Function of Specific K+ Channels in Sustained
High-Frequency Firing of Fast-Spiking Neocortical Interneurons
A.
Erisir,1
D.
Lau,2
B.
Rudy,2 and
C. S.
Leonard1
1Department of Physiology, New York Medical
College, Valhalla 10595; and 2Department of
Physiology and Neuroscience and Department of Biochemistry, New York
University School of Medicine, New York, New York 10016
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ABSTRACT |
Erisir, A.,
D. Lau,
B. Rudy, and
C. S. Leonard.
Function of Specific K+ Channels in Sustained
High-Frequency Firing of Fast-Spiking Neocortical Interneurons.
J. Neurophysiol. 82: 2476-2489, 1999.
Fast-spiking GABAergic interneurons of the neocortex and hippocampus
fire high-frequency trains of brief action potentials with little
spike-frequency adaptation. How these striking properties arise is
unclear, although recent evidence suggests K+ channels
containing Kv3.1-Kv3.2 proteins play an important role. We investigated
the role of these channels in the firing properties of fast-spiking
neocortical interneurons from mouse somatosensory cortex using a
pharmacological and modeling approach. Low tetraethylammonium (TEA)
concentrations (
1 mM), which block only a few known K+
channels including Kv3.1-Kv3.2, profoundly impaired action potential repolarization and high-frequency firing. Analysis of the spike trains
evoked by steady depolarization revealed that, although TEA had little
effect on the initial firing rate, it strongly reduced firing frequency
later in the trains. These effects appeared to be specific to Kv3.1 and
Kv3.2 channels, because blockade of dendrotoxin-sensitive Kv1 channels
and BK Ca2+-activated K+ channels, which also
have high TEA sensitivity, produced opposite or no effects.
Voltage-clamp experiments confirmed the presence of a Kv3.1-Kv3.2-like
current in fast-spiking neurons, but not in other interneurons.
Analysis of spike shape changes during the spike trains suggested that
Na+ channel inactivation plays a significant role in the
firing-rate slowdown produced by TEA, a conclusion that was supported
by computer simulations. These findings indicate that the unique
properties of Kv3.1-Kv3.2 channels enable sustained high-frequency
firing by facilitating the recovery of Na+ channel
inactivation and by minimizing the duration of the
afterhyperpolarization in neocortical interneurons.
 |
INTRODUCTION |
Inhibitory GABAergic interneurons play essential
roles in cortical function. They are implicated in the formation and
reorganization of receptive fields, in the refinement of cortical
connections during development, and in the generation and spread of
cortical rhythmical activity (Chagnac-Amitai and Connors
1989
; Freund and Buzsaki 1996
; Gilbert
1993
; Gray 1994
; Jacobs and Donoghue
1991
; Jones 1993
; Martin 1988
;
Sillito 1984
; Singer and Gray 1995
;
Steriade 1997
; Traub et al. 1996
;
Vidyasagar et al. 1996
). Moreover, their dysfunction may
be responsible for promoting seizure activity (Hosford
1995
; Jefferys and Whittington 1996
).
Understanding the mechanisms underlying the electrical activity of
cortical GABAergic interneurons is therefore critical for understanding
both the normal functioning and pathophysiological processes of the
cerebral cortex.
Cortical GABAergic interneurons display diverse intrinsic
electrophysiological properties, morphology, connectivity, and
neurochemical features (Connors and Gutnick 1990
;
Huettner and Baughman 1988
; Kawaguchi and Kubota
1998
; Keller 1995
). The interneurons that contain the Ca2+ binding protein parvalbumin
constitute more than half of the cortical interneurons (Kubota
and Kawaguchi 1994
), and a strong correlation between
parvalbumin expression and the "fast-spiking" (FS) phenotype has
been established in rat neocortex and hippocampus by immunocytochemical
staining and by single-cell RT-PCR (Cauli et al. 1997
;
Freund and Buzsaki 1996
; Kawaguchi 1995
;
Kawaguchi and Kubota 1997
). These neurons are
characterized, in vitro and in vivo, by a striking ability to fire
sustained high-frequency trains of brief duration action potentials
with little spike-frequency adaptation in response to sustained
depolarizing inputs (Azouz et al. 1997
; Baranyi
et al. 1993
; Connors and Gutnick 1990
;
McCormick et al. 1985
; Mountcastle et al.
1969
). These distinctive firing properties suggest that FS
neurons may express distinct types of ion channels compared with other
interneurons and pyramidal cells (Huettner and Baughman
1988
), although a provocative analysis of realistic neuronal
models suggests that the differential expression of ion channels may
not be necessary to account for the diversity of cortical neuron firing
properties (Mainen and Sejnowski 1996
). In a whole cell
patch-clamp study of dissociated cortical neurons, Hamill et al.
(1991)
found that FS neurons had significantly larger K+ currents than pyramidal cells and suggested that a
higher K+ channel density might contribute to the
differences in firing properties. Recently, the findings that the
products of two potassium channel genes, Kv3.1 and Kv3.2, are
prominently expressed in parvalbumin-containing fast-spiking cortical
interneurons have refined this view and focused interest on the
possibility that channels formed from these subunits play a special
role in fast-spiking (Chow et al. 1999
; Du et al.
1996
; Lenz et al. 1994
; Martina et al.
1998
; Massengill et al. 1997
; Moreno et
al. 1995
; Perney et al. 1992
; Sekirnjak et al. 1997
; Weiser et al. 1995
).
Kv3.1 and Kv3.2 channels display unusual properties when expressed in
heterologous expression systems. They are fast-activating delayed
rectifiers that require large membrane depolarizations (above
10 mV)
to produce significant activation and they deactivate very quickly on
repolarization (for review see Rudy et al. 1999
). Their
rates of deactivation are at least 7-10 times faster than those of
other known voltage-gated K+ channels (Coetzee et
al. 1999
), except for Kv1.7, a nonneuronal member of the Kv1
family that deactivates only two to three times slower than Kv3
channels (Kalman et al. 1998
). Based on these properties
and their distribution patterns in CNS neurons, it has been proposed
that Kv3.1 and Kv3.2 channels function in the repolarization of action
potentials of short-duration and in facilitating high-frequency firing
(Du et al. 1996
; Lenz et al. 1994
;
Martina et al. 1998
; Massengill et al.
1997
; Moreno et al. 1995
; Perney et al.
1992
; Perney and Kaczmarek 1997
;
Sekirnjak et al. 1997
; Wang et al. 1998
;
Weiser et al. 1995
). Consistent with this hypothesis, we
and others have shown that low doses of 4-aminopyridine (4-AP), which
block heterologously expressed Kv3.1-Kv3.2 channels, block a similar
current and impair spike repolarization in cortical fast-spiking
interneurons (Du et al. 1996
; Massengill et al.
