calcium current recovered to within 70% of the control after washout.
Nifedipine (10 µM), nimodipine (3 µM), and nimodipine (10 µM)
reduced the HVA calcium current by 18.8 ± 3.5%
(n = 7), 20.8 ± 4.3% (n = 4),
and 23 ± 1.7% (n = 5), respectively (Fig.
8). There were no significant differences
between these values (P > 0.05). These results are
consistent with those published in previous studies (Deak et al.
1998; Potier and Rovira 1999
) and it can be
concluded that at these concentrations, both nifedipine and nimodipine
cause a complete inhibition of the current carried by L-type channels
in these cells.
|
Nimodipine (3 µM) also caused a small reduction (Fig. 8B)
of the LVA calcium ion current, with a greater effect at 10 µM (Fig. 8C). Nimodipine (10 µM) has previously been observed to
inhibit an LVA current component (Avery and Johnston
1996). In this study the
sIAHP was recorded at a holding
potential of
50 mV. At this potential, the LVA current is expected to
be inactivated and is therefore unlikely to contribute to the
generation of the sIAHP.
Effect of ryanodine
Ryanodine was applied at a maximal concentration of 10 µM
(Tanabe et al. 1998). A steady block of 29.4 ± 6.1% (n = 5; Fig. 9) of
the sIAHP was achieved at ~20 min.
Because of the slow onset of action, particular care was taken to
select cells that showed very little rundown of the
sIAHP (as shown in Fig.
9B). These findings are consistent for a role of CICR in the
activation of the sIAHP in cultured
neurons. There were no obvious effects of ryanodine on the duration or
amplitude of the action potential, confirming previous studies
(Sandler and Barbara 1999
).
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DISCUSSION |
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Using perforated patches to reduce rundown, we have demonstrated
that a sIAHP can be detected in 50-60%
of cultured rat hippocampal pyramidal cells. The
sIAHP peaks ~600 ms after a train of
action potentials and has an average decay time constant of ~1.3 s.
The sIAHP was abolished by application
of Cd2+, a blocker of voltage-gated calcium ion
channels, indicating that the sIAHP is
activated by a rise in intracellular calcium. As reported for
hippocampal slices, the sIAHP was
insensitive to 100 nM apamin (Lancaster and Nicoll 1987)
and 1 mM TEA (Lancaster and Adams 1986
) but was
suppressed by application of 1 µM noradrenaline and 3 µM muscarine.
The time course and pharmacologic characteristics of the
sIAHP in the cultured pyramidal cells
appear to be similar to those reported for pyramidal cells in slices
(see Storm 1990
). The amplitude of the
sIAHP was significantly increased in
the presence of 100 nM charybdotoxin, which is probably a consequence of the widening of action potentials (Fig. 3F). The
sIAHP was also discovered to be
relatively insensitive to D-tubocurarine. In view of the
insensitivity of the sIAHP to apamin
and D-tubocurarine, of the SK channels so far identified,
only the SK1 channel pharmacology closely matches the pharmacology of
the sIAHP. Thus as suggested by
Vergara et al. (1998)
, it is possible that SK1 underlies
the sIAHP.
Application of 10 µM ryanodine reduced the
sIAHP in cultured neurons by ~30%
suggesting that CICR plays a role in the activation of the
sIAHP. This finding is in agreement
with the results of Tanabe et al. (1998), who observed
~55% inhibition with 10-100 µM ryanodine, but differs from those
of Torres et al. (1996)
and Zhang et al.
(1995)
, neither of whom detected any effect of ryanodine at a
concentration of 20 µM. However, Torres et al. (1996)
and Tanabe et al. (1998)
observed a reduction of the
sIAHP with thapsigargin, an indirect
inhibitor of CICR.
