Next Article 
Volume 17, Number 17,
Issue of September 1, 1997
pp. 6493-6503
Copyright ©1997 Society for Neuroscience
-Adrenergic Stimulation Selectively Inhibits Long-Lasting
L-Type Calcium Channel Facilitation in Hippocampal Pyramidal
Neurons
Robin K. Cloues,
Steven J. Tavalin, and
Neil V. Marrion
Vollum Institute, Oregon Health Sciences University, Portland,
Oregon 97201-3098
ABSTRACT
- INTRODUCTION
- MATERIALS AND METHODS
- RESULTS
- DISCUSSION
- FOOTNOTES
- REFERENCES
ABSTRACT 
L-type calcium channels are abundant in hippocampal pyramidal
neurons and are highly clustered at the base of the major dendrites. However, little is known of their function in these neurons.
Single-channel recording using a low concentration of permeant ion
reveals a long-lasting facilitation of L-type channel activity that is
induced by a depolarizing prepulse or a train of action potential
waveforms. This facilitation exhibits a slow rise, peaking 0.5-1 sec
after the train and decaying over several seconds. We have termed this behavior "delayed facilitation," because of the slow onset. Delayed facilitation results from an increase in opening frequency and the
recruitment of longer duration openings. This behavior is observed at
all membrane potentials between 
20 and 60 mV, with the induction
and magnitude of facilitation being insensitive to voltage.
-Adrenergic receptor activation blocks induction of delayed
facilitation but does not significantly affect normal L-type channel
activity. Delayed facilitation of L-type calcium channels provides a
prolonged source of calcium entry at negative membrane potentials. This
behavior may underlie calcium-dependent events that are inhibited by
-adrenergic receptor activation, such as the slow
afterhyperpolarization in hippocampal neurons.
Key words:
calcium channel;
L-type;
modulation;
facilitation;
hippocampus;
single channel
INTRODUCTION
A
number of high-voltage-activated Ca channel subtypes are found in CNS
neurons, including the dihydropyridine-sensitive L-type Ca channel,
which was first described in ventricular heart muscle (for review, see
Tsien et al., 1988
; McCleskey, 1994). Recent work has identified
possible roles for some Ca channel subtypes in neurons (Takahashi and
Momiyama, 1993; Wheeler et al., 1994), but little is known of the
function of L-type Ca channels in the CNS or their modulation by
endogenous neurotransmitters.
L-type Ca channels are prevalent in hippocampal pyramidal neurons,
contributing ~30-50% of total calcium current (Mintz et al., 1992;
Swartz and Bean, 1992; McDonough et al., 1996). The majority of L-type
channels are distributed on the surface of both the cell body and the
proximal dendrites (Westenbroek et al., 1990). The somatic distribution
of these channels is highly uneven, with the majority being localized
to the base and proximal portions of both the apical and basal
dendrites (Ahlijanian et al., 1990; Westenbroek et al., 1990). Their
abundance and location suggest that L-type calcium channels likely play
a crucial role in control of dendritic excitability and in addition
provide calcium for intracellular effectors. For example, activation of
L-type calcium channels provides the intracellular calcium increase for generation of the slow afterhyperpolarization (AHP) in these neurons (Rascol et al., 1991; Moyer et al., 1992). Therefore, modulation of
L-type Ca channel activity may have dramatic effects on pyramidal cell
excitability.
Cardiac L-type Ca channels exhibit distinct gating modes, characterized
either by short openings and low open probabilities [P(o)]
or by long openings and high P(o) (Hess et al., 1984). High
P(o) activity is promoted by the dihydropyridine (DHP)
calcium channel agonist BAY K 8644, and low P(o) behavior is
favored by DHP antagonists (Hess et al., 1984). In addition, strong
prepulse depolarizations can drive the L-type channel from its normal
rapid gating pattern to one characterized by long openings and high P(o) (Pietrobon and Hess, 1990). Such behavior, termed
prepulse facilitation, has also been observed for L-type channels in
CNS neurons. For example, cerebellar granule cells possess an L-type calcium channel that exhibits reopening behavior after membrane depolarization (Slesinger and Lansman, 1991, 1996; Forti and Pietrobon, 1993). Prepulse facilitation of L-type calcium channels has also been
observed in hippocampal pyramidal neurons (Fisher et al., 1990;
Thibault et al., 1993; Kavalali and Plummer, 1994, 1996).
Prepulse facilitation of L-type Ca channels in cerebellar and
hippocampal neurons is complete within 100-200 msec after the prepulse. This relatively slow process can be modestly prolonged by
lowering the permeant ion concentration from 110 to 20 mM
(Thibault et al., 1993). In this study we report a longer-lasting form
of facilitated L-type calcium channel activity that is induced by either a depolarizing prepulse or a train of action potential waveforms. Unlike previously described examples, the facilitation is
slow to rise and is extremely long-lasting, decaying over several seconds. We have termed this phenomenon "delayed facilitation." In
addition, we show that induction of delayed facilitation is blocked by
activation of -adrenergic receptors. Delayed facilitation provides a
long-lasting source of calcium entry at negative membrane potentials.
This behavior may underlie prolonged calcium-dependent events that are
inhibited by -adrenergic stimulation, such as the slow AHP.
MATERIALS AND METHODS
Acutely dissociated hippocampal CA1 neurons were obtained as
described previously (McDonough et al., 1996). Briefly, Sprague Dawley
rats (9-14 d old) were anesthetized with halothane and decapitated.
Hippocampi were rapidly dissected and cut into 300-400-µm-thick slices. Slices were incubated at 37°C in saline containing (in mM): Na2SO4, 82;
K2SO4, 30; HEPES, 10; and
MgCl2, 5, pH 7.4, with added protease type XXIII (3 mg/ml) for nine min, bubbled with O2. Tissue slices were
then transferred to solution containing trypsin inhibitor (1 mg/ml) and
bovine serum albumin (1 mg/ml) for 1 min and finally rinsed in saline
solution containing no enzyme. The CA1 region was microdissected and
triturated into Falcon Primaria dishes as needed.
