859
J Physiol 580.3 (2007) pp 859–882
BK potassium channels facilitate high-frequency firing
and cause early spike frequency adaptation in rat CA1
hippocampal pyramidal cells
Ning Gu, Koen Vervaeke and Johan F. Storm
Institute of Basal Medicine, Department of Physiology and Centre of Molecular Biology and Neuroscience, University of Oslo, PB 1103 Blindern,
N-0317 Oslo, Norway
Neuronal potassium (K+ ) channels are usually regarded as largely inhibitory, i.e. reducing
excitability. Here we show that BK-type calcium-activated K+ channels enhance high-frequency
firing and cause early spike frequency adaptation in neurons. By combining slice electrophysiology and computational modelling, we investigated functions of BK channels in regulation
of high-frequency firing in rat CA1 pyramidal cells. Blockade of BK channels by iberiotoxin
(IbTX) selectively reduced the initial discharge frequency in response to strong depolarizing
current injections, thus reducing the early spike frequency adaptation. IbTX also blocked the
fast afterhyperpolarization (fAHP), slowed spike rise and decay, and elevated the spike threshold.
Simulations with a computational model of a CA1 pyramidal cell confirmed that the BK
channel-mediated rapid spike repolarization and fAHP limits activation of slower K+ channels (in
particular the delayed rectifier potassium current (I DR )) and Na+ channel inactivation, whereas
M-, sAHP- or SK-channels seem not to be important for the early facilitating effect. Since the
BK current rapidly inactivates, its facilitating effect diminishes during the initial discharge, thus
producing early spike frequency adaptation by an unconventional mechanism. This mechanism
is highly frequency dependent. Thus, IbTX had virtually no effect at spike frequencies < 40 Hz.
Furthermore, extracellular field recordings demonstrated (and model simulations supported)
that BK channels contribute importantly to high-frequency burst firing in response to excitatory
synaptic input to distal dendrites. These results strongly support the idea that BK channels play
an important role for early high-frequency, rapidly adapting firing in hippocampal pyramidal
neurons, thus promoting the type of bursting that is characteristic of these cells in vivo, during
behaviour.
(Received 16 December 2006; accepted after revision 9 February 2007; first published online 15 February 2007)
Corresponding author J. F. Storm: Department of Physiology and Centre of Molecular Biology and Neuroscience
(CMBN), University of Oslo, PB 1103 Blindern, N-0317 Oslo, Norway. Email: jstorm@medisin.uio.no
Potassium (K+ ) channels are by far the most diverse class
of ion channels, and are important for regulating neuronal
excitability and signalling activity in a variety of ways
(Hille, 2001). Since these channels mediate outward K+
currents and increase the membrane conductance, they
tend to hyperpolarize the cell and attenuate the effects
of excitatory stimuli. Potassium channels are therefore
normally regarded as inhibitory, i.e. they reduce neuronal
excitability. The clinical relevance of the dampening effect
of K+ channels is illustrated by the several familial forms
of diseases in humans that are caused by genetic K+
channel defects, including benign neonatal convulsions
(Steinlein, 2001) and episodic ataxia (Browne et al. 1994).
This paper has online supplementary material.
C 2007 The Authors. Journal compilation C 2007 The Physiological Society
Accordingly, genetic suppression of K+ channel activity in
mice also causes epilepsy (Smart et al. 1998; Peters et al.
2005). Also pharmacological blockade of K+ channels,
e.g. with 4-aminopyridine (Rutecki et al. 1987) or barium
(Hotson & Prince, 1981), readily causes epileptic seizures.
A variety of inhibitory K+ channel effects have been
described in rodent CA1 hippocampal pyramidal cells,
which currently is one of the most intensely studied
neuronal types in the mammalian brain (Storm, 1990;
Johnston et al. 2000). For example, A- and D-type K+
channels suppress the initial excitability and cause a
delay in the onset of spiking (Gustafsson et al. 1982;
Storm, 1988). A-type channels also strongly suppress
dendritic excitability and the impact of dendritic excitatory
synapses (Hoffman et al. 1997; Ramakers & Storm, 2002).
Calcium-activated K+ channels of the sAHP type cause
DOI: 10.1113/jphysiol.2006.126367
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860
N. Gu and others
profound feed-back inhibition of the action potential
frequency during repetitive firing (Madison & Nicoll, 1982,
1984; Pedarzani & Storm, 1993). Also Kv7/KCNQ/M-type
potassium channels contribute importantly to spike
frequency adaptation in these cells (Madison & Nicoll,
1984; Gu et al. 2005; Peters et al. 2005). Calcium-activated
K+ channels of the SK type have been reported to have a
similar role (Stocker et al. 1999), but this appears not to be
the case in CA1 pyramidal cells under normal conditions
(Gu et al. 2005). However, SK channels have been shown
to be activated by Ca2+ spikes (Gu et al. 2005) or by Ca2+
influx through synaptically activated NMDA channels (Cai
et al. 2004; Ngo-Anh et al. 2005).
BK channels are Ca2+ - and voltage-activated potassium
channels with large conductance (also called BKCa , KCa 1.1,
MaxiK, Slo) (Blatz & Magleby, 1987; Marty, 1989; Sah &
McLachlan, 1992; Vergara et al. 1998). These channels
have been shown to mediate rapid spike repolarization
and fast afterhyperpolarization (fAHPs) in many types of
neurons (Adams et al. 1982; Storm, 1987a,b; Lancaster &
Nicoll, 1987; Schwindt et al. 1988; Womack & Khodakhah,
2002). The BK channels were originally also reported to
reduce the frequency of action potentials and to limit
epileptiform bursts in rat hippocampal CA1 pyramidal
neurons (Lancaster & Nicoll, 1987; Alger & Williamson,
1988). However, during previous experiments more than
a decade ago, it was noticed that blockade of the BK
channels in rat CA1 pyramidal cells slowed down the
initial discharge frequency in response to current injection
(J. F. Storm, unpublished observations). At that time,
this effect was attributed to suppression of the BK
channel-dependent rapid spike repolarization and fAHP
(Storm, 1987a,b). These effects would be expected to
increase inactivation of the spike-generating transient
Na+ current (I NaT ) and activate more of the slower K+
currents, thereby enhancing refractoriness and reducing
excitability during the immediate aftermath of the first
action potential (Shao et al. 1999a). Thus, BK channels
seemed to be able to facilitate high-frequency firing in these
neurons.
Recently, the idea of a facilitory effect of BK channels
was supported by a study of epilepsy in humans. It was
discovered that an abnormal increase in the BK channel
conductance, caused by a gain-of-function mutation in
the BK channel α-subunit, underlies human epilepsy
and paroxysmal movement disorder (Du et al. 2005).