1997
). In a recent study, Martina et al. (1998)
confirmed the differential expression of Kv3.1 and Kv3.2 transcripts in
fast-spiking hippocampal basket cells and showed that the major
component of the K+ current in these cells is similar to
heterologously expressed Kv3.1 and Kv3.2 currents.
The role that these channels play in repetitive firing is less clear.
In a study of cultured neocortical neurons (Massengill et al.
1997
), 4-AP (0.1 mM) reduced the firing rate of neurons expressing Kv3.1 transcripts, but these cells had very low maximal firing rates (25 spikes/s) compared with those reported for FS neurons
in slices at the same temperature (104 spikes/s) (Cauli et al.
1997
), leaving open the role of Kv3.1-Kv3.2 channels in high-frequency firing. In hippocampal basket cells, application of 4-AP
(0.2 mM) in the presence of Ca2+-channel blockade
interfered with repetitive firing and produced large
spike-afterdepolarizations, at least at the single current strength
reported (Martina et al. 1998
). These observations,
along with a recent report showing that tetraethylammonium (TEA),
presumably by blocking Kv3 channels, affected the ability of auditory
neurons to respond to high-frequency stimuli (Wang et al.
1998
), are consistent with a general role in high-frequency
firing. However, a systematic study of the effects of Kv3.1-Kv3.2
channel blockade on the firing patterns of cortical interneurons, which
may lead to an understanding of the mechanisms by which these channels
regulate fast-spiking, is still lacking.
We have therefore utilized a pharmacological and computer modeling
approach to investigate the specific roles played by a Kv3.1-Kv3.2-like
current in the generation of the FS phenotype in neocortical
interneurons. The data showed that a Kv3.1-Kv3.2-like current is
necessary to maintain sustained, but not early high-frequency firing.
Analysis of the spike shape changes occurring during a train of action
potentials suggested that Kv3.1-Kv3.2 currents facilitate sustained
high-frequency firing by limiting the accumulation of Na+
channel inactivation, an hypothesis that was supported by computer modeling.
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METHODS |
Brain slices were prepared from 14- to 32-day-old C57/Bl6 mice
(Taconic Farms, Germantown, NY). All procedures complied with National
Institutes of Health guidelines for ethical use of animals. Following
the induction of deep anesthesia with Halothane, the mice were
decapitated and the brains were rapidly removed into an ice-cold,
oxygenated Ringer solution that contained (in mM) 121 NaCl, 2.5 KCl,
1.25 NaH2PO4, 2 CaCl2, 1 MgCl, 26 NaHCO3, 20 dextrose, and 4.2 lactic acid. The cerebrum was
blocked at a coronal or parasagital plane, and vibratome-sectioned into
250- to 300-µm-thick sections. Somatosensory cortex slices were
incubated at 35°C for 20 min in oxygenated Ringer solution and then
were stored at room temperature, until they were transferred to a
submerged recording chamber, which was perfused at 3-5 ml/min with the
same Ringer solution at room temperature. Drugs were applied by
superfusion. Dendrotoxin I and K (DTX), and tetrodotoxin (TTX) were
purchased from Alomone Labs (Jerusalem, Israel), TEA from Research
Biochemicals, and charybdotoxin (CTX) from Sigma (St. Louis, MO);
iberiotoxin (IbTX) was a gift from Dr. Maria L. Garcia (Merck Laboratories).
Neurons were visualized at ×160-200 magnification with near infrared
light (>775 nm) transillumination, using a nuvicon tube camera
(VE-1000, Dage, Michigan City, IN) and the DIC optics of a fixed-stage
microscope (BX50WI, Olympus, Melville, NY). Cells were selected for
whole cell recording from mainly layer II/III on the basis of a
nonpyramidal shape and multipolar dendrites. Neurons were recorded in
current-clamp mode for the analysis of action potential shape and
repetitive firing properties, using an electronic bridge amplifier
(Axoclamp 2A, Axon Instruments, Foster City, CA) with the output filter
set at 10 kHz. Voltage-clamp measurement of ionic currents was obtained
from outside-out macro-patches pulled from neurons that were first
characterized under current-clamp conditions using an Axopatch 200A
amplifier (Axon Instruments). Macro-patches were obtained by slowly
backing the pipette from the cell surface along the long axis of the
pipette while monitoring the uncompensated capacitative transients and
cell input resistance (Keros and McBain 1997
). Once
resealing occurred, the pipette tip was further withdrawn to just above
the surface of the slice. Ringer containing TTX (1 µM) was superfused
to block voltage-gated sodium currents. Recorded currents were filtered
at 5 kHz with a four-pole Bessel filter. Pipettes fabricated from
Corning 7052 glass were used directly for current-clamp recordings and
were coated with silicone elastomer (Sylgard 184, Dow, Midland, MI) and
fire-polished immediately before use for macro-patch voltage-clamp recordings. Current-clamp studies were conducted using a pipette solution containing (in mM) 144 K-gluconate, 0.2 EGTA, 3 MgCl2, 10 HEPES, 0.3 NaGTP, and 4 Na2ATP.
Voltage-clamp studies were conducted with a pipette solution containing
(in mM) 100 K-gluconate, 10 K4-bis-(o-aminophenoxy)-N,N,N',N'-tetraacetic
acid (BAPTA), 5 KCl, 3 MgCl2, 10 HEPES, 0.3 NaGTP, and 4 Na2ATP to limit any contribution to the current from
Ca2+-activated K+ currents that might have been
present. Biocytin (0.1%; Sigma) was added to the pipette solution just
before use. Potentials were recorded with respect to a Ag/AgCl
reference electrode located near the outflow of the chamber. Liquid
junction potentials were estimated to be
13 and
11 mV for each
internal solution, and because the difference was small, the values of
membrane potentials were not corrected.