Experiments using the membrane-permeant cAMP analog
8-(4-chlorophenylthio)-adenosine 3
:5-cyclic monophosphate (8-CPT)
cAMP used cultured hippocampal neurons. These experiments were
completed before acutely dissociated neurons were used routinely.
Delayed facilitation was observed with these neurons (Craig and
Marrion, 1995) and did not differ from that reported in this study.
Hippocampal cells were dissociated from neonatal (1-3 d) Sprague
Dawley rats and maintained in culture as described previously (Legendre
and Westbrook, 1990).
Before recording, cells were incubated for 20-30 min in a modified
Krebs' solution containing (in mM): NaCl, 150; KCl, 5; CaCl2, 1; MgCl2, 1; HEPES, 10;
and glucose, 5, pH 7.4, supplemented with 
-conotoxins MVIIC (5 µM) and GVIA (1 µM) to block P-, Q-, and
N-type Ca channels (McCleskey et al., 1987; McDonough et al., 1996).
During recording, cells were superfused (15 ml/min) with an external
solution containing (in mM): potassium aspartate, 125; KCl,
35; MgCl2, 5; HEPES(Na), 10; EGTA, 10; and
CaCl2, 5.64 (to give an estimated free concentration
of 60 nM) (Fabiato and Fabiato, 1979), pH 7.4, with CsOH.
Cells in this solution had ~0 mV membrane potential. All potentials
are expressed as the negative of the potential imposed on the pipette.
Cell-attached patch recordings (Hamill et al., 1981) were made using
thick-walled (1.5 mm outer diameter, 0.5 mm inner diameter) quartz
electrodes (7-10 m
) containing (in mM):
BaCl2, 10; tetraethylamonnium chloride, 135; and
HEPES, 10, pH 7.4. Single-channel currents were recorded with an
Axopatch 200A amplifier (Axon Instruments) and digitized onto video (94 kHz sampling frequency, 37 kHz bandwidth; VR10B, Instrutech Corp.) for
later analysis. Single-channel records were filtered at 1-2 kHz with
an eight pole Bessel filter (Frequency Devices) and acquired at 100 µsec intervals for analysis using Pulse (Heka, distributed by
Instrutech Corp.). Single channels were analyzed using MacTAC (Skalar
Instruments, distributed by Instrutech Corp.). The "50% threshold"
technique was used to estimate event amplitudes and durations, with
each transition inspected visually before being accepted. Open duration
histograms, constructed from openings to level 1, were binned
logarithmically (20 bins/decade) plotted against the square root
transformation of the ordinate (number of events/bin), and the
distribution was fitted by a sum of exponential probability density
functions using the maximum likelihood method. With this type of
representation, peaks in the histogram correspond to the time constant
of the exponential (Sigworth and Sine, 1987). Events <166 µsec were
missed, because after filtering they did not reach the 50% threshold.
These missed events were not corrected for.
Channel P(o) was first estimated as NP(o), the
product of the open probability × the number of channels.
NP(o) was calculated as the sum of (dwell time × level
number) divided by the total time. N was estimated as the
number of simultaneously open channels after induction of delayed
facilitation, and finally P(o) was obtained by dividing
NP(o) by N. P(o) was calculated within
200 msec time segments using ReadEvents 1.36 (Dr. Scott Eliasof, Vollum Institute). However, it is apparent from data such as those illustrated in Figure 2 that an estimate of N is difficult. We have
observed patches that did not display superimposed openings on
induction of delayed facilitation or during standard activation (by a
depolarizing voltage pulse from 60 mV to 0 mV) but exhibited multiple
openings after addition of BAY K 8644. However, because we have found
that BAY K 8644 could not be completely washed from the experimental rig, its use was limited to a period of experiments demonstrating that
the channel class underlying delayed facilitation is L-type. Thus, the
value of N is probably an underestimate, thereby giving an
overestimate of patch P(o) but with accurate determinations of the reported changes in P(o). This may be reflected in
the range of P(o) values observed under control conditions
in Figures 1, 3, and 6. However, L-type
channels are known to exhibit activity at negative membrane potentials
(Marchetti et al., 1995; Magee et al., 1996; Rubart et al., 1996). For
example, whole-cell Ca current and intracellular Ca measurements in
hippocampal CA1 pyramidal neurons have demonstrated a DHP
antagonist-sensitive Ca current active at membrane potentials negative
to 60 mV (Magee et al., 1996). The DHP-sensitive Ca channels were
found to be located most densely on the soma and proximal apical
dendrite of these neurons (Magee et al., 1996), the subcellular
location of L-type Ca channels (Ahlijanian et al., 1990; Westenbroek et
al., 1990). These data suggest that L-type channels exhibit a low level
of activity at very negative membrane potentials.
Fig. 2.
A train of action potential waveforms induce
delayed facilitation of L-type channels. Selected traces of 5 sec
sweeps of channel activity for control (left) and after
a train of action potential waveforms (right) at 20
mV. The patch appeared to contain two channels. Low P(o)
activity was seen in control sweeps. Delayed facilitation was observed
after the 50 Hz. train of an action potential waveform
(inset). Each action potential waveform consisted of a
ramp to a peak of +50 mV, followed by successive ramps to +20 and 10
mV. The potential was returned to 70 mV by an additional ramp (to
mimic an afterhyperpolarization). The membrane potential was slowly
ramped back to 60 mV before the next waveform was initiated (see
Materials and Methods for details). Delayed facilitation was evoked by
10 action potential waveforms in 200 msec, giving a frequency of 50 Hz.
Openings with a curtailed amplitude reflect short duration openings
with apparent amplitude that is clipped at the bandwidth used (2 kHz).
Expanded traces for behavior observed in control jumps and after the
train of action potential waveforms are shown below. The expanded
traces were taken from the sweeps marked with a bar and
*. As is seen in open duration histograms (see Fig. 6), delayed
facilitation is caused by an increase in opening frequency and an
increase of longer duration openings.
[View Larger Version of this Image (47K GIF file)]
Fig. 1.