Similarly, dentate granule neurons from mice lacking
the β4 BK channel subunit show a gain-of-function
for BK channels that sharpen action potentials, thereby
facilitating high-frequency firing and leading to temporal
lobe seizures (Brenner et al. 2005). Thus, for the first
time, epilepsy was found to be caused by an increase in a
potassium current, rather than by a decrease. Furthermore,
BK channel-deficient mice have recently been shown to
have reduced EEG power (Storm et al. 2006).
J Physiol 580.3
In the meantime, other reports have also lent support
to the notion that suppression of BK channel activity
can indirectly convey a reduction of neuronal excitability.
Thus, our group found that mice lacking BK channels
showed a reduction of fAHP and firing activity in
cerebellar Purkinje neurons (Sausbier et al. 2004).
Furthermore, in cultured neurons from the cerebral cortex
of mice, bursting activity induced by various procedures
was inhibited by the BK channel blocker iberiotoxin
(IbTX) (Jin et al. 2000).
Previously, certain other K+ channels, in particular
the Kv3 channels with their unusually fast activating
and deactivating kinetics, have been found to facilitate
sustained high-frequency firing, by mediating rapid
spike repolarization and deep, fast afterhyperpolarizations
(Rudy et al. 1999). Thus, blockade of Kv3 channels in
interneurons resulted in a cumulative Na+ channel
inactivation, thus slowing down the firing rate (Erisir et al.
1999; Lien & Jonas, 2003).
Nevertheless, the effects of BK channel activity
on excitability and the underlying mechanisms have
previously not been examined in a systematic manner,
although BK channels are believed to mediate rapid
spike repolarization and fAHPs in many types of neurons
(Adams et al. 1982; Storm, 1987a, 1990; Lancaster &
Nicoll, 1987; Schwindt et al. 1988; Takahashi, 1990; Gho
& Ganetzky, 1992; Sah & McLachlan, 1992; Gola & Crest,
1993; Hu et al. 2001; Womack & Khodakhah, 2002; Sah
& Faber, 2002; Zhang et al. 2003; Sausbier et al. 2004).
Earlier studies have provided seemingly conflicting results.
Thus, in one study, blockade of BK channels by a low
dose of tetraethylammonium (TEA) or charybdotoxin
(ChTX) was found to reduce the fAHP amplitude and
increase the initial discharge frequency during a train of
action potentials in hippocampal CA1 pyramidal neurons
(Lancaster & Nicoll, 1987). Furthermore, BK channel
blockade by TEA or ChTX was found to increase the
duration of epileptiform bursts and suppress the associated
transient AHP (Alger & Williamson, 1988). Later, it was
suggested that when the cell is depolarized, BK channels
may contribute to the normal post-burst medium afterhyperpolarization (mAHP) and early spike frequency
adaptation (Storm, 1989, 1990; Williamson & Alger, 1990).
Increased excitability was reported to result from BK
channel blockade also in dissociated Purkinje neurons
(Swensen & Bean, 2003), rat dorsal root ganglia neurons
(Zhang et al. 2003), and spontaneously firing vestibular
nucleus neurons (Nelson et al. 2003). This is in apparent
contrast to our own previous results, as well as the reduced
Purkinje cell excitability and reduced EEG power found in
neurons from BK channel knock-out mice (Sausbier et al.
2004; Storm et al. 2006), and the human epilepsy caused by
a BK channel gain-of-function mutation (Du et al. 2005).
For these reasons, we have undertaken a systematic
examination of the effects of BK channels on the
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BK channels enhance high-frequency firing and adaptation
J Physiol 580.3
excitability and firing pattern of CA1 hippocampal
pyramidal cells, as well as the underlying mechanisms.
We found that blockade of the BK channels significantly
slows down the early high-frequency firing, indicating
that BK channels facilitate high-frequency firing in
these cells. Furthermore, by combining experiments and
computational modelling, we found that these effects
are mediated through fast spike repolarization, fAHP
generation and modulation of other K+ channels and
Na+ channel inactivation. This provides an example of
how a fast calcium-activated potassium channel type can
enhance neuronal excitability through interaction with
other ion channels. Some of the data have already been
published in abstract form (Gu et al. 2006; Vervaeke et al.
2006a).
Methods
Slice electrophysiology
Slices for sharp-electrode intracellular recording and
field potential recording. Young adult male Wistar rats
(4–7 weeks of age) were deeply anaesthetized with
Suprane before decapitation. Transverse hippocampal
slices (400 μm) were cut with a Vibroslicer (Campden
Instruments, UK) and maintained in an interface chamber
filled with artificial cerebral spinal fluid (aCSF) containing
(mm): 125 NaCl, 25 NaHCO3 , 1.25 KCl, 1.25 KH2 PO4 ,
1.5 MgCl2 , 1.0 CaCl2 , 16 glucose and saturated with
95% O2 –5% CO2 . The aCSF was heated to 30◦ C during
recording, and the temperature was kept constant within
±0.5◦ C. The experimental procedures were approved by
the responsible veterinarian of the Institute, in accordance
with the statute regulating animal experimentation given
by the Norwegian Ministry of Agriculture 1996.
Sharp-electrode intracellular recording. Slices were
transferred to a recording chamber maintained at
30 ± 0.5◦ C, where they were submerged and perfused
with aCSF of the composition described above, except
that the concentration of CaCl2 was 1.6 mm and KCl
was 3.5 mm. Sharp electrodes were filled with 4.0 mm
potassium acetate (resistance 80–140 M, pH 7.35). Intracellular recordings were obtained from CA1 pyramidal
neuron somata with the ‘blind’ method. Currentclamp recordings were performed using an Axoclamp
2A amplifier (Molecular Devices Corporation, USA) in
bridge mode. Only cells with a stable resting membrane
potential more negative than −60 mV and stable action
potential amplitudes (> 80 mV) were included.
Extracellular field potential recording. Extracellular field
potentials were recorded submerged in a chamber
maintained at 31 ± 0.5◦ C, with low resistance glass
micropipettes filled with aCSF (as described above,
861
except that the CaCl2 concentration was 2.0 mm) and
coupled to an Axoclamp 2B amplifier (Molecular Devices
Corporation). A tungsten stimulation electrode was placed
in the middle of the stratum radiatum while the recording
electrode was placed in the pyramidal layer, ∼200–400 μm
apart. The stimuli, consisting of a brief current pulse
(100 μs duration, repeated every 30 s) elicited two to
four population spikes. dl-APV (50 μm) and bicuculline
(5 μm) were routinely added to the extracellular solution.
The extracellular recording solution was the same as
described above except that the CaCl2 concentration was
2.0 mm and the K+ concentration was 4.0 mm.
Data acquisition, storage and analysis
The data were acquired using pCLAMP 7.0 or 9.0
(Molecular Devices Corporation) at a sampling rate
of 5–20 kHz, analysed and plotted with pCLAMP 9.0
and Origin 7.5 (Microcal). Values are expressed as
mean ± s.e.m. Two-tailed Student’s t test was used for
statistical analysis (P = 0.05). The P values are given in
the figure legends.