Membrane currents and voltages were controlled and recorded with a
computer running PCLAMP7 software (Axon Instruments). Current and
voltage signals were sampled at 20 kHz, and analysis of these waveforms
was performed using Igor Pro (Wavemetrics) software. Action potential
shape parameters were measured from action potentials evoked by
just-suprathreshold 200-ms current steps from a membrane potential near
70 mV. Spike amplitude was measured as the difference between the
peak and the threshold of the action potential. Spike threshold was
determined by finding the potential at which the second derivative of
the voltage waveform exceeded 3 times its standard deviation in the
period preceding spike onset. The afterhyperpolarization (AHP) was
measured as the difference between the spike threshold and voltage
minimum following the action potential peak. Maximum rates of rise and
decay of the action potential were computed from the maximum and
minimum of the smoothed first derivative of the voltage waveform. Spike
width was measured at half the spike amplitude. Spike times were
measured by determining the time at which the rising phase of the
action potential crossed a fixed threshold potential. Instantaneous
frequency (1/interspike interval) was computed from trains of action
potentials evoked by 600 ms duration pulses for the 1st, 2nd, 4th, and
the last intervals. Steady-state firing rate was computed as the
average of instantaneous frequency for the last five intervals of a
train. Instantaneous frequency for the 1st, 2nd, 4th, and the last
intervals along with steady-state firing rate were plotted as a
function of the injected current strength to construct rate-frequency
curves. Current strength was increased at 50-pA increments until spike failure occurred within the 600-ms duration pulse. The maximum steady-state firing rate was computed by averaging the steady-state firing rates from trains evoked by the three current strengths before
that which produced spike failure. Spike frequency adaptation was
measured during the first 200 ms of such trains [A200 = (Freq1st
Freq200
ms)/Freq1st] and the reported A200 was
an average of the A200 values measured from the traces used
to compute the maximum steady-state firing. Statistical analyses were
conducted using the program DataDesk 6 (Data Descriptions, Ithaca, NY).
Results are reported as means ± SE.
For histochemical characterization of recorded cells, slices were fixed
in 4% paraformaldehyde for 2 days at 4°C and then stored
refrigerated in 0.01 M phosphate-buffered saline (PBS) containing 30%
sucrose and 0.05% sodium azide. A freezing microtome was used to
resection slices at 50- to 100-µm thickness before incubation in a
monoclonal parvalbumin antibody (Sigma; 1:400 dilution in PB containing
1% bovine serum albumin, 0.75% Triton at room temperature for 2 days), followed by FITC-conjugated secondary antibody (anti-mouse, 1:50
dilution in PBS; Fisher Chemicals) to visualize Parvalbumin, and by
Texas Red conjugated avidin (1:50 in PBS containing 0.7% Triton) to
visualize the biocytin-filled cell. Sections were then mounted on
gelatin subbed slides and examined on a confocal microscope.
A computer model of an FS cell was implemented using Nodus 3.2 software
running on a Power Macintosh computer. The neuron consisted of an
isopotential sphere with 16 µm diam, a membrane capacitance
(Cm) of 1.0 µF/cm2, a membrane
resistance (Rm) of 10 k
· cm2, a cytoplasmic resistance
(Ri) of 100
· cm, and a resting
membrane potential of
70 mV. The leakage conductance was chosen to be 10 nS with a reversal potential of
70 mV to approximate the average input resistance of recorded FS neurons. The voltage dependence of each
current was modeled using a Hodgkin-Huxley formulation for a transient
Na+ current, a Kv3.1-Kv3.2, current and a Kv1.3 current.
The Na+ current model was derived from currents recorded
from hippocampal basket cells (Martina and Jonas 1997
)
and neocortical interneurons (Huguenard et al. 1988
). A
two-state Kv3.1-Kv3.2 model was derived from fits of Kv3.1 currents
expressed in HEK293 cells (Rudy and Leonard, unpublished data), and the
Kv1.3 model was derived from "n"-type currents measured in human
T-lymphocytes (Cahalan et al. 1985
). The maximum value
of gNa was 900 nS, of
gKv3.1-Kv3.2 was 1,800 nS, and of
gKv1.3 was 1.8 nS. The Na+
conductance was proportional to m3h, the Kv3.1-Kv3.2
conductance was proportional to n2, and the Kv1.3
conductance was proportional to n4. The rate constants for
the Na current were
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The rate constants for the Kv3.1-Kv3.2 current were
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The rate constants for the Kv1.3 current were
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RESULTS |
Interneurons in mouse somatosensory cortex have diverse firing
properties
Based on responses to current injection, the recorded nonpyramidal
cells were classified into two groups: fast-spiking and regular-spiking-nonpyramidal (RSNP) cells, the latter of which also
included cells showing a low-threshold response similar to that
described in rat frontal cortex (Kawaguchi and Kubota
1997
, 1998
). FS neurons (Fig.
1A; n = 19)
had an average resting potential of
60.6 ± 1.9 (SE) mV and an
average input resistance of 110.4 ± 11 M
. They had
short-duration action potentials (0.60 ± 0.04 ms) and large AHPs
(
16.4 ± 1.2 mV). In response to sustained current injection, FS
cells began firing repetitively with abrupt onset and were able to fire
at high frequencies with relatively little spike frequency adaptation
(A200= 0.34 ± 0.02) that occurred mainly
over the first few intervals (Fig. 1, B and C).
The instantaneous firing rate increased monotonically with current
strength (Fig. 1D), and in some cases, this relation reached
a clear plateau at current strengths below those that caused spike
failure. The average maximum steady-state firing rate for FS neurons
was 123.1 ± 11 Hz.

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Fig. 1.
Properties of fast-spiking (FS) neurons recorded from layer II/III
somatosensory cortex of mouse. A: FS neurons had brief
duration action potentials and showed little variation in shape between
the 1st and 2nd spikes. Action potentials were evoked by
just-suprathreshold currents (400 and 500 pA). Traces are superimposed
and aligned on the 1st spike. B: repetitive firing of FS
neurons for 3 current steps of increasing magnitude (300-500 pA).
Repetitive firing had an abrupt onset in FS cells. In this case, the FS
neuron fired only a single spike in response to a 300-pA depolarization
but then fired at ~50 spikes/s in response to a 400-pA current step.
Spike amplitude decreased slightly following the 1st spike.
C: instantaneous firing frequency was plotted as a
function of time from onset of the current pulse for selected current
strengths (solid lines). FS neurons showed only a small amount of spike
frequency adaptation. Dashed line indicates the frequency-latency curve
for the minimum current strength that produced spike failure during the
pulse. D: instantaneous frequency (1/interspike
interval) was plotted as a function of injected current. The
frequencies of the 1st, 2nd, and 4th intervals and the steady-state
frequency (SS) were monotonically proportional to the current strength.
The amount of adaptation increased with current intensity, and the
relation between firing rate and current showed evidence of reaching a
plateau at the highest frequencies. E1 and
E2: biocytin-labeled FS neurons (E1) were
immunoreactive for parvalbumin (E2). Data from
A-E are all from the same FS neuron. Scale bar = 40 µm.
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It has been shown in quantitative studies both in rat (Sekirnjak
et al. 1997
; Weiser et al. 1995
) and in mouse
(Chow et al. 1998
, 1999
) that all
parvalbumin-containing neurons in the layers I-IV of the neocortex
express Kv3.1 proteins, whereas all PV-containing neurons in layers
V-VI coexpress both Kv3.1 and Kv3.2 proteins, probably in heteromeric
complexes. We therefore used post hoc immunohistochemistry to
parvalbumin (Kawaguchi and Kubota 1998
) to confirm that
the cells having fast-spiking characteristics are Kv3.1-Kv3.2
containing cells (Fig. 1, E1 and E2).