Delayed facilitation of L-type calcium channel
activity by a depolarizing prepulse. A, Stability plot
of L-type channel activity in a cell-attached patch, showing
P(o) for each 6 sec sweep. The patch appeared to contain
a single channel. The patch potential was stepped to 30 mV for 6 sec
from a holding potential of 60 mV, either directly (Control,
thin bar) or immediately after a prepulse to +40 mV (200 msec
duration) (Prepulse, thick bar). Channel activity was
low throughout the control sweeps and was dramatically augmented after
the depolarizing prepulse. Sweeps were considered facilitated if the
patch P(o) measured over the entire 6 sec sweep exceeded
two times the mean P(o) of control sweeps (dashed
line). B, Selected sweeps as indicated in
A. Little channel activity was observed on changing the
patch potential from 60 to 30 mV (control, left
traces). After the prepulse to +40 mV, there was a large
increase in L-type activity. Short duration events were observed
throughout the 6 sec sweep.
[View Larger Version of this Image (47K GIF file)]
Fig. 3.
Trains of action potential waveforms are more
suited to promoting delayed facilitation than a depolarizing prepulse.
Top, Time plot of patch P(o) for each 6 sec postpulse potential of 50 mV. Holding potential was 60 mV. The
patch appeared to contain two channels. Delayed facilitation was not
observed with a 200 msec prepulse to +40 mV but was observed with a 50 Hz train of an action potential waveform (see Fig. 2,
inset, for waveform schematic and Materials and Methods
for description). Bottom, Selected traces of the 6 sec
time segment used to calculate patch P(o). Low
P(o) activity was seen for both control and after a prepulse. Delayed facilitation was observed after the 50 Hz train of an
action potential waveform.
[View Larger Version of this Image (27K GIF file)]
Fig. 6.
Time-dependent increase in P(o)
results partly from an increase in long duration openings.
A, Top, Open probability in 200 msec time
segments averaged over a 5 sec voltage pulse to 20 mV. Under control
conditions (mean of 10 sweeps) the P(o) was low
throughout the voltage pulse (left). A 200 msec prepulse
to +40 mV (right) caused a dramatic increase in
P(o) with a rise time of ~600 msec and a subsequent
decay (mean of nine facilitated sweeps). The rising phase of the
waveform was interpolated with a continuous line, and
the decay was fit with an exponential function (
~1.5 sec).
Bottom, Open duration histograms show that openings under control conditions were of very short duration ( ~0.4 msec) (0% of long openings), and that the prepulse recruited longer duration
openings (13% of events), best fit by an exponential of ~4 msec.
B, Top, P(o) plots of control (mean of 14 sweeps) and after facilitation (mean of 10 sweeps) measured at 50 mV. As seen at 20 mV, a prepulse induced a time-dependent increase in
P(o). The rising phase of the waveform was interpolated
with a continuous line, and the decay of facilitated
P(o) increase was fit with an exponential function ( ~1.3 sec). Bottom, Open duration histograms show that
the prepulse-induced increase in P(o) was accompanied by
an increase in longer duration openings (long duration openings
contributed 6% of events in control sweeps, which increased to 11% in
facilitated sweeps). Holding potential was 60 mV for both patches.
P(o) measurement began 250 msec after the prepulse to
exclude the fast decaying form of prepulse facilitation (see
text).
[View Larger Version of this Image (30K GIF file)]
Delayed facilitation was evoked by three different voltage protocols: a
200 msec prepulse to +40 mV, a 100 Hz train of 10 rectangular voltage
pulses to +40 mV (5 msec duration, separated by 5 msec intervals), and
a 50 Hz train of 10 action potential waveforms. Each action potential
waveform consisted of a 200 µsec ramp from 60 mV to a peak of +50
mV, followed by a 400 µsec ramp to +20 mV, a 400 µsec ramp to 10
mV, and ending with a 2 msec ramp to 70 mV. The potential was
returned to 60 mV by an additional 17 msec ramp (to mimic an
afterhyperpolarization) before the next waveform was initiated. The
half-maximal duration of each waveform was 1 msec (see Fig.
2, inset).
Single channel conductance was estimated as the slope of the
current-voltage relationship for L-type Ca channels recorded with 10 mM Ba2+ as the charge carrier.
Current-voltage curves were generated using the current amplitude
determined from gaussian fits to amplitude histograms from individual
patches (see above). Open times of L-type channels in the absence of a
DHP agonist were extremely short (see Figs. 6 and 8), and only events
that exceeded a certain duration were used to provide an estimate of
single-channel amplitude. For example, when data were Bessel-filtered
at 1 kHz, the rise time of the eight pole Bessel filter was ~400
µsec. Under these conditions, only events that exceeded a 1 msec
duration were included, thereby excluding those events in which the
full amplitude was not resolved. All statistical tests were unpaired,
two-tailed Student's t test or ANOVA (Origin, Microcal
Software Inc.). Values are given as mean ± SEM.
Fig. 8.
Inhibition of delayed facilitation by
-adrenergic receptor stimulation. Cell-attached patch recording of
delayed facilitation of L-type channel activity. The patch appeared to
contain two channels. A, Top, Under control conditions,
a 200 msec prepulse to +40 mV induced facilitation of L-type channel
openings at 40 mV. Bottom, Open duration histogram of
all events after the prepulse. The distribution was best fit by the sum
of two exponentials with time constants 0.3 and 2.5 msec.
B, Top, In the presence of the -adrenergic agonist isoproterenol (1 µM), the prepulse
failed to evoke high P(o) L-type channel activity.
Bottom, Open state analysis of events after the prepulse
were best fit by a single exponential distribution ( ~0.3 msec).
C, Top, The -adrenergic receptor antagonist
propranolol (10 µM) was applied in the continued presence
of isoproterenol. Approximately 5 min after the antagonist was added,
delayed facilitation was observed after the prepulse. Bottom, Open duration histogram of events after the
prepulse, in the presence of isoproterenol and propranolol. Recovery
from the effect of isoproterenol was apparent with the return of the longer time constant ( ~3.1 msec).