The interspike interval (ISI) was the time measured
between the peaks of two consecutive action potentials
(APs) within a spike train. The 1st ISI is the time
between the peaks of the 1st and 2nd APs, and so on.
The instantaneous firing frequency of an ISI is f = 1/ISI
(Hz). The cell input resistance was calculated by dividing
the steady-state voltage response by the current pulse
amplitude (Rinput = V /I).
Chemicals and drugs
The BK-channel blocker iberiotoxin (IbTX) was
obtained from Dr Hans-Guenther Knaus, Innsbruck
Medical University, Austria. The M-channel blocker
XE991 was obtained from DuPont pharmaceutical
company and NeuroSearch, Denmark. DNQX (6,7dinitroquinoxaline-2,3-dione)
and
TEA
(tetraethylammonium) were purchased from Tocris Cookson
Ltd (UK). Apamin was purchased from Latoxan (France).
The remaining chemicals, including forskolin, were
purchased from Sigma-Aldrich Norway AS (Oslo).
Substances were bath-applied by adding them to the
perfusion medium.
Computer simulations
Computer simulations were performed using the
Surf-hippo neuron simulator, version 3.5a (Graham,
2004). Surf-Hippo is written in Lisp and was run on a
Linux workstation. For integrating the circuit equations,
Surf-Hippo uses a variant of the Crank Nicholson
method as described by Hines (1984). The variable
time-step method was used to speed up the simulations
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862
N. Gu and others
(Borg-Graham, 2000), with a maximum voltage error
criterion of 0.05 mV.
We used a computer model that is derived from
the Borg-Graham CA1 pyramidal neuron model
(Borg-Graham, 1999), which has been refined through
collaborative studies by using our experimental data on
discharge patterns, AHPs, subthreshold resonance and
oscillations (Shao et al. 1999a; Hu et al. 2002; Gu et al.
2005; Vervaeke et al. 2006b). This model reproduces
quite accurately the spiking behaviour of these neurons
(cf. Shao et al. 1999a) as well as the AHPs following
single action potentials or spike trains (Gu et al. 2005;
Vervaeke et al. 2006b). We refer to the full description
of the model in the supplementary material. Briefly, the
cell was represented by five compartments, comprising
an isopotential soma (diameter 20 μm) and a dendritic
cable (total length 800 μm and diameter 5 μm) consisting
of four segments of equal length. Dendritic spines
were not explicitly modelled, but their contribution to
the linear dendritic load was modelled by raising the
dendritic segment capacitance and reducing the dendritic
membrane resistance (Rall et al. 1992). The soma and
the dendritic segments had a membrane resistance of 20
and 6 k cm2 , respectively, while the capacitance was 1.0
and 1.5 μF cm−2 . A uniform intracellular resistance of
100 cm was assumed (Stuart & Spruston, 1998). With
these values, the model was able to account for the input
resistance and whole-cell capacitance measured in our
experiments. The active conductances included persistent
and transient Na+ currents (I NaP , I NaT ), four voltage-gated
potassium currents (I A , I D , I DR , I M ), a fast inactivating
Ca2+ - and voltage-dependent K+ current (I BK ) (Shao et al.
1999a), two voltage-gated calcium currents (I CaN and
I CaL ), a hyperpolarization-activated non-specific cation
current (I h ) and a slow Ca2+ -activated potassium current:
the sAHP current (I sAHP ).
The soma was divided into three subcellular
compartments to model the differential and localized
intracellular Ca2+ dynamics. The outer membrane shell
consists of two juxta-membrane shells to reproduce the
Ca2+ channel dependence of the BK- and sAHP current,
in particular a co-localization of the channels underlying
I CaN and I BK (Borg-Graham, 1987). The rest of the somatic
volume is occupied by a core compartment. As Ca2+ ions
enter through the calcium channels they diffuse among
the subcellular compartments and undergo instantaneous
buffering while a Ca2+ pump causes efflux of Ca2+ ions
into the extracellular space. [Ca2+ ] in all compartments
was initially set to 50 nm.
Calculation of all currents was based on the extended
Hodgkin–Huxley formalism with rate constants derived
from a single barrier thermodynamic gating particle
model (Borg-Graham, 1987). Exceptions are I NaT and I BK ,
which were calculated according to a more generalized
Markov scheme (Shao et al. 1999a; Borg-Graham, 1999;
J Physiol 580.3
Vervaeke et al. 2006b). The simulation temperature was
30◦ C.
In Fig. 10, the accumulative I BK inactivation was
removed by increasing the transition rate from the
inactivated state (I) to the closed state (C). This was done
by shifting the V 1/2 of the squeezed exponential describing
this transition from −120 mV to −50 mV (eqn (1)).
⎛
−1 ⎞−1
V − V1/2
⎠
αI→C (V ) = ⎝τ0 + α0 + exp
k
(1)
In Fig. 10D, the recovery of inactivation of I NaT was
varied by changing the V 1/2 of the squeezed exponential
describing the transition from the inactivated (I) to the
closed state 1 (C1) to −53, −55, −57 and −59 mV
for the panels from left to right, respectively. The
excitatory synapse was calculated as I syn = g syn (E rev – V m ),
with reversal potential (E rev ) of 0 mV and the unitary
synaptic conductance (g syn , peak conductance 0.3 μS)
was described by a difference of exponentials with time
constant of 0.1 ms for the rising phase and 5.0 ms for the
decay phase. This synapse was inserted 300 μm from the
soma.
Results
Intracellular recordings
Somatic intracellular recordings with sharp microelectrodes were obtained from 53 CA1 pyramidal neurons,
in stratum pyramidale of rat hippocampal slices from
young adult male Wistar rats (4–7 weeks of age). All
cells were recorded in current-clamp mode, in normal
aCSF (with [K+ ]o = 3.5 mm and [Ca2+ ]o = 1.6 mm) at
30.0 ± 0.5◦ C. The average resting membrane potential
(V rest ) and input resistance (Rinput ) were −68.2 ± 1.6 mV
and 40.7 ± 2.1 M, respectively. To standardize our tests,
the membrane potential prior to stimulation of the cell
was normally maintained at a constant level (−60 mV) by
injection of DC current (Figs 1–5, 7 and 9), but tests at
−70 mV were also performed.
TEA reduced the early high-frequency firing, but did
not affect low-frequency firing
Several potassium channels that are important for spike
repolarization and spike frequency control in mammalian
central neurons can be reversibly blocked by TEA. This
is true for several voltage-gated K+ channels such as Kv3
(Rudy & McBain, 2001), some Kv7 channels (KCNQ or
M-channels) (Hadley et al. 2000) and the Ca2+ -activated,
voltage-dependent BK channels (Storm, 1987a; Lancaster
& Nicoll, 1987).
TEA, being a small, water-soluble molecule, has the
advantage that it readily diffuses in and out of the slice,
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J Physiol 580.3
BK channels enhance high-frequency firing and adaptation
producing rapid effects and recovery upon washout.