These findings also confirmed the strong correlation between
parvalbumin expression and the FS phenotype as previously established
in rat neocortex (Kawaguchi and Kubota 1998
).
In comparison to FS cells, RSNP neurons (Fig.
2; n = 37) had
significantly longer duration action potentials (1.71 ± 0.062 ms;
P < 0.001) and smaller amplitude AHPs (0.1 ± 0.7 mV; P < 0.001), showed much greater spike
frequency adaptation (0.75 ± 0.01; P < 0.001), and had a much lower maximum frequency of firing (24.8 ± 1.1; P < 0.001). RSNP neurons also showed
substantial spike broadening (27.8 ± 4.5%; P < 0.001) between the first and second action potentials of a train
that was greater than that observed for FS neurons (4 ± 0.9%;
P < 0.001). Biocytin labeling of some of these
RSNP cells (n = 12) verified that they were
nonpyramidal in morphology (data not shown), but none were positive for
parvalbumin.

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Fig. 2.
Properties of layer II/III regular-spiking-nonpyramidal (RSNP) neurons
compared with FS neurons. A: RSNP neurons had broader 1st
action potentials and showed greater spike broadening between the 1st
and 2nd spike than did FS neurons (compare with Fig.
1A). B: just-subthreshold current
produced a "low-threshold" response for this RSNP neuron.
Suprathreshold current produced repetitive firing at comparatively
lower frequencies and with substantially more spike frequency
adaptation than observed for FS neurons (compare to Fig.
1B). C: instantaneous firing frequency
vs. time from onset of current pulse ( ) revealed a large amount of
frequency adaptation and a lower minimum current producing spike
failure (- - -). D: the relation between instantaneous
frequency and current plateaued for all intervals at lower frequencies
than those observed for FS neurons. Data from A-D are
from the same neuron. E: summary of spike and firing
parameters for RSNP ( ) and FS ( ) neurons. The
afterhyperpolarization (AHP) amplitude of the 1st spike, width of 2nd
action potential (2nd width), and the fraction of adaptation at 200 ms
(A200) are plotted against the maximum steady-state firing
frequency (Firing Rate). These data revealed that neurons were
naturally grouped into 2 populations. FS and RS indicate the loci of
the neurons illustrated in Fig. 1 and Fig. 2, respectively.
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A scatter-plot comparison of spike-shape parameters and maximal average
firing frequency revealed a bimodal distribution for all cells with
little overlap (Fig. 2E). Thus FS and RSNP neurons were
reliably distinguished on the basis of firing frequency, adaptation,
and spike shape parameters. Properties of FS and RSNP cells are
summarized in Table 1.
Low concentrations of TEA produce striking changes in the action
potential of FS neurons
To investigate the effects of blocking Kv3.1-Kv3.2 channels,
we exploited their relatively high sensitivity to TEA (extracellular application 1 mM TEA blocks >80% of Kv3.1-Kv3.2 channels expressed in
mammalian heterologous expression systems) (Chandy and Gutman 1995
; Grissmer et al. 1994
;
Hernández-Pineda et al. 1999
; Rudy et al.
1999
; Vega-Saenz de Miera et al. 1994
). Bath
application of 1 mM TEA produced a nearly complete and reversible
elimination of the AHP (Fig.
3A) in all FS neurons tested
(
12.5 ± 1.6 vs. 0.8 ± 1.9 mV, P < 0.001;
n = 7). TEA also increased the action potential width
from 0.64 ± 0.04 to 1.16 ± 0.08 ms (P < 0.001), suggesting that the current(s) generating the AHP play an
important role in repolarizing the action potential. This was supported by the observation that the maximum spike repolarization
rate of the first spike during a train of action potentials generated by a just suprathreshold stimulus was reduced from
148.7 ± 28.6 to
62.7 ± 10.3 mV/ms (P < 0.001), whereas the
maximum spike depolarization rate of the first spike
remained unaffected (295.7 ± 33.6 vs. 295.5 ± 43.6 mV/ms;
P = 0.995). These findings indicate that one or more
K+ currents with a high sensitivity to TEA play
an important role in repolarizing the action potential and generating
the AHP of FS neurons.

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Fig. 3.
Tetraethylammonium (TEA; 1 mM) reversibly inhibits the AHP and slows
repetitive firing of FS neurons. A: action potential of
FS neuron before (control), during (TEA), and after (Wash) the
application of 1 mM TEA. Low concentrations of TEA broadened the action
potential, decreased the maximum rate of repolarization, and blocked
the AHP. B1: TEA reversibly slowed the firing frequency
evoked by a constant current pulse. Notice the smaller AHP achieved
during the interspike interval. B2: 1 mM TEA also
increased the susceptibility to spike failure. A current pulse of 1,350 pA evoked a train of action potentials under control conditions
(left). Spike amplitude decreased but achieved a stable
amplitude. The same current pulse in the presence of 1 mM TEA resulted
in a more rapid reduction of spike amplitude and failure
(right). C: instantaneous frequency vs.
current for the frequencies measured in the 1st interval (1st;
and ) and the steady-state (SS;
and ). Control conditions, and . TEA (1 mM; and )
produced a large reduction in the steady-state firing rate but only
negligible changes in the firing rate measured from the 1st interval.
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TEA impairs the ability of FS neurons to fire high-frequency trains
of action potentials
Rather than increasing the firing rate, as might be
expected from blocking the AHP, TEA reversibly decreased the
steady-state firing rate of FS neurons (Fig. 3B). This
occurred at all current strengths tested, indicating that strong
depolarization could not overcome the effect of channel blockade. The
average maximum steady-state firing rate was reduced (from 104.6 ± 10.8 to 65.6 ± 10 Hz; P < 0.001), and spike
failure occurred at lower current strengths than observed in the
control condition. Interestingly, no systematic reduction in the
instantaneous firing rate of the first interval was observed (Fig.
3C), and in two cases, an initial burst of action potentials
occurred at high current strengths (data not shown). Instead, the TEA
suppression of firing rate developed during the spike train, and only
reached a maximum by ~10 intervals (Fig.
4, top). This resulted in a
large increase in the amount of spike-frequency adaptation after the
application of TEA (from 0.37 to 0.61; P < 0.001).
