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The Journal of Neuroscience, March 1, 2003, 23(5):1593
Afterhyperpolarization Regulates Firing Rate in Neurons of the
Suprachiasmatic Nucleus
Robin K.
Cloues and
William A.
Sather
Department of Pharmacology, University of Colorado Health Sciences
Center, Denver, Colorado 80262
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ABSTRACT |
Cluster I neurons of the suprachiasmatic nucleus (SCN), which are
thought to be pacemakers supporting circadian activity, fire
spontaneous action potentials that are followed by a monophasic afterhyperpolarization (AHP). Using a brain slice preparation, we have
found that the AHP has a shorter duration in cells firing at higher
frequency, consistent with circadian modulation of the AHP. The AHP is
supported by at least three subtypes of KCa channels, including apamin-sensitive channels, iberiotoxin-sensitive channels, and channels that are insensitive to both of these antagonists. The
latter KCa channel subtype is involved in rate-dependent
regulation of the AHP. Voltage-clamped, whole-cell
Ca2+ channel currents recorded from SCN neurons were
dissected pharmacologically, revealing all of the major high-voltage
activated subtypes: L-, N-, P/Q-, and R-type Ca2+
channel currents. Application of Ca2+ channel
antagonists to spontaneously firing neurons indicated that
predominantly L- and R-type currents trigger the AHP. Our findings
suggest that apamin- and iberiotoxin-insensitive KCa channels are subject to diurnal modulation by the circadian clock and
that this modulation either directly or indirectly leads to the
expression of a circadian rhythm in spiking frequency.
Key words:
suprachiasmatic nucleus; calcium channel; calcium-activated potassium channel; action potential; spontaneous
activity; afterhyperpolarization; circadian rhythm
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Introduction |
The suprachiasmatic nuclei
(SCN) of the hypothalamus contain the primary circadian clock
that controls various physiological and behavioral rhythms in mammals,
including rhythms in body temperature and sleep-wake behavior (Moore
and Eichler, 1972
; Klein et al., 1991). Single SCN neurons are
autonomous pacemaker cells that harbor the fundamental molecular
workings of the circadian clock, an interconnected set of
transcriptional/translational negative and positive feedback loops that
produce coordinated, rhythmic changes in expression of clock genes
(Dunlap, 1999; Reppert and Weaver, 2001). In keeping with the role
of a 24 hr pacemaker, firing of SCN neurons follows the
circadian clock, with higher spike activity during the day and lower
activity at night (Inouye and Kawamura, 1979; Gillette, 1991; Jagota et
al., 2000). Even an isolated SCN neuron in culture, lacking any
synaptic input, will sustain spontaneous and rhythmic firing (Welsh et
al., 1995). Such circadian oscillation in firing frequency is known to
be critical for transmittal of time information because suppression of
action potentials via selective application of tetrodotoxin to the SCN
abolishes the circadian rhythm of many organismal behaviors (Schwartz et al., 1987; Earnest et al., 1991; Schwartz, 1991; Newman et
al., 1992). Little is known, however, regarding the mechanism that
links the molecular clockworks to rhythmic electrophysiological output.
A prerequisite for understanding how the rhythm of the core of the
clock is transduced into a rhythm in spike rate is the identification
of ion channels responsible for spontaneous, rhythmic firing. Some of
these have been identified. Thus it is known that a slowly inactivating
Na+ current generates an interspike
depolarization that brings the membrane potential to firing threshold
(Pennartz et al., 1997). Additional ion channels, including T-type
Ca2+ channels, L-type
Ca2+ channels, and
hyperpolarization-activated cation channels (Ih), may assist in generating the interspike depolarization (Akasu et al.,
1993; Jiang et al., 1995; Pennartz et al., 2002).
Other channels involved in spontaneous, rhythmic firing have remained
uncharacterized. After action potential repolarization, SCN neurons
exhibit an intermediate-duration afterhyperpolarization (AHP) that may
contribute to regulation of firing rate. Because the AHP in other
spontaneously active central neurons is a key determinant of cellular
excitability (Aizenman and Linden, 1999; Bevan and Wilson, 1999) and is
subject to modulation by intracellular messengers (Sah, 1996), the AHP
represents a potential target for regulation by the circadian clock.
In this study, we have found that the duration of the AHP in SCN
neurons firing at higher frequencies is significantly shorter than in
more slowly firing neurons, consistent with circadian modulation. To
better understand the nature of the changes in the AHP, we undertook a
pharmacological analysis of the ion channels supporting the AHP in
cluster I SCN neurons, which are the most abundant neuronal type in the
SCN and are thought to be important in the output of these nuclei
(Pennartz et al., 1998). This analysis classifies
KCa channels that underlie the AHP, identifies
the Ca2+ channels that trigger the AHP,
and highlights particular subtypes of KCa and
Ca2+ channels as candidates for circadian regulation.
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Materials and Methods |
Preparation of brain slices. Hypothalamic slices
containing the SCN were obtained from Fisher 344 or Sprague
Dawley rats (17-25 d postnatal) that had been maintained on a
12 hr light/dark cycle for at least 1 week before they were
killed. Animals were anesthetized with halothane and
decapitated, and the brain was quickly removed and placed in ice-cold
artificial CSF (ACSF) of the following composition (in
mM): 126 NaCl, 3 KCl, 1.5 MgCl2, 2.4 CaCl2, 1.2 NaH2PO4, 11 glucose, 25.9 NaHCO3, continuously gassed with 95% O2-5% CO2, pH 7.4. Coronal hypothalamic slices (250 µm) were cut on a vibratome and
incubated in ACSF at room temperature (~23-25°C) for at least 45 min before recording. Experiments were performed primarily on neurons
taken from rats killed during their subjective day, although a subset
of experiments was performed with neurons from subjective night rats.
Recordings were made from all regions of the SCN.
Current-clamp recordings. Slices were placed in a recording
chamber mounted on the stage of an upright microscope
(Nikon Eclipse E600 FN). Single SCN neurons in slices were
visualized using a water-immersion objective lens [40×, 0.8 numerical
aperture (NA)], differential interference contrast optics, and a 0.9 NA condenser. An infrared-sensitive video camera was used to display
the field of view on a video monitor. To measure the AHP, action
potential firing rate, and other properties of membrane potential
changes in SCN neurons, recordings were made using the current-clamp
mode of the Axopatch 200B amplifier. The bathing solution, ACSF, was bubbled continuously with 95% O2-5%
CO2. Recordings were obtained at room temperature
(20-23°C). For Co2+ substitution
experiments, a HEPES-based ACSF was used that contained (in
mM): 126 NaCl, 3 KCl, 2.4 CaCl2 (or CoCl2), 1.5 MgCl2, 26 HEPES, 11 glucose, pH 7.4, bubbled with
100% O2. The pipette solution contained (in
mM): 135 potassium gluconate, 10 KCl, 10 HEPES, 0.5 EGTA, 2 MgATP, pH 7.4. The pipette was mounted on a piezoelectric manipulator bolted onto the fixed stage of the microscope, and whole-cell recordings were obtained at room temperature using an
Axopatch 200B amplifier (Axon Instruments, Foster City,
CA). Data were filtered at 2 kHz (
3 dB; four-pole low-pass Bessel filter integral to the Axopatch 200B amplifier) and sampled at 5 kHz
using an ITC-16 analog-to-digital converter (Instrutech, Great Neck, NY) and Pulse software (HEKA, distributed by
Instrutech). In a few experiments, 10 mM BAPTA was substituted for EGTA. A liquid junction potential of 12.5 mV was measured for the solutions used in current-clamp recording (Neher, 1992); data were not adjusted for this junction potential.