In our experiments, bath-application of a low dose of
TEA (100 μm) produced clear, reversible effects on spike
repolarization and frequency in all the CA1 pyramidal
cells tested (Fig. 1). From a membrane potential of
−60 mV, we injected two depolarizing current pulses: one
brief and strong (50 ms, 0.8 nA), the other longer but
weaker (100 ms, 0.2 nA), in order to evoke high- and
low-frequency firing, respectively (Fig. 1Aa). A 6 s interval
between the two pulses ensured that the slow afterhyperpolarization (sAHP) following the first pulse fully
decayed before the onset of the second. At the start of
each experiment, the current intensities were adjusted to
evoke five spikes within 50 ms (strong pulse), followed by
four spikes within 100 ms (weak pulse), and the intensities
then remained constant for the rest of the experiment.
Interestingly, application of 100 μm TEA always reduced
the initial discharge frequency in response to the strong
pulse – an effect that may seem unexpected for a K+
channel blocker (Fig. 1A, left, insets). Thus, the initial spike
863
frequencies were significantly reduced by TEA: both the
instantaneous frequency for the first interspike interval
(ISI) (Fig. 1B and C), and the average frequency of the 1st
to the 3rd ISIs (Fig. 1D and E). Thereby, the 2nd to 4th
action potentials were delayed, often preventing the 5th
action potential from occurring within the 50 ms pulse
(Fig. 1Ab, left). The effects of TEA were fully reversible
(Fig. 1B and D). This demonstrated that the K+ channel
blocker TEA actually reduced the excitability of these
neurons, in response to strong stimulation. In contrast, the
response to the weaker pulse was not noticeably changed by
100 μm TEA (Fig. 1Ab, right; Fig. 1B and D, open symbols;
Fig. 1C and E, right). These results seem to indicate
that TEA-sensitive potassium channels can enhance the
excitability and increase the firing rate of CA1 pyramidal
neurons. However, this effect is dependent on the discharge
frequency, as it was only seen during high-frequency spike
trains.
What is the mechanism underlying the enhanced
excitability? TEA (100 μm) did not significantly change
Aa
1st - 3rd ISI
Control
st
1st - 3rd ISI
nd
1 AP
2 AP
-60mV
TEA
b
-60mV
Washout
c
20 mV
20 ms
-60mV
High freq.
Low freq.
TEA 100uM
C
200
(1st ISI)
frequency (Hz)
300
100
0
0
10
20
Time (min)
30
(1st ISI)
B
frequency (Hz)
0.5 nA
250
200
150
100
50
0
High freq.
*
Low freq.
NS
Ctrl TEA Ctrl TEA
TEA 100uM
(1st - 3rd ISI)
150
High freq.
Low freq.
100
50
0
0
10
20
Time (min)
30
200
150
(1st - 3rd ISI)
200
Averaged freq. (Hz)
E
D
Averaged freq. (Hz)
Figure 1. Tetraethylammonium (TEA) reduced
high-frequency discharge
Sharp intracellular electrode recordings from rat CA1
pyramidal cells. Aa, repetitive firing at two different
frequencies was evoked by somatic injection of depolarizing
current pulses. Left: a strong (0.8 nA) 50 ms long pulse
evoked 5 action potentials (APs). Right: a weaker (0.2 nA)
100 ms long pulse evoked 4 APs. The current pulses, which
were separated by a 6 s interval, were repeated at constant
intensities throughout the experiment, while the
background membrane potential was kept at −60 mV by
injecting steady depolarizing current. Ab, bath application
of TEA (100 μM) clearly reduced the high-frequency firing
rate and spike number (left: the number of APs was
reduced from 5 to 4), but had no significant effect on low
firing frequency (right).
Ac, washout of TEA for 10 min reversed the effect. B, time
course of the 1st ISI firing frequency. (•: high frequency; e:
low frequency). C, summary data showing that 100 μM TEA
significantly reduced the 1st ISI high firing frequency
(∗ : n = 7; washout: n = 4; P < 0.05), but had no effect on
low-frequency firing (NS: n = 7; washout: n = 4; P > 0.05).
D, time course of the averaged firing frequency of the
1st–3rd interspike intervals (ISIs): •, high frequency; e, low
frequency. E, summary data showing that 100 μM TEA
significantly reduced the mean firing frequency of 1st–3rd
ISI in response to the strong (0.8 nA, 50 ms) pulse
(∗∗ : n = 7; washout: n = 4; P < 0.01), but had no effect on
the low firing frequency in response to the weak (0.2 nA,
100 ms) pulse (NS: n = 7; washout: n = 4; P > 0.05).
High freq.
**
Low freq.
NS
100
50
0
Ctrl TEA Ctrl TEA
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864
N. Gu and others
the holding current (i.e. the steady current required to hold
the cell at −60 mV), in any of the cells tested (n = 7). Nor
did TEA significantly change input resistance of the cells,
as measured by injecting negative current pulses (n = 4).
However, TEA (100 μm) produced a significant spike
broadening and blockade of the fast afterhyperpolarization
(fAHP) in all cells tested. (P < 0.01, n = 7), in accordance
with previous results (Storm, 1987a,b; Shao et al. 1999a).
Figure 2 shows the effect of IbTX on the early firing
during a 50 ms spike train, evoked by a strong depolarizing
current pulse (0.8–1.2 nA, 50 ms). Again, the current
intensity was adjusted to trigger five spikes during each
pulse under control conditions, but thereafter the intensity
was kept constant. Application of IbTX (100 nm) slowed
the repolarization of the first two action potentials and
increased the duration of the 1st ISI (Fig. 2Aa–c), thus
reducing the instantaneous firing frequency (Fig. 2C).
However, IbTX did not significantly change the cell input
resistance, nor the DC current required to hold the
background potential at −60 mV (n = 7, P > 0.05, data
not shown). Similar results were obtained in all cells
tested (Fig. 2E, ∗ P < 0.05, n = 6). Figure 2D shows that
the frequency of the initial discharge (1st to 3rd ISIs)
was reduced by BK channel blockade, thus reducing the
number of spikes during the 50 ms pulse from 5 to 4 (see
Fig. 2Bb). Similar effects were observed in all cells tested
The BK channel blocker iberiotoxin (IbTX) reduced
high-frequency firing
Since TEA blocks several K+ channel types, more selective
blockers are needed to identify the channel types mediating
its effects. To test whether BK channels, which are sensitive
to TEA in the micromolar range (Storm, 1987a), were
involved, we used the highly selective BK channel blocker
iberiotoxin (IbTX) (Galvez et al. 1990).