These results suggest that the processes underlying firing frequency
suppression took time to accumulate. One such process is
Na+ channel inactivation, which was also
implicated by the observation of spike failure at lower current
strengths in the presence of TEA. Consistent with a role for such a
mechanism, we observed a decrease in the maximum depolarization slope
of each action potentials in a train that was much larger in the
presence of TEA and that had a time course that matched the TEA
produced changes in firing frequency (Fig. 4, bottom). These
effects of TEA were not due to a direct action on
Na+ channels because the drug did not affect the
maximum depolarization slope of the first action potential in a spike
train (see previous section) but did decrease it for the second action
potential (249.6 ± 42.9 vs. 209.5 ± 39.2 mV/ms;
n = 7; P < 0.01, measured with just
suprathreshold current pulses).

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Fig. 4.
Firing rate slowdown produced by TEA accumulated during the spike train
in tandem with a decrease in spike depolarization rate. The difference
between the instantaneous frequency for a control train and one evoked
in the presence of 1 mM TEA (1,100-pA current step) is plotted as a
function of interval number (top) and required ~10
intervals to reach a steady level. This slowdown was correlated with a
progressive decrease in the maximum depolarization rate of the action
potential. This decrease in spike depolarization rate was greatly
accentuated in the presence of TEA, suggesting a larger accumulation of
Na+ channel inactivation following blockade of
TEA-sensitive channels.
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Given that Kv3 channels are blocked by TEA with a
Kd ~200 µM, it was expected that
the effects of TEA on action potential shape and repetitive firing
would be dose-dependent over the range of 0.1-1 mM, if they were
mediated by antagonizing Kv3 channels (Fig.
5). The amount of inhibition of the AHP,
the degree of action potential broadening, the amount of inhibition of
spike repolarization rate, and the suppression of steady firing rate,
were all dose-dependent and approached saturation as the TEA
concentration reached 1 mM. Finally, spike failure during repetitive
firing occurred at lower current strengths as the TEA concentration
increased over this range (Fig. 5B2), whereas only minor
effects were observed on the early intervals (Fig. 5B1) at
all concentrations of TEA.

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Fig. 5.
TEA actions on spike shape and repetitive firing occur at low doses.
A: 0.1, 0.5, and 1 mM TEA resulted in progressively
larger effects on the AHP, spike width, and maximum spike
repolarization rate with little effect on the amplitude or rate of rise
of the action potential. B1: for this cell there was no
effect of TEA on the instantaneous frequency measured from the 1st
interspike interval for firing frequencies lower than ~150 spikes/s
(<400 pA). For larger current strengths, TEA produced a slight
increase in the instantaneous frequency that was dose dependent and
reversible. B2: the same range of TEA concentration produced
a progressively larger decrease in the steady-state firing rate.
Similarly, spike failure during repetitive firing occurred at
progressively lower current strengths with increased TEA dose. This is
indicated by the last point of each curve, which marks the greatest
current strength that did not produce failures. These data are
consistent with TEA acting on channels having a
Kd near that reported for Kv3 channels (200 µM).
|
|
Specific K+ channel types mediate the effects of low
TEA concentrations on FS interneurons
Three additional heterologously expressed K+
channels are known that have TEA sensitivities similar to that of Kv3
channels (Kd ~ 200 µM): the large
conductance, Ca2+-activated
K+ (BK) channels containing proteins of the Slo
family (Kd 80-330 µM), and two
voltage-gated K+ channels, Kv1.1
(Kd ~ 300 µM) and KCNQ2 (90%
blocked by 1 mM TEA) (Coetzee et al. 1999
). Because
KCNQ2 subunits form very slowly activating and deactivating channels
(time constants of hundreds of milliseconds to seconds) (Yang et
al. 1998
), which would not be significantly activated during
short action potentials, they were not further examined. Although there
are no known specific toxins for Kv3.1 and Kv3.2 channels, we were able
to examine the contribution of the other two known TEA-sensitive
K+ channels using available specific toxin antagonists.
DTX, which blocks several Kv1 channels including Kv1.1 (Chandy
and Gutman 1995
; Coetzee et al. 1999
;
Robertson et al. 1996
), produced an irreversible
increase in background synaptic activity (Fig.
6A) but had no significant
effects on action potential shape (Fig. 6B). Nevertheless,
DTX produced significant increases in steady-state firing
rate (maximum steady-state firing rate increased from 156.6 ± 14.0 to 172.6 ± 13.5 Hz; P < 0.05). This
increase was apparent at both initial and late intervals and over a
large range of currents (Fig. 6C). Closer examination of the
voltage trajectories between action potentials during steady-state
firing revealed that the AHP recovered more rapidly following the
application of DTX (Fig. 6D). These data are consistent with
an effect of DTX on a K+ current contributing to
the late portion of the AHP and indicate that DTX-sensitive channels do
not significantly contribute to spike repolarization and the early
phase of the AHP.

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Fig. 6.
Dendrotoxin I and K (DTX) and iberiotoxin (IbTX) do not mimic the
effects of TEA on FS neurons. A: DTX-I (100 nM)
application resulted in an increase of spontaneous synaptic potentials.
B: 3 superimposed action potentials before (Control;
solid lines) and after (DTX; dashed lines) DTX-I application indicate
that DTX had no significant effects on spike shape parameters.
C1: response of an FS neuron to 250-pA current in normal
Ringer. C2: response of FS to the same current pulse
after 15 min application of 100 µM DTX-I. In contrast to the effects
of TEA, DTX significantly increased the firing frequency of FS neurons.
C3: instantaneous frequency vs. current pulse amplitude
for the 1st interval ( and ) and the
steady state ( and ) before ( and ) and after DTX ( and ).
DTX significantly increased the firing rate for both the early and late
intervals in the train. D: superimposed truncated action
potentials from an FS cell recorded before ( ) and after (- - -) DTX
application. DTX resulted in a slightly faster decay of the AHP.
E1: IbTX had no significant effect on spike shape or on
repetitive firing (E2) of a representative FS neuron.
|
|
Application of IbTX (10-50 nM, n = 5) or CTX (100 nM,
n = 1), which block BK channels (Coetzee et al.
1999
), did not produce significant changes in the spike shape
or repetitive firing properties of FS neurons (Fig. 6E).
This finding was further supported by our observation that blocking
Ca2+ channels with bath application of both
cadmium (500 µM) and nickel (500 µM) produced only minor changes in
the AHP or in the maximum steady-state firing rate (n = 2; data not shown). The changes in spike shape and repetitive firing
parameters produced by TEA, DTX, and IbTX/CTX are summarized in Fig.
7.

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Fig. 7.
Actions of TEA (1 mM), DTX, and IbTX/charybdotoxin (CTX) are summarized
as percent change from control. A: none of the agents
had significant effects on spike amplitude (Spike Amp.) or on the
maximum rate of rise of the 1st action potential (Max. Rising Slope).