[View Larger Version of this Image (33K GIF file)]
All reagents were obtained from Sigma, except -conotoxin MVIIC
(Bachem), -conotoxin GVIA (Peninsula Laboratories), nimodipine and
isoproterenol (Research Biochemicals), CaCl2 (Fluka), BAY K
8644 and HEPES (Calbiochem), and CsOH (Aldrich, WI).
RESULTS
Delayed facilitation of L-type channel activity
Delayed facilitation of L-type calcium channels was identified
using cell-attached patch recordings from acutely dissociated CA1
hippocampal pyramidal neurons with a low concentration of Ba2+ or Ca2+ as the permeant ion
(5-10 mM). Prepulse depolarization (+40 mV) dramatically
enhanced L-type channel activity at negative membrane potentials (27 of
55 patches) (Fig. 1). In the absence of a prepulse (control), channels
exhibited a very low P(o). After a 200 msec depolarization
to +40 mV, the peak P(o) was increased 14.2 ± 3.6-fold (n = 17). When measured over a 6 sec sweep, prepulses
increased total patch P(o) by 7.4 ± 1.5 (n = 22). It is not known whether channel activity was
required during the prepulse for delayed facilitation to be observed,
because openings could not be resolved at +40 mV (during the
prepulse).
Delayed facilitation required a low concentration of permeant ion
(5-10 mM) and was never seen with 110 mM
Ba2+ in the recording solution (also see Thibault et
al., 1993). Whole-cell current activation curves were used to measure
the effect of charge screening of Ba2+ on L-type
channel voltage dependence. An increase of Ba2+
concentration from 15 to 110 mM produced a depolarizing
shift of 14.5 ± 2.7 mV (n = 5) in the position of
the activation curves. Correction of this voltage shift did not reveal
"hidden" delayed facilitation of L-type channels (n = 26). Therefore, delayed facilitation was only observed when low, more
physiological concentrations of permeant ion were used.
Delayed facilitation was often preceded by a more rapidly decaying form
of facilitated L-type channel activity (26 of 55 patches). This fast
facilitation consisted of longer duration openings than observed in the
absence of a prepulse and decayed within 100-200 msec (not shown).
Fast facilitation appeared to be kinetically distinct from delayed
facilitation and decayed before the peak of delayed facilitation was
observed. In addition, they frequently occurred in separate patches,
with 13 patches exhibiting only delayed facilitation, whereas 14 patches displayed both forms. Of those patches that did not exhibit
delayed facilitation, ~45% displayed fast facilitation (12 of 28 patches). Fast facilitation possesses a time course similar to that
described previously for L-type channels (Kavalali and Plummer, 1994,
1996) and was not examined in detail in this study. All subsequent data
are presented with analysis started 250 msec after the prepulse or
termination of the burst of action potential waveforms, thereby
excluding this form of facilitation from analysis (see Fig. 6
legend).
Delayed facilitation of L-type calcium channels could be evoked either
by a long rectangular voltage step (200 msec) (Fig. 1) or by a train of
short (5 msec) duration voltage pulses (see Fig. 5). Both protocols
featured a voltage excursion to +40 mV, which is close to the peak of
the action potential measured in hippocampal neurons (Lancaster and
Adams, 1986). Delayed facilitation was also induced by a 50 Hz train of
action potential waveforms (Fig. 2) (9 of 15). This result is of major
physiological significance. It indicates that a train of only 10 action
potential waveforms, at a lower frequency than the likely maximum
firing frequency (100 Hz; Lancaster and Adams, 1986), evokes robust
delayed facilitation at 20 mV. In three of three patches that did not
exhibit delayed facilitation with a 200 msec rectangular prepulse, we
were able to produce it when the protocol was switched to a train of
action potential waveforms. Figure 3
shows an example of such a patch in which repeated depolarizing
prepulses (+40 mV, 200 msec duration) failed to evoke delayed
facilitation. However, after the protocol was changed to the train of
action potential waveforms (see Fig. 2, inset) delayed
facilitation was observed at 50 mV in ~30% of sweeps (Fig. 3).
Induction of delayed facilitation at 60 mV by action potential
waveforms was also observed using 5 mM
Ca2+ as the charge carrier (two of three patches)
(data not shown). Delayed facilitation with Ca2+ was
indistinguishable from that recorded using Ba2+;
i.e., peak P(o) occurred with a delay and decayed over the 6 sec voltage excursion. There was also an increase in the relative contribution of longer duration openings in facilitated sweeps, as
observed with Ba2+ (see below). These data indicate
that the physiological charge carrier through L-type Ca channels can
sustain delayed facilitation, and that changes in membrane potential
that occur during a burst of action potentials may be more suited to
promoting L-type channel facilitation than standard rectangular voltage
pulses.
Fig. 5.
Ensemble current of delayed facilitation induced
by a train of voltage pulses. Ensemble current generated by a 100 Hz
train of rectangular voltage pulses to +40 mV of 5 msec duration,
separated by 5 msec intervals. Inset, Voltage protocol
in more detail. Holding potential was 60 mV, and post-train voltage
was 30 mV. Ensemble is an average of 27 sweeps. A slow rise of inward
current was observed, peaking ~600 msec after the termination of the
train. The rising phase of the waveform was interpolated with a
continuous line, and the decay was fit with an
exponential time course (the arrow marks the start point
of the exponential fit; ~1.6 sec). The dashed line
represents zero current.
[View Larger Version of this Image (25K GIF file)]
N-, P-, and Q-type channel currents were blocked by preincubating cells
with -conotoxins GVIA and MVIIC (McCleskey et al., 1987; McDonough
et al., 1996). Under these conditions the channel activity observed
during delayed facilitation was blocked by the DHP antagonist
nimodipine (1 µM; n = 4) (results not
shown). In addition, the channel activity observed after preincubation
with -conotoxins GVIA and MVIIC was sensitive to the DHP agonist BAY K 8644 (5 µM; n = 7) (Fig.