For experiments examining the contribution of ryanodine receptors to
the control of AHP amplitude, perforated-patch recording with
amphotericin B (Sigma, St. Louis, MO) was used. The
perforated-patch method was used in these experiments because it
facilitated the long-duration, stable recordings that were needed to
study the effects of the slowly acting, bath-applied ryanodine (Vilchis et al., 2000). A stock solution of amphotericin B (1 mg/ml, in dimethylsulfoxide) was diluted into a potassium gluconate-based pipette
solution (1:200) that was otherwise identical to that used in the
ruptured-patch current-clamp recordings. The patch pipette tip was
filled with amphotericin B-free potassium gluconate-based solution via
tip immersion, and then the pipette was back-filled with the
amphotericin B-containing solution. After obtaining a high-resistance
seal between pipette and cell membrane, access resistance was monitored
by regular examination of the current response to a voltage step (+10
mV). Because amphotericin B partitioned into the pipette-encircled
patch, access resistance dropped within 10 min to a stable level of
~40 M
.
Voltage-clamp of Ca2+ channel
currents. For recording whole-cell
Ba2+ currents, the extracellular solution
contained (in mM): 100 NaCl, 3 KCl, 0.15 MgCl2, 5 BaCl2, 17 tetraethylammonium chloride (TEA-Cl), 0.5 4-aminopyridine, 5 CsCl, 26 HEPES, 10 glucose, 0.03 bicuculline methiodide, and 0.001 tetrodotoxin, pH 7.4. A reservoir containing this solution was bubbled
continuously with 100% oxygen, and a gravity-driven system was used to
perfuse the oxygenated solution through the recording chamber at 3-5
ml/min. Whole-cell recording pipettes pulled from borosilicate glass
(GC120F-10; Warner Instruments, Hamden, CT) had
resistances of 4-6 M when filled with a solution containing (in
mM): 120 TEA-Cl, 9 EGTA, 2 MgATP, and 9 HEPES, pH 7.4. After seal formation
(Rseal > 3 G), neurons were
allowed to stabilize for 300-500 sec before data were recorded. Series resistance compensation and capacitance transient cancellation were
performed using the circuitry of the amplifier. Data were filtered at 2 kHz and sampled at 4 kHz. Linear leak and residual capacitance currents
were removed on-line by subtracting scaled and inverted current
responses to hyperpolarizing voltage steps of one-quarter the amplitude
of the depolarizing test pulse. A liquid junction potential of +1.4 mV
was measured for the solutions used in voltage-clamp recording (Neher,
1992); the reported data have not been corrected for this junction
potential. All experiments were performed at room temperature.
In all SCN whole-cell voltage-clamp recordings, a series resistance of
typically 20-60 M was compensated by ~80%. Whole-cell Ba2+ current amplitude ranged up to ~300
pA, so in the well clamped compartment of the cell the maximal error in
command voltage attributable to pipette series resistance was estimated
as 1.2-3.6 mV for the largest (300 pA) currents. Although SCN neurons
are electrotonically compact compared with many central neurons,
Ba2+ currents originating from distal
processes of SCN neurons were unlikely to have been adequately clamped.
An indication of this was provided by examination of the kinetics of
Ca2+ channel currents. Comparison of
activation and deactivation rates of whole-cell
Ba2+ currents obtained from slice
recordings with those recorded from dissociated SCN neurons [prepared
as described by Huang (1993)] showed that activation times measured in
slices (
act = 2.4 ± 0.2 msec;
n = 12; 10 mV) were longer than those measured in
dissociated neurons (act = 1.1 ± 0.3 msec; n = 5; 10 mV). Measured deactivation times were also significantly longer in duration for slices:
deact = 1.7 ± 0.2 msec in slices versus
0.4 ± 0.3 msec in dissociated neurons. As a consequence of the
limited quality of voltage clamp in the slice recording configuration,
we restricted our analysis of Ca2+ channel
currents in SCN slices to peak current amplitudes: the relatively slow
rates of activation and inactivation for
Ca2+ channels make peak current amplitude
somewhat less sensitive to clamp quality than other parameters.
Pharmacological analysis of channels. Channel antagonists
used to identify KCa channels in SCN neurons are
in general highly specific (Gribkoff et al., 1997; Vergara et al.,
1998; Kaczorowski and Garcia, 1999; Sah and Davies, 2000). Iberiotoxin
(dose = 100 nM; IC50 = 1-5 nM) (Galvez et al., 1990; Candia et al.,
1992) is a very selective blocker of type I, but not type II,
BKCa channels (Gribkoff et al., 1997; Meera et
al., 2000). Apamin (dose = 100 nM;
IC50 = 0.06-1 nM) (Hughes
et al., 1982; Blatz and Magleby, 1986) blocks many, but perhaps not
all, neuronal SKCa channels. Charybdotoxin
(dose = 100 nM; IC50 = 10 nM) (Anderson et al., 1988), another blocker
of type I but not type II BKCa channels, was
tested in some experiments. Interpretation of the effects of this
latter blocker was complicated by the fact that it is, in addition, a
potent antagonist of some intermediate conductance KCa channels (IKCa) and
some voltage-gated potassium channels. At the doses used for
iberiotoxin and apamin, essentially complete block of sensitive
channels was expected, whereas insensitive KCa
subtypes and other ion channels would not have been affected.
The stability of Ca2+ channel currents
recorded from slices was advantageous for pharmacological analysis of
Ca2+ channel types present in SCN neurons.
During the first 300-500 sec after breakthrough, voltage-clamped
Ba2+ currents carried by
Ca2+ channels usually increased in
amplitude and then became nearly constant; peak current amplitude
diminished by only 2 ± 0.02% over the next 420 sec (n
= 9 neurons). Consequently,
Ca2+ channel antagonists were applied
after a stabilization period of generally 300-500 sec after the start
of whole-cell recording. To achieve a steady level of block, channel
blocking agents were usually applied for 180-300 sec via the bath
perfusion system (3-5 ml/min). Delays in onset of antagonist action of
up to ~50 sec were often observed, reflecting the slow solution
exchange time and the time needed for antagonists to diffuse into the
tissue. Considering the time needed to achieve steady block relative to the rate of run-down in peak current amplitude, run-down had only a
minor effect on our measurements.