Aa
Control
AP1
J Physiol 580.3
c
IbTX
b
AP1
AP2
AP2
Overlay
AP2
AP1
20 mV
2 ms
fAHP
fAHP
Ba
c
b
20 mV
20 ms
0.5 nA
D
IbTX 100nM
200
150
100
0
5
10 15 20
Time (min)
25
200
200
frequency (Hz)
250
E
Averaged 1st-3rd ISI
frequency (Hz)
1st ISI frequency (Hz)
C
IbTX 100nM
150
100
50
0
5
10 15
Time (min)
20
25
control
IbTX
*
**
150
100
50
0
1st ISI
1st-3rd ISI
Figure 2. The BK channel blocker IbTX reduced early high-frequency firing
A and B, typical sharp intracellular electrode recordings from a rat CA1 pyramidal cell. Ba, a high-frequency
train of 5 action potentials (APs) was evoked by a 0.9 nA, 50 ms long depolarizing current pulse, starting from a
membrane potential of −60 mV (maintained by steady depolarizing current injection). Bb, 13 min after application
of iberiotoxin (IbTX, 100 nM), the cell fired only 4 APs in response to an identical current pulse. A shows the expanded
traces of the 1st and 2nd APs taken from B, showing that the 2nd was significantly delayed after application of
IbTX (Ac). Blockade of BK channel broadened the action potentials and eliminated the fast afterhyperpolarizations
(fAHPs; arrow). Summary time course showing that 100 nM IbTX reduced the instantaneous firing frequency of
the 1st ISI from 169 ± 5 Hz to 146 ± 7 Hz (C, n = 6), and the averaged 1st−3rd ISI frequency from 122 ± 4 Hz
to 103 ± 3 Hz (D, n = 6). E, summary data from all cells tested (n = 6): comparison of the 1st ISI frequencies and
averaged 1st−3rd ISI frequencies before and after application of IbTX (n = 6, each. ∗ P = 0.016, ∗∗ P = 0.001).
C 2007 The Authors. Journal compilation C 2007 The Physiological Society
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BK channels enhance high-frequency firing and adaptation
J Physiol 580.3
(Fig. 2E, ∗∗ P < 0.01, n = 6). (Wash-out of IbTX was not
attempted, since previous experience indicates that this
cannot normally be obtained in slices; Shao et al. 1999a.)
These experiments are in agreement with previous findings
(J. F. Storm, unpublished data), supporting the hypothesis
that blockade of BK channels, rather than increasing
the excitability of the CA1 hippocampal neurons, tends
to reduce their initial firing rate during high-frequency
discharge.
frequency greater than 150 Hz. However, the experiments
with TEA, suggest that blockade of BK channels does
not affect the firing rate at lower frequencies (Fig. 1).
Nevertheless, the apparent lack of effect in that case might
be due to a balance between opposing effects of different
currents blocked by TEA, e.g. M-current (Hadley et al.
2000) and BK current. To test these hypotheses, we used a
similar protocol as in Fig. 1A (right), injecting weak pulses
(0.2–0.4 nA) that evoked four action potentials within
100 ms (Fig. 3B). With this procedure, application of IbTX
(100 nm) did not produce any significant change in the
1st ISI; there was neither a prolongation nor a shortening
of the interval (Fig. 3Aa–c), in contrast to the effect
of IbTX on the high-frequency response (Fig. 2Aa–c).
Nevertheless, IbTX clearly slowed the repolarization of the
first two action potentials (Fig. 3Ac), and also decreased
Blockade of BK channels failed to affect the early
firing at low frequency
The above results suggest that BK channel activity
promotes high-frequency firing, at least at average
frequencies greater than 100 Hz and, for the 1st ISI, at
Aa
Control
IbTX
b
AP2
AP1
865
AP1
Overlay
c
AP1
AP2
AP2
20 mV
5 ms
Ba
b
c
20 mV
20 ms
−60 mV
0.5 nA
E
D
IbTX 100nM
100
50
0
0
5
10
15
20
25
150
150
frequency (Hz)
150
Averaged 1st-3rd ISI
frequency (Hz)
1st ISI frequency (Hz)
C
IbTX 100nM
100
50
0
100
Time (min)
5
10 15 20
Time (min)
25
NS
50
0
0
control
IbTX
NS
1st ISI
1st-3rd ISI
Figure 3. Application of IbTX had little or no effect on low-frequency firing
A and B, typical sharp intracellular electrode recordings from a rat CA1 pyramidal cell. Ba, the cell fired a train of 4
APs in response to a 0.4 nA, 100 ms long depolarizing current pulse, with the preceding membrane potential held
at −60 mV by steady-state current. Aa–c show expanded traces taken from 1st to 2nd action potentials of Ba–c.
Ab and Bb were recorded 14 min after bath application of 100 nM IbTX. Bc, overlaid traces showing that IbTX
did not significantly change the firing rate of the cell. The current pulse was constant (0.4 nA). Ac, IbTX caused
spike broadening of the two first spikes, but the interspike interval (ISI) was not changed. C, summary time course
from all the cells tested shows that IbTX did not significantly change the instantaneous firing frequency of the
1st ISI: (before IbTX: 68.1 ± 8.5 Hz; after application of IbTX: 65.2 ± 8.5 Hz; n = 4). D, time course of averaged
1st–3rd ISI frequency (41.3 ± 4.1 and 40.8 ± 3.4 Hz, before and after application of IbTX, respectively) (n = 4).
E, summary graph showing no significant IbTX effect on either the 1st ISI or the averaged 1st–3rd ISI frequency
(NS: P > 0.05).
C 2007 The Authors. Journal compilation C 2007 The Physiological Society
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866
N. Gu and others
the spike frequencies in response to strong depolarizing
pulses in these experiments as shown in Fig. 2, confirming
that the toxin blocked BK channels. Similar results were
obtained in all cells tested in these ways (Fig. 3E, n = 6 for
the 1st ISI and the 1st to 3rd ISIs, NS; P > 0.05). Figure 3C
and D summarizes the average time courses for all the cells
tested in this way.
All the experiments presented in Figs 1–3 were
performed while holding the cell at a slightly depolarized
potential: −60 mV. However, it is possible that this
depolarization might increase the resting inactivation
of the Na+ channels, leading to a larger effect of BK
blockade than at the normal resting potential of these cells,
which is close to −70 mV. To test for this possibility, we
repeated such experiments at a background potential of
−70 mV. We found that the effects of IbTX at −70 mV
were essentially the same as at −60 mV. Thus, also
at −70 mV, IbTX application significantly reduced the
initial high-frequency firing frequency in response to
strong current injection (n = 6, P < 0.01; for 1st ISI, the
frequency was reduced from 203 ± 14 to 152 ± 19 Hz;
for the 1st to 3rd ISI, the average frequency was reduced
from 144 ± 10 to 109 ± 10 Hz). Also, at −70 mV, IbTX
failed to affect the low-frequency firing in response to
weak current injection (average frequency of 1st to 3rd
ISI: 79.3 ± 7.5 Hz before IbTX; 77.4 ± 6.3 Hz after IbTX
application; P > 0.05, n = 4).
