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The Journal of Neuroscience, August 20, 2003, 23(20):7525-7542
 Previous Article | Next Article 
Apamin-Sensitive Small Conductance Calcium-Activated Potassium Channels, through their Selective Coupling to Voltage-Gated Calcium Channels, Are Critical Determinants of the Precision, Pace, and Pattern of Action Potential Generation in Rat Subthalamic Nucleus Neurons In Vitro
Nicholas E. Hallworth,1
Charles J. Wilson,2 and
Mark D. Bevan3
1University of Tennessee, Anatomy and
Neurobiology, Memphis, Tennessee 38163, 2Division of
Life Science, University of Texas, San Antonio, Texas 78294, and
3Department of Physiology, Feinberg School of
Medicine, Northwestern University, Chicago, Illinois 60611-3008
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Abstract
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Distinct activity patterns in subthalamic nucleus (STN) neurons are
observed during normal voluntary movement and abnormal movement in Parkinson's
disease (PD). To determine how such patterns of activity are regulated by
small conductance (SK) calcium-activated potassium channels (KCa)
and voltage-gated calcium (Cav) channels, STN neurons were recorded
in the perforated patch configuration in slices, [which were prepared from
postnatal day 16 (P16)-P30 rats and held at 37°C] and then treated with
the SK KCa channel antagonist apamin or the SK KCa
agonist 1-ethyl-2-benzimidazolinone or the Cav channel antagonists
 -conotoxin GVIA (Cav2.2-selective) or nifedipine
(Cav1.2-1.3-selective). In other experiments, fura-2 was introduced
as an indicator of intracellular calcium dynamics.
A component of the current underlying single-spike afterhyperpolarization
was sensitive to apamin, phase-locked to calcium entry via Cav2.2
channels, and necessary for precise, autonomous, single-spike oscillation. SK
KCa/Cav2.2 channel coupling did not underlie
spike-frequency adaptation but limited activity in response to current
injection by encoding the accumulation of intracellular calcium, maintained
the characteristic sigmoidal frequency-intensity relationship and generated a
post-train afterhyperpolarization. In addition, SK KCa channels
terminated rebound burst activity more effectively in neurons with
short-duration bursts (<100 msec) than neurons with long-duration bursts
(>100 msec), presumably through their activation by Cav3
channels. Cav1.2-1.3 channels were not strongly coupled to SK
KCa channels and therefore supported secondary range and
long-duration rebound burst firing. In summary, SK KCa channels
play a fundamental role in autonomous, driven, and rebound activity and oppose
the transition from autonomous, rhythmic, single-spike activity to burst
firing in STN neurons.
Key words: basal ganglia; oscillation; afterhyperpolarization; rebound burst; apamin; EBIO
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Introduction
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The glutamatergic subthalamic nucleus (STN) is a key component of the basal
ganglia, a collection of subcortical brain nuclei involved in motor, sensory,
associative, and limbic functions (for review, see
Wise et al., 1996 ). Under
resting conditions, the tonic activity of the STN drives, in part, the tonic
activity of its target nuclei. During movement, sporadic high-frequency
activity of the STN plays a pivotal role in the response of the basal ganglia
to cortical and thalamic activation (Kitai
and Deniau, 1981; Matsumara et
al., 1992; Fujimoto and Kita,
1993; Wichmann et al.,
1994; Mouroux et al.,
1995). In Parkinson's disease (PD), abnormal, rhythmic, bursting
activity of the STN plays a key role in the expression of symptoms
(Bergman et al., 1994; Levy et
al., 2000,
2002). Indeed, therapies like
high-frequency electrical stimulation of the STN have been successful in
ameliorating the symptoms of PD (for review, see
Benabid et al., 2001).
In recent years, several groups have defined intrinsic membrane properties
of STN neurons in vitro that are likely to contribute to their
pattern of activity and integration of synaptic input in vivo
(Nakanishi et al., 1987;
Beurrier et al., 1999,
2000;
Bevan and Wilson, 1999;
Song et al., 2000;
Wigmore and Lacey, 2000;
Otsuka et al., 2001;
Bevan et al., 2002;
Zhu et al., 2002;
Baufreton et al., 2003;
Do and Bean, 2003). These
properties include: (1) autonomous, rhythmic single-spike activity, (2) the
ability to fire at high frequencies (100-500 Hz), (3) a primary range of
activity (<40 Hz) that is approximately half as sensitive to input as a
secondary range of activity (>40 Hz), and (4) heterogeneous rebound burst
activity. Further analyses of the ion channels underlying these properties
will be critical for our understanding of the function and dysfunction of the
STN and for the development and refinement of treatments for PD that are based
on the modulation of STN activity.
In recent whole-cell patch clamp recording studies of STN neurons, calcium
entry via voltage-gated calcium (Cav) channels predominantly
activated small conductance (SK) calcium-activated potassium channels
(KCa) channels, which played a pivotal, largely suppressive role in
shaping activity or activated nonspecific cation channels, which augmented
activity (Bevan and Wilson,
1999; Beurrier et al.,
1999,
2000;
Otsuka et al., 2001). Because
the whole-cell configuration of the patch-clamp technique can introduce alien
calcium buffers and anions that disrupt intracellular calcium dynamics
(Neher and Augustine, 1992;
Zhang et al., 1995;
Helmchen and Tank, 1999;
Velumian and Carlen, 1999) and
block intrinsic ion channels (Zhang et
al., 1994; Velumian et al.,
1997), we applied the perforated patch-clamp technique, with
gramicidin as the pore-forming substance
(Myers and Haydon, 1972;
Abe et al., 1994;
Kyrozis and Reichling, 1995)
to study in a more definitive manner the role of apamin-sensitive SK
KCa channels and their coupling to Cav channels in STN
neurons. In other cases, we deliberately introduced the calcium indicator
fura-2 to track the dynamic changes in intracellular calcium
(Grynkiewicz et al., 1985;
Lev-Ram et al., 1992) that are
presumably related to the activation of SK KCa channels.
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Materials and Methods
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Electrophysiology
Slice preparation. Electrophysiological studies were performed on
brain slices prepared from 43 16- to 30-d-old Sprague Dawley rats. Animals
were deeply anesthetized with a mixture of ketamine and xylazine and perfused
transcardially with 10-40 ml of ice-cold modified artificial CSF (ACSF), which
had been bubbled with 95% O2 and 5% CO2 and contained
(in mM): 230 sucrose, 2.5 KCl, 1.25 Na2HPO4,
0.5 CaCl2, 10 MgSO4, and 10 glucose. The brain was then
rapidly removed, blocked in the sagittal plane, glued to the stage of a
vibratome (VT 1000S; Leica, Nussloch, Germany), and immersed in ice-cold
modified ACSF. Slices containing the STN were cut at a thickness of 300 µm
and subsequently transferred to a holding chamber, where they were submerged
in ACSF, which was bubbled continuously with 95% O2 and 5%
CO2, maintained at room temperature, and contained (in
mM): 126 NaCl, 2.5 KCl, 1.25 Na2HPO4, 2
CaCl2, 2 MgSO4, and 10 glucose.
Visualized recording. Single slices were transferred to the
recording chamber and perfused continuously (2-3 ml/min) with oxygenated ACSF
at 37°C. Somatic recordings were made using patch pipettes pulled from
standard-wall borosilicate glass (Warner Instruments, Hamden, CT) on a P-97
Flaming-Brown micropipette puller (Sutter Instruments, Novato, CA). Pipettes
were filled with a solution containing (in mM): 134.1
K-MeSO4, 0.9 KCl, 3.6 NaCl, 1 MgCl2·6
H2O, 10 HEPES, 0.1 Na4EGTA, 0.4 Na3GTP, and 2
Mg1.5ATP. The pH and osmolarity of the pipette solution were 7.3
and 290-300 mOsm, respectively. Gramicidin was added to the pipette solution
at an approximate concentration of 20 µg/ml <1 hr before seal formation
was attempted. The resistance of filled pipettes was between 2.5 and 5
M . Gramicidin was applied because it forms pores that are permeable
only to monovalent cations and small neutral molecules like water and thus
leaves intact natural calcium dynamics and ion channel properties
(Myers and Haydon, 1972;
Abe et al., 1994;
Kyrozis and Reichling, 1995).
A 40x water-immersion objective (Axioskop; Zeiss, Oberkochen, Germany)
was used to examine each slice using infragradient contrast video microscopy
(Dodt et al., 1999)
(Infra-patch Workstation; Luigs and Neumann, Ratingen, Germany). Perforated
recordings were made in current-clamp and voltage-clamp mode using an EPC
9/2.C amplifier (Heka, Lambrecht, Germany), which was operated using Pulse 8.5
software (Heka). Signals were low-pass filtered at a frequency (1.7-33.3 kHz)
that was one-third the frequency of digitization (5-100 kHz). Junction
potentials were not corrected in the perforated configuration because, in
contrast to the whole-cell configuration, the composition of ions in the
recorded neuron cannot be assumed to be the same as electrode solution
(Marty and Neher, 1983;
Neher, 1992; Bevan et al.,
2000,
2002). Deliberate or
accidental establishment of the whole-cell configuration was recognized as a
sudden drop in series resistance, an increase in the amplitude of recorded
action potentials, and an  6 mV offset in membrane potential. The latter
was measured as an increase in the threshold for the generation of action
potentials that accompanied break-in. The value of the offset was similar to
the empirically calculated junction potential between the electrode solution
and the external media of 8.7 mV (Neher,
1992; Barry, 1994;
Bevan et al., 2000,
2002). The voltages reported
in this paper are therefore likely to be <3 mV more depolarized than the
actual voltages.
While fast capacitive transients of the pipette were nulled on-line, there
was no compensation of series resistance or whole-cell capacitance, thus
voltage errors attributable to series resistance were subtracted off-line as
described previously (Bevan et al.,
2000).
Drugs. Drugs were diluted in ACSF and bath-applied. Fast synaptic
transmission was blocked by the continuous application of 50 µM
D-(-)-2-amino-5-phosphonopentanoic acid (APV), 20 µM
6,7-dinitroquinoxaline-2,3-dione (DNQX), and 20µM SR 95531
hydrobromide (GABAzine) so that the action of SK and Cav channel
drugs on intrinsic properties could be studied in isolation from effects on
synaptic transmission. Synaptic transmission receptor blockers,
1-ethyl-2-benzimidazolinone (EBIO) and nifedipine were obtained from Tocris
(Ellisville, MO). Apamin (1 pM-100 nM) and
-conotoxin GVIA (1 µM) were obtained from Sigma (St.
Louis, MO).
Data analysis. Data were analyzed using Pulsefit (Heka) and Origin
5.0 (Microcal, Northampton, MA). Descriptive statistics refer to the mean
± SEM. Frequency distributions of the experimental data were compared
with normal distributions of similar means and SDs, which were constructed
using random numbers generated by Statview 5.0 (SAS, Cary, NC), using the
Kolmogorov-Smirnov (K-S) test. Although datasets were typically not
significantly different from a normal distribution, our sample sizes were
small. We therefore applied nonparametric statistics, which are subject to
fewer assumptions concerning the distribution of data. The means of paired and
unpaired experimental datasets were thus compared using the Wilcoxon signed
rank (WSR) test and the Mann-Whitney U test (M-W U)
respectively, and probability values <0.05 were considered significant.
The dose-response relationship for apamin was characterized in Origin
(Microcal) by fitting a single Langmuir isotherm to the plot of mean peak
apamin-sensitive current at each concentration of drug. The best fit was
assessed over iterative trials in which the variables representing the Hill
coefficient and the half-maximal response were permitted to vary.
The frequency and statistical significance of any periodic discharge
features induced by apamin were determined using the Lomb algorithm
(Kaneoke and Vitek, 1996).
Lomb periodograms represent power spectra of the autocorrelograms constructed
using 121 spikes of recorded data.
Calcium imaging
Slices were prepared with, stored in, and perfused with ACSF as described
above. Slices were viewed under infrared illumination (780 ± 30 nm)
using the same CCD camera used for calcium imaging and an Olympus (Melville,
NY) BX50WI upright microscope, equipped with DIC optics anda40x (0.8 NA)
water immersion objective. Micropipettes had resistances of 4-6 M and
were filled with a solution containing (in mM): 128.7
K-MeSO4, 2.3 KCl, 10 HEPES, 0.4 Na3GTP, 1 MgATP, 1
Na2ATP, and 0.05-0.2 fura-2 (Na-salt). The pH and osmolarity of the
pipette solution were 7.3 and 290-300 mOsm, respectively. Current-clamp
recordings were made using an active bridge amplifier (Neurodata IR283; Cygnus
Technology, Delaware Water Gap, PA). Electrical and optical data were
collected synchronously using a single computer. Electrical records were
digitized at 16 bit resolution at 10 kHz and corrected for a 5 mV liquid
junction potential (see above). Optical recordings were obtained using a
cooled CCD camera (EEV37; Photometrics, Tucson, AZ) in frame transfer mode.
Frame rates of 20-50 Hz were used, depending on the size of the field of view.
Fluorescence measurements were made using an excitation wavelength of 380 nm
and were corrected for bleaching and autofluorescence. To correct for
bleaching, fluorescence measurements were collected while the recorded neuron
was held hyperpolarized. These measurements were then filtered at 3 Hz and
subtracted from the fluorescence measurements, which were made when the cell
was generating action potentials. Autofluorescence was corrected by
subtracting the fluorescence of a nearby region of the slice, which was devoid
of fura-containing elements, from the value of F at the beginning of
each trial. Changes in the fluorescence of fura-2 are presented as
% F/F, which for changes of <50% are related in an
almost linear manner to calcium concentration
(Lev-Ram et al., 1992). All
measurements made in this study were within this range. At an excitation
wavelength of 380 nm, the fluorescence of fura-2 decreases with increasing
calcium concentration. Therefore, for the sake of clarity, the measurements
are plotted as -%F/F so that decreases in fluorescence have the same
polarity as increases in calcium concentration.
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Results
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SK KCa channels underlie a major component of single-spike
afterhyperpolarization in STN neurons
To evoke and study in a quantitative manner the currents underlying
single-spike afterhyperpolarization, a protocol based on principles outlined
by Lancaster and Adams (1986)
was applied. This technique has been used successfully to elucidate currents
underlying afterhyperpolarization in other systems
(Storm, 1989;
Wolfart et al., 2001;
Wolfart and Roeper, 2002;
Faber and Sah, 2002). To
induce a single unclamped action potential, the membrane was depolarized from
a holding potential of -65 to 20 mV for 10 msec. The membrane potential was
then returned to -65 mV, and the resulting currents were recorded. This
protocol evoked an outward current that apparently peaked 38.9 ± 5.9
msec after the termination of the voltage step and decayed within 100-200 msec
(n = 16) (Fig.
1A). Because the slow decay of this current was similar
to those observed for currents carried by SK KCa channels, the
current was tested with varying concentrations of the selective SK
KCa channel blocker apamin (for review, see
Sah, 1996;
Vergara et al., 1998;
Bond et al., 1999;
Sah and Faber, 2002). As
predicted, the current was inhibited by low concentrations of apamin
(Fig. 1A). To further
analyze the apamin-sensitivity of the single-spike afterhyperpolarization, the
apamin-sensitive current was calculated for each concentration of apamin by
subtracting the spike-evoked current obtained in apamin from the spike-evoked
current observed in control media. At all drug concentrations, the peak
amplitudes of subtracted currents were not significantly different from normal
distributions of similar means and variance that were generated from random
numbers (K-S test; 10 pM: 16.7 ± 4 pA, n = 8,
p = 0.65; 100 pM: 21.3 ± 6 pA, n = 7,
p > 0.99; 500 pM: 42.1 ± 7 pA, n = 8,
p > 0.99; 1 nM: 78.7 ± 9 pA, n = 6,
p > 0.99; 10 nM: 76.8 ± 13 pA, n = 5,
p > 0.99; 100 nM: 79.0 ± 12 pA, n = 14,
p > 0.99). Population-subtracted currents were used to construct a
dose-response curve, which was reasonably well described by a Hill coefficient
of 1.04, was half-maximal at 246 pM, and was clearly saturated
at 1 nM. The sensitivity of single spike afterhyperpolarization is
therefore somewhat intermediate to the sensitivity of identified SK2
KCa channels in slice preparations of cerebellar Purkinje neurons
(135 pM; Cingolani et al.,
2002) and identified SK3 KCa channels in isolated
superior cervical ganglion neurons (2.3 nM;
Hosseini et al., 2001) and
slice preparations of dorsal vagal neurons (2.2 nM;
Pedarzani et al., 2000) and
dopaminergic substantia nigra neurons (9.2 nM;
Wolfart et al., 2001). Thus,
these data suggest that a major component of the single-spike
afterhyperpolarization in STN neurons was mediated by SK KCa
channels that were composed of relatively apamin-sensitive subunits
(Ishii et al., 1997) and is
consistent with the presence of mRNA encoding SK2 KCa and SK3
KCa channels in STN neurons
(Stocker and Pedarzani,
2000).
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Figure 1. A major component of single-spike afterhyperpolarization is mediated by SK
KCa channels. A, B, In voltage-clamp, a single unclamped
action potential was induced by stepping from a holding potential of -65 to 20
mV for 10 msec. The membrane potential was then returned to -65 mV, and the
current underlying afterhyperpolarization was studied. The single spike-evoked
current was sensitive to apamin. B, Under current-clamp, the
reduction in apamin-sensitive current manifested itself as a reduction in
single-spike afterhyperpolarization and as a general depolarization (same cell
as in A). Note also the steepening of the voltage trajectory in the
interspike interval and the rise in the threshold for action potential
generation. C, Application of the selective SK channel agonist EBIO
markedly increased the magnitude of current that was evoked by the protocol
described in A. D, Current-clamp recording of the same
neuron in C. EBIO increased the magnitude and duration of
single-spike afterhyperpolarization, which led a reduction in the frequency of
autonomous oscillation.
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In some cells, apamin application also further revealed a spike-evoked
slowly deactivating inward tail current
(Fig. 1A). In the
absence of the slowly deactivating inward tail current, the apamin-sensitive
outward current peaked at 6.0 ± 1.3 msec (n = 8) and
deactivated with a time constant of 60.2 ± 6.4 msec (n = 8),
which were similar to the kinetics of previously reported spike-evoked
apamin-sensitive currents (for review, see
Sah, 1996;
Sah and Faber, 2002).
In current-clamp mode, the blockade of SK KCa channels reduced
single-spike afterhyperpolarization and led to a general depolarization
(Fig. 1B).
Furthermore, the voltage trajectory associated with the interspike interval
was steeper, and the threshold for action potential generation was higher in
the presence of apamin (Fig.
1B).
Although apamin is a widely used selective SK KCa channel
antagonist, it does not wash out easily from brain slices, and its effects are
typically irreversible. Therefore, to provide further evidence that the
effects of apamin were attributable to SK KCa channel blockade and
not run-down in treated neurons, we used EBIO, which selectively enhances the
calcium sensitivity of SK KCa channels
(Xia et al., 1998;
Syme et al., 2000;
Pedarzani et al., 2001;
Wolfart et al., 2001;
Cingolani et al., 2002;
Faber and Sah, 2002). We
predicted that the effects of SK KCa channel activation would be
mostly opposite to the effects of SK KCa channel blockade. Under
conditions of fast synaptic transmission blockade, bath application of 200
µM EBIO increased the mean peak current underlying single-spike
afterhyperpolarization by a factor of 1.72 (WSR test; control: 28.2 ±
4.0 pA; EBIO: 48.5 ± 5.6 pA, n = 7, p = 0.018)
(Fig. 1C). In
current-clamp mode, activation of SK KCa channels with 200
µM EBIO markedly increased the afterhyperpolarization that
followed autonomously generated action potentials
(Fig. 1D).
SK KCa channels influence the precision of autonomous
rhythmic single-spike activity in STN neurons
Under perforated recording conditions and in the presence of synaptic
transmission blockers, blockade of SK KCa channel current with
apamin reduced single-spike afterhyperpolarization and the rhythmicity of
autonomous single-spike activity (Fig.
2). The precision of firing was assessed using the coefficient of
variation (CV) of 100 interspike intervals of spontaneous activity recorded in
current-clamp mode. In accordance with previous studies, STN neurons
discharged in a rhythmic single-spike manner in control media
(Fig. 2A)
(Overton and Greenfield, 1995;
Beurrier et al., 1999,
2000;
Bevan and Wilson, 1999;
Wigmore and Lacey, 2000;
Baufreton et al., 2001,
2003;
Bevan et al., 2002;
Zhu et al., 2002;
Do and Bean, 2003) but never
exhibited the spontaneous rhythmic burst activity that has been observed by
some groups (Beurrier et al.,
1999,
2000,
2001; Baufreton et al.,
2001,
2003). The subsequent
application of 500 pM apamin reduced single-spike
afterhyperpolarization, which increased the rate of activity and decreased the
precision of firing (Fig.
2B). Saturating concentrations of apamin led to a
profound reduction of the single-spike afterhyperpolarization, which was
accompanied by a further decrease in rhythmicity
(Fig. 2C). The
apamin-sensitivity of periodicity (CV) suggested that the precision of firing
was regulated in part by relatively apamin-sensitive SK KCa
channels (Fig. 2D).
Thus, the mean CV measured in control media (0.074 ± 0.005; n
= 27) was not significantly different (WSR test) from the mean CV in 10
pM apamin (0.074 ± 0.007; n = 8; p =
0.8886) or the mean CV in 100 pM apamin (0.08 ± 0.02;
n = 7; p = 0.2367). However, the application of 500
pM apamin resulted in a mean CV that was significantly different
from control (WSR test; 0.20 ± 0.10, n = 8, p =
0.0173). Further blockade of SK KCa channels produced similar
disruptions: the mean CV in 1 nM apamin (WSR test; 0.19 ±
0.07, n = 7, p = 0.018), the mean CV in 10 nM
apamin (WSR test; 0.20 ± 0.06, n = 5, p = 0.0431),
and the mean CV in 100 nM apamin (WSR test; 0.36 ± 0.09,
n = 19, p = 0.002) were all significantly different from
that obtained in control ACSF. Although the mean CV appeared to increase at
concentrations beyond 1 nM, these differences were not significant.
The CV in 1 nM apamin was not significantly different from that in
10 nM (M-W U test; n = 12, p = 0.6847)
or in 100 nM (M-W U test; n = 12, p =
0.0686). Thus, the disruption in CV displayed sensitivity to apamin that was
similar to the apamin sensitivity of single-spike afterhyperpolarization that
was assessed with the hybrid clamp protocol.
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Figure 2. SK KCachannels are critical for the rhythmicity of autonomous
activity. A-C, Activity of a representative neuron under control
conditions and after treatment with apamin. Interspike interval histograms
generated from 100 interspike intervals of the activity associated with these
conditions are shown to the right. A, Precise single-spike firing was
observed before drug application (CV = 0.05; frequency = 21.8 Hz). B,
C, Partial inhibition (B) and complete inhibition (C)
of the current with 500 pM apamin and 100 nM apamin,
respectively, reduced the precision and increased the frequency of activity
(500 pM apamin: CV = 0.1, frequency = 28.1 Hz; 100 nM
apamin: CV = 0.18, frequency = 27.5 Hz). D, Population CV data.
Nonparametric paired comparisons revealed that the mean CV in control was not
significantly different from the mean CV in 10 pM apamin or the
mean CV in 100 pM apamin. The mean CV in control was however
significantly different from the mean CV in concentrations of apamin  500
pM. Calibration in C also applies to A and
B.
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In general, within a cell, the firing rate did not change significantly
with apamin application until partial blockade (500 pM) was
achieved (Fig. 2B).
Firing rate was consistently higher at this concentration (WSR test; control:
12.4 ± 1.09 Hz, n = 27; 500 pM: 15.1 ± 2.6
Hz, n = 8, p = 0.0357). Further application of higher
concentrations of apamin often led to such profound disruptions in firing
pattern and elevation of spike threshold (Figs.
1,
2) that the mean firing rate
was not significantly different from control.
Application of 200 µM EBIO did not significantly alter the
precision of spontaneous firing in STN cells as measured by the coefficient of
variance (WSR test; control: 0.066 ± 0.007; EBIO: 0.05 ± 0.007,
n = 5, p = 0.138), but it did lead to a decrease in the mean
firing rate of these cells (WSR test; control: 13.5 ± 3.0 Hz; EBIO: 8.2
± 2.1 Hz, n = 5, p = 0.0422)
(Fig. 1D).
An earlier study suggested that Cav channels contributed to
rhythmicity in STN neurons by passing calcium, which in turn activated SK
KCa channels (Bevan and Wilson,
1999). These Cav channels appear to be predominantly
activated at suprathreshold voltages
(Bevan and Wilson, 1999;
Song et al., 2000). Indeed,
the time course of single spike-evoked SK KCa current, the
trajectory of the interspike interval associated with autonomous activity and
the effects of apamin on autonomous activity suggest that calcium entry via
Cav channels mainly occurs during and subsequent to action
potential generation. To determine directly whether calcium entry during
spontaneous activity was primarily associated with the generation of action
potentials, we studied calcium dynamics directly using imaging with fura-2.
The temporal resolution of this method (15-25 msec, depending on the magnitude
of the field of view) is typically not adequate to determine whether calcium
entry begins just before or during the action potential, nor can it reveal the
time to peak of calcium levels. To overcome this limitation, we developed a
method for enhancing temporal resolution for repetitive events like rhythmic
firing. The method is illustrated for autonomous activity in
Figure 3 and is based on the
superimposition of data from multiple events. Calcium transients associated
with action potentials during spontaneous firing were collected at the highest
possible temporal resolution (25 msec in this example). Individual cycles of
membrane potential and calcium signal during spontaneous activity were
extracted and aligned on the action potential. Because sampling of the calcium
signal occurred asynchronously with the action potential, the calcium samples
were out of register with each other in the superimposition. Thus, each cycle
of the spontaneous activity provided a set of calcium measurements at
different times. Assuming that the calcium transient is the same for each
action potential (Helmchen et al.,
1996), then superimposing the data from different action
potentials gives a time series with resolution increased approximately by a
factor of the number of superimposed spikes. In the example in
Figure 3 and in three other
neurons that were studied with this technique, it was apparent that calcium
levels in STN neurons did not begin to rise before the action potential and
reached their peak during the early phase of the spike afterhyperpolarization.
This is consistent with calcium entry via Cav channels with
activation and deactivation kinetics slower than those of the action potential
currents. The calcium current was activated by the depolarization of the
action potential but outlasted the action potential, that is, the calcium
current was mostly a tail current of the action potential. If the calcium
entering Cav channels is solely responsible for the calcium
transient, then the time required for deactivation of the calcium current can
be approximated as the time required for the calcium influx to plateau
(20 msec). The time course of calcium decay after the action potential
was approximately exponential (441.2 ± 217.2 msec; n = 4), and
calcium levels returned to approximately baseline levels (0%) before the
occurrence of the next action potential in the series. Arresting the
spontaneous activity in four of four neurons tested by hyperpolarizing current
injection did not produce detectable decreases below background levels of
calcium (data not shown). Taken together, our observations suggest that action
potential generated calcium dynamics rather than background calcium levels are
important for the activation of SK KCa channels in STN neurons.
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Figure 3. Calcium dynamics faithfully track the autonomous generation of action
potentials. A, B, Simultaneous electrical (A) and
fluorescent (B) recordings from the soma of a spontaneously active
STN neuron. Oscillations in membrane potential are phase-locked to
oscillations in intracellular calcium levels. C, Using the
superimposition of multiple cycles of autonomous activity and related calcium
dynamics, more precise temporal resolution of calcium dynamics was achieved
(see Results). Calcium levels fell to baseline levels immediately before the
generation of an action potential, rose during and after the generation of an
action potential, and reached their maximum during the single-spike
afterhyperpolarization. Calibration in B also applies to
A.
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The decay of intracellular calcium that was measured with imaging was much
slower than the decay of SK KCa current measured using the hybrid
clamp protocol. Similar observations have been made in other neurons and are
thought to arise from the fact that the average level of intracellular calcium
is not a precise reflection of the calcium concentration that is present in
the vicinity of SK KCa channels
(Wilson and Callaway,
2000).
SK KCa channels influence the sensitivity of STN neurons
to depolarizing input
STN neurons are capable of maintaining high firing rates in response to
synaptic input or current injection (Kitai
and Deniau, 1981; Rouzaire-Dubois and Scarnati,
1985,
1987;
Nakanishi et al., 1987;
Fujimoto and Kita, 1993;
Mouroux et al., 1995;
Bevan and Wilson, 1999). The
putative role of SK KCa channels in high-frequency firing was
investigated by comparing driven firing in control media and in the presence
of apamin (Fig. 4A-D).
Neurons were driven for 500 msec from rest with ascending steps of 20 pA of
injected current. When the relationship between frequency and current
(f-I relationship) was plotted, STN neurons exhibited a sigmoidal
function that was similar to an earlier report that used the whole-cell
recording technique (Fig.
4B) (Bevan and
Wilson, 1999). The f-I curve was composed of a
low-sensitivity primary range up to 40 Hz, a more sensitive secondary
range, and a tertiary range at which the frequency of discharge began to
saturate. In control conditions, STN cells fired up to 500 Hz and displayed,
on average, a secondary range to primary range slope ratio of 1.93 ±
0.14 (n = 21). Partial and saturating blockade of apamin-sensitive
current increased the firing rate of STN neurons during periods of applied
positive current (Fig.
4A,B). This manifested itself as both a leftward shift in
the secondary range (Fig.
4Bi) and as an increase in the gradient of the primary
range (Fig. 4Bii). The
first effect was quantified within each cell as the normalized amount of
current needed to achieve the half-maximal firing rate
(Fig. 4C). As
illustrated in Figure 4, A and
Bi, and shown for the population in
Figure 4C,
concentrations of 1 nM apamin or greater significantly reduced this
amount (WSR test; 10 pM: 1.07 ± 0.04, n = 8,
p = 0.0747; 100 pM: 1.02 ± 0.06, n = 7,
p = 0.8658; 500 pM: 0.97 ± 0.03, n = 8,
p = 0.3980; 1 nM: 0.74 ± 0.05, n = 7,
p = 0.018; 10 nM: 0.70 ± 0.06, n = 6, p
0.0277; 100 nM: 0.77 ± 0.04, n = 14, p =
0.0019). In the primary range, partial and saturating blockade of the
apamin-sensitive current led to an increase in the responsiveness of STN cells
to current injection (Fig.
4A,B,D). Although the normalized slope of the primary
range was not significantly changed by the application of 100 pM
apamin or less (WSR test; 10 pM: 1.04 ± 0.05, n =
8, p = 0.5754; 100 pM: 1.00 ± 0.04, n = 6,
p = 0.9165), the application of 500 pM apamin or more led
to a significant increase in this gradient (WSR test; 500 pM: 1.22
± 0.08, n = 6, p = 0.0277; 1 nM: 1.58
± 0.14, n = 7, p = 0.018; 10 nM: 1.86
± 0.15, n = 6, p = 0.0277; 100 nM: 1.68
± 0.11, n = 13, p = 0.0015). As complete inhibition
of the apamin-sensitive current had no significant effect on the normalized
slope of the secondary range in STN neurons (100 nM: 0.98 ±
0.07, n = 13, p = 0.4802), the difference between the slopes
of the primary and secondary ranges decreased, and a linearization transpired,
such that the ratio of the slope of the primary range over the slope of the
secondary range was significantly higher as it approached 1 (100
nM: 0.95 ± 0.07, n = 13, p = 0.0015).
Thus, current mediated through apamin-sensitive channels normally acts to
limit the excitability of STN neurons and plays a critical role in maintaining
the characteristic shape of the f-I relationship. The
dose-sensitivity of these effects (Fig.
4A-D) is suggestive that they were mediated in large part
by channels containing relatively apamin-sensitive subunits.
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Figure 4. SK KCa channels influence the sensitivity of firing to
depolarizing input. A-D, Driven firing was augmented in a
dose-dependent manner by the application of apamin. Apamin increased the
frequency of firing in response to current injection in both the primary and
secondary firing ranges. The f-I relationship was shifted leftwards
(Bi, C) and the gradient of the primary range (B, D)
resembled that of the secondary range. Thus, apamin at concentrations >500
and 100 pM significantly decreased the current required for
half-maximal firing (C) and increased the gradient of the primary
range (D), respectively. E, F, Driven firing was markedly
reduced by the application of EBIO. Fi, The f-I relationship
of the neuron in E was shifted right by EBIO. Fii, The
gradients of primary and secondary range firing were reduced by SK
KCa channel activation, but the ratio of the sensitivities of
primary to secondary range firing were unaltered.
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Enhancement of SK KCa channel activity with EBIO produced
effects on high-frequency discharge that were opposite to treatment with
apamin. Mean firing rates were decreased as compared with control rates in
response to equivalent amounts of injected current
(Fig. 4E,F). This
manifested itself as a rightward shift in the f-I relationship for
these cells (Fig.
4E,F). The normalized amount of current required to
achieve the half-maximal firing rate was increased after application of EBIO
(WSR test; 1.31 ± 0.04, n = 5, p = 0.0431).
Activation of SK KCa channels also led to a decrease in the
responsiveness of STN cells to current injection in both the primary and
secondary ranges of the f-I relationship
(Fig. 4E,F). The
normalized slopes of the primary (0.65 ± 0.05; n = 5) and
secondary ranges (0.77 ± 0.07; n = 5) were significantly
reduced by EBIO (WSR test; p = 0.0431)
(Fig. 4E,F). In
contrast to the linearization observed in the presence of apamin, the relative
changes in both slopes were similar in EBIO, such that the ratio of the slope
of the primary range to the slope of the secondary range in control media was
not altered by application of the drug (WSR test; control: 0.50 ± 0.06;
EBIO: 0.43 ± 0.05, n = 5, p = 0.3452)
(Fig. 4E,F).
SK KCa channels do not determine intratrain
spike-frequency dynamics in STN neurons
Although SK KCa channels dictated the overall sensitivity of STN
neurons to depolarizing input, they did not control firing dynamics within
periods of driven firing (Fig.
5). At firing rates in the secondary and tertiary range of the
f-I curve, STN neurons displayed an initial increase in firing rate
when driven (Fig.
5B-D). At all levels of driven firing, STN neurons also
displayed a subsequent minor but consistent spike-frequency adaptation
(Fig. 5). Both of these
properties were unchanged by the application of saturating concentrations of
apamin in seven of seven neurons tested. Furthermore, comparison of interval
and instantaneous frequency plots from driven firing of similar frequencies
under control conditions and in the presence of apamin revealed that
intratrain spike-frequency dynamics were virtually identical (n = 7).
Similarly, spike-frequency dynamics during periods of driven firing were
relatively unchanged after activation of these channels with 200
µM EBIO (Fig.
4E,F). When traces of similar mean frequency were
compared, EBIO did not increase spike-frequency adaptation. Taken together
these data suggest that intratrain spike-frequency dynamics are primarily
controlled by channels other than SK KCa channels.
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Figure 5. Intratrain spike-frequency dynamics are not controlled by SK KCa
channels. A-D, Comparison of firing of similar frequency in control
conditions and in the presence of apamin revealed that the pattern of spiking
within a driven train was not altered by apamin. The speed-up in firing, which
was followed by minor spike-frequency adaptation at high frequencies of
activity was present in control conditions and in the presence of apamin.
Calibration in C also applies to A and B.
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SK KCa channels, in part, underlie post-train
afterhyperpolarization in STN neurons
High-frequency firing also led to an accumulation in the depth and duration
of post-train apamin-sensitive afterhyperpolarization in STN neurons
(Fig. 6). Thus, the interval
between a period of high-frequency firing and the resumption of spontaneous
firing increased as a function of both the current used to drive the cell and
the mean frequency realized during the driven period
(Fig. 6A,C,D,F,G,I).
Under control conditions, the time to the first spike after driven firing was
approximately linearly related to the mean frequency of driven activity
(Fig. 6A,C,D,F,G,I).
Across 14 cells examined, the mean slope of this relationship was 3.28
± 0.67 msec/Hz. Saturating concentrations of apamin widely abolished
the post-train afterhyperpolarization but had one of two opposite effects on
the resumption of spontaneous activity. Complete blockade of SK KCa
channels with saturating concentrations of the drug prolonged the time to the
first spike after driven firing in 6 of 15 neurons examined
(Fig. 6A-C). In five
neurons, blockade of the SK KCa channels led to a shortening of the
interval between the termination of driven firing and the first subsequent
action potential (Fig.
6D-F). In the remaining four cells, apamin treatment
produced such great variability in the resumption of spontaneous activity that
a clear pattern was not observed. These mixed responses indicate that SK
KCa channels are important for the smooth and uniform resumption of
spontaneous activity after high-frequency activity in STN neurons. In the
absence of SK KCa channels, the resumption of activity is less
uniform, across the population and within individual neurons. The involvement
of SK KCa channels in post-train afterhyperpolarization was
confirmed by the application of 200 µM EBIO, which increased the
post-train afterhyperpolarization and the time for the resumption of firing
after driven firing in five of five neurons
(Fig. 6G-I).
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Figure 6. SK KCa channels in part underlie post-train
afterhyperpolarization. A-I, High-frequency firing led to the
accumulation of post-train apamin-sensitive afterhyperpolarization, which
delayed the resumption of spontaneous activity. A-F, Post-train
afterhyperpolarization was reliably reduced in duration and magnitude by the
application of apamin. The abolition of apamin-sensitive post-train
afterhyperpolarization either increased (A-C) or reduced
(C-E) the time for the resumption of spontaneous activity. Thus, in
the absence of SK KCa channel activation, the manner in which
spontaneous activity resumes after high-frequency activity is more
heterogeneous. G-I, EBIO consistently increased the magnitude and
duration of post-train afterhyperpolarization and the time for the resumption
of spontaneous activity. Calibration in A also applies to B.
Calibration in D also applies to E. Calibration in
G also applies to H.
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The role of SK KCa channels during repeated cycles of
high-frequency activity
The build-up of post-train afterhyperpolarization has been suggested as one
possible mechanism that might underlie the therapeutic value of high-frequency
stimulation of the STN for the treatment of PD
(Bevan and Wilson, 1999;
Dostrovsky and Lozano, 2002).
To test this proposition further, we drove cycles of high-frequency activity
in STN neurons with repeated injections of current in control conditions and
in the presence of apamin (Fig.
7). In agreement with the data described above, apamin treatment
produced faster firing on each cycle of current injection, reduced post-train
afterhyperpolarization between cycles of current injection, but had no effect
on the decline in spiking during successive cycles of activity
(Fig. 7). These data suggest
that during cycles of high-frequency activity there will be a build-up of
spike-generated afterhyperpolarization, which will reduce activity between
cycles of current injection. In addition, a second apamin-insensitive process
leads to a gradual reduction in firing during repeated cycles of current
injection. The accommodation and broadening of action potentials during
successive cycles of activity suggest that the slow inactivation of
Nav channels may be responsible
(Do and Bean, 2003).
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Figure 7. SK KCa channels underlie the build-up of post-train
afterhyperpolarization but do not underlie the reduction in intratrain spike
frequency during successive cycles of driven activity. A-E, An STN
neuron was driven with repeated cycles of current injection: 150 pA for 100
msec, which was repeated five times with an interval of 100 msec. The
post-train afterhyperpolarization, which increased during successive cycles of
activity (A) was apamin-sensitive (B). The successive
decline in intratrain firing was, however, apamin-insensitive and was
associated with the marked accommodation and broadening of action potentials
(A-E). Calibration in A also applies to B.
Calibration in C also applies to D.
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Intracellular calcium accumulates during and decays slowly after
driven repetitive firing
The slow decay of intracellular calcium during autonomous activity, the
slow decay of single spike-evoked apamin-sensitive SK KCa channel
current, and the accumulation of post-train apamin-sensitive
afterhyperpolarization suggested that during high-frequency repetitive
activity intracellular calcium accumulates and leads to the persistent
activation of SK KCa channels. Combined calcium imaging with fura-2
and electrical recording were used to directly test this hypothesis
(Fig. 8). Spontaneous activity
was arrested during these experiments by the constant injection of
hyperpolarizing current to maintain a baseline level of intracellular calcium.
These experiments confirmed that intracellular calcium accumulated and did not
return to baseline levels during repetitive firing of frequencies greater than
10 Hz. Furthermore, the accumulation of intracellular calcium was
dependent on the frequency of action potential generation during 1 sec periods
of driven activity. After each period of driven firing, calcium levels
gradually fell to baseline levels over a period of several seconds. A linear
relationship was observed between the peak calcium level and frequencies of
driven activity >10 Hz (Fig.
8). Similar relationships for action potential generation and
calcium fluorescence were observed in each of 10 neurons tested.
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Figure 8. Intracellular calcium levels accumulate during driven repetitive activity.
Combined electrical and fluorescent measurements indicated that intracellular
calcium accumulated when repetitive firing was driven at frequencies of >10
Hz. Intracellular calcium did not return to baseline levels during repetitive
firing but did return slowly to baseline levels after driven firing over a
period of several seconds. There was a linear relationship between the number
of action potentials generated by current injection and the peak level of
intracellular calcium for firing frequencies >10 Hz.
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SK KCa channels preferentially determine the duration and
intensity of rebound activity in STN neurons with short-duration rebound
bursts
Using previously published criteria (Bevan et al.,
2000,
2002), the majority of STN
neurons generated a rebound burst of activity in response to the removal of a
500-msec-long period of hyperpolarizing current injection (n = 23/30;
77%). Based on the findings of Bevan et al.
(2002), rebound burst
responses were scrutinized in neurons in which the membrane potential was
driven to a peak hyperpolarization of -79.0 ± 3 mV because this
represents a potential of key physiological significance, the mean equilibrium
potential of GABA-A IPSPs in STN neurons. In this study, 52% of neurons
(n = 12/23) displayed burst rebound responses at peak
hyperpolarizations in this range. Using the classifications outlined in Bevan
et al. (2002), 58% of these
cells fired bursts of <100 msec in duration (n = 7/12; mean
duration = 33.7 ± 9.5 msec), and 42% fired bursts with durations
>100 msec (n = 5/12; mean duration = 310.9 ± 130.1 msec).
These mixed responses imply heterogeneity in the intrinsic burst-generating
mechanisms of STN neurons. Thus, burst responses were compared after the
application of apamin to assess whether this heterogeneity could be accounted
for by differences in apamin-sensitive current
(Fig. 9). The mean duration of
burst responses elicited from peak hyperpolarizations of -79.0 ± 3 mV
was increased 10-fold for the entire population of short-duration burst
response neurons (mean duration = 304.1 ± 119.2; n = 7;
p = 0.018) (Fig.
9A). Whereas the mean burst duration for all neurons with
burst responses longer than 100 msec was also significantly higher in
saturating apamin (mean duration = 466.0 ± 140.5; n = 5;
p = 0.0431) (Fig.
9B), the relative increase in duration was much smaller
in these neurons. Indeed, the mean fold-increase in burst duration after
apamin application was significantly higher for neurons with rebound durations
<100 msec (M-W U test; duration <100 msec: mean fold-increase =
9.23 ± 3.1, n = 7; duration >100 msec: mean fold-increase =
1.71 ± 0.2, n = 5; p = 0.028). SK KCa
channels therefore strongly curtailed burst duration in one subpopulation of
STN neurons (short-duration rebounding cells) but were less influential in
another subpopulation (long-duration rebounding cells). It might be
hypothesized that long-duration rebounding cells simply express fewer SK
KCa channels, which exert a minor influence on rebound responses.
However, the size of the apamin-sensitive single-spike afterhyperpolarization
(as measured in voltage-clamp mode as per
Fig. 1) was not significantly
different in neurons with short-duration or long-duration rebound bursts (M-W
U test; short-duration = 55.6 ± 6.9 pA; long-duration 99.0
± 31.3 pA; p = 0.5637). Similarly, the effects of SK
KCa channel blockade on autonomous activity (M-W U test;
short-duration: CV = 0.39 ± 0.14, n = 7; long duration: CV =
0.27 ± 0.12 n = 4; p = 0.3447) and driven firing (M-W
U test; short duration: normalized current for half maximal firing =
0.75 ± 0.069, n = 3; long duration: normalized current for
half maximal firing = 0.84 ± 0.10, n = 7, p = 0.4759)
were similar in neurons with short- and long-duration rebound bursts.
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Figure 9. SK KCa channels differentially sculpt rebound activity in
neurons with short- and long-duration rebound bursts. A, The
application of 100 nM apamin to a neuron with a rebound burst
response of <100 msec extended greatly the duration and intensity of
rebound activity at all levels of preceding hyperpolarization. B, In
contrast, more modest effects on rebound activity were observed when 100
nM apamin was applied to a neuron with a long-duration rebound
burst response (> 500 msec) in control conditions. C, Application
of EBIO consistently shortened the duration of rebound bursts evoked after
hyperpolarization to a range of membrane potentials. Calibration in C
also applies to A and B.
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The capability of SK channels to control rebound activity in STN neurons
was confirmed by the application of 200 µM EBIO, which strongly
truncated rebound burst responses (Fig.
9C). When comparing population data for responses
elicited with peak hyperpolarizations in the -76 to -82 mV range, burst
duration was significantly shortened by EBIO (WSR test; control: 230.8
± 97.7 msec; EBIO: 61.7 ± 33.4 msec, n = 5, p
= 0.0431). In summary, SK KCa channels therefore play a critical
role in determining the nature of rebound activity, but factors that determine
their activation during rebound activity but not autonomous or driven activity
are heterogeneous.
Rebound responses are associated with action potential-independent
calcium entry
The heterogeneous effects of SK KCa channels blockade on rebound
bursting, but not other forms of activity, suggest that calcium dynamics
during rebound activity are different from those associated with autonomous or
driven activity. During autonomous and driven activity, the majority of
calcium entry was associated with action potentials. We hypothesized that
during rebound activity there is likely to be an additional recruitment of
Cav channels that activate below spike threshold. By adjusting the
holding potential or the degree of hyperpolarization produced during 500-1000
msec hyperpolarizing current injections, rebound responses that were below the
threshold for action potential generation were generated. Under these
conditions, we clearly observed that calcium entry was associated with the
early and late phases of subthreshold rebound responses in each of seven
neurons that were examined (Fig.
10). These and previous findings (Beurrier et al.,
1999,
2000;
Bevan and Wilson, 1999;
Song et al., 2000;
Otsuka et al., 2001;
Bevan et al., 2002;
Baufreton et al., 2003) point
to the involvement of Cav channels in the underlying rebound
depolarization. These channels recover from inactivation when hyperpolarized
below the potentials associated with autonomous oscillation and driven firing
and are strongly activated at subthreshold voltages. The early component of
the rebound depolarization has been shown previously to be mediated, in part,
by Cav channels that inactivate rapidly, are highly
nickel-sensitive, and are therefore presumably derived from the
Cav3 class (Beurrier et al.,
1999,
2000;
Song et al., 2000;
Otsuka et al., 2001). The
long-lasting component of the rebound depolarization appears to be mediated in
part by slowly inactivating/noninactivating, nifedipine-sensitive
Cav1.2-1.3 channels (Beurrier
et al., 1999; Otsuka et al.,
2001). The calcium imaging presented here confirms that calcium
entry is indeed associated with the early and late phases of subthreshold
rebound depolarization (Fig.
10).
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Figure 10. Action potential-independent calcium entry during rebound activity. A,
B, Combined fluorescent and electrical recordings subthreshold and
suprathreshold rebound responses in an STN neuron (inset). A, After
the termination of a hyperpolarizing current step, a low-threshold calcium
spike was generated without accompanying action potentials. The calcium spike
was associated with a marked increase in intracellular calcium in both the
soma (black trace) and dendrites (gray trace). B, In the same neuron,
the early phase of a suprathreshold rebound response was followed by a later
and longer lasting subthreshold response that was also associated with an
increase in calcium in the soma and dendrites of the STN neuron. Calibration
in B also applies to A.
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SK KCa channels are critical determinants of the pattern
of rhythmic activities in STN neurons
Spontaneous bursting activity in STN neurons has been observed occasionally
(Beurrier et al., 1999,
2000,
2001; Baufreton et al.,
2001,
2003). When it has been
observed, gluconate was used as the intracellular anion, and/or GABA-A
receptors were concurrently blocked with the methiodide salt of bicuculline.
In recent years it has been shown that both the intracellular application of
gluconate and the extracellular application of bicuculline methiodide block SK
KCa channels and SK KCa channel-mediated functions
(Zhang et al., 1994;
Johnson and Seutin, 1997;
Seutin et al., 1997;
Velumian et al., 1997;
Debarbieux et al., 1998;
Aizenman and Linden, 1999;
Stocker et al., 1999). We
therefore tested whether rhythmic burst activity could be induced in neurons
recorded in the perforated configuration by the application of apamin. In
these and all other slice experiments in this study, we used antagonists of
fast synaptic transmission that are not known to block SK KCa
channels. Rhythmic burst activity in STN neurons has been most commonly
generated by the injection of negative holding current and hyperpolarization
to voltages not normally associated with spontaneous oscillation (Beurrier et
al., 1999,
2000,
2001; Baufreton et al.,
2001,
2003). Thus, we applied tonic
hyperpolarizing current in an attempt to induce this form of activity. In the
perforated patch recordings reported here and previously (Bevan et al.,
2000,
2002), spontaneous burst
activity was never observed in control conditions either at rest or when the
membrane was hyperpolarized with negative current
(Fig. 11A). In
contrast, after the application of apamin, burst activity was induced by
equivalent levels of hyperpolarizing current in 5 of 8 (63%) STN neurons
(Fig. 11B). This
activity was quantified using the CV of the distribution of 120 interspike
intervals and the Lomb periodogram of the autocorrelation of 121 spikes. For
the five neurons that developed burst activity, their mean CV measures
increased significantly (WSR test; control: 0.25 ± 0.02; 10
nM apamin: 2.1 ± 0.40, n = 5, p = 0.0431),
and the interspike interval histograms constructed from data in the presence
of apamin displayed counts at long interval times that were not apparent in
the interspike interval histograms constructed using control data. In
addition, the predominant frequencies of activity identified by Lomb
periodograms indicated a shift in control conditions from a frequency (7.06
± 2.7 Hz) that was close to the mean frequency of single-spike activity
(4.54 ± 0.91) to a frequency (0.75 ± 0.1 Hz) that was dissimilar
to the mean frequency of activity (6.4 Hz ± 1.39) but matched the
frequency of burst activity in apamin
(Fig. 11). These data indicate
that rhythmic low-frequency burst activity can be induced in STN neurons by
concurrent SK KCa channel blockade and somatic hyperpolarizing
current injection.
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Figure 11. SK KCa channel blockade and hyperpolarization transform rhythmic
single-spike activity into rhythmic burst activity. A, B, Tonic,
slow, rhythmic single-spike activity (A) was transformed into tonic,
rhythmic burst activity when SK KCa channels were blocked
(B). C, D, Note also that after SK KCa channel
blockade, the predominant frequency in the Lomb periodogram switched from a
frequency that was similar to the mean frequency of single-spike activity
(C) to a frequency that was similar to the frequency at which bursts
occurred (D). The horizontal line in the Lomb periodograms denotes a
level of significance of p = 0.05. Calibration in A also
applies to B.
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Cav2.2 channels but not Cav1.2-1.3 channels are
strongly coupled to SK KCa channels. Cav1.2-1.3 channels
underlie, in part, secondary range firing and long-duration rebound
activity
On the basis of the previous data and the observation that the major
contributors of high voltage-activated calcium current in STN neurons are
Cav2.2 channels (52%, Song et
al., 2000), we hypothesized that these channels are strongly
coupled to SK KCa channels. Using similar analytical and current
injection protocols to those described above, we therefore compared
autonomous, driven, and rebound activity in neurons under control conditions
and in the presence of the selective Cav2.2 channel antagonist
-conotoxin GVIA (1 µM). The effects on autonomous
activity and driven activity were similar to the effects of apamin. Thus,
Cav2.2 channel blockade reduced the magnitude of single-spike
afterhyperpolarization in all 21 neurons tested, reduced the rhythmicity of
autonomous activity (WSR test; CV in control media = 0.074 ± 0.007; CV
in -conotoxin GVIA = 0.165 ± 0.035, n = 21, p
< 0.0001) (Fig.
12A,B), reduced the normalized current to reach half
maximal firing (WSR test, 0.88 ± 0.5, n = 20, p =
0.0064) (Fig. 12D),
and increased the normalized gradient of the primary range (WSR test; 1.56
± 0.11, n = 19, p = 0.0005)
(Fig. 12C,D). In
contrast to the effects of SK KCa channel blockade,
Cav2.2 channel blockade also reduced the maximal firing frequency
(Fig. 12C,D). These
data indicate that Cav2.2 channels, in addition to activating SK
KCa channels, also contribute a significant inward current that
augments high-frequency activity. The effects of -conotoxin GVIA on
autonomous activity (M-W U test; short-duration: CV = 0.107 ±
0.007, n = 9; long-duration: CV = 0.147 ± 0.020, n =
6; p = 0.099), driven high-frequency activity (M-W U test;
short duration: normalized current for half maximal firing = 0.96 ±
0.065, n = 9; long-duration: normalized current for half maximal
firing = 0.83 ± 0.12, n = 5; p = 0.317), and primary
range activity (M-W U test; short-duration: normalized slope of the
primary range = 1.41 ± 0.13, n = 9; long-duration: normalized
slope of the primary range = 1.71 ± 0.33, n = 4; p =
0.2801) were similar in neurons with short-or long-duration rebounds.
Interestingly, the duration of rebound activity was not increased by
Cav2.2 channel blockade in neurons with short-duration rebounds
(WSR test; control = 36.1 ± 5.9 msec; -conotoxin GVIA = 41.0
± 9.6 msec; n = 9; p = 0.594) or long-duration
rebounds (WSR test; control = 348.95 ± 109.5 msec; -conotoxin
GVIA = 295.02 ± 107.89 msec; n = 6; p = 0.4631), a
clear difference to the effects of apamin, which suggests another source of
calcium must activate SK KCa channels during rebound activity
(Fig. 12E). Taken
together these data suggest that differential coupling of SK KCa
channels to Cav2.2 channels is unlikely to account for diversity of
rebound activity in STN neurons.
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Figure 12. SK KCa channels are strongly coupled to Cav2.2
channels. A, B, Rhythmic autonomous oscillation and single-spike
afterhyperpolarization were disrupted by the application of -conotoxin
GVIA in a manner that was similar to the effects of apamin. C, D,
Cav2.2 channel blockade also increa | |
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Abstract
Introduction