Although the Ca2+ channel antagonists used
here are not ideal in their specificity, these blockers were
nonetheless effective in identifying Ca2+
channel isoforms that activate the action potential AHP. The classical
L channel antagonist nimodipine (dose = 10 µM;
IC50 = 50 nM under our recording
conditions) (Marchetti et al., 1995), the N channel antagonist
-conotoxin-GVIA (-CTx-GVIA) (dose = 3 µM;
IC50 
0.7 nM) (Boland et al.,
1994), and the P channel antagonist -agatoxin-IVA (-Aga-IVA)
(dose = 200 nM; IC50 = 1.5 nM) (Mintz et al., 1992) were applied by bath perfusion at
concentrations 133-4300× IC50 values and
therefore were expected to block at least 99% of current carried by
these Ca2+ channel subtypes. Nonclassical
L channels based on the CaV1.3 (
1D) subunit may not have been fully blocked
by 10 µM nimodipine (Xu and Lipscombe, 2001). Q-type
channels are blocked by -Aga-IVA, but these channels are less
sensitive to this antagonist, so that ~70% of Q-type current was
expected to be blocked at the dose used (dose = 200 nM; IC50 = 90 nM)
(Randall and Tsien, 1995). However, there was no significant
difference between a mixture of blockers containing -Aga-IVA
and one containing -conotoxin-MVIIC (-CTx-MVIIC) (dose = 3 µM) suggesting that the contribution of Q-type current
is not large in SCN neurons. R-type current was defined as the
Ba2+ current not blocked by a combination
of nimodipine, -CTx-GVIA, -Aga-IVA, and mibefradil. We further
dissected the R-type current according to block by the toxin SNX-482,
which blocks 1E-based Ca2+ channels
(CaV2.3; dose = 200 nM;
IC50 = 15 nM) (Newcomb et al., 1998)
and the less specific blocker Ni2+
(dose = 30 µM; IC50 = 4-30
µM) (Tottene et al., 1996; Zamponi et al., 1996). In this
concentration range, Ni2+ would not be
expected to inhibit N- or P/Q-type currents but would block a portion
of L-type currents (IC50 = 60 µM)
(Zamponi et al., 1996). Ni2+ also
blocks one of the three T-type Ca2+
channel isoforms, but SCN neurons do not express the
Ni2+-sensitive T channel isoform
CaV3.3 (1H) (Lee et al.,
1999; Talley et al., 1999). Because of the difficulty in accurately
voltage clamping SCN neurons, we did not attempt to estimate the
contribution of T channel current in these cells. The modestly
selective blocker mibefradil was included in the mixture that was used
to estimate the contribution of R-type
Ca2+ channels in whole-cell
Ba2+ currents and to examine the
contribution of Ca2+ channel subtypes to
AHP activation. By using interpolated IC50 values
appropriate for our recording conditions (~100-800 nM), a 500 nM dose of mibefradil was expected to block 38-83%
of T current in SCN neurons (McDonough and Bean, 1998; Martin et al., 2000). Higher doses of mibefradil were not used because this drug interacts with other Ca2+ channel
subtypes, albeit with lower potency (Bezprozvanny and Tsien, 1995;
Martin et al., 2000).
Stock solutions of nimodipine, ryanodine, and picrotoxin were made by
dissolving these ion channel antagonists in 100% ethanol. Stock
solutions for all other ion channel antagonists were made using
distilled water. Stocks were stored at 20°C and in the dark.
()-Bicuculline methiodide was not prepared as a stock solution because of its chemical instability; instead this agent was dissolved in the bath solution on a daily basis, before the start of experiments.
Channel antagonists were obtained from the following sources:
picrotoxin, ()-bicuculline methiodide, -CTx-GVIA, apamin, iberiotoxin, charybdotoxin, and ryanodine from Sigma;
-Aga-IVA, -CTx-MVIIC, and SNX-482 from Peptide Institute (Osaka,
Japan); nimodipine from Research Biochemicals (Natick, MA); and
mibefradil was a generous gift from Dr. Jean-Paul Cloze and Dr. Eric
Ertel (F. Hoffmann-La Roche, Basel, Switzerland).
Mean values are reported together with their SEs, and all error bars
indicate SEs of the mean. The number of experiments, n,
indicates in all cases the number of neurons studied. Student's t test was used to determine statistical significance.
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Results |
Spontaneous neuronal firing rate correlates with AHP duration
SCN neurons spontaneously fire action potentials over a range of
frequencies (0-15 Hz), with average firing rates being higher during
the daytime and lower during the nighttime (Inouye and Kawamura, 1979;
Gillette, 1991; Jagota et al., 2000). Clock cells are not tightly
synchronized with one another, however, and the circadian time at which
peak firing occurs can be considerably out of phase among SCN neurons.
We therefore used comparisons of action potential waveforms recorded
from cells firing spontaneously at different rates, regardless of
circadian time, as a strategy to identify components of the action
potential that might be regulated in a circadian manner. From these
comparisons, we identified an altered AHP waveform as a major factor
regulating firing frequency: the AHP became shorter in duration as
spike frequency increased (Fig.
1A). In addition, the
action potential width was significantly broader in slower firing
neurons, but other action potential properties remained unchanged: the
depolarizing ramp preceding the action potential upstroke, interspike
membrane potential, action potential height, and maximum
hyperpolarization after the action potential were not significantly
different between fast and slow firing neurons (Table
1).
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Figure 1.
Firing properties of cluster I SCN neurons under
whole-cell current clamp. A, Action potential waveforms,
averaged from four to six spikes, from three different cells firing at
the rates indicated. Note that in cells with faster firing rates, the
duration of the AHP was briefer. Records of action potentials were
shifted along the voltage axis to superimpose action potential
threshold. B, Single action potentials recorded in
control conditions, in the presence of 30 µM bicuculline
methiodide and in the presence of 30 µM
Cd2+ are superimposed. Cd2+ was
used as a measure of the Ca2+-dependent AHP.
Bicuculline did not cause a reduction in the amplitude of the AHP.
C, Positive current injection (20 pA, as indicated below
current-clamp record), evoked a train of action potentials that did not
exhibit spike frequency adaptation. In many, but not all neurons, there
was a progressive diminution in the amplitude of action potentials that
followed the initial spike.
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Under current-clamp conditions, action potential discharge was followed
by a monophasic AHP in the majority of neurons (98 of 110) studied
(Fig. 1A,B). These kinds of neurons
have been classified previously as cluster I neurons (Pennartz et al.,
1998). Cells with a biphasic AHP (cluster II and III neurons) were not included in our analysis. Identification of cluster I neurons was
confirmed by testing for the absence of spike frequency adaptation during a train of action potentials evoked by injection of steady positive current (Fig. 1C) (Pennartz et al., 1998).
In this work, we defined AHP as the
Ca2+-dependent component of the
hyperpolarization that followed each action potential (Fig. 1B). We measured the
Ca2+-dependent component by blocking
Ca2+ entry with 30 µM Cd2+, which
blocks virtually all Ca2+ channel current
in SCN neurons (93 ± 2%; n = 10). The average difference between maximum hyperpolarization before and after Cd2+ application was 10.0 ± 2.1 mV
(n = 10). We compared this result with
experiments in which external Ca2+ was
replaced with Co2+. Using this method, the
amplitude of the Ca2+-dependent AHP was
11.3 ± 1.0 mV (n = 5), which was not
significantly different from measurements using
Cd2+ (p > 0.1). We
also tested the ability of a Ca2+ chelator
(10 mM BAPTA, included in the whole-cell patch
pipette solution) to occlude the effect of
Cd2+. Consistent with expectation,
Cd2+ had a negligible effect on the AHP in
cells loaded with BAPTA (average difference between maximum
hyperpolarization before and after Cd2+
application was 2.6 ± 0.6 mV; n = 9),
indicating that the Ca2+-dependent AHP was
abolished by high intracellular BAPTA. These results indicate that 30 µM Cd2+ blocks the
entry of Ca2+ required for activation of
KCa channels and that
Cd2+ block provides a practical index of
AHP amplitude.