The experiments and model simulations in this study
were mainly performed at temperatures close to 30◦ C
(range: 29–31◦ C), as is common for hippocampal slice
recordings. To test whether the observed effects also occur
at physiological temperatures, we also performed a few
experiments at ∼37.5◦ C. At this recording temperature,
the maximal early spike rate was higher (up to ∼300 Hz),
but IbTX still significantly reduced the early discharge rate
by more than 20%. Thus, in the two cells tested, the average
initial firing frequency for 1st to 3rd ISIs was reduced, with
convincing time courses, from 194 to 153 Hz (by 22%)
and from 263 to 208 Hz (by 21%), respectively (data not
shown).
These results indicate that the effect of BK channels
on neuronal excitability is frequency dependent. However,
why did the BK channels only facilitate the high-frequency
firing while having no detectable effect on the
low-frequency firing? To investigate this, we took a closer
look at the first spikes during high-frequency discharge.
Effects of IbTX on action potential slopes during early
high-frequency firing
Figure 4Aa shows a typical example of the first two action
potentials of a high-frequency spike train (same procedure
as in Fig. 2; 1st ISI, 5.7 ms). In normal medium, there
was always a clear fast AHP (arrow) after the first spike.
Application of IbTX eliminated this fAHP, and the 1st
J Physiol 580.3
ISI was increased from 5.7 to 7.2 ms (the frequency was
reduced from 175 to 139 Hz; Fig. 4Ab). In parallel, the
decay of the first two spikes were slowed by IbTX (Fig. 4Ac),
due to loss of repolarizing BK current (Storm, 1987a; Shao
et al. 1999a). Similar results were obtained in all five cells
tested (summarized in Fig. 4Da and b, and Fa and b).
We then switched our attention to the rising slope of the
action potentials, reasoning that the IbTX-induced spike
broadening and loss of the fAHP might affect sodium
channel inactivation. Since the upstroke of the spikes is
due to the charging of the cell membrane capacitance by
the voltage-dependent transient Na+ current, the rising
slope of the spike reflects the size of the net Na+ current
during the fast depolarization. Figure 4Ba and b shows
superimposed traces of the two first spikes; the insets
show the rising slopes at an expanded time scale. While
the rising slopes of the first spike were not affected by
IbTX (insets of Fig. 4Ba, summary time course in Ca, and
summary data in Cb), the rising slope of the 2nd spike
was significantly reduced by IbTX (see inset of Fig. 4Bb,
summary time course in Ea). Such an effect was observed
during high-frequency spike trains in all five cells tested
in this way (Fig. 4Eb, P < 0.05). In contrast, IbTX had
no detectable effect on the rising slope of 1st and 2nd
action potentials during low-frequency firing, i.e. four
spikes in 100 ms, as in Fig. 3 (n = 5; P > 0.05 for both
1st and 2nd spike, data not shown). Furthermore, during
high-frequency firing, IbTX shifted the threshold of the
2nd action potential to a more depolarized potential (on
average 3.6 mV shift; n = 5, P < 0.05), while the threshold
of the 1st spike remained unchanged (n = 5, P > 0.05).
These results indicate that blockade of BK channels
reduced the rate-of-rise and elevated the threshold of
the second action potential in the train, presumably
because broadening of the first spike combined with fAHP
blockade caused increased Na+ channel inactivation and
reduced recovery before the second spike. In contrast,
IbTX did not affect the upstroke of the first spike itself,
indicating that the toxin did not directly affect the sodium
channels.
f–I curves: IbTX reduced the spike frequency gain
in the high-frequency range
To determine the BK channel contribution to spike
frequency regulation at a range of different firing
frequencies, a series of depolarizing current pulses of
different intensities were injected into each cell before and
after IbTX application. The resulting discharge frequencies
(f ) versus injected current (I) were plotted as f–I curves
(Fig. 5A and B). At the lowest frequencies (< 25 Hz),
IbTX had no detectable effect on the f–I plots (Fig. 5A
and B; •, control; e, IbTX). At higher frequencies,
however, the f–I curves obtained after IbTX application
showed a clear decrease in slope compared with the curves
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BK channels enhance high-frequency firing and adaptation
J Physiol 580.3
Modelling f–I curves: BK channels facilitate
high-frequency firing
obtained in the same cells in normal medium, before
IbTX was applied. This was seen both for the f–I curves
representing the instantaneous frequency of the first ISI in
the train (Fig. 5A) and for those representing the averaged
frequencies of the first three interspike intervals (Fig. 5B).
Thus, for both types of plots, the effect of IbTX was
stronger at higher intensities of injected current, i.e. the
effect of BK channel blockade increased with the overall
spike frequency (Fig. 5A and B). These results confirm that
BK channels enhance the excitability of CA1 pyramidal
cells by promoting high-frequency initial discharge, in a
manner that is itself frequency dependent.
Ca
Control
fAHP
b
Da
7.2 ms
IbTX
20 mV
c
b
AP1 rise slope (%)
AP2
AP1
5.7 ms
1 ms
Ea
AP1
AP2 rise slope (%)
Overlay
Ba
To explore the mechanisms underlying the BK
channel-induced increase in firing frequency, we
performed numerical simulations using a computer
model of a rat CA1 pyramidal neuron derived from a
previous model (Borg-Graham, 1999). Our model is
described in detail in Methods (Computer simulations)
and further justification of the parameter values is
given in Vervaeke et al. (2006b). Briefly, the model
contained the following active conductances: persistent
and transient Na+ currents (I NaP , I NaT ), four voltage-gated
AP1 decay slope (%)
Aa
867
AP2
b
IbTX
IbTX
20 mV
1 ms
▲
AP2 decay slope (%)
Fa
200
200
IbTX 100nM
150
100
100
50
50
0
0
5
200
150
10
15
Time (min)
20
25
b
0
200
100
50
50
0
5
200
150
10
15
Time (min)
20
25
200
100
50
50
5
200
10
15
Time (min)
20
25
b
50
10
15
Time (min)
IbTX
20
**
150
50
5
*
CTRL
100
0
IbTX
0
100
0
CTRL
200
IbTX 100nM
150
**
150
IbTX 100nM
0
IbTX
0
b
100
0
CTRL
150
IbTX 100nM
100
0
NS
150
25
0
CTRL
Figure 4. Effects of IbTX on the 1st and 2nd action potential slope during early high-frequency firing
Aa–c, recordings of the first two action potentials during a high frequency spike train, elicited by a brief, depolarizing
current step (0.8 nA, 50 ms). Aa, in normal medium, the ISI between the 1st and 2nd spikes was 5.7 ms (firing
rate: 175 Hz). Note the fAHP following the 1st AP (arrow). Ab, application of the BK channel blocker IbTX (100 nM)
delayed the onset of the 2nd action potential (ISI: 7.2 ms; frequency: 139 Hz). Ac, overlaid traces of Aa and Ab,
comparing the 1st and 2nd APs evoked by identical current pulses (0.8 nA) before and after application of IbTX. Ba,
the decay slope of the 1st AP was reduced after application of IbTX (dashed line), whereas the rising slope remained
unchanged (see inset at expanded time scale, X: 0.2 ms; Y: 50 mV). Bb, both the rising slope (see inset) and the
repolarization slope of the 2nd AP were slowed down by IbTX (dashed line), and there was a slight elevation of
the spike threshold (). Ca–Fa, normalized average time courses (%) from all the cells tested: IbTX effects on the
rising slope (Ca) and decay slope (Da) of the 1st AP, and the rising slope (Ea) and decay slope (Fa) of the 2nd AP.
