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The Journal of Neuroscience, September 1, 2001, 21(17):6553-6560
Modulation of Excitability by  -Dendrotoxin-Sensitive Potassium
Channels in Neocortical Pyramidal Neurons
John M.
Bekkers and
Andrew J.
Delaney
Division of Neuroscience, John Curtin School of Medical Research,
Australian National University, Canberra, ACT 0200, Australia
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ABSTRACT |
Many neurons transduce synaptic inputs into action potentials (APs)
according to rules that reflect their intrinsic membrane properties.
Voltage-gated potassium channels, being numerous and diverse
constituents of neuronal membrane, are important participants in
neuronal excitability and thus in synaptic integration. Here we address
the role of dendrotoxin-sensitive "D-type" potassium channels in
the excitability of large pyramidal neurons in layer 5 of the rat
neocortex. Low concentrations of 4-aminopyridine or  -dendrotoxin
(-DTX) dramatically increased excitability: the firing threshold for
action potentials was hyperpolarized by 4-8 mV, and the firing
frequency during a 1-sec-long 500 pA somatic current step was doubled.
In nucleated outside-out patches pulled from the soma, -DTX
reversibly blocked a slowly inactivating potassium current that
comprised ~6% of the total. This current first turned on at voltages
just hyperpolarized to the threshold for spiking and activated steeply
with depolarization. By assaying -DTX-sensitive current in
outside-out patches pulled from the axon and primary apical dendrite,
it was found that this current was concentrated near the soma. We
conclude that -DTX-sensitive channels are present on large layer 5 pyramidal neurons at relatively low density, but their strategic
location close to the site of action potential initiation in the axon
may ensure that they have a disproportionate effect on neuronal
excitability. Modulation of this class of channel would generate a
powerful upregulation or downregulation of neuronal output after the
integration of synaptic inputs.
Key words:
action potential; D-current; dendrite; dendrotoxin; neocortex; potassium channel; pyramidal neuron
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INTRODUCTION |
Many classes of neurons function as
transducers that convert patterns of (input) synaptic potentials into
patterns of (output) action potentials (APs), which then propagate
information to other neurons. This transduction process, called
synaptic integration, is often highly complex and depends on, among
other things, the types and distribution of ion channels that populate
the neuronal membrane (Häusser et al., 2000 ). One of the major
determinants of AP firing is voltage-gated potassium channels, which
tend to dampen or pace electrogenic activity (Hille, 1992). Thus, to
fully understand synaptic integration, it is important to know the
disposition of the various types of voltage-gated potassium channels
that are found on the somata and dendrites of neurons.
In recent years synaptic integration has been intensively studied in
the large pyramidal neurons with somata that are located in
layer 5 of mammalian neocortex (Stuart et al., 1997b). These neurons
are attractive subjects because of their unusually accessible apical
dendrites and their importance as the main output neurons of the
cortex. To date, the function and distribution of
Na+ channels (Stuart and Sakmann, 1994),
Ih channels (Williams and Stuart,
2000; Berger et al., 2001) and two types of voltage-gated K+ channels
(IA and delayed rectifier channels)
(Bekkers, 2000a,b; Korngreen and Sakmann, 2000) have been studied
in this type of cell.
Another member of the class of voltage-gated
K+ channels, frequently reported in other
types of neurons, is the D-current
(ID), which was originally identified
by its ability to delay the firing of APs after a depolarizing current
step (Storm, 1988). ID is now more
commonly defined by its slow inactivation kinetics and sensitivity to
low concentrations of 4-aminopyridine (4-AP) and dendrotoxin
(DTX) (Storm, 1993; Coetzee et al., 1999). 4-AP (50-100 µM) was reported to have no effect (Bekkers,
2000a), and dendrotoxin little effect (Korngreen and Sakmann, 2000), on
nucleated outside-out patches pulled from the somata of large layer 5 pyramidal neurons, suggesting that ID
is only weakly expressed in these cells (Albert and Nerbonne, 1995).
However, an immunocytochemical study has shown that the major apical
dendrites of large layer 5 neurons are particularly enriched in
Kv1.2 subunits, whereas the cell bodies are relatively spared of
Kv1.2 staining (Sheng et al., 1994). The Kv1.2 subunit, probably in
combination with other Kv proteins and Kv subunits, is thought to
comprise ID channels (Coetzee et al.,
1999). Thus, the earlier work might have missed significant
ID current by confining the search to
somatic membrane. We therefore decided to revisit this question by
exploring the possibility that ID is
preferentially expressed in the dendrites.
We find that these large layer 5 cortical neurons do, indeed, contain
ID-like channels, as revealed by a
marked increase in excitability in 4-AP or -DTX and by the presence
of a small -DTX-sensitive current in outside-out patches.
Surprisingly, despite the immunocytochemical evidence, these channels
tend to be concentrated close to the soma in the axon and primary
apical dendrite. With this distribution, the channels are ideally
placed to influence neuronal output.
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MATERIALS AND METHODS |
Neocortical slices (300 µm thick) were prepared from 14- to
21-d-old Wistar rats using standard techniques that were approved by
the Animal Experimentation Ethics Committee of the Australian National
University (Bekkers, 2000a). Large layer 5 pyramidal neurons in
sensorimotor cortex were identified and patch electrodes were
positioned using infrared videomicroscopy. Experiments used slices in
which the primary apical dendrites were most nearly parallel to the cut
surface, to minimize damage to the dendritic tree. Recordings were made
with a MultiClamp 700A computer-controlled patch-clamp amplifier (Axon
Instruments, Foster City, CA), the headstage of which contains both an
Axopatch-like voltage-clamp circuit and an Axoclamp-like voltage
follower for true current clamp. In voltage-clamp mode, linear leak and
capacitance currents were removed using an on-line subtraction
procedure, as described previously (Bekkers, 2000a). When recording
from nucleated outside-out patches, 80-90% series resistance
compensation was applied, minimizing steady-state voltage errors to <1
mV. Currents were filtered at 2 kHz and digitized at 10 kHz. Current
clamp recordings in whole-cell mode used bridge balance and capacitance
neutralization, which were checked throughout the experiment. Families
of current injections, done at 4 min intervals, were repeated at least
three times in control and test solutions, and the measurements in each
condition were averaged together. Voltages were filtered at 10 kHz and
digitized at 20-50 kHz. Patch electrodes had resistances of 3-5 M
(whole-cell or nucleated patches) or 7-12 M (outside-out patches).
The usual bath solution comprised (in mM): 125 NaCl, 3 KCl,
25 NaHCO3, 1.25 NaH2PO4, 1 MgCl2, 2 CaCl2, 25 glucose,
pH 7.4, when saturated with 95% O2/5%
CO2, plus 10 µM CNQX to inhibit excitatory synaptic transmission. In experiments with -DTX, 0.1% bovine serum albumin was also added (Martina et al., 1998). In some
experiments (e.g., see Fig. 5), a high-TEA bath solution was used, in
which 30 mM tetraethylammonium chloride substituted for 30 mM of the NaCl. Internal solution comprised (in
mM): 135 potassium methylsulfate, 7 NaCl, 5 EGTA, 2 MgATP,
0.3 GTP, 10 HEPES, adjusted to pH 7.2 with KOH. All voltages have been
corrected for the measured junction potential of these solutions ( 7
mV). Drugs were dissolved in bath solution and applied either by
Picospritzer (General Valve, Fairfield, NJ) or by perfusing the bath
volume >10 times. Control experiments confirmed that Picospritzer
("puffer") application of external solution alone (without drugs)
had no effect on any of the measurements. Also, in experiments using the puffer to apply -DTX to outside-out patches (see Fig. 7), correct application of the toxin was checked at the end of the day by
confirming that the puffer produced the expected increase in whole-cell
excitability (see Fig. 2). All compounds were obtained from Sigma (St.
Louis, MO), apart from CNQX (Tocris Cookson, Bristol, UK) and -DTX
(Alomone, Jerusalem, Israel). All experiments were performed at room
temperature (22-25°C).
Analysis was done using AxoGraph (Axon Instruments). Instantaneous AP
frequency was calculated from the reciprocal of the time between
successive AP peaks. The voltage threshold for AP firing was determined
as the interpolated membrane potential
(Vm) at which
dVm/dt equaled 20 V/sec; this criterion gave the best agreement with threshold
determinations done by eye (e.g., see Fig. 3). AP half-width was
measured at half the difference between the firing threshold and the
peak of each AP. Input resistance was estimated from the steady-state
voltage response to a long (1 sec) hyperpolarizing current step (100 pA). Amplitudes of currents in nucleated and outside-out patches were
measured by averaging over a window 100-150 msec after the
beginning of the voltage step to the test potential. Inhibition by
-DTX was calculated by dividing the mean amplitude measured during
toxin application by the average amplitudes measured before and after
toxin application (e.g., see Figs. 4B, 5A,
7A). To generate activation plots, currents were converted
to conductances using the measured reversal potential for the delayed
rectifier current in nucleated patches from these neurons (85 mV)
(Bekkers, 2000a). The plots were fitted to the Boltzmann equation,
f(V) = Gmax/(1 + exp((V1/2 V)/k)), where Gmax is the maximal conductance,
V1/2 is the voltage at which activation is half-maximal, and k is the slope factor. To
generate normalized activation plots (see Fig. 5), the data from each
cell were normalized by the fitted
Gmax, and all cells were averaged together. Statistical comparisons used the t test. Errors
are given as ± SEM, with n the number of cells or patches.
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RESULTS |
4-AP (100 µM) increases neuronal excitability
A defining property of D-type potassium current in hippocampal
pyramidal neurons is its sensitivity to both -dendrotoxin and low
concentrations of 4-aminopyridine (Storm, 1988; Wu and Barish, 1992;
Golding et al., 1999). We began by examining the effect of 100 µM 4-AP on the firing properties of large layer 5 cortical pyramidal neurons.
Excitability was assayed by injecting 1-sec-long steps of current at
the soma under current clamp (Fig.
1A). Adding 100 µM 4-AP to the bath consistently increased the
excitability of all neurons tested. This occurred despite any
significant change in both the resting potential (73.5 ± 0.9 mV
in control, 72.2 ± 0.8 mV in 4-AP; n = 6;
p > 0.16, paired t test) and the input resistance (42.7 ± 2.7 M in control, 46.1 ± 3.0 M in
4-AP; p > 0.08) of the neurons. Excitability changes
were quantified in two ways (Fig.
1B,C). Changes in the current
required for firing action potentials were determined by
plotting the number of APs evoked during each current step versus the
amplitude of this injected current (Fig. 1B).
Addition of 100 µM 4-AP shifted the plot to the
left, indicating that less current was required to generate the same
number of APs in 4-AP. The mean threshold current (the minimum injected
current that fires APs) was 221 ± 18 pA in control solution and
110 ± 14 pA in 100 µM 4-AP
(n = 6 cells; significantly different,
p < 0.01).
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Figure 1.
Bath application of 100 µM 4-AP
increases the excitability of large layer 5 cortical pyramidal neurons.
A, Action potentials (APs) elicited by
1-sec-long 200 pA (left) or 500 pA
(right) current steps in the same neuron before
(top) or after (bottom) addition of 100 µM 4-AP to the external solution. For this neuron,
200 pA was just suprathreshold for the firing of an AP in control
solution. Note that all membrane potentials have been corrected for the
measured liquid junction potential (7 mV; see Materials and Methods).
B, Mean number of APs elicited by 1-sec-long current
steps to the values given on the x-axis ( ,
Control;  , 4-AP; n = 6 cells; ±SEM). 4-AP reduces the amount of current required to fire
APs. C, Mean instantaneous AP frequency (i.e.,
reciprocal of interval between adjacent APs) versus the interval
number, measured during a 1-sec-long 500 pA current step. Same data set
and symbols as in B. 4-AP increases the firing rate of
APs during a fixed current step.
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The second measure of excitability quantified the rate of AP firing
during a current step of fixed amplitude (500 pA). The instantaneous AP
frequency, calculated from time intervals between successive APs, was
plotted versus the interval number in the train (Fig. 1C).
Addition of 100 µM 4-AP significantly increased the AP frequency throughout the train.
The neuron in Figure 1A is representative of the
majority of neurons in this study (24/28 tested) in that it fired a
burst of two or three APs at the start of larger current steps, then fired regularly without accommodating. This behavior is typical of
large layer 5 neurons in rats of this age (Kasper et al., 1994; Williams and Stuart, 1999). Of the remaining neurons in the sample, three weakly accommodated and one showed strong burst firing; these
were not included in the analysis.
-DTX (1-2 µM) also increases excitability
Low concentrations of 4-AP may partially block the A-type
potassium current in pyramidal neurons, causing changes in excitability and confounding the effect of ID
blockade (Hoffman et al., 1997; Bekkers, 2000a; Korngreen and Sakmann,
2000). Accordingly, we repeated the above experiments with the more
selective ID blocker, -dendrotoxin.
-DTX was applied by either bath perfusion (at 1 µM) or
puffer near the soma (at 2 µM); identical results were
obtained for each. As for 4-AP, 1-2 µM -DTX increased
excitability in all neurons tested (Fig.
2) without affecting either the resting
potential (72.5 ± 1.0 mV in control, 73.1 ± 1.0 mV in
toxin; n = 4; p > 0.28, paired
t test) or the input resistance (35.6 ± 4.8 M in
control, 37.0 ± 3.4 M in toxin; p > 0.45).
The toxin reduced the mean current required to first evoke an AP
(283 ± 40 pA in control, 153 ± 16 pA in -DTX;
n = 4; p < 0.01) (Fig.
2B) and increased the firing rate of APs during a 500 pA current step (Fig. 2C). Thus, 1-2
µM -DTX appeared to act like 100 µM 4-AP in enhancing the excitability of these
neurons.
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Figure 2.
Bath or puffer application of 1-2
µM -dendrotoxin (-DTX)
increases excitability, like 100 µM 4-AP.
A, Data recorded in the same neuron before
(top) and after (bottom) puffing 2 µM -DTX near the soma. B,
C, Summary plots like those in Figure 1 (,
Control; , -DTX;
n = 4 cells; ±SEM). Like 4-AP, -DTX reduces the
current required to fire APs and increases AP firing rate.
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Effects of 4-AP and -DTX on individual action potentials
To further compare the effects of 4-AP and -DTX, the properties
of individual APs were measured (Fig. 3).
Both 4-AP and -DTX hyperpolarized by 4-8 mV the firing threshold
for APs (Fig. 3A, dashed lines, B), an
effect that accounts for the reduced current needed to fire APs after
drug perfusion (Figs. 1B, 2B). In
contrast, the two drugs differed in their effect on the AP half-width:
100 µM 4-AP significantly broadened (by
0.15-0.59 msec on average, depending on spike number) all but the
first AP in the train, whereas 2 µM -DTX had
no effect on half-width (Fig. 3C) (p < 0.02 for all except the first AP in 4-AP; p > 0.37 for all APs in -DTX; n = 5 cells). Finally, the
afterhyperpolarization after each AP was not significantly affected by
either 4-AP or -DTX (62.0 ± 0.2 mV in control, 61.6 ± 0.1 mV in 4-AP, 63.6 ± 0.2 mV in -DTX; p > 0.05; n = 5 cells).
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Figure 3.
4-AP (left panels) and -DTX
(right panels) both hyperpolarize the firing threshold
for APs, but differ in their effects on AP half-width.
A, APs near the start of a 1-sec-long 500 pA current
step, recorded before (thin line) and after
(thick line) application of 100 µM 4-AP
(left) or 2 µM -DTX
(right). Each panel is from a different neuron.
Horizontal dashed lines indicate the firing thresholds
for later APs with and without drugs. B, Mean firing
threshold for each AP in a train during a 1-sec-long 500 pA current
step versus the AP number. , Control; , 100 µM 4-AP (left panel; n = 5 cells; ±SEM) or 2 µM -DTX (right
panel; n = 4 cells; ±SEM). Both 4-AP and
-DTX hyperpolarize the firing threshold throughout the train.
C, Mean half-width for each AP in the train, for the
same data set as in B. 4-AP significantly increases the
half-width of all but the first AP (left panel),
whereas -DTX has no effect (right panel).
Insets show the sixth AP in an illustrative train,
recorded in control (thin line) or in drug solution
(thick line).
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These results suggest that 100 µM 4-AP and 2 µM -DTX act in a similar, but not identical, manner to
increase the excitability of large layer 5 cortical pyramidal neurons.
The different effects on AP half-width may reflect nonspecific actions
of 4-AP on the A-current (Storm, 1993). Therefore, the remainder of our
experiments used only the more selective
ID blocker, -DTX.
-DTX partially inhibits a K+ current in
nucleated outside-out patches
Evidence presented later in this paper suggests that
-DTX-sensitive K+ channels are located
on or near the soma. Accordingly, we used nucleated outside-out patches
pulled from the soma to look for this current.
Nucleated patches were voltage clamped at 67 mV, stepped to 117 mV
for 500 msec to remove resting inactivation of all potassium currents
(Bekkers, 2000a), and then stepped to a test potential of +3 mV. This
elicited a brief inward Na+ current
followed by a slow outward K+ current
(Fig. 4A,
insets) (leak and capacitance currents
subtracted). Figure 4A plots the amplitudes of both
Na+ and K+
currents versus time for one experiment. After a baseline period, 2 µM -DTX was puffed onto the patch (Fig.
4A, bar), causing a small reduction in the
K+ current (Fig. 4A,
). The puffer solution also contained 0.5 µM
TTX as a positive control; complete blockade of the
Na+ current (Fig. 4A,
 ) confirmed that the toxins were fully bathing the patch. Control
experiments established that puffer solution without toxins had no
effect. Thus, in this patch, 2 µM -DTX blocked a small (~90 pA) outward current (unlabeled trace in
top inset).
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Figure 4.
Two micromolar -DTX has a small inhibitory
effect on K+ current recorded in voltage-clamped
nucleated outside-out patches. A, Amplitudes of outward
K+ current (top, ) and inward
Na+ current (bottom,  ) measured
simultaneously in a nucleated patch during voltage-clamp steps from
117 mV to +3 mV, plotted against time during the experiment.
K+ current amplitude was averaged over a
window 100-150 msec after the step onset, to avoid A-current;
Na+ current was measured at the peak. Puffer
application of 2 µM -DTX (horizontal
bar) caused a small inhibition of the K+
current, whereas 0.5 µM tetrodotoxin
(TTX) included in the puffer solution fully
blocked the Na+ current, confirming that the toxins
were bathing the patch. Insets show, on slow
(top) and fast (bottom) time bases, the
currents measured at the numbered time points. Unlabeled
trace in the top inset is the -DTX-sensitive
current, obtained by subtraction. B, Mean normalized
K+ current () and Na+ current
() measured in 11 experiments of this sort. The dashed
line is drawn by eye to emphasize the small mean inhibition
produced by 2 µM -DTX: 6% on average.
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The averaged, normalized time course plot from eight nucleated patches
is shown in Figure 4B. -DTX (2 µM) caused a mean inhibition of the outward
current of 6 ± 3% () compared with the average of amplitudes
before and after toxin application. Thus, nucleated outside-out patches
do contain an -DTX-sensitive current, but it is a minor component of
the total K+ current.
Activation properties of the -DTX-sensitive current in
nucleated patches
Because the -DTX-sensitive current is small, it was not
practical to obtain complete activation families of this current by
subtracting records obtained in the same nucleated patch before and
after application of -DTX, because of rundown and other
instabilities. Instead, we compared averaged, normalized activation
plots for different patches in either the presence or absence of
-DTX; any differences between them would reflect the
-DTX-sensitive component.
We first tried this strategy in normal bath solution. Activation
families of K+ currents were recorded in
the presence or absence of -DTX (2 µM applied by
puffer), and their amplitudes were measured over a window
100-150 msec after the start of the test pulse. This is on the plateau
of the -DTX-sensitive current (Fig. 4A,
inset) and avoids the fast A-current that is present in
these patches (Bekkers, 2000a). After conversion to conductance,
activation plots were fitted to the Boltzmann equation (Materials and
Methods). It was found that the fit parameters in the two conditions
were not significantly different (p > 0.12, unpaired t test; n = 7 cells in each
condition). This result may arise if the -DTX-sensitive current is
too small to reliably measure against a large background of the delayed
rectifier, IK.
To explore this possibility, we repeated the experiment in bath
solution containing 30 mM TEA to partially block
IK and so increase the relative size
of -DTX-sensitive current. [The latter current is not affected by
TEA (Storm, 1988.)] It was first confirmed that 2 µM -DTX reversibly blocked a
K+ current in nucleated patches in TEA
solution (Fig. 5A). The mean amplitude of the current blocked by -DTX in TEA-containing bath solution (35 ± 7 pA; n = 6 patches) was similar
to that in normal bath solution (40 ± 15 pA; n = 8 patches). However, because IK was
reduced by ~85% in 30 mM TEA (Korngreen and
Sakmann, 2000), the percentage block by -DTX, relative to the total
amount of slow K+ current, was much larger
in TEA [28 ± 7%; n = 6 (Fig. 5A);
cf. 6% in normal bath solution (Fig. 4B)]. Thus,
TEA emphasized the -DTX-sensitive current and facilitated
measurement of its properties.
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Figure 5.
Bath application of TEA reduces the amount of the
delayed rectifier current (IK) in
nucleated patches, facilitating measurement of the activation
properties of -DTX-sensitive current. A, Averaged,
normalized amplitudes of K+ current measured in
nucleated patches bathed in external solution containing 30 mM TEA plus 0.4 µM TTX, plotted against time
during the experiment. Currents were evoked by voltage-clamp steps from
117 to +43 mV, and their amplitudes were measured by averaging over a
window 100-150 msec after the start of the step. Puffer
application of TEA-TTX solution plus 2 µM -DTX
(horizontal bar) reversibly inhibited the
K+ current by 28% on average. Inset
shows typical currents recorded in one patch, averaged during the
control and wash periods (trace a) and during the period
of toxin application (trace b). Unlabeled
trace is the subtraction of trace b from
a, the -DTX-sensitive current. B,
Families of voltage-clamped K+ currents recorded in
nucleated patches in TEA-TTX bath solution in the absence
(top) and presence (bottom) of 2 µM -DTX, applied by puffer. The pulse protocol is
shown at the top. Square brackets over
the current traces indicate the window over which the amplitude was
measured for the activation plots (100-150 msec after the start of the
test pulse). Note that the recordings were obtained from different
patches. C, Averaged, normalized activation plots in the
absence (; n = 7 patches) and presence (;
n = 5 patches) of 2 µM -DTX, all
in TEA-TTX solution. Superimposed smooth curves are the
results of fits of the Boltzmann function with the indicated fit
parameters. D, Activation plot for the
-DTX-sensitive current, obtained by scaling down
the + -DTX data in C by 28%, subtracting
this from the -DTX data, and renormalizing the result. The
superimposed continuous curve is a Boltzmann fit to the
points, giving the indicated fit parameters. The dashed
curve is the mean activation plot for
IK measured in nucleated patches from the
same neurons, reported in Bekkers (2000a).
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Activation families in 30 mM TEA were measured in separate
patches in the presence or absence of 2 µM -DTX (Fig.
5B). The averaged, normalized activation plot in each
condition was fitted to the Boltzmann equation (Fig. 5C).
The activation plot was significantly shifted to the right by addition
of -DTX (V1/2 = 4.8 mV,
k = 25.7 mV in control, n = 7 patches;
V1/2 = 22.1 mV, k = 26.0 mV in -DTX, n = 5 patches; p < 0.01, unpaired t test). The activation plot for
-DTX-sensitive current was calculated by subtracting the "+
-DTX" activation data from the " -DTX" data, after
scaling down the amplitude of the former by 28% (the mean inhibition
produced by 2 µM -DTX in TEA) (Fig.
5A). The result of this calculation, after renormalization,
is shown in Figure 5D. Fit of the Boltzmann equation to this
plot gave V1/2 = 22.4 mV,
k = 18.9 mV (Fig. 5D, continuous
curve). This is similar to the peak activation plot for
IK measured in the same cells (Fig.
5D, dashed curve) (from Bekkers,
2000a).
Note that the activation plot shown in Figure 5C (+ -DTX,
open symbols) is not the same as the peak activation plot
for IK (Fig. 5D,
dashed curve), because the former was measured 100-150 msec
after the peak of IK and is confounded
by the voltage-dependent inactivation of
IK.
-DTX-sensitive channels are functionally different from
IK channels
-DTX-sensitive channels resemble
IK channels both in their steady-state
activation properties (Fig. 5D) and their relatively slow
kinetics (Figs. 4A, 5A). Do these
similarities extend to their role in the firing of these neurons? To
address this question, we compared the effects of blockade of these two
kinds of K+ current. As noted above,
-DTX blocked ~6% of the slow K+
current in nucleated patches in normal bath solution (Fig.
4B). Six percent block of
IK was achieved by adding 0.32 mM TEA to the normal external solution,
using the reported IC50 value for TEA blockade of
IK in these patches (5 mM)
(Korngreen and Sakmann, 2000). The firing properties of the
same neuron were then compared before and after addition of 0.32 mM TEA, using the same measures as were used
earlier for -DTX.
This low concentration of TEA had a weak effect on the excitability of
neurons, reducing by 16% the mean current injection required to first
evoke an AP [267 ± 21 pA in control, 224 ± 12 pA in TEA;
n = 7 cells; p < 0.05 (Fig.
6A,B);
cf. 46% reduction by -DTX (Fig. 2B)]. In
striking contrast with the result for -DTX (Fig. 3B), TEA
had no significant effect on the firing threshold for APs
(n = 7 cells; p < 0.05) (Fig.
6C); however, TEA did prolong AP half-widths (1.09 ± 0.02 msec in control, 1.46 ± 0.04 msec in TEA; p > 0.1) (Fig. 6A), contrasting with -DTX, which
had no effect on half-widths (Fig. 3C). Similar results were
obtained for another cell to which 1.1 mM TEA was
applied (sufficient to block ~18% of
IK).
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Figure 6.
-DTX-sensitive channels are functionally
different from TEA-sensitive IK channels.
A, APs near the start of a 500 pA current step, recorded
in the same neuron before (Con, thin
line) and after (TEA, thick line)
perfusion of bath solution containing 0.32 mM TEA. This
concentration of TEA blocks ~6% of
IK, which is the same percentage of
the total slow K+ current as is blocked by 2 µM -DTX. The horizontal dashed line
indicates that the AP firing threshold is little affected by TEA (see
C); however, spike broadening is clearly apparent in
TEA, probably because of its inhibition of the fast
Ca2+-activated K+ current,
IC. B, Mean number of APs
elicited by 1-sec-long current steps to the indicated values (,
Control; , TEA; n = 7 cells). TEA (0.32 mM) has only a weak effect on the
current required to fire APs. For comparison, the dashed
line shows the effect of 2 µM -DTX, from
Figure 2B. C, Mean firing
threshold for each AP in a train during a 1-sec-long 500 pA current
step, plotted against the AP number. Same symbols and data set as in
B. For comparison, the dashed line shows
the effect of -DTX, from Figure 3B. Unlike -DTX,
TEA has no significant effect on the firing threshold.
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In summary, both -DTX and low concentrations of TEA blocked part of
a delayed rectifier-like K+ current in
these neurons, yet these drugs had very different effects on the
behavior of APs. These results strongly suggest that TEA-sensitive
IK channels and -DTX-sensitive
channels are functionally distinct. A possible explanation is that the
two channel types differ subtly in their kinetics or voltage dependence (Fig. 5D). This was hard to study in detail, given the small
size of the -DTX-sensitive currents and the difficulties of the
pharmacological method for separating them. We therefore turned to
another possibility and asked whether these functional differences
might reflect differences in channel distributions.
-DTX-sensitive channels tend to be concentrated near
the soma
In three cells, puffer application of 2 µM -DTX
to the primary apical dendrite (~100 µm from the soma) had little
effect on neuronal excitability (data not shown). In the same cells, puffing -DTX close to the soma increased excitability as usual (Fig.
2). In a different experiment, bath perfusion of 100 µM 4-AP caused the usual increase in excitability in a neuron in which the
primary apical dendrite had been amputated 160 µm from the soma
[using the method described in Bekkers (2000c)]. Both of these
experiments suggest that distal membrane does not contain a high
density of -DTX-sensitive current, or if it does, this current
contributes little to the spiking behavior initiated by somatic current
injections. To further examine this issue, the axodendritic density of
-DTX-sensitive channels was mapped.
Conventional outside-out patches were pulled from known locations on
the axon and primary apical dendrite of large layer 5 pyramidal
neurons. For each patch, a time course plot was acquired before,
during, and after application of 2 µM -DTX using a
puffer (Fig. 7A). The
amplitude of -DTX-sensitive current was estimated by subtracting
averaged currents obtained during toxin application from the averages
of currents before and after the toxin (Fig. 7A,
inset).
View larger version (24K):
[in this window]
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|
Figure 7.
-DTX-sensitive channels, assayed in
conventional outside-out patches, are concentrated near the soma.
A, Amplitude of K+ current measured
during a voltage-clamp step from a prepulse (117 mV for 500 msec) to
a test pulse of +43 mV (repeated 5 times and averaged), plotted against
time during the experiment. Amplitude was averaged over a window
100-150 msec after the step onset. -DTX (2 µM) was
applied by puffer (horizontal bar). Labeled
traces in the inset are averages of the currents
enclosed by square brackets in the time course plot; the
unlabeled trace is the subtraction of trace
b from a. This patch was pulled from the
primary apical dendrite 105 µm from the soma. B,
Amplitude of -DTX-sensitive current (each point obtained from a
separate outside-out patch using the averaged-subtraction method
described in A) plotted against the distance from the
soma that the patch was pulled. Patches were from either the axon ()
or the primary apical dendrite (). The continuous straight
line is given by I = 3.2 0.009 d, where I is the mean amplitude of the
-DTX-sensitive current in picoamperes, and d is the
distance from the soma in micrometers.
|
|
The amplitude of toxin-sensitive current in each patch was plotted
against the distance from the soma to the point on the axon or dendrite
from which the patch was pulled (Fig. 7B). The mean
amplitude of the -DTX-sensitive current declined with distance along
the dendrite from the soma (slope 0.9 pA/100 µm) (Fig. 7B, fitted straight line). The mean current was
3.6 ± 1 pA in the axon (n = 5 patches), 2.2 ± 1 pA (n = 14) in the proximal 100 µm of the
dendrite, and 0.9 ± 0.4 pA (n = 8) in the
dendrite 200-300 µm from the soma. Thus, the toxin-sensitive current
is small but shows a tendency to concentrate in the axon and proximal apical dendrite. This contrasts with delayed rectifier potassium current, which is both larger and more uniformly distributed along the
primary apical dendrite of these neurons (13.4 ± 2.0 pA over 0-100 µm, 10.5 ± 2.1 pA over 200-300 µm) (Bekkers,
2000b).
| |
DISCUSSION |
In this paper we have shown that -DTX-sensitive
ID-like potassium channels are present
on large pyramidal neurons in layer 5 of rat neocortex, albeit at a
relatively low density. Despite their scarcity, blockade of these
channels had a powerful effect on the excitability of the neurons.
Addition of -DTX hyperpolarized the firing threshold for APs by ~5
mV (Fig. 3) and roughly doubled the firing frequency of APs during
prolonged 500 pA current injections (Fig. 2). By assaying the amount of
-DTX-sensitive current present in outside-out patches pulled from
the axon and primary apical dendrite, we showed that these
ID-like channels tended to be
concentrated close to the soma (Fig. 7). Given that the usual site of
AP initiation is in the axon of these neurons (Stuart et al., 1997a),
-DTX-sensitive channels are strategically located for influencing
neuronal excitability.
ID was first described by Storm (1988)
in hippocampal CA1 pyramidal neurons. It was distinguished by its slow
inactivation, sensitivity to 40 µM 4-AP, and
ability to delay the onset of AP firing during long current steps. A
more recent criterion is its sensitivity to -DTX, which has been
shown to block the same slowly inactivating current as 50-100
µM 4-AP in hippocampal (Wu and Barish, 1992;
Golding et al., 1999) and cortical neurons (Foehring and Surmeier,
1993; Locke and Nerbonne, 1997). Even within this class of
4-AP/-DTX-sensitive currents there is a marked diversity in
biophysical properties (Wu and Barish, 1992; Foehring and Surmeier, 1993; Albert and Nerbonne, 1995; Bossu et al., 1996; Lüthi et al., 1996; Massengill et al., 1997; Budde and White, 1998). Thus, ID has been defined as a
dendrotoxin-sensitive voltage-sensitive current with variable kinetics
and voltage dependence that is also sensitive to low concentrations of
4-AP (Coetzee et al., 1999). It probably encompasses various subunit
combinations, most likely Kv1.2 with other subunits (Coetzee et al.,
1999).
Given that 4-AP also blocks the A-current (Storm, 1993), we preferred
to use -DTX as the defining antagonist because of its presumed
greater specificity. Indeed, subtle pharmacological differences between
100 µM 4-AP and 2 µM -DTX are apparent
from their different effects on AP half-width (Fig. 3C).
This may be attributable to partial blockade of the A-current by
micromolar 4-AP, as has been reported (Storm, 1987, 1988).
Alternatively, it is possible that ID
in these neurons is inhibited differently by 4-AP and -DTX, or that
several variants of ID are present and
are differentially inhibited by these compounds. Notwithstanding these
ambiguities, we use the term "ID"
as a convenient shorthand for the current blocked by -DTX.
The small size of ID in nucleated
patches from large layer 5 pyramids (~6% of total slow
K+ current in normal external solution)
(Fig. 4B) may explain why we did not detect
ID in earlier studies on these neurons
(Bekkers, 2000a), although others have reported small effects of DTX-I
(Korngreen and Sakmann, 2000) and micromolar concentrations of 4-AP
(Albert and Nerbonne, 1995) on this cell type. Our result agrees with immunocytochemical staining showing a relative paucity of Kv1.2 on the
somata of these neurons (Sheng et al., 1994). On the other hand, Sheng
et al. (1994) found a high density of Kv1.2 along the length of the
apical dendrites of cortical neurons. It was surprising, therefore, to
discover that the dendritic density of
ID, assayed in outside-out patches,
was not markedly greater than on the soma and, indeed, tended to
decrease with distance from the soma (Fig. 7B). The
discrepancy might be explained by the absence from the dendrites of an
additional subunit(s) that must coassemble with Kv1.2 to produce a
functional channel in these cells (cf. Grissmer et al., 1994).
Alternatively, developmental factors may be important: Sheng et al.
(1994) used adult rats, whereas our experiments used 14- to 21-d-old
animals. Upregulation of Kv1 mRNA in rat brain beyond the first 2 postnatal weeks has been reported (Swanson et al., 1990).
The voltage-dependence of activation of -DTX-sensitive current is
similar to that of the delayed rectifier current,
IK, which is the dominant slow
K+ current in these neurons (Fig.
5D) (Bekkers, 2000a). Both start to activate at around 60
mV, negative to the threshold for AP firing (approximately 54 mV in
control conditions) (Fig. 3B). The activation of
ID at subthreshold potentials can also
be inferred from the observation that the threshold for the first AP in
a train is significantly hyperpolarized by -DTX (Fig.
3B); that is, -DTX has an effect on APs before the
membrane potential has depolarized for the first time above AP
threshold. After activation of ID, its
slow inactivation kinetics ensures that it never fully turns off during
the train, suppressing excitability and lengthening the interspike
interval (Erisir et al., 1999). Blockade of
ID thus enhances excitability and
firing frequency. Interestingly, this enhanced excitability is
manifested differently in regular spiking pyramidal neurons in
subiculum and CA1 of rat hippocampus: blockade of
ID converts these neurons into a
distinct burst-firing mode (Staff et al., 2000), in contrast to our
results for the majority of large layer 5 pyramidal neurons, which
showed only an increased frequency of regular firing (Fig.
2C).
Despite the similarity of ID to
IK, blockade of
ID (with -DTX) and blockade of a
similar amount of IK (with TEA) had
dissimilar effects on excitability. One difference was that TEA
significantly broadened the APs, whereas -DTX did not. This is most
easily explained by the nonspecific (for present purposes) inhibitory effect of TEA on the fast calcium-activated
K+ current,
IC. This inhibition is well known to
produce spike broadening (Storm, 1987; Schwindt et al., 1988). The
other difference was that TEA had no effect on the threshold for AP
firing, in contrast to -DTX (Fig. 6C; compare Fig.
3B). It is possible that subtle differences in the
voltage-dependent properties of ID and
IK can explain this. For example, the
V1/2 for peak activation of
ID is slightly hyperpolarized from
that for IK [22.4 mV, cf. 17.0 mV
(Fig. 5D and Bekkers, 2000a)]. This will emphasize the
effect of ID near spike threshold,
which is a voltage range in which small conductance changes can have
large effects on the trajectory of the membrane potential.
Activation kinetics may also be an important difference between
ID and
IK. In other cell types,
ID has been reported to activate more
rapidly than IK (Wu and Barish, 1992;
Bossu et al., 1996; Locke and Nerbonne, 1997). However, estimates of
the activation kinetics of subtraction-isolated currents are very
sensitive to rundown, and none of the above studies used bracketing, as
we did, to minimize these errors. In our data,
ID exhibited both slow (Figs.
4A, 7A) and fast [followed by slow (Fig.
5A)] activation kinetics. This variability may be intrinsic
to ID, or it may reflect difficulties
with the pharmacological subtraction method. If the activation of
ID is indeed faster than that of
IK, this may allow ID to make a larger contribution
during the brief depolarization produced by an AP.
Functional differences between IK and
ID may also stem from their
contrasting dendritic distributions. Whereas
IK is uniformly distributed (Bekkers,
2000b), ID tends to be concentrated
near the soma (Fig. 7B) (although it remains possible that
ID density in the basal dendrites,
where we could not measure, is high). This may adapt
ID to the role of modulating spike
threshold in the axon. Such a role could be further enriched if
ID is subject to neuromodulation.
Indeed, activation of metabotropic glutamate receptors has recently
been reported to accelerate the inactivation of
ID in CA1 pyramidal neurons,
increasing neuronal excitability (Wu and Barish, 1999).
In summary, we have shown that ID is
present, although at low density, in large layer 5 pyramidal neurons of
the rat neocortex. The strategic location of
ID near the site of AP initiation in the axon ensures that it will have a powerful effect on the regulation of neuronal excitability and thus on the coupling between synaptic input and AP output.
| |
FOOTNOTES |
Received April 5, 2001; revised June 22, 2001; accepted June 26, 2001.
The work was supported by recurrent funding from the John Curtin School
of Medical Research. We thank Drs. Pankaj Sah and Greg Stuart for
comments on this manuscript.
Correspondence should be addressed to Dr. J. M. Bekkers, Division
of Neuroscience, John Curtin School of Medical Research, GPO Box 334, Canberra, ACT 2601, Australia. E-mail:
John.Bekkers{at}anu.edu.au.
A. J. Delaney's present address: Vollum Institute L474, Oregon
Health Sciences University, 3181 S.W. Sam Jackson Park Road, Portland,
OR 97201.
| |
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Oscillatory and Intrinsic Membrane Properties of Guinea Pig Nucleus Prepositus Hypoglossi Neurons In Vitro
J Neurophysiol,
July 1, 2006;
96(1):
175 - 196.
[Abstract]
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D. Guan, J. C. F. Lee, T. Tkatch, D. J. Surmeier, W. E. Armstrong, and R. C. Foehring
Expression and biophysical properties of Kv1 channels in supragranular neocortical pyramidal neurones
J. Physiol.,
March 1, 2006;
571(2):
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[Abstract]
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N. Keren, N. Peled, and A. Korngreen
Constraining Compartmental Models Using Multiple Voltage Recordings and Genetic Algorithms
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December 1, 2005;
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[Abstract]
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B. D. Burrell and C. L. Sahley
Serotonin Mediates Learning-Induced Potentiation of Excitability
J Neurophysiol,
December 1, 2005;
94(6):
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[Abstract]
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S. Khavandgar, J. T. Walter, K. Sageser, and K. Khodakhah
Kv1 channels selectively prevent dendritic hyperexcitability in rat Purkinje cells
J. Physiol.,
December 1, 2005;
569(2):
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[Abstract]
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B. L. Moss, A. D. Fuller, C. L. Sahley, and B. D. Burrell
Serotonin Modulates Axo-Axonal Coupling Between Neurons Critical for Learning in the Leech
J Neurophysiol,
October 1, 2005;
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[Abstract]
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L. L. Scott, P. J. Mathews, and N. L. Golding
Posthearing Developmental Refinement of Temporal Processing in Principal Neurons of the Medial Superior Olive
J. Neurosci.,
August 31, 2005;
25(35):
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[Abstract]
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L. Yan, J. Herrington, E. Goldberg, P. M. Dulski, R. M. Bugianesi, R. S. Slaughter, P. Banerjee, R. M. Brochu, B. T. Priest, G. J. Kaczorowski, et al.
Stichodactyla helianthus Peptide, a Pharmacological Tool for Studying Kv3.2 Channels
Mol. Pharmacol.,
May 1, 2005;
67(5):
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[Abstract]
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[PDF]
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S. Kroner, J. A. Rosenkranz, A. A. Grace, and G. Barrionuevo
Dopamine Modulates Excitability of Basolateral Amygdala Neurons In Vitro
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March 1, 2005;
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[Abstract]
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B. E. McKay, M. L. Molineux, W. H. Mehaffey, and R. W. Turner
Kv1 K+ Channels Control Purkinje Cell Output to Facilitate Postsynaptic Rebound Discharge in Deep Cerebellar Neurons
J. Neurosci.,
February 9, 2005;
25(6):
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[Abstract]
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F. R. Fernandez, W. H. Mehaffey, M. L. Molineux, and R. W. Turner
High-Threshold K+ Current Increases Gain by Offsetting a Frequency-Dependent Increase in Low-Threshold K+ Current
J. Neurosci.,
January 12, 2005;
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363 - 371.
[Abstract]
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X. Chen and D. Johnston
Properties of single voltage-dependent K+ channels in dendrites of CA1 pyramidal neurones of rat hippocampus
J. Physiol.,
August 15, 2004;
559(1):
187 - 203.
[Abstract]
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R. H. Cudmore and G. G. Turrigiano
Long-Term Potentiation of Intrinsic Excitability in LV Visual Cortical Neurons
J Neurophysiol,
July 1, 2004;
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341 - 348.
[Abstract]
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E. S. L. Faber and P. Sah
Opioids Inhibit Lateral Amygdala Pyramidal Neurons by Enhancing a Dendritic Potassium Current
J. Neurosci.,
March 24, 2004;
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[Abstract]
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W. Shen, S. Hernandez-Lopez, T. Tkatch, J. E. Held, and D. J. Surmeier
Kv1.2-Containing K+ Channels Regulate Subthreshold Excitability of Striatal Medium Spiny Neurons
J Neurophysiol,
March 1, 2004;
91(3):
1337 - 1349.
[Abstract]
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E S L. Faber and P. Sah
Ca2+-activated K+ (BK) channel inactivation contributes to spike broadening during repetitive firing in the rat lateral amygdala
J. Physiol.,
October 15, 2003;
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[Abstract]
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F. W. Hopf, M. G. Cascini, A. S. Gordon, I. Diamond, and A. Bonci
Cooperative Activation of Dopamine D1 and D2 Receptors Increases Spike Firing of Nucleus Accumbens Neurons via G-Protein {beta}{gamma} Subunits
J. Neurosci.,
June 15, 2003;
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[Abstract]
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Y. Dong and F. J. White
Dopamine D1-Class Receptors Selectively Modulate a Slowly Inactivating Potassium Current in Rat Medial Prefrontal Cortex Pyramidal Neurons
J. Neurosci.,
April 1, 2003;
23(7):
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[Abstract]
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H. M Brew, J. L Hallows, and B. L Tempel
Hyperexcitability and reduced low threshold potassium currents in auditory neurons of mice lacking the channel subunit Kv1.1
J. Physiol.,
April 1, 2003;
548(1):
1 - 20.
[Abstract]
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C.-C. Lien and P. Jonas
Kv3 Potassium Conductance is Necessary and Kinetically Optimized for High-Frequency Action Potential Generation in Hippocampal Interneurons
J. Neurosci.,
March 15, 2003;
23(6):
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[Abstract]
[Full Text]
[PDF]
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D. V Vasilyev and M. E Barish
Regulation of an inactivating potassium current (IA) by the extracellular matrix protein vitronectin in embryonic mouse hippocampal neurones
J. Physiol.,
March 15, 2003;
547(3):
859 - 871.
[Abstract]
[Full Text]
[PDF]
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P. D. Dodson, M. C. Barker, and I. D. Forsythe
Two Heteromeric Kv1 Potassium Channels Differentially Regulate Action Potential Firing
J. Neurosci.,
August 15, 2002;
22(16):
6953 - 6961.
[Abstract]
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Z.-L. Mo, C. L Adamson, and R. L Davis
Dendrotoxin-sensitive K+ currents contribute to accommodation in murine spiral ganglion neurons
J. Physiol.,
August 1, 2002;
542(3):
763 - 778.
[Abstract]
[Full Text]
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C.-C. Lien, M. Martina, J. H Schultz, H. Ehmke, and P. Jonas
Gating, modulation and subunit composition of voltage-gated K+ channels in dendritic inhibitory interneurones of rat hippocampus
J. Physiol.,
January 15, 2002;
538(2):
405 - 419.
[Abstract]
[Full Text]
[PDF]
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E. K. Lambe and G. K. Aghajanian
The Role of Kv1.2-Containing Potassium Channels in Serotonin-Induced Glutamate Release from Thalamocortical Terminals in Rat Frontal Cortex
J. Neurosci.,
December 15, 2001;
21(24):
9955 - 9963.
[Abstract]
[Full Text]
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TOP
ABSTRACT
INTRODUCTION