4). Before addition of BAY K 8644, channel openings were observed during depolarizing voltage pulses from
60 to 0 mV (Fig. 4Ai) and after a burst of action
potential waveforms (Fig. 4Cii). Short duration channel
activity was elicited by a 200 msec voltage pulse to 0 mV (holding
potential, 60 mV) and was observed throughout the pulse. Generation
of an ensemble current gave a waveform that showed some decay during
the voltage pulse (Fig. 4Aiii). Measurement of
channel openings >1 msec in duration (see Materials and Methods) over
a voltage range of 20 to 10 mV gave a slope conductance of 10.5 pS
(Fig. 4B). Delayed facilitation was evoked by a burst
of action potential waveforms, revealing that this patch contained at
least two channels (Fig. 4Ci,ii) of similar amplitude to
events evoked by a voltage pulse (Fig. 4B). After
addition of BAY K 8644 (5 µM), voltage pulses to 0 mV
(holding potential, 60 mV) produced superimposed channel openings of
long duration (Fig. 4Aii). Generation of an ensemble current showed that BAY K 8644 had greatly augmented the channel activity, producing a large current that showed little decay during the
voltage pulse (Fig. 4Aiv). Long duration openings
were also observed during 5 sec pulses either with or without a
preceding burst of action potential waveforms (Fig.
4Ciii,iv). Estimation of channel slope conductance in the
presence of BAY K 8644 was 15.5 pS (Fig. 4B) (mean
conductance, 14.3 ± 0.9 pS; n = 4). The increase
in single-channel conductance observed in the presence of BAY K 8644 has been reported previously (Mantegazza et al., 1995). The conversion
of short duration channel activity to long duration openings
characteristic of DHP agonist-modified channels indicates that the
channel underlying delayed facilitation is the L-type channel. However,
because DHP agonists affect the gating of this channel (Fig. 4), all
remaining data were obtained in the absence of BAY K 8644.
Fig. 4.
Channels underlying delayed facilitation are
sensitive to the DHP agonist BAY K 8644. A, Inward
channel openings evoked by repeated depolarizing voltage pulses from
60 mV to 0 mV recorded from a cell-attached patch with 10 mM Ba2+ as the charge carrier. Channel
openings are downward. This patch appeared to contain
two channels. i, In the absence of a DHP agonist, single-level channel openings were brief and were observed throughout the 200 msec depolarization. Generation of an ensemble current (average
of 23 sweeps) gave a waveform that showed little decay during the
depolarization (iii). ii, In the presence
of the DHP agonist BAY K 8644 (5 µM), channel openings
were of long duration and were observed to the second level. Generation
of an ensemble current (average of 27 sweeps) showed that BAY K 8644 had greatly augmented channel activity (iv).
B, Current-voltage relationship of channel amplitude
observed in the absence and presence of BAY K 8644. Channel amplitude
was obtained by gaussian fits to amplitude histograms obtained by
visual inspection of each opening (see Materials and Methods) evoked by
a family of depolarizing voltage pulses to 40 to 10 mV (holding
potential, 60 mV). Continuous lines represent the
least squares fit to the data. In the absence of BAY K 8644 (
),
channel slope conductance was 10.5 pS. In the presence of BAY K 8644 (
), the channel slope conductance increased to 15.5 pS (see
Results). Channel amplitude observed in the absence of BAY K 8644 during 5 sec pulses to 20 (see below), with (
) or without (
) a
train of action potential waveforms superimposed on the control
I/V. In the presence of BAY K 8644, the increase in channel amplitude was also observed for openings evoked during 5 sec
pulses to 20 (see below), with (
) or without (
) a train of
action potential waveforms. C, i, iii, Records evoked by
a 5 sec voltage pulse from 60 mV to 20 mV (see Fig. 2 for protocol) in the absence (i) and presence
(iii) of BAY K 8644 (5 µM). ii, iv, Records evoked by a 5 sec voltage pulse to 20 mV (holding potential, 60 mV) preceded by the train of action potential waveforms (see Fig. 2 for protocol) in the absence (ii) and
presence (iv) of BAY K 8644 (5 µM).
i, ii, Delayed facilitation was evoked by a train of
action potential waveforms and was observed at 20 mV, with few
openings seen in the absence of the action potential waveform train.
Induction of delayed facilitation demonstrated that the patch contained
two channels. iii, iv, In the presence of BAY K 8644, long duration openings were observed during 5 sec pulses to 20 (see
below), with or without a train of action potential waveforms. Note the
addition of BAY K 8644 caused the short duration openings to be
replaced by openings characteristic of DHP-agonist modified channels.
In addition, note that normal channel gating was observed between
bursts of DHP-modified behavior (iv, upper trace).
[View Larger Version of this Image (37K GIF file)]
The slope conductance of L-type channels exhibiting delayed
facilitation and recorded in the absence of DHP agonist was 10.7 ± 1.9 pS (n = 3) (see Materials and Methods), close to
that observed for L-type calcium channels in ventricular heart muscle
(Hess et al., 1986). No significant difference in conductance was
observed between L-type channels in patches that exhibited delayed
facilitation and those that did not (p > 0.05).
In addition, channel activity observed in control sweeps (i.e., without
the prepulse) and those activated by a voltage step from 60 mV (200 msec duration) had conductances identical to the channels that
displayed delayed facilitation. Delayed facilitation was observed only
in patches that possessed L-type channels activated by depolarization
from 60 mV to 0 mV, which suggests that delayed facilitation results from an increase in activity of the standard L-type channel, rather than recruitment of a novel channel type (see Discussion).
Time-dependent and voltage-independent properties of
delayed facilitation
The time course of delayed facilitation can be best described by a
generated ensemble current. Figure 5
illustrates an ensemble of L-type channel activity, induced by a train
of short-duration rectangular voltage pulses. The onset of facilitated
channel activity was slow to rise, peaking ~600 msec after
termination of the train (Fig. 5). The behavior subsequently decayed
with an exponential time course ( ~1.6 sec). Although the time
course of delayed facilitation varied between patches, it consistently
peaked >500 msec after the prepulse and decayed by the end of the 6 sec test potential.
Delayed facilitation was attributable to a time-dependent increase in
P(o) (Fig. 6) and was observed
at membrane potentials between 20 and 60 mV. Under control
conditions, at both 20 (Fig. 6A) and 50 mV (Fig.