Cb–Fb, summary graph from all the cells tested, showing that BK channel blockade significantly reduced rising
slope of the 2nd AP· Eb (∗ : n = 5, P < 0.05), as well as the decay slopes of both 1st AP (Db,∗∗ : n = 5, P < 0.01)
and 2nd AP (Fb, ∗∗ : n = 5, P < 0.01). In contrast, IbTX had no significant effect on the rising slope of the 1st AP
(Cb, NS: n = 5, P > 0.05).
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IbTX
868
N. Gu and others
potassium currents (I A , I D , I DR , I M ), a fast inactivating
Ca2+ - and voltage-dependent K+ current (I BK ), two
voltage-gated calcium currents (I CaN and I CaL ), a
hyperpolarization-activated non-specific cation current
(I h ) and a slow Ca2+ -activated potassium current
(I sAHP ). Also, the soma was divided into three subcellular
compartments to model localized intracellular Ca2+
dynamics.
By varying the BK channel density, we explored the
effects on the input–output properties of these cells by
simulating f–I curves (G BK : 0, 10, 20, 40, 80, 130, 200,
400, 600 and 800 pS μm−2 ). Depolarizing current pulse
injections of increasing amplitude were simulated at the
soma (pulse duration: 1 s). Before each current pulse, the
J Physiol 580.3
membrane potential was set to −60 mV by steady current.
Increasing the BK channel density clearly promoted higher
firing frequencies, both for the 1st ISI (Fig. 5C) and for the
average frequency of the first three intervals (Fig. 5D).
Reduction of the firing frequency by blocking
BK channels is not dependent on activation
of M-, sAHP- or SK-channels
Several mechanisms might contribute to the reduced
firing frequency after blocking BK channels. In particular,
the broadened action potential probably causes more
Ca2+ influx to occur, in particular since Ca2+ influx
predominantly happens during the spike repolarization
B
200
175
175
Average frequency
1st -3rd ISI (Hz)
200
150
125
100
75
50
NS
25
0
C
*
0.1
0.2
**
0.3
0.4
0.5
Frequency
1st ISI (Hz)
250
225
200
175
150
125
100
75
50
25
0
GBK = 800 pS/ m
GBK = 130 pS/ m
2
2
GBK = 0 pS/ m
2
0.05 0.10 0.15 0.20 0.25 0.30 0.35
I (nA)
*
NS
125
100
75
50
25
D
I (nA)
control
IbTX
150
0
0.6
0.1
0.2
0.3
0.4
0.5
0.6
I (nA)
Average frequency
1st - 3rd ISI (Hz)
Frequency
1st ISI (Hz)
A
250
225
200
175
150
125
100
75
50
25
0
GBK = 800 pS/ m
GBK = 130 pS/ m
2
2
GBK = 0 pS/ m
2
0.05 0.10 0.15 0.20 0.25 0.30 0.35
I (nA)
Figure 5. BK channel effects on spike frequencies: results from IbTX experiments and computer
simulations
A, action potential frequency of the first interspike interval (1st ISI) in response to a series of 1.0 s long depolarizing
current pulses (0.1–0.6 nA, in steps of 0.05 nA) in normal medium (•) and after adding 100 nM IbTX ( e). The
plot shows the mean firing frequencies from all CA1 hippocampal pyramidal cells tested (n = 7). IbTX clearly
reduced the high-frequency firing in response to strong current pulses (≥ 0.2 nA; ∗ P < 0.05; ∗∗ P < 0.01), but had
no significant effect on the low firing rate in response to 0.15 nA pulses (NS: P > 0.05). B, similar to A, but showing
the average frequencies of the first three intervals (1st–3rd ISI). IbTX significantly reduced the spike frequencies
in response to current pulses > 0.35 nA (∗ : n = 7, P < 0.05), but had no significant effect at the lower current
intensities (0.15–0.3 nA; NS: n = 7, P > 0.05). C and D, computer simulations with a CA1 pyramidal cell model
showed that BK channels promote high-frequency firing. The f–I relations of the 1st ISI (C) and of the average of
the first 3 interspike intervals (D, 1st–3rd ISI) are plotted for different BK channel conductance densities (GBK : 0,
10, 20, 40, 80, 130, 200, 400, 600 and 800 pS μm−2 ). Current pulses of increasing amplitude were applied at
the soma (pulse duration: 1 s). The membrane potential prior to each current pulse was held at −60 mV by steady
current injection. The continuous lines represent the standard model with IBK present (130 pS μm−2 ) or blocked
(0 pS μm−2 ).
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BK channels enhance high-frequency firing and adaptation
J Physiol 580.3
phase. Therefore, blockade of BK channels would be
expected to increase the Ca2+ -activated K+ current which
underlies the slow afterhyperpolarization (I sAHP ), which
would reduce the firing frequency. We used the model to
predict the effects of interactions between I BK and I sAHP
on the f–I curves (Fig. 6A and B). Blocking I sAHP only
affected the lower frequency range of the f–I curve because
the sAHP conductance is rather small. Thus, at higher
frequencies this conductance is negligible compared with
the total conductance during the interspike trajectory. Our
simulations with and without I sAHP showed that the BK
channels induced a virtually identical increase of firing
frequency in the upper frequency range, whether I sAHP was
present or not. Thus, effects mediated by I sAHP could not
account for the observed effects of BK channel blockade
by IbTX (Fig. 5A and B).
Alternatively, the spike broadening could activate more
M-current (I M , mediated by KCNQ/Kv7 channels) which
underlies the medium afterhyperpolarization (mAHP) in
these cells and is known to strongly dampen excitability
(Storm, 1989; Peters et al. 2005; Gu et al. 2005). Therefore,
we also explored the role of I M on the input–output
A
225
Control
no IBK
200
no IsAHP
175
no IsAHP + no IBK
150
125
100
75
50
25
0
225
Control
no IBK
200
no IsAHP
175
no IsAHP + no IBK
250
Average Frequency
ISI-1-3 (Hz)
Frequency
ISI-1 (Hz)
properties by simulating f–I curves. Blocking I M in the
model strongly increased the firing frequency, in particular
in the lower frequency range (Fig. 6C and D), as reported
previously (Madison & Nicoll, 1984; Storm, 1989; Gu et al.
2005; Peters et al. 2005). Nevertheless, the inclusion of BK
channels still induced virtually identical increase in firing
frequencies in the upper frequency range whether I M was
present or not.