This study also provides further information on the role of L- and
N-type calcium channels in generation of the
sIAHP. Nifedipine, at both 1 µM and
10 µM, inhibited the sIAHP by only
30%. Another L-type calcium channel blocker, nimodipine, at
concentrations that specifically inhibit calcium channels, also reduced
the sIAHP by <50%. It should be
noted that in the acute cell preparation both nifedipine and nimodipine
inhibited the HVA calcium current to similar extents (Fig. 8)
suggesting both drugs inhibit L-type calcium channels maximally at the
concentrations used. This suggests that because L-type channels were
maximally inhibited by these concentrations, L-type calcium channels
are not exclusively concerned in the activation of the
sIAHP in cultured hippocampal
pyramidal cells. This conclusion is in keeping with studies employing
hippocampal slices (Moyer et al. 1992; Rascol et
al. 1990
) in which inhibition of L-type calcium channels
produced only partial inhibition of the sAHP. In organotypic slice
cultures, however, the L-type calcium channel blocker, isradipine,
produced almost complete inhibition of the
sIAHP, suggesting a dominant role for L-type
calcium channels (Tanabe et al. 1998
). Therefore the
source of calcium for the generation of the sAHP may differ in
different preparations.
At a high concentration (10 µM), nimodipine was found to abolish the
sIAHP, despite blocking HVA calcium
channels to the same extent as 10 µM nifedipine and 3 µM nimodipine
(Fig. 8). One factor contributing to the complete block of the
sIAHP is likely to be the inhibitory
effect on action potentials (Fig. 7) resulting in a reduction in
calcium entry into the neurons. Nimodipine, at this concentration, may
also be inhibiting the potassium channel underlying the
sIAHP. Dihydropyridines have been
found to inhibit potassium channels in various other preparations (for
example Ellory et al. 1992; Mlinar and Enyeart
1994
). Thus in this study, nimodipine appears to have multiple
actions that are not shared by nifedipine.
Although N-type channel inhibitors have been shown to have no effect on
the sAHP in rat hippocampal slices (Rascol et al. 1990)
or in rat hippocampal organotypic slice cultures (Tanabe et al.
1998
), in our cultured cells
-conotoxin GVIA (100 nM) irreversibly inhibited the sIAHP by
35% (Fig. 5B). This suggests that N-type calcium channels
also have a role in activation of the
sIAHP. This observation coupled with
the finding that L-type calcium channel blockers only partially reduced
the sIAHP in these cells supports the
conclusion that calcium entry via L-type calcium channels is not solely
responsible for the activation of the
sIAHP. Application of nifedipine and
-conotoxin together reduced the sIAHP by 70 ± 3% (Fig.
5B), significantly <100%. Because cadmium completely
abolished the sIAHP, the indication is
that calcium entry via other types of HVA calcium channels may also
play a role in activation of the
sIAHP.
It should be noted that in rat neocortical pyramidal cells
(Pineda et al. 1998) and in guinea-pig nucleus basalis
neurons (Williams et al. 1997
), an apamin-insensitive
sIAHP is activated predominantly by
calcium entry via N-type calcium ion channels. Thus it is clear that
the types of calcium channels involved in the activation of the
apamin-insensitive sIAHP vary between
neurons and may depend on the particular calcium channels present in
the vicinity of the potassium channels responsible for the
sIAHP.
In conclusion, we have found that the
sIAHP can be successfully recorded
from cultured hippocampal pyramidal cells and that activation by a
train of action potentials, the time course and inhibition by muscarine
and noradrenaline are very similar to the findings in hippocampal
slices (Sah 1996; Storm 1990
). Thus cultured neurons provide a useful alternative to hippocampal slices for
the further study of hippocampal physiology and pharmacology. In
addition, in cultured rat hippocampal pyramidal cells, calcium entry
via both the L- and N- type calcium ion channels contributes to the
activation of the sIAHP. Taken
together with published work, it is clear that the coupling of the
potassium channel underlying the sAHP to calcium sources is not well
established and requires further studies.
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ACKNOWLEDGMENTS |
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We are grateful to Prof. D. H. Jenkinson and Drs. T.G.J. Allen and D.C.H. Benton for helpful discussions and Dr. S. J. Marsh for help with the photography of the cells.
This work was supported by the Wellcome Trust. M. Shah is a Medical Research Council scholar.
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FOOTNOTES |
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Address for reprint requests: D. G. Haylett, Dept. of Pharmacology, University College London, Gower Street, London WC1E 6BT, UK.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 13 September 1999; accepted in final form 11 January 2000.
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REFERENCES |
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