6B), the patch P(o) was low throughout a 5 sec sweep. After a prepulse to +40 mV (200 msec duration), patch
P(o) slowly increased to a plateau of ~0.1-0.15 and
decayed back toward control levels with an exponential time course ( ~1.5 sec) (Fig. 6). This waveform was observed at each voltage tested, with no obvious effect of membrane potential on the time to
peak or the decay rate of delayed facilitation.
The time-dependent increase in patch P(o) resulted both from
an increased channel opening frequency (evident in Figs. 1, 2, 3, 4) and an
increase in the proportion of long duration openings. Under control
conditions, patches exhibited short duration channel open times that
were best described by a single exponential ( ~0.4 msec) or by two
exponentials with a small percentage of longer duration openings ( ~3 msec) (Fig. 6A,B). On facilitation by a voltage
prepulse (+40 mV prepulse, 200 msec duration), an increase in the
contribution of longer duration openings was observed. Open time
histograms of all events in facilitated sweeps were best described by
the sum of two exponentials ( ~0.4 and 2-5 msec), with a
significant increase in openings (ANOVA, p < 0.01) described by the longer time constant (Fig. 6A,B).
This observation was the same at each membrane potential tested (20
to 50 mV) and was unaffected by the membrane potential (see
below).
The induction of delayed facilitation was not affected by the
postprepulse potential (Fig. 6). In addition, the membrane potential at
which delayed facilitation was observed did not have a marked effect on
the contribution of longer duration openings. Figure 7A shows the contribution of
the longer time constant to the open duration histogram of facilitated
openings. At each potential, either a 200 msec voltage prepulse to +40
mV or a train of action potential waveforms evoked a significant
increase over control activity in the proportion of longer duration
openings (ANOVA, p < 0.01). In contrast, there was no
significant difference in the contribution of longer duration openings
at each membrane potential (ANOVA, p > 0.1) (Fig.
7A).
Fig. 7.
Delayed facilitation of L-type channel activity is
not markedly voltage-dependent. A, The contribution of
the exponent with the longer time constant is plotted for control and
facilitated sweeps. Longer duration openings contributed ~1% of
events in control sweeps. After facilitation by a 200 msec prepulse to
+40 mV, longer openings constituted ~5% of all events. The increase in the number of longer duration openings was significant at each voltage (one-way ANOVA, p < 0.01). This increase
in longer duration openings was the same at each membrane voltage
(n = 3-5 for each voltage). B, The
magnitude of the increase in peak P(o), induced by a
prepulse to +40 mV (200 msec duration), or a train of action potential
waveforms was not obviously voltage-sensitive. The ratio of the peak
P(o) observed in 200 msec time segments to the mean P(o) observed during the 6 sec control sweep is plotted
as mean ± SEM. The effect of postprepulse displayed no obvious
voltage dependence (one-way ANOVA, p > 0.1).
C, The induction of delayed facilitation was not
dependent on the postprepulse potential. The ratio of facilitated
sweeps relative to the total number of sweeps is plotted. There was no
significant difference in the number of facilitated sweeps observed at
each potential (one-way ANOVA, p > 0.1).
D, Open-state kinetics of L-type channels are not
voltage-dependent. Plotted are time constants (short and long) obtained
from the fitting of open duration histograms (for example, see Fig. 6).
Time constants were obtained from control sweeps (open
triangles, open circles superimposed with closed
circles), facilitated sweeps (closed triangles, closed
circles), and standard activation voltage pulses from a holding
potential of 60 mV (closed and open
diamonds). Channel open times obtained under all conditions were voltage-insensitive (n = 3-6 for each
voltage).
[View Larger Version of this Image (20K GIF file)]
The effect of the postpulse membrane voltage on induction of delayed
facilitation was assessed by quantitation of the peak P(o)
increase. This was measured as the ratio of the facilitated peak
P(o) and the mean P(o) observed in control
sweeps. Figure 7B shows that there was no significant
difference in the magnitude of the peak P(o) change at each
potential (ANOVA, p > 0.1). In addition, the number of
sweeps within an experiment that displayed delayed facilitation was
unaffected by the postpulse membrane potential (ANOVA,
p > 0.1) (Fig. 7C).
L-type calcium channels exhibited a biexponential open time
distribution, with prepulse depolarization promoting more long open
time events. Mean open time constants were voltage-insensitive in both
control and facilitated sweeps (Fig. 7D). The open time kinetics of control and facilitated behavior were compared with those
of L-type channels activated by a standard activation protocol (150 msec depolarizing voltage steps from 60 mV). Open duration histograms
of L-type channel activity evoked by both protocols showed two
exponential components, with comparable time constants. As was observed
for control and prepulse-induced activity, time constants obtained from
standard activation steps were also voltage-insensitive (Fig.
7D; see Fig. 9 for schematic representation of protocols). This indicates that delayed facilitation does not affect L-type channel
open times.
Fig. 9.
Selective inhibition of delayed facilitation by
-adrenergic receptor activation or addition of a cAMP analog.
A, The P(o) measured in 6 sec sweeps at
40 or 50 mV after a prepulse to +40 mV is normalized to control
(without the prepulse). In the absence of isoproterenol, the prepulse
induced a 5.0 ± 1.3-fold increase in P(o) during
facilitated sweeps (n = 5). In different paired
experiments (before addition of 8-CPT cAMP), the prepulse induced a
7.2 ± 0.83-fold increase in P(o) during
facilitated sweeps (n = 4). The increase in patch
P(o) was inhibited by the addition of the -adrenergic
receptor agonist isoproterenol (1 µM) or the
membrane-permeant analog of cAMP 8-CPT cAMP (1 mM). Results
are from paired data, with the effect of either isoproterenol or 8-CPT
cAMP being compared with delayed facilitation evoked before their
application. The two control bars, in isoproterenol and in 8-CPT cAMP,
have been normalized to the first control (in the absence of
treatment), showing that neither isoproterenol nor 8-CPT cAMP had an
effect on control behavior. B, The P(o) observed during a 200 msec voltage pulse from 60 mV to 0 mV in the
presence of isoproterenol was normalized to the P(o)
observed in the absence of agonist. Isoproterenol had no significant
effect on the channel activity observed during this standard activation protocol (n = 4).