In order to test experimentally whether I M and I sAHP
were important for the BK channel effect on firing
frequency, we first blocked I M and I sAHP and then
performed the same type of experiment as shown in Figs 1,
2 and 4. Thus, we evoked a high-frequency spike train
by injecting a current pulse, and blocked BK channels
by adding 100 nm IbTX. (In three experiments, I M and
I sAHP were blocked by 10 μm XE991 and 50 μm forskolin,
respectively; in one experiment, I M and I sAHP were both
blocked by 20 μm carbachol; all drugs were bath-applied.)
In all the four cells tested in this way, IbTX caused a
pronounced reduction in the initial spike frequency, and
the effect had a convincing time course (Fig. 7Da). For
the 1st and 2nd ISI, the effect was essentially the same as
B
250
150
125
100
75
50
25
0
0.05 0.10 0.15 0.20 0.25 0.30 0.35
0.05 0.10 0.15 0.20 0.25 0.30 0.35
I (nA)
225
Control
no IBK
200
no IM
175
no IM + no IBK
150
125
100
75
50
25
0
225
Control
no IBK
200
no IM
175
no IM + no IBK
250
Average Frequency
ISI-1-3 (Hz)
250
Frequency
ISI-1 (Hz)
I (nA)
D
C
869
150
125
100
75
50
25
0
0.05 0.10 0.15 0.20 0.25 0.30 0.35
0.05 0.10 0.15 0.20 0.25 0.30 0.35
I (nA)
I (nA)
Figure 6. Computer simulations: reduction in firing frequency by BK channel blockade is not dependent
on activation of I M or I sAHP
A and B, f–I relations of the first interspike interval (A, ISI-1) and of the average of the first 3 interspike intervals
(B, ISI-1–3) from simulations with IsAHP present (dashed lines) or blocked (continuous lines). The f–I relations were
simulated as described in Fig. 5. C and D, similar to A and B, but here the f–I relations were plotted from simulations
with IM present (dashed lines) or blocked (continuous lines).
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870
N. Gu and others
seen without blockade of I M and I sAHP (Figs 1, 2 and 4),
thus confirming the prediction of the model: that I M and
I sAHP are not important for the ability of BK channels to
facilitate high-frequency firing. However, in the cells where
I M and I sAHP had been blocked, the IbTX-induced spike
broadening and the depolarization during the interspike
intervals were even more severe (Fig. 7A and B) than with
I M and I sAHP intact (Figs 1, 2 and 4), probably because
I M , and even I sAHP , may contribute to repolarization of
late, broadened spikes and during ISIs. As the spikes late
in the train became severely blunted and broadened, it
was difficult to interpret the effects on the late firing
frequency.
Finally, we also tested the possibility that Ca2+ -activated
+
K channels of the SK type underlie the reduction in
firing frequency in response to BK channel blockade. In
six cells the SK channels were blocked by 100 nm apamin
after (n = 4) or before (n = 2) adding 100 nm IbTX. In
the cells that were first treated with IbTX, apamin had
no detectable effect on the spike frequency, indicating
that SK channels did not contribute appreciably to spike
frequency regulation in spite of the IbTX-induced spike
broadening (n = 4, data not shown). Conversely, in the
cells first treated with apamin, the IbTX-induced reduction
in spike frequency was undiminished, thus confirming that
SK channels did not mediate this effect (n = 2, data not
shown). These results confirm and extend our previous
finding that SK channels do not contribute appreciably to
the mAHP or spike frequency regulation in CA1 pyramidal
cells (Gu et al. 2005).
Control
IbTX
1st ISI
A
J Physiol 580.3
20mV
2 ms
-60 mV
0
5
10
15
Time (min)
b
250
200
IbTX 100nM
150
100
50
0
5
10
15
Time (min)
b
IbTX 100nM
250
200
150
100
50
0
0
5
10
15
Time (min)
20
100
**
50
Control
IbTX
250
200
**
150
100
50
0
20
300
Control
IbTX
150
0
20
2nd AP rise slope
(mV/ms)
2nd AP rise slope
(mV/ms)
1st ISI frequency
(Hz)
1st AP decay slope
(- mV/ms)
50
0
Da
IbTX 100nM
100
0
Ca
b
150
1st ISI frequency
(Hz)
1st AP decay slope
(- mV/ms)
Ba
Control
IbTX
300
250
200
150
100
50
0
*
Figure 7. Blockade of BK channel reduced the
burst firing frequency in the presence of XE991
and forskolin
A, sample traces from a sharp-electrode current-clamp
recording showing the first three action potentials
(1st–3rd spikes) evoked by a 0.8 nA current pulse,
before and after bath-application of 100 nA IbTX. The
perfusion media contained XE991 and forskolin, to
block Kv7/KCNQ/M- and sAHP-currents, respectively.
Ba–Da, time courses of the effects of IbTX (100 nA,
same experiment as in A) on the decay slope of the 1st
spike (Ba), the rise slope of the 2nd spike (Ca), and the
instantaneous firing frequency of 1st ISI (Da). Similar
results were observed in all the cells tested with IbTX
(100 nA, n = 3), as shown by the column graphs in Bb,
Cb and Db (∗ P < 0.05, ∗∗ P < 0.01).
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J Physiol 580.3
BK channels enhance high-frequency firing and adaptation
871
Analysing the roles of ionic currents during repetitive
firing: modelling with different BK channel densities
I (nA)
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I (nA)
Vm (mV)
A
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GBK: 130 pS μm−2
No IBK
voltage
20 mV
5 ms
IBK
INa
INa (I-state)
IA
I (nA)
2
I (nA)
4
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IDR
0
dV/dt (mV/ms)
Next, we used the model to study the roles of various ionic
currents during repetitive firing (Fig. 8). A short spike
train evoked by injection of a 50 ms long depolarizing
current pulse at the soma (mean spike frequency ∼200 Hz)
was simulated with three different BK channel densities:
(1) with a BK channel density that probably is close to
normal (130 pS μm−2 ; continuous black lines in Fig. 8)
and was used in our standard model (Fig. 6); (2) with an
increased BK channel density (600 pS μm−2 : dotted lines);
and (3) without any BK current (red lines). Figure 8 shows
the BK current (I BK ; previously dubbed I CT , by Storm,
1990 and Shao et al. 1999a), the sodium current (I Na ), the
inactivation state of I Na (I-state), the A-current (I A ), the
delayed rectifier potassium current (I DR ), the M-current
(I M ), and the slow AHP current (I sAHP ). I D is not plotted
in Fig. 8 because it did not contribute appreciably to spike
repolarization under the conditions simulated here. It is a
not a large current and is largely inactivated at potentials
positive to ∼−65 mV. Therefore, the remaining I D was too
small to contribute appreciably.
At the presumed normal BK channel density
(130 pS μm−2 ; black lines), the first spike in the train
is mainly repolarized by I BK , I DR and I A , but later in
the spike train, I BK and I A inactivate while I DR plays a