In most experiments, bicuculline methiodide (30 µM) or
picrotoxin (100 µM) was used to block
GABAA receptors because SCN neurons make
extensive GABAergic connections with one another (Strecker et al.,
1997). Although bicuculline methiodide is known to block some kinds of
KCa channels (Khawaled et al., 1999), in the work described here, no significant reduction in AHP amplitude was observed
after application of bicuculline methiodide (Fig. 1B) or picrotoxin. In the presence of bicuculline methiodide, the AHP
averaged 7.8 ± 0.4 mV (n = 7), and in the
presence of picrotoxin the AHP averaged 6.7 ± 0.8 mV
(n = 5). These values were not significantly different from the AHP amplitude measured in the absence of
GABAA receptor antagonists
(p > 0.1). In reporting the results of
subsequent experiments, we have therefore combined experimental results
from slices treated with bicuculline methiodide, with picrotoxin, or with no GABAA receptor antagonist.
To begin probing for factors controlling AHP duration and firing
frequency, we examined the effects of blocking
Ca2+ influx on AHP waveform in neurons
firing at different spontaneous rates (Fig.
2). In the example shown in Figure
2A, block of Ca2+ influx
by Cd2+ had a much greater effect on the
AHP in a rapidly firing neuron (11 Hz, right) than in a
slowly firing neuron (1.3 Hz, left). Subtraction of the
action potential waveform recorded in the presence of
Cd2+ from that recorded in control
conditions (Fig. 2A, inset) revealed that
the Ca2+-dependent AHP decayed more
quickly in the faster firing neuron. Paradoxically, the
Ca2+-dependent AHP was also larger in
amplitude in the faster firing neuron. Among neurons firing at
different spontaneous rates, the duration and amplitude of their
Cd2+-subtracted AHP waveforms were highly
correlated with neuronal firing rate (Fig. 2B,C).
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Figure 2.
Changes in the Ca2+-dependent
AHP over a range of firing frequencies. A, Comparison of
two cells, one firing at 1.3 Hz (left panel) and
one firing at 11 Hz (right panel). After several
minutes of spontaneous firing in control conditions, 30 µM Cd2+ was applied to slices.
Ensemble-averaged action potential waveforms (4-6 action potentials
for each condition) are shown superimposed. Insets show
subtraction of waveforms recorded in 30 µM
Cd2+ from control waveforms
(Cd2+-subtracted AHP). Solid line in
the inset indicates 0 mV. Calibration: 10 mV, 10 msec.
B, Half-time for decay of the
Cd2+-subtracted AHP as a function of spontaneous
firing frequency. For each neuron analyzed, decay half-time
(t1/2) was measured as the time from
maximum hyperpolarization to 50% of that value, and the measured
t1/2 value was plotted versus the
spontaneous firing frequency in that particular neuron. The
t1/2 versus frequency data were fit with a
linear regression, using a maximum likelihood estimate. Correlation
coefficient was r = 0.67. C,
Amplitude of the Cd2+-subtracted AHP as a function
of spontaneous firing frequency. Amplitude of the subtracted AHP was
measured from maximum hyperpolarization to 0 mV. Correlation
coefficient for the regression fit was r = 0.66.
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Ca2+-sensitive K+ channels
underlying the AHP of cluster I neurons
Subtype-specific antagonists were used to identify the kinds of
KCa channels that produce the AHP in cluster I
SCN neurons. In this procedure, blockers of various kinds of
KCa channels were applied to spontaneously firing
SCN neurons, and after establishment of a stable reduction in AHP
amplitude, the AHP was completely blocked by applying 30 µM Cd2+. Percentage
reduction in AHP amplitude was calculated according to the
equation: percentage reduction = [(AHPcontrol AHPKCa-antagonist)/(AHPcontrol AHPCd)] × 100%. As shown in Figure
3A, iberiotoxin (100 nM), a blocker of certain large conductance
KCa channels (BKCa
channels), reduced AHP amplitude by ~40%. A peptide antagonist
similar to iberiotoxin in action, charybdotoxin (100 nM), reduced AHP amplitude by a roughly similar
amount (28 ± 12%; range, 0-58%; n = 6; data not shown). Also illustrated in Figure 3A is the effect on
AHP amplitude of apamin (100 nM), a blocker of
certain small conductance KCa channels
(SKCa channels): apamin reduced AHP amplitude by ~20%. Coapplication of apamin and iberiotoxin reduced AHP amplitude by only ~55%, suggesting that SCN neurons possess some
KCa channels that are insensitive to both apamin
and iberiotoxin. Because 30 µM
Cd2+ may also have actions on channels
other than voltage-gated Ca2+ channels
(Bekkers, 2000), however, we tested the prediction that block of the
AHP by apamin and iberiotoxin would be smaller than block by a mixture
designed to block Ca2+ channels, including
L-, N-, P/Q-, R-, and T-type Ca2+
channels. Figure 3B shows that the
Ca2+ channel mixture further reduced AHP
amplitude after application of the combination of apamin and
iberiotoxin, consistent with the idea that the AHP of SCN neurons is
supported by at least three subtypes of KCa
channels: apamin-sensitive channels, iberiotoxin-sensitive channels,
and channels insensitive to both antagonists. In addition, the wide
range in percentage block by iberiotoxin or apamin suggests that there
is considerable cell-to-cell variability in the expression of
KCa channels (Fig. 3, legend).
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Figure 3.
SCN neurons possess at least three classes of
KCa channels. A, Example records and group
data from experiments using blockers of KCa channels.
Spontaneous action potentials were recorded under control conditions
and in the presence of iberiotoxin (100 nM), apamin (100 nM), or both of these channel blockers.
Cd2+ (30 µM) was added at the end of
each experiment to fully block the Ca2+-dependent
AHP, thus establishing a baseline from which to estimate the
contribution to the AHP of KCa channels that were sensitive
to apamin, sensitive to iberiotoxin, or insensitive to both blockers.
Superimposed records were always obtained from the same neuron, but
each panel of records was recorded from a different neuron. Iberiotoxin
reduced AHP amplitude by 40 ± 5%, with a range of 18-55%
(n = 6); apamin reduced AHP amplitude by 18 ± 5%, with a range of 8-37% (n = 6); and the
combination of apamin and iberiotoxin reduced AHP amplitude by 54 ± 10%, with a range of 32-91% (n = 5).
B, Further diminution in the AHP by the
Ca2+ channel blocker mixture after application of
apamin and iberiotoxin. The Ca2+ channel mixture
contained 10 µM nimodipine, 3 µM
-CTx-GVIA, 200 nM -Aga-IVA, 30 µM
Ni2+, and 500 nM mibefradil, which will
block most of the Ca2+ entry into SCN neurons (see
also Fig. 5). Example action potentials in the various conditions are
superimposed on the left, and a bar chart
showing the percentage reduction in AHP amplitude is shown on the
right. The reductions in AHP amplitude produced by the
two sets of blockers (apamin + iberiotoxin,
Ca2+ channel
cocktail) were statistically different from one another
at the p < 0.05 level.