TEA (1 mM) significantly slowed the maximum rate of spike
repolarization (Max. Repol. Slope). Neither DTX nor IbTX/CTX had
significant effects on the Max Repol. Slope. TEA significantly reduced
the amplitude of the AHP (AHP Amp.). Neither DTX nor IbTX/CTX had
significant effects on the AHP amplitude. TEA significantly increased
the width of the action potential (Spike Width). Neither DTX nor
IbTX/CTX had significant effects on the Spike Width. TEA significantly
slowed the maximum average steady-state firing rate (Max. Freq.). DTX
significantly increased the Max. Freq., whereas IbTX/CTX had no
significant effect. TEA significantly increased the amount of
spike-frequency adaptation measured at 200 ms (A200).
Neither DTX nor IbTX/CTX had a significant effect.
*P < 0.01, **P < 0.001, ***P < 0.0001.
|
|
Low concentrations of TEA block a Kv3.1-Kv3.2-like current in FS
neurons but not in RSNP neuron
To confirm that Kv3.1-Kv3.2-like currents were present in
neocortical FS neurons and to determine their contribution to the total
somatic K+ current, membrane currents were
recorded from outside-out macropatches pulled from the somata of
physiologically identified FS neurons. The use of outside-out
macropatches was imperative because the very large whole cell currents
recorded from these neurons precluded adequate voltage control and
temporal resolution. Macropatches were also pulled from RSNP neurons
for comparison (Fig. 8). Patches obtained
from FS neurons had significantly larger outward currents (534.6 ± 140.5 pA steady state, n = 5) than those from RSNP
neurons (182.7 ± 64.2 pA steady state, n = 5;
P < 0.05). TEA (1 mM) blocked the majority of the
current (69.3 ± 8.4%, n = 4) from FS neurons but
only a smaller portion of the current from RSNP neurons (23.3 ± 9.8%, n = 4; P < 0.01). Moreover, the
tail currents measured at
40 mV from FS neurons decayed much more
rapidly than those from RSNP neurons (Fig. 8, A2 and
B2). To examine the voltage dependence of the TEA-sensitive
component, current-voltage (I-V) curves were constructed
from subtraction currents (Fig. 8, A3 and B3). In
FS neurons, the resulting TEA-sensitive current showed significant
activation at potentials more positive than
20 mV, whereas the
current from an RSNP neuron activated at more negative potentials (Fig.
8C). The tail currents of the TEA-sensitive components were
also very different (Fig. 8D): the current from the FS
neuron deactivated as a single fast exponential (
= 5.9 ms),
which compared well to that of Kv3.1 (
~ 3 ms) and Kv3.2
(
~ 6 ms) channels measured in heterologous expression
systems (Grissmer et al. 1994
; Hernández-Pineda et al. 1999
). In contrast, a
double exponential function with longer time constants was necessary to
fit the TEA-sensitive current from the RSNP neuron. Thus the
TEA-sensitive currents obtained from the somatic membrane of FS neurons
behaved like Kv3.1-Kv3.2 currents, whereas those from layer II/III RSNP
neurons, which do not express Kv3.1-Kv3.2 subunits, did not. These data confirm the conclusions from studies in other neurons showing that
native Kv3.1 and Kv3.2 channels have properties remarkably similar to
those in heterologous expression systems (Du et al. 1996
; Hernández-Pineda et al. 1999
;
Martina et al. 1998
; Rudy et al. 1999
;
Wang et al. 1998
) and suggest that factors such as associated subunits or cell-specific postranslational modifications do
not significantly change the electrophysiological properties of native
neuronal channels containing Kv3 proteins.

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Fig. 8.
Outside-out macro-patches from the somata of FS neurons had a large
Kv3.1-Kv3.2-like current, whereas those from RSNP neurons did not.
A1: currents from an FS neuron in control Ringer ( )
and after the addition of 1 mM TEA (- - -). Voltage jumps from 70
to +40 mV resulted in a large outward current that was largely blocked
by 1 mM TEA. B1: results from similar experiments
performed on macro-patches from RSNP neurons indicated, as shown in
this example, that RSNP neurons had smaller outward currents and that a
smaller fraction of that outward current was blocked by 1 mM TEA.
A2 and B2: the time course of tail
currents from FS (A2) and RSNP (B2)
macro-patches were measured in response to a voltage jump from +40 to
40 mV. Both sets of tails were fit well by double exponential
functions, but the dominant time constant was close to 5 ms for the FS
neuron, whereas it was close to 40 ms for the RS neuron.
A3 and B3: the TEA-sensitive currents,
computed by subtraction, were larger in macro-patches from FS neurons
than those from RS neurons. C: the current-voltage
(I-V) relation of the TEA-sensitive current from FS
neurons ( ) showed significant current at voltages above
20 mV and was similar to Kv3.1-Kv3.2 currents in heterologous
expression systems. In patches from RSNP neurons, the TEA-sensitive
current ( ) activated at more negative potentials.
D: the time course of the TEA-sensitive tail current
from an FS neuron was fit well by a single exponential having a 5.9 ms
time constant and was similar to that reported for Kv3.1-Kv3.2
channels. In contrast, the time course of the TEA-sensitive tail
current from an RSNP neuron was dominated by an exponential with a much
slower time constant (62.7 ms) unlike that reported for Kv3.1-Kv3.2
channels.
|
|
Kv3.1-Kv3.2 channels enabled high-frequency firing by speeding the
recovery of sodium-conductance inactivation while minimizing the
duration of the afterhyperpolarization
The previous experiments suggest that Kv3.1-Kv3.2 currents
facilitate sustained high-frequency firing of FS neurons, in part, by
reducing the amount of Na+ channel inactivation
that accumulates during the spike train. To test this mechanism
further, we constructed a single compartment Hodgkin-Huxley-like
model. The model included voltage-gated Na+
channels, Kv3.1-Kv3.2 channels, and Kv1-like channels (see
METHODS) and was studied under current-clamp conditions
with 200-ms current pulses (Fig. 9).
Under control conditions, when none of the channels were blocked,
depolarizing currents produced repetitive firing with an abrupt onset
(initial steady-state frequency 62 Hz). Like our recorded FS neurons,
the model displayed fast spiking with slight, early, spike frequency
adaptation. The firing rate varied monotonically with injected current
strength, and this relation approached an asymptote as current
increased, as observed for FS neurons (data not shown). Blocking the
Kv3.1-Kv3.2 channels in the model mimicked the effects of low TEA
concentrations on FS neurons: the AHP was decreased, the action
potential broadened, and repolarization slowed. This result supports
the conclusion that a Kv3.1-Kv3.2 current strongly contributes to spike
repolarization and the AHP. In addition, blockade of the Kv3.1-Kv3.2
current decreased the repetitive firing rates for all current strengths (Fig. 9, B and D), and spike failure occurred at
lower current strengths. In contrast, blocking the Kv1-like current had
no effect on the action potential shape but produced an increase,
rather than decrease in the firing rate (Fig. 9, C and
D), as we observed experimentally when we applied DTX.