[View Larger Version of this Image (26K GIF file)]
In summary, the data presented in Figures 6 and 7 show that the
induction of delayed facilitation, the magnitude and time course of the
change in P(o), and the observed increase in long duration
openings are insensitive to the postpulse voltage.
Modulation of delayed facilitation
The hippocampus receives projections of noradrenaline-containing
fibers from the locus coeruleus. The diffuse nature of these projections implicates noradrenaline as a neuromodulator. Activation of
-adrenergic receptors in dentate gyrus granule cells augments the
whole-cell calcium current, an effect mimicked by membrane-permeant cAMP analogs (Gray and Johnston, 1987). In contrast, application of the
-adrenergic receptor agonist isoproterenol (1 µM) to
cell-attached patches in this study inhibited delayed
facilitation of L-type channel activity (five of six patches) (Fig.
8). Before application of isoproterenol
(control), a 200 msec prepulse to +40 mV induced delayed facilitation
of L-channel activity. Analysis of channel open times after the
prepulse showed that the distribution was best fitted by the sum of two
exponentials (Fig. 8; see Fig. 6 for comparison). Under control
conditions, channel open time was best described by a single
exponential (, ~0.25 msec) (not shown). Approximately 1 min after
the application of isoproterenol, prepulse-induced facilitation was
inhibited (Fig. 8). Channel open time was now best described by a
single exponential with a short open time constant, similar to control
sweeps without the prepulse. A reversal of the effect of isoproterenol
was observed after addition of the -adrenergic receptor antagonist
propranolol (10 µM) in the continued presence of
isoproterenol (two of three patches). Recovery was apparent by the
return of high P(o) behavior after the prepulse and a return
of the longer time constant in the open duration histogram (Fig. 8).
Open state kinetics in the presence of isoproterenol were the same as
those without prepulse, suggesting that application of isoproterenol
prevented the induction of delayed facilitation by a depolarizing
prepulse.
Isoproterenol selectively inhibited delayed facilitation of L-type
channel activity. Figure 9 shows mean
results of the effect of isoproterenol and the membrane-permeant analog
of cAMP (8-CPT cAMP; 1 mM) on the P(o) of
control and delayed facilitated channels and the effect of
isoproterenol on channel activity evoked by a standard activation
protocol. In paired experiments, a 200 msec prepulse to +40 mV evoked
either a 5.0 ± 1.3-fold (isoproterenol experiments,
n = 5) or a 7.2 ± 0.83-fold (8-CPT cAMP
experiments, n = 4) P(o) increase during 6 sec sweeps to 40 or 50 mV. Both isoproterenol (1 µM)
and 8-CPT cAMP (1 mM) completely abolished induction of
delayed facilitation, leaving a P(o) that was not significantly different from control sweeps (p > 0.05, paired Student's t test) (Fig. 9). In contrast,
both isoproterenol and 8-CPT cAMP had no effect on control L-type
channel activity observed in the six second sweeps. Finally,
isoproterenol (1 µM) produced a small, but not
significant, enhancement of activity evoked by a 200 msec voltage pulse
from 60 to 0 mV (Fig. 9). These data demonstrate that stimulation of
-adrenergic receptors inhibits delayed facilitation of L-type
calcium channels, while having little effect on standard L-type channel
activity.
DISCUSSION
Delayed facilitation of L-type channel activity possesses some
unusual properties. In addition to being observed at very negative membrane potentials, close to the resting membrane potential of the
cells, delayed facilitation exhibited a very slow time course, not
reaching a peak until 0.5-1 sec after termination of the prepulse (Fig. 5). It was evoked by a single voltage prepulse, close to the peak
of the action potential, and may be preferentially induced by a short
train of action potentials (Figs. 2, 3). The time-dependent change in
P(o) occurred at all membrane potentials and was caused by
an increase in channel opening frequency and the recruitment of longer
duration events (Figs. 6, 7). Finally, the induction of delayed
facilitation was blocked by activation of -adrenergic receptors
(Figs. 8, 9).
The slow rise and longevity of delayed facilitation makes this behavior
distinct from previously described examples of prepulse facilitation of
L-type channels in hippocampal neurons (Fisher et al., 1990; Thibault
et al., 1993; Kavalali and Plummer, 1994, 1996). Two mechanisms have
been proposed for the more rapid decaying form of prepulse
facilitation: either an activation of a distinct DHP-sensitive channel
(Kavalali and Plummer, 1994, 1996) or a channel-related phenomenon such
as relief of inactivation or loss of a blocking ion (Thibault et al.,
1993). In this study, the conductance of channels underlying delayed
facilitation was not significantly different from the conductance of
channels evoked by short depolarizing voltage pulses (Fig. 4). Delayed
facilitation was observed in four patches that showed openings to a
single level. This data would suggest that delayed facilitation
represents an increase in activity of the standard L-type channel,
rather than the recruitment of a novel DHP-sensitive channel. However, this suggestion must be considered with caution, because it is extremely difficult to determine the number of channels present in a
patch (see Materials and Methods). The marked delay and slow rise time
of delayed facilitation suggest that second messenger-mediated modulation of the channel may be responsible. This hypothesis is
supported by the block of delayed facilitation by -adrenergic receptor activation. This inhibition was mimicked by a
membrane-permeant cAMP analog, suggesting that inhibition is mediated
by a phosphorylation event. Therefore, delayed facilitation may be
promoted by dephosphorylation. It is proposed that the presence of a
phosphate group prevents induction of delayed facilitation, and that
the channel is tonically phosphorylated at a site inaccessible to a
phosphatase. A train of action potentials or a voltage prepulse would
induce a conformational change, allowing access to the
phosphatase. The decay of delayed facilitation may reflect the action
of an associated kinase. However, it is also possible that delayed
facilitation does not require either kinase or phosphatase activity,
and that -adrenergic receptor activation simply prevents induction
of delayed facilitation.
Inhibition of delayed facilitation by -adrenergic stimulation
is opposite to what is expected, because both the L-type
calcium current recorded in ventricular myocytes (Bean et al., 1986)
and high-voltage-activated (HVA) calcium current in dentate granule cells (Gray and Johnston, 1987) were found to be enhanced by
-adrenergic agonists. These effects were mimicked by
membrane-permeant analogs of cAMP and were assumed to result from
activation of PKA. In addition, a voltage-dependent facilitation of
L-type calcium currents in skeletal muscle required activation of PKA
and was blocked by its specific inhibitor PKI (Sculptoreanu et al.,
1993). However, delayed facilitation exhibits a slow rising phase and
is much longer-lasting than facilitation in skeletal muscle, suggesting that the two systems are not comparable. The reported effect of -adrenergic stimulation on HVA calcium current in dentate granule cells (Gray and Johnston, 1987) has not been repeated. In addition, attempts to repeat this effect using hippocampal neurons maintained in
culture have not been successful (R. A. Craig and N. V. Marrion, unpublished observation). The reason for this discrepancy is
unclear. However, two separate gene products [C (Snutch et al., 1991)
and D class (Williams et al., 1992)] encode subtypes of neuronal
L-type channels, and both of these classes of channels are present in hippocampal neurons (Hell et al., 1993a). Furthermore, two isoforms of
the 
1 subunit of the C class channel co-exist in rat brain, and only
one is a substrate for PKA (Hell et al., 1993b). It is possible that
this variability underlies the observed differences in modulation.
However, it should be noted that a small, but not statistically
significant, enhancement of activity evoked by a standard activation
protocol was observed (Fig. 9). It is possible that this observation is
related to the effect reported by Gray and Johnston (1987).
The time course of delayed facilitation is remarkably close to
that of the slow AHP in hippocampal pyramidal neurons. L-type channels
are activated by somatically generated action potentials (Magee and
Johnston, 1995), whereas the slow AHP is observed after a train of
these action potentials. Under voltage clamp, both are activated by the
same voltage protocol and display a slow rising phase and decay at
similar rates (Lancaster and Adams, 1986; Sah and Issacson, 1995).
Previous studies have suggested that calcium-activated small
conductance potassium channels (SK channels) underlying the slow AHP
are intimately linked to L-type Ca channels. The slow AHP is blocked by
DHP antagonists (Rascol et al., 1991; Moyer et al., 1992), indicating
that L-type Ca channels provide the calcium for activation of the slow
AHP. The number of L-type channels (Thibault and Landfield, 1996) and
the amplitude of the slow AHP (Moyer et al., 1992) both increase with
age. In addition, learning-induced reductions in the slow AHP are
reported (de Jonge et al., 1990), and block of L-type channels with
nimodipine enhances associative learning (Deyo et al., 1989).
The present finding that delayed facilitation is blocked by
-adrenergic receptor activation may shed new light on the modulation of the slow AHP in hippocampal neurons. The time course of the slow AHP
is suggested to be controlled by diffusion of intracellular calcium
accumulated during the preceding burst of action potentials (Lancaster
and Adams, 1986; Lancaster and Nicoll, 1987). -Adrenergic modulation
of the slow AHP, which is mediated by activation of PKA (Pedarzani and
Storm, 1993), has been assumed to occur at a step subsequent to the
entry of Ca (Nicoll, 1988; Knöpfel et al., 1990). This assumption
results from the observations that both calcium action potentials and
the resulting intracellular calcium transients are unaffected by
agonists that suppress the slow AHP (Madison and Nicoll, 1982;
Knöpfel et al., 1990). However, the intracellular calcium
transients are only 10-50 nM in amplitude (Knöpfel
et al., 1990). The three classes of SK channels cloned, which include
SK1, the mRNA for which is present in hippocampal pyramidal neurons,
are half-maximally activated by ~700 nM Ca (Köhler
et al., 1996). This value is similar to the Ca sensitivity of the
apamin-sensitive SK channel found in rat adrenal chromaffin cells
(K0.5, ~800 nM) (Park,
1994). Therefore, the measured intracellular calcium transients are
probably too small to account for activation of SK channels that
underlie generation of the slow AHP. It seems likely that local
intracellular calcium increases are responsible for activation of SK
channels in hippocampal neurons, with the measured intracellular
calcium transients representing residual calcium not removed by
cellular sequestration and pump processes. Therefore, it seems likely
that if these calcium transients principally arise from calcium entry
during the depolarization used to evoke the slow AHP (with only a minor
component arising from delayed facilitation), they would not be
apparently affected by the receptor activation that suppresses the slow
AHP. However, activation of potassium channels by the intracellular
photolytic release of Ca from DM-nitrophen is apparently sensitive to
-adrenergic receptor stimulation (Lancaster and Zucker, 1994). It is
not known whether the potassium channels activated by this method were
SK channels. In addition, because repeated photolysis causes depletion
of Ca-loaded DM-nitrophen, and calcium release was not monitored
(Lancaster and Zucker, 1994), the lack of a reversible effect of the
applied agonist makes it possible that the apparent response could
result from this depletion. The results presented here raise the
possibility that the time course of the slow AHP is determined by
delayed facilitation. The selective modulation of delayed facilitation by -adrenergic receptor activation, with little or no effect on the
channel activity evoked during membrane depolarization (Fig. 9), would
mean that an effect on calcium spikes and residual intracellular
calcium transients would not be expected. Therefore, modulation of
delayed facilitation of L-type calcium channels may underlie the
suppression of the slow AHP by -adrenergic receptor activation.
FOOTNOTES
Received Feb. 6, 1997; revised June 4, 1997; accepted June 10, 1997.
This work was supported by National Institutes of Health Grant NS29806
(N.V.M.). We thank Drs. D. Shepherd and B. Hirschberg for critical
reading of this manuscript. In addition, we thank Robin A. Craig for
initiating this project and providing data using cultured hippocampal
neurons.
Correspondence should be addressed to Neil V. Marrion, Vollum
Institute, Oregon Health Sciences University, 3181 Southwest Sam
Jackson Park Road, Portland, OR 97201-3098.
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