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We examined whether the iberiotoxin-sensitive portion of the AHP
changed with firing frequency, as observed for the
Cd2+-subtracted AHP (Fig. 2). When we
applied 100 nM iberiotoxin to SCN neurons firing
spontaneously between 0.2 and 8.2 Hz (n = 16) and
measured the amplitude and decay kinetics of the iberiotoxin-subtracted potential, we found no correlation between firing rate and either decay
half-time (correlation coefficient r = 0.08; Table 1)
or amplitude (r = 0.25).
Ca2+ channel subtypes present in neurons of
the SCN
Subtypes of Ca2+ channels present in
SCN neurons were identified using antagonists effective against
particular Ca2+ channel subtypes. In these
voltage-clamp experiments, Ba2+ was
substituted for Ca2+ as the permeant ion
to suppress Ca2+-sensitive currents.
Figure 4A-D
illustrates the action of subtype-selective Ca2+ channel antagonists on whole-cell
Ba2+ current recorded from voltage-clamped
SCN neurons in hypothalamic slices. As shown in Figure
4A, the L channel antagonist nimodipine partially
blocked Ba2+ current, indicating that
L-type Ca2+ channels carried a fraction of
whole-cell Ca2+ channel current. For a
series of control neurons, total Ba2+
current remained stable over the typical duration of experiments (5-15
min), as shown by the data points and dashed line in Figure 4A. N-type and P/Q-type
Ca2+ channels were also identified in SCN
neurons, on the basis of partial block of whole-cell
Ba2+ current by high concentrations of
-CTx-GVIA and -Aga-IVA (Fig. 4B,C).
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Figure 4.
Pharmacological identification of
Ca2+ channel currents in SCN neurons.
A, Plot of peak inward Ba2+ current
versus time for a single SCN neuron recorded in a hypothalamic slice.
The L channel antagonist nimodipine (10 µM) was applied
during the period indicated by the black bar. Nimodipine
blocked 29% of total Ba2+ current in this neuron
(filled circles). Asterisks
connected by a dotted line mark the mean
Ba2+ current recorded from seven neurons to which no
antagonist was applied. Inset shows current records
obtained at the times indicated by the numbers. The slow
time course of the tail currents reflects in part the speed of voltage
clamp for SCN neurons in slices. B, Application of the N
channel antagonist -CTx-GVIA (3 µM) to a neuron in
another slice blocked 30% of total Ba2+ current in
this example. C, Application of the P/Q channel
antagonist -Aga-IVA (200 nM) to a neuron in another
slice blocked 47% of total Ba2+ current in this
example. D, A combination of nimodipine
(nimod.) (10 µM), -CTx-GVIA (3 µM), -Aga-IVA (200 nM), and mibefradil
(mibef.) (500 nM) was added to block L-, N-,
P/Q-, and T-type Ca2+ channels, revealing a
prominent component of antagonist-resistant current (R-type
Ca2+ current). In all examples currents were
activated every 5 sec by 50 msec voltage steps from 80 to 10 mV
(voltage at which peak inward current was obtained).
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Figure 4D shows that application of a combination of
antagonists designed to block L-, N-, P/Q-, and T-type channels (10 µM nimodipine, 3 µM
-CTx-GVIA, 200 nM -Aga-IVA, and 500 nM mibefradil) blocked only half of the total
Ba2+ current in an SCN neuron. At the
antagonist concentrations used, classical L-, N-, and P-type channels
were expected to be fully blocked. Approximately 70% of any Q-type
current present was expected to have been blocked by 200 nM -Aga-IVA. We tested whether the current
remaining in the presence of L-, N-, P-, and T-type channel blockers
was carried by Q-type channels by substituting -CTx-MVIIC (3 µM) for -Aga-IVA in the mixture. This
concentration of -CTx-MVIIC would be expected to fully block both P-
and Q-type Ca2+ channels over the time
that it was applied (>10 min) (Randall and Tsien, 1995). We found that
the mixture containing -CTx-MVIIC did not block significantly more
Ba2+ current (52 ± 6%;
n = 4) than the mixture containing -Aga-IVA (51 ± 4%; n = 12; p > 0.1). Thus, most
of the current resistant to block by the combination of nimodipine,
-CTx-GVIA, -Aga-IVA (or -CTx-MVIIC), and mibefradil was, by
definition, R type (Zhang et al., 1993; Randall and Tsien, 1995).
In some neuronal cell types, R-type current is carried, at least in
part, by 1E-based
Ca2+ channels (Wang et al., 1999; Foehring
et al., 2000; Tottene et al., 2000; Lee et al., 2002). We therefore
tested the effect of SNX-482, a toxin selective for
1E-based Ca2+
channels, on whole-cell Ba2+
current. Figure 5A illustrates a cell in which a large
portion of Ba2+ current (77%) was blocked
by a combination of nimodipine, -CTx-GVIA, -Aga-IVA, and
mibefradil. The remaining current was not affected by application of
SNX-482 (200 nM) but could be blocked by
addition of Cd2+ (30 µM), a nonselective blocker of all
voltage-gated Ca2+ channel subtypes. In
seven cells tested, SNX-482 blocked <10% of current remaining in the
presence of L-, N-, P/Q-, and T-type channel blockers, suggesting that
1E-based Ca2+
channels are not significantly expressed in SCN neurons.
R-type current can also be blocked with some selectivity (see Materials
and Methods) by the divalent cation Ni2+
(Zamponi et al., 1996; N'Gouemo and Rittenhouse, 2000; Tottene et al.,
2000), and so we tested Ni2+ on the
current resistant to block by the combination of nimodipine, -CTx-GVIA, -Aga-IVA, and mibefradil. As illustrated in Figure 5B,
Ni2+ (30 µM)
blocked ~75% of the resistant Ba2+
current, confirming the identification of R-type current in these neurons. In the presence of nimodipine, -CTx-GVIA, -Aga-IVA, and
mibefradil, Ni2+ blocked 40 ± 6%
(n = 7) of the original total
Ba2+ current, a percentage not
significantly different from Ni2+ block in
the absence of the other four Ca2+ channel
antagonists (45 ± 6%; n = 6;
p > 0.1). This observation suggests that 30 µM Ni2+ does not
block Ca2+ currents other than R type in
SCN neurons. A summary of the Ca2+ channel
antagonist data is illustrated in Figure 5C.
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Figure 5.
SCN neurons possess a large R-type
Ca2+ current. A, Plot of peak inward
Ba2+ current versus time for an experiment designed
to test the sensitivity of R-type Ca2+ current to
the 1E channel blocker SNX-482. A combination of
nimodipine (10 µM), -CTx-GVIA (3 µM),
-Aga-IVA (200 nM), and mibefradil (500 nM)
was added to block L-, N-, P/Q-, and T-type Ca2+
channels. After a washout period, SNX-482 (200 nM) was
applied but had no effect on the current. At the end of the experiment,
complete block of Ca2+ channels was produced by
applying 30 µM Cd2+. Example records
obtained at various times during the experiment are illustrated in the
inset. B, In a similar experiment, the R
current antagonist Ni2+ (30 µM) was
applied, which blocked 75% of the current remaining after application
of blockers of L-, N-, P/Q-, and T-type channels. C,
Summary of average percentage block by Ca2+ channel
antagonists (same concentrations as described in A). In
C1, the various antagonists were applied either alone or
in various combinations. As a percentage of total
Ba2+ current, nimodipine blocked 18 ± 3%,
with a range of 0-33% (n = 18);
-CTx-GVIA blocked 27 ± 1%, with a range of 24-30%
(n = 6); -Aga-IVA blocked 24 ± 4%, with a range of 15-47% (n = 8).