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Fig. 9.
Blockade of a Kv3.1-Kv3.2 current in a computer model of FS neurons
mimics effects of TEA on spike shape and repetitive firing in FS
neurons. A: spike trains resulting from 200 ms duration
current pulses (0.3, bottom; and 0.5 nA,
top) injected into the FS model cell. Spike frequency
was related to the strength of current injection. Action potentials in
the model showed a small amplitude decrease during the pulse and some
early frequency adaptation as observed for FS neurons.
B: spike trains resulting from the model after blocking
Kv3.1-Kv3.2 channels. The AHP was greatly attenuated (compare to dashed
line). Spike frequency for a given current strength also slowed, as
observed experimentally with application of low doses of TEA to FS
neurons. At higher current strengths, a greater spike amplitude
decrease occurred following Kv3.1-Kv3.2 blockade, and spike failure
occurred at a lower current strength than for control conditions (data
not shown). C: blockade of the Kv1.3 current in the model,
after restoring all of the Kv3.1-Kv3.2 current, restored the AHP
amplitude and produced an increase in firing rate. Dashed line in
A-C is a 80 mV reference to help compare the
magnitude of the AHP in each condition. D: instantaneous
frequency vs. time after pulse onset (Latency) for spike trains evoked
by 0.3-nA current steps in each of the conditions illustrated in
A-C. E, top: membrane potential
(Vm) response to the 0.3-nA current pulse
are superimposed at higher temporal resolution for the conditions in
A-C to show the changes in spike shape and repetitive
firing that occurred. Bottom: the corresponding time
course of Na-channel inactivation (hNa) for
these conditions. Spike broadening and AHP reduction produced by
blockade of Kv3.1-3.2 resulted in a greater degree of inactivation and
less recovery from inactivation than observed in either the Control
condition or with just Kv1.3 blocked. This supports the hypothesis that
the spike slowing following Kv3.1-3.2 blockade results from greater
accumulation of Na-channel inactivation.
|
|
How these opposite effects of K+ channel blockade
on firing rate arose was investigated by examining the changes in
channel parameters during action potential trains. The spike-broadening produced by blocking Kv3.1-Kv3.2 channels resulted in greater sodium
channel inactivation occurring during the action potential (hNa; Fig. 9E). Moreover,
due to the blockade of the AHP, the rate of recovery from
Na+ channel inactivation following the action
potential was slowed down, resulting in a significant decrease in the
amount of recovery during the interspike interval. Thus fewer
Na+ channels were available to depolarize the
neuron in the period leading up to the next spike. A greater membrane
depolarization was required before there was enough
Na+ current to begin the next spike and the next
spike was delayed. No such effect was observed on blockade of the
Kv1-like current. Because this current contributed little to shaping
the action potential, there was no significant effect on the amount of
Na+ channel inactivation or on its rate of
recovery from inactivation (Fig. 9E). Rather, because the
Kv1-like current deactivated slowly, it was not completely turned off
during the brief AHP. Moreover, because it activates near spike
threshold, it actually grows prior to the next action potential and
functions to lengthen the interspike interval. Hence, blocking the
Kv1-like current shortens the AHP without decreasing the peak
amplitude, thereby increasing the firing rate.
Although our model qualitatively reproduces the repetitive firing
behavior of FS neurons, there are some quantitative differences. For
example, the slow-down in firing frequency produced by Kv3 blockade in
the model (Fig. 9D) occurred faster than the slow-down observed in our TEA experiments. This may result from differences between the actual and modeled Na+ channel kinetics or from
the presence of additional conductances not included in the model.
Knowledge of these factors is required before a more accurate model is attempted.
 |
DISCUSSION |
Using pharmacological and modeling approaches we have demonstrated
that the action potential and repetitive firing properties of
fast-spiking interneurons in the mouse somatosensory cortex are
powerfully shaped by a K+ current closely similar
to Kv3.1and Kv3.2 currents. We found that submillimolar concentrations
of TEA disrupted the fast-spiking phenotype and that this action of TEA
on FS neurons was highly specific. Selective toxins, which antagonize
other K+ channels having a high sensitivity to
TEA, had either no effect or facilitated high-frequency firing of FS
neurons. Of all K+ channels known (Coetzee
et al. 1999
), only those containing subunits of the Kv3
subfamily could account for the results of our pharmacological experiments. We also found that the majority of the somatic
K+ current from FS (but not RSNP) neurons
resulted from a current that strongly resembles the current expressed
by Kv3.1 and Kv3.2 proteins in mammalian heterologous expression
systems (Grissmer et al. 1994
;
Hernández-Pineda et al. 1999
; Rudy et al.
1999
). Taken together, the data strongly support the idea that
Kv3.1-Kv3.2 channels play a dominant role in repolarizing the action
potential and enabling high-frequency firing in neocortical FS neurons, a conclusion that was further supported and extended by our computer simulations.
Just how do Kv3.1 and Kv3.2 channels function in FS neurons? Based on
the requirement for membrane depolarization above
20 mV to achieve
significant activation of heterologously expressed Kv3.1-Kv3.2
channels, it has been suggested that these channels, when expressed in
sufficient numbers, could repolarize action potentials without
influencing their threshold, in contrast to K+
channels that activate at more negative potentials (Kanemasa et
al. 1995
; Weiser et al. 1994
,
1995
). Our findings strongly support this view of Kv3.1
and Kv3.2 channel function. Blockade of a Kv3.1-Kv3.2-like current by
low concentrations of TEA profoundly slowed action potential
repolarization in FS neurons without changing the threshold, the
maximum depolarization rate, or the spike amplitude. These data imply
that the Kv3.1-Kv3.2-like channels become sufficiently activated
during the brief spike to contribute selectively to action potential
repolarization. This point is directly supported by experiments in
which transfected HEK293 cells expressing Kv3.1 or Kv3.2 channels were
voltage clamped to an action potential waveform (Rudy et al.
1999
). No current was seen until after the action potential
reached its peak. The results of our computer simulations mimicked
these findings for FS neocortical neurons, and a recent study of
hippocampal basket cells indicates that a native 4-AP-sensitive
current, which may arise from Kv3.1 and/or Kv3.2 channels, can be
activated by brief action potentials (Martina et al.
1998
).
In addition to firing brief action potentials, FS neurons have large
AHPs compared with other neocortical interneurons and pyramidal cells.