Combined application of nimodipine, -CTx-GVIA, -Aga-IVA, and
mibefradil blocked 51 ± 4%, with a range of 31-77%
(n = 12); substitution of -Aga-IVA with
-CTx-MVIIC (3 µM) blocked 52 ± 6%, with a range
of 36-62% (n = 4). Combined application of
nimodipine, -CTx-GVIA, -Aga-IVA, mibefradil, and
Ni2+ (cocktail) blocked
76 ± 4% (n = 6) of total
Ba2+ current. At 30 µM,
Cd2+ applied alone blocked 93 ± 2%
(n = 10) of total Ba2+
current in SCN neurons. In C2, nimodipine, -CTx-GVIA,
-Aga-IVA, and mibefradil were applied together to neurons first,
followed by SNX-482 and then Cd2+. The combination
of nimodipine, -CTx-GVIA, -Aga-IVA, and mibefradil blocked
57 ± 6%, with a range of 44-77% (n
= 6) of total Ba2+ current. After
application of this combination of blockers, addition of SNX-482
blocked only 6 ± 2% (n = 6) of total
Ba2+ current, and subsequent application of 30 µM Cd2+ blocked the remaining current
(25 ± 5%; n = 6). In C3, the
experiment was conducted in an identical manner, with the exception
that application of the L-, N-, P/Q-, and T-type blockers was followed
by Ni2+ and then Cd2+. The
combination of nimodipine, -CTx-GVIA, -Aga-IVA, and mibefradil
blocked 44 ± 7%, with a range of 31-50% (n
= 6) of total Ba2+ current. After
application of this combination of blockers, addition of
Ni2+ blocked 52 ± 3% (n = 6) of total Ba2+ current, and subsequent application
of 30 µM Cd2+ blocked the remaining
current (15 ± 5%; n = 6).
Ni2+ added alone to SCN neurons blocked 45 ± 6%, with a range of 29-66% (n =
6).
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Effect of Ca2+ channel blockers on the AHP
In some neurons, tight coupling exists between specific classes of
Ca2+ channels and
KCa channels (Davies et al., 1996; Marrion and
Tavalin, 1998; Pineda et al., 1998). We therefore tested the effect of individual Ca2+ channel antagonists on the
AHP amplitude in spontaneously firing SCN neurons (Fig.
6). The N-, P/Q-, and T-type
Ca2+ channel blockers -CTx-GVIA (3 µM), -Aga-IVA (200 nM), and mibefradil (500 nM) had few or negligible effects on AHP amplitude
(<10%; n = 6 for each), suggesting minimal
participation of these kinds of channels in activating the AHP. In
contrast, the L-type Ca2+ channel
antagonist nimodipine (10 µM) had a significant
effect on peak AHP, reducing the amplitude by ~30%.
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Figure 6.
Action of Ca2+ channel blockers
on AHP amplitude. A, Ca2+ channel
antagonists were applied to spontaneously firing neurons in SCN slices.
Examples from individual experiments are shown, superimposing three
records from each experiment (control, block by an
isoform-specific antagonist, and block by
Cd2+). B, A summary of
the results from these experiments is presented as a bar
chart. Doses used for individual antagonists were 10 µM nimodipine, 3 µM -CTx-GVIA,
200 nM -Aga-IVA, and 500 nM
mibefradil. Nimodipine reduced AHP amplitude by 31 ± 2%, with a
range of 17-51% (n = 6); the blockers
-CTx-GVIA, -Aga-IVA, and mibefradil reduced AHP amplitude by
<10% (n = 6 for each).
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We also tested whether R-type current was involved in triggering the
AHP (Fig. 7A,B). A combination
of antagonists that block non-R-type Ca2+
current (nimodipine, -CTx-GVIA, -Aga-IVA, and mibefradil) only partially suppressed the AHP (46 ± 5%; n = 5),
suggesting that R-type current activated KCa
channels responsible for the remainder of the AHP. Indeed, block of R
current by Ni2+ (30 µM) partially suppressed the AHP (~20%).
Coapplication of the L- and R-type blockers nimodipine and
Ni2+ had effects that were additive,
reducing the AHP by ~50%. This block was not significantly different
from that produced by application of a mixture containing all
Ca2+ channel blockers (nimodipine,
-CTx-GVIA, -Aga-IVA, mibefradil, Ni2+; 63% ± 5%; n = 7;
p > 0.1).
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Figure 7.
Participation of R-type current in AHP activation.
A, Coapplication of antagonists for L-, N-, P/Q-, and
T-type Ca2+ channels blocked ~50% of the AHP,
suggesting that R-type Ca2+ channels must also
participate in activation of KCa channels. Application of
Ni2+ (30 µM) reduced AHP amplitude and
was additive with nimodipine (10 µM). A mixture of
antagonists that included nimodipine, -CTx-GVIA, -Aga-IVA,
mibefradil, and Ni2+ blocked the AHP by a similar
amount as Ni2+ and nimodipine applied together.
B, Summary of AHP reduction by combinations of
Ca2+ channel blockers. A combination of nimodipine
(10 µM), -CTx-GVIA (3 µM), -Aga-IVA
(200 nM), and mibefradil (500 nM) inhibited the
AHP by 46 ± 5%, with a range of 31-56% (n = 5). Ni2+ added alone (30 µM) reduced
AHP amplitude by 20 ± 2%, with a range of 10-25%
(n = 6). Inhibition of the AHP by
coapplication of nimodipine plus Ni2+ (50 ± 8%; range, 17-90%; n = 8) was comparable with
application of a mixture containing nimodipine, -CTx-GVIA,
-Aga-IVA, mibefradil, and Ni2+ (63 ± 5%;
range, 49-78%; n = 7). C, Plot of
normalized AHP amplitude versus time for four cells in which ryanodine
(10 µM), a blocker of Ca2+-induced
Ca2+ release, was applied for >30 min. Long-term
recordings were made using the perforated-patch method to prevent
rundown of the AHP.
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Because Ca2+ release via ryanodine
receptors triggers the AHP in some kinds of neurons, we tested the
effect of ryanodine, an inhibitor of
Ca2+-induced
Ca2+ release (Sutko et al., 1985; Akita
and Kuba, 2000), on the AHP of cluster I SCN neurons (Fig.
7C). Measured from spike threshold, maximum
hyperpolarization after a spike was diminished by 1.4 ± 0.5 mV
(n = 4) after 30 min of exposure to 10 µM ryanodine. This was not statistically
different from the reduction in afterhyperpolarization magnitude
observed after 30 min in control conditions (1.4 ± 1.0 mV;
n = 3). Thus release of
Ca2+ from ryanodine-sensitive stores
appears to play a minimal or no role in generating the AHP in cluster I
SCN neurons, despite the importance of ryanodine receptors in mediating
light-induced phase delays in the SCN (Ding et al., 1998).
Effects on firing rate resulting from pharmacological antagonism of
the AHP
In many kinds of neurons, the magnitude and duration of the AHP
are important factors in setting interspike interval and, thereby,
neuronal firing rate. For example, reduction in the AHP by the action
of neurotransmitters can increase firing rate (Madison and Nicoll,
1982; Pedarzani and Storm, 1995). The relationship between firing
frequency of SCN neurons and duration of the
Ca2+-dependent AHP (Fig.
2B) predicts that reducing AHP duration would increase firing rate. We therefore examined how pharmacological antagonism of the AHP affected spike frequency in spontaneously active
cluster I SCN neurons (Fig.
8A). In these
experiments, Ca2+ channel blockers and
KCa channel blockers were tested. Slices were
bathed in bicuculline methiodide (30 µM) or
picrotoxin (100 µM) to preclude secondary
effects of Ca2+ or
KCa channel blockers on firing rate, effects that
could otherwise have arisen via alteration of
Ca2+-dependent release of GABA. In control
experiments, spontaneous firing rates were found to be stable during
the initial 15 min after breakthrough, and we therefore confined our
measurements to this time period.
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Figure 8.
Effect on firing rate of Ca2+
channel and KCa channel antagonists. A,
Records (2 sec duration) of spontaneous firing in an SCN neuron under
control conditions and after application of a mixture of
Ca2+ channel blockers in the bath solution. The
Ca2+ channel mixture contained nimodipine (10 µM), -CTx-GVIA (3 µM), -Aga-IVA (200 nM), mibefradil (500 nM), and
Ni2+ (30 µM). B,
Normalized firing rate in the presence of various channel blockers.
Black bars represent results for Ca2+
channel antagonists, and gray bars represent results for
KCa channel antagonists. All firing rates with antagonist
were compared with a baseline firing rate in that neuron measured just
before antagonist application. In control recordings, firing frequency
did not significantly change over the first 15 min of whole-cell
recording; at 15 min, firing frequency was the same as that measured
just after establishment of whole-cell recording (striped
bar labeled control; p > 0.1; n = 5). The duration of experiments testing
antagonist action on firing frequency was in all cases <15 min.
Individual selective Ca2+ channel antagonists were
applied at a dose equal to that used in the Ca2+
channel blocker mixture. Apamin and iberiotoxin were applied at 100 nM. Cd2+ was applied at 30 µM. Firing rate experiments were performed in the
presence of 30 µM bicuculline methiodide. Sample size
ranged from n = 6-10, except for experiments with
Cd2+, for which n = 16. Paired
Student's t test; **p < 0.01.
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KCa channel antagonists did not alter firing
rate. Neither block of SKCa channels by apamin
(100 nM) nor block of BKCa channels by iberiotoxin (100 nM) had a statistically significant
effect on spike frequency (Fig. 8B), nor did
individual subtype-selective blockers of
Ca2+ channel subtypes alter firing rate.
Applied alone, nimodipine, -CTx-GVIA, -Aga-IVA, mibefradil, or
Ni2+ did not significantly affect firing
rate (Fig. 8B). However, application of a combination
of nimodipine plus Ni2+ had a significant
effect on firing rate (p < 0.01;
n = 6), although the effect was, unexpectedly, a
decrease in rate. Simultaneous application of all five
subtype-selective Ca2+ channel blockers
also slowed firing rate (p < 0.01;
n = 7). In contrast, complete blockade of
Ca2+ entry by
Cd2+ (30 µM)
increased firing rate ~1.5-fold (see Discussion).
Effects of channel blockers on spike parameters
Blockers of Ca2+ channels and
KCa channels were examined for their effects on
spike parameters other than the AHP: interspike potential
(VIP), determined as the plateau
region of membrane potential between action potentials; spike
amplitude, measured from VIP to peak
depolarization; and spike width, measured at half amplitude. None of
the blockers tested significantly affected VIP, indicating that changes in firing
rate could not be attributed to changes in
VIP (Table
2). This was confirmed for
Cd2+ by the fact that membrane potential
was not affected when this blocker was applied, whereas spiking was
suppressed by tetrodotoxin (1 µM):
VIP was 40 ± 3.1 mV before
Cd2+ application and 39 ± 2.4 mV
during Cd2+ treatment
(p > 0.1; n = 6).
Action potential amplitude was not affected by
KCa channel blockers nor by
Ca2+ channel blockers except for
Cd2+, which substantially attenuated spike
height (Table 2). A possible explanation for the
Cd2+ effect is that block of the AHP,
complete for Cd2+ but only partial for the
other blockers (even the Ca2+ channel
blocker mixture) (Fig. 7), prevented normal recovery from inactivation
of voltage-gated Na+ channels. In contrast
to VIP and spike amplitude, spike
width was more sensitive to blockers (Table 2). Action potentials were broadened by the BKCa channel blocker
iberiotoxin, consistent with the known role of
BKCa channels in action potential repolarization. Action potentials were also broadened by the N-channel blocker -CTx-GVIA, by the five-component Ca2+
channel blocker mixture (nimodipine, -CTx-GVIA, -Aga-IVA,
Ni2+, and mibefradil), and by the
nonselective Ca2+ channel blocker,
Cd2+, presumably because
BKCa channel activity was reduced consequent to
Ca2+ channel block.
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Discussion |
Firing frequency of neurons is commonly modulated by spike
afterhyperpolarizations (Barrett and Barrett, 1976; Yarom et al., 1985;
Madison and Nicoll, 1986; Sah, 1996). For SCN neurons, in which
rhythmic modulation of firing communicates circadian information to the
organism, the role of the AHP in modulating spike rate has previously
received little attention. Here we have focused on cluster I neurons of
the SCN, which are the most abundant cells present in SCN (Pennartz et
al., 1998) and exhibit rhythmic modulation of firing frequency (Welsh
et al., 1995). Our examination of action potential waveforms from
cluster I neurons firing at different rates revealed that the AHP
duration shortens as spike frequency increases.
Three classes of KCa channels in SCN neurons
Evidence that KCa channels regulate rhythmic
firing of SCN neurons has been sparse. Injection of apamin into brain
ventricles has been found to disrupt normal circadian behaviors
(Gandolfo et al., 1996), and charybdotoxin and apamin block
serotonin-induced phase shifting of SCN firing (Prosser et al., 1994).
Our pharmacological dissection of KCa channel
current shows that cluster I neurons express three major groups of
KCa channels. SKCa and
BKCa channels were identified on the basis of
partial block of the AHP by apamin and iberiotoxin. Apamin selectively
blocks SK2 and SK3 subtypes of SKCa channels,
whereas iberiotoxin selectively blocks type I
BKCa channels. Coapplication of nearly saturating
concentrations of apamin and iberiotoxin reduced the amplitude of the
AHP by only ~55%, however, indicating that a large fraction of the
channels underlying the AHP are insensitive to these blockers.
SK1-based channels, which under some conditions have been found to be
insensitive to apamin and iberiotoxin (Kohler et al., 1996; Sah, 1996;
Vergara et al., 1998; Kaczorowski and Garcia, 1999; Sah and
Davies, 2000), are not expressed in SCN neurons (Stocker and Pedarzani,
2000). Other channels that might support the apamin- and
iberiotoxin-insensitive KCa channel current in
SCN neurons include iberiotoxin-insensitive BKCa
channels (Meera et al., 2000), intermediate conductance
IKCa channels, or the essentially
uncharacterized channels that underlie the apamin- and
iberiotoxin-insensitive slow afterhyperpolarization in hippocampal
pyramidal neurons (IsAHP) (Sah and Faber,
2002).
Four types of high voltage-gated Ca2+ channels
in SCN neurons
On the basis of sensitivity to Ca2+
channel antagonists, our work shows that SCN neurons possess four
principal components of high-voltage activated
Ca2+ channel current: L-, N-, and
P/Q-currents, which had been previously identified in these neurons
(Huang, 1993; Chen and van den Pol, 1998), and also R-type current. The
precision in our measurement of relative current magnitude for the
various Ca2+ channel subtypes was
compromised by the imperfect selectivity of the specific channel
antagonists, the incomplete block of individual Ca2+ channel subtypes, and the inability
to voltage clamp distal processes of SCN neurons. Nonetheless, our
pharmacological dissection of components of
Ca2+ channel current yielded an estimate
for the size of the L-type component that is similar to that reported
for dissociated SCN neurons (Huang, 1993).
Our analysis revealed that R-type current was the largest current
component in SCN neurons from slices. The majority of R current was
blocked by low concentrations of Ni2+,
although a proportion (~15% of total
Ca2+ channel current) remained that was
insensitive to this concentration of Ni2+.
The nonblocked fraction might represent
Ni2+-insensitive R current (Schramm et
al., 1999) or other Ca2+ channel
components incompletely blocked at the antagonist doses used. Because
SNX-482 did not block R current, 1E-based
Ca2+ channels are not likely to support
the R current of SCN neurons. R current identified here may correspond
to the large component of Ca2+ current
that was found in previous studies to be insensitive to the combination
of nimodipine and -CTx-GVIA (Huang, 1993).
Coupling of Ca2+ channels to KCa
channels in SCN neurons
In some neurons, tight coupling exists between specific
Ca2+ channel subtypes and
KCa channel subtypes (Davies et al., 1996; Marrion and Tavalin, 1998; Borde et al., 2000; Martinez-Pinna et
al., 2000), whereas in other neurons, multiple
Ca2+ channel subtypes contribute to AHP
activation (Williams et al., 1997; Pineda et al., 1998). In cluster I
neurons of the SCN, both L- and R-type
Ca2+ currents are involved in AHP
activation. N and T channels make small contributions as well, and P/Q
channels appear not to contribute. That large-amplitude N and P/Q
currents contributed little or nothing to AHP activation suggests that
Ca2+ channel subtypes are differentially
distributed on SCN neurons, with L- and R-type channels located in
closest proximity to KCa channels.
Physiological significance
Neurons studied here were in the daytime phase of the circadian
cycle, but we suggest that the relationship between firing frequency
and AHP waveform may contribute to circadian control of firing
frequency in SCN neurons. This proposed mechanism for circadian
regulation of KCa channel activity and the AHP
might arise from circadian changes in the expression, splicing,
post-translational modification, or regulation of specific
KCa channels.
In faster firing SCN neurons, a shortened interspike interval appears
to rely in part on speeded decay of the
Cd2+-sensitive component of the AHP, yet
the Cd2+-sensitive component of the AHP
was larger and action potentials were narrower in faster firing
neurons, suggesting that peak KCa channel
activity is augmented in faster firing neurons. Thus in faster firing
neurons the number of KCa channels open at the
peak of the AHP is increased, but the AHP terminates more rapidly. These two changes in the AHP might be accomplished in various ways. For
example, the kinetics of KCa channel gating might
be speeded globally such that the maximal number of channels open simultaneously is increased (greater AHP magnitude) and yet the duration of the AHP is shortened. Alternatively, the increased magnitude of the AHP in faster firing neurons might be attributable to
an increase in the number of KCa channels
available to open, and, in addition, SCN channels active in the range
50 to 60 mV and with a reversal potential positive to that of the
AHP might exhibit increased activity (e.g., hyperpolarization-activated cation channels (Akasu et al., 1993) or
Na+ channels (Pennartz et al., 1997).
Increased activity of these latter kinds of channels might mask the
full duration of KCa channel activity and
terminate the AHP more rapidly. The fact that faster firing neurons did
not hyperpolarize more than slower firing neurons, although the
net size of the Cd2+-subtracted AHP was
greater in faster neurons, is consistent with the idea that these
channels might compensate for the hyperpolarizing influence of
KCa channels. In this case, circadian dependence on the activity of these channels, as well as of
KCa channels, may be involved in determining the
duration of the AHP and the frequency of firing.
In spontaneously firing SCN neurons, full suppression of the
Ca2+-dependent AHP by application of
Cd2+ increased firing rate (Fig.
8B), whereas partial suppression of the AHP by
mixtures of specific Ca2+ channel blockers
had the opposite effect. This unexpected contrast could arise if
voltage-gated Ca2+ channels participate
both in bringing neurons above firing threshold and in triggering the
AHP. In this scenario, the net effect on firing rate of block of
Ca2+ channels would reflect the balance
between a slowed trajectory through firing threshold (decreased firing
rate) versus a reduced AHP duration (increased rate). In the case of
Cd2+ block, we speculate that the effect
of full suppression of the AHP dominates over the slowed trajectory
through threshold, so that neurons fire faster. With the
Ca2+ channel blocker mixtures, we
speculate that slowed trajectory through threshold dominates over the
effect of partial suppression of the AHP, resulting in a net decrease
in neuronal firing.
The molecular mechanism of the frequency-dependent change in AHP
waveform appears to be based in part on KCa
channels that are insensitive to either apamin or iberiotoxin, because
application of these specific antagonists did not alter firing
frequency. Additionally, there was no correlation between parameters of
the iberiotoxin-sensitive component of the AHP and firing rate. We conclude that other KCa channel types, perhaps
iberiotoxin-insensitive BKCa channels (Meera et
al., 2000) or IKCa channels, are modulated in a
manner that diurnally alters the firing rate of SCN neurons. For these
KCa channels, circadian modulation might occur
via second messenger-mediated modification of channel function, via
changes in the coupling of Ca2+ entry
(Pennartz et al., 2002) to channel activation, or by altered expression
of channels or associated regulatory proteins. A notable recent finding
regarding this latter point is that transcription of Slob,
which encodes a Ca2+-binding protein that
interacts with and regulates the activity of the Slo
KCa channel (Schopperle et al., 1998), is under
circadian control in Drosophila (Claridge-Chang et al.,
2001; McDonald and Rosbash, 2001). More recently, expression of the
mammalian ortholog of the BK-type Slo channel,
Kcnma1, has also been found to be under circadian control
(Panda et al., 2002). The convergence on KCa
channels of molecular and electrophysiological approaches underscores
the likely importance of these channels in the mechanism of circadian
firing of SCN neurons.
TOP
ABSTRACT
Introduction