Our data strongly indicate that the Kv3.1-Kv3.2-like current is also
responsible for this large AHP, because it was abolished by 1 mM TEA
but not by blockers of other known channels having comparable
sensitivities to TEA. Although a large AHP is often associated with
central neurons that fire slowly (Henderson et al. 1982
;
Leonard and Llinás 1990
; Yarom et al.
1985
), where it functions to slow firing, we found that the
large AHP (and brief action potential) generated in FS neurons
functions to enable high-frequency firing. This function appears to
result directly from the voltage dependence and rapid deactivation
kinetics of native Kv3.1 and Kv3.2 channels and the apparently low
levels of Ca2+-activated K+
currents and other K+ currents having slower
deactivation kinetics and more negatively shifted voltage dependencies.
Our studies suggest that an important mechanism by which
Kv3.1-Kv3.2-like channels enable fast spiking is by limiting the impact of Na+ channel inactivation on repetitive
firing. In trains of action potentials, the interspike interval is
established, in part, by the amount of Na+
channel inactivation that accumulates during the train. By keeping action potentials brief, Kv3.1-Kv3.2 currents reduce the amount of
Na+ channel inactivation that occurs during the
action potential. This was evident in our simulations where a
substantial increase in the amount of Na+ channel
inactivation occurred following spike broadening produced by
Kv3.1-Kv3.2 channel blockade. Kv3.1-Kv3.2 currents also function to
speed recovery from Na+-channel inactivation by
generating a large AHP. Results from our simulations support this idea
because blockade of Kv3.1-Kv3.2 currents both slowed the recovery of
Na+ channel inactivation and reduced the amount
of recovery that occurred after an action potential. A role for
Na+ channel inactivation in decreasing firing
frequency was also evident in our FS recordings. TEA greatly enhanced
the reduction in depolarization rate that occurred with successive
spikes in a train.
It is also worth noting that another factor by which the blockade of
Kv3 channels could slow firing frequency is by an increased activation
of other possible conductances in response to the broadening of the
action potential. The contribution of these possible factors remains to
be investigated.
Finally, as was evident in our simulations, the large magnitude of the
AHP in FS neurons also functions to terminate the Kv3.1-Kv3.2 current,
which, because of its rapid deactivation rates, and positive activation
voltage, minimizes the duration of the refractory period. The situation
was completely different for K+ channels that
activate at more negative potentials and have slower deactivation
kinetics in our simulations. Due to these factors, the Kv1-like
conductance decayed little during the interspike interval and
contributed to delaying the onset of the next spike. Hence blocking
that current in the model produced an increase in spike
frequency. Collectively, these results strongly suggest that the
particular activation range and fast deactivation kinetics of
Kv3.1-Kv3.2 channels function to enable sustained high-frequency firing
in FS neurons.
Kv3 proteins are found in the somata of many other neurons capable of
high-frequency firing, including some, but not all, GABAergic neurons,
suggesting a similar role in facilitating high-frequency firing for
these neurons. Kv3 genes are also prominently expressed in many neurons
that process sensory information, including many auditory structures
(Perney and Kaczmarek 1997
; Perney et al. 1992
; Rudy et al. 1992
; Weiser et al.
1994
, 1995
). For example, Kv3.1 and Kv3.3
transcripts are found in neurons of the medial nucleus of the trapezoid
body (MNTB). These neurons do not fire sustained high-frequency trains
of action potentials in response to steady current injection, however,
they can fire action potentials entrained to very high-frequency inputs
(>600 Hz), which preserves the timing information contained in
auditory signals (Brew and Forsythe 1995
). Clearly, the
presence of Kv3 channels alone is not sufficient for the generation of
sustained high-frequency firing. Nevertheless, Kv3 channels do appear
to function in the high-frequency firing of these neurons because low
concentrations of TEA reduced their ability to follow stimulus
frequencies >200 Hz. (Wang et al. 1998
). The different
firing properties between MNTB neurons and neocortical FS neurons could
be explained, in part, by the different levels of
low-voltage-activating DTX-sensitive K+ channels
(Brew and Forsythe 1995
; Wang et al.
1998
).
K+ channel diversity is a main factor
contributing to the diversity of the electrophysiological properties of
neurons, and it also contributes to the specificity of neuromodulator
actions (Adams and Galvan 1986
; Baxter and Byrne
1991
; Hille 1992
; Kaczmarek and Levitan
1987
; Llinas 1988
; Rudy 1988
).
The large number of K+ channel subunits
discovered in the last 10 years unexpectedly exceeds the diversity
predicted from electrophysiological studies of native
K+ currents. Over 100 different pore-forming
subunits of mammalian K+ channels have been
discovered (Coetzee et al. 1999
). Interactions among
different subunits and other factors suggest the existence of hundreds
if not thousands of different types of K+
channels. A significant challenge lies in integrating this molecular diversity into a physiological context. Kv3 channels represent a case
in point. Before the isolation of Kv3 cDNAs and the characterization of
Kv3 channels in heterologous expression systems, the existence of these
channels in neurons as a separate channel type was apparently undetected. Kv3 channels had not been separated from other components of the K+ current, and some of the initial papers
on the cloning of Kv3 cDNAs suggested that their properties in neurons
might be different (McCormack et al. 1990
; Rudy
et al. 1991
; Vega-Saenz de Miera et al. 1992
).
The characterization of the electrophysiological and pharmacological
properties of Kv3 currents in heterologous expression systems and the
delineation of their expression patterns in the CNS provided clues that
have allowed the isolation of native Kv3 currents in neurons and the
generation of hypotheses as to their functional roles in the CNS. We
now provide strong evidence in favor of the hypothesis that Kv3.1-Kv3.2
channels play specific roles in the generation of sustained
high-frequency firing in cortical interneurons. It is expected that
similar strategies with other cloned K+ channel
proteins whose neuronal roles are unknown will result in the discovery
of additional previously unknown native K+
channels and novel mechanisms to regulate neuronal function.
 |
ACKNOWLEDGMENTS |
We thank Dr. S. Hestrin for help and advice about recording from
neocortical interneurons.
This research was supported by National Institute of Neurological
Disorders and Stroke Grant NS-27881 and National Science Foundation
Grant IBN989606 to C. S. Leonard and National Institute of
Neurological Disorders and Stroke Grants NS-30989 and NS-35215 and
National Science Foundation Grant IBN9630832 to B. Rudy.
 |
FOOTNOTES |
Address for reprint requests: C. S. Leonard, Dept. of Physiology,
New York Medical College, Valhalla, NY 10595.
The costs of publication of this article were defrayed in part by the
payment of page charges. The article must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
Received 21 June 1999; accepted in final form 26 August 1999.
 |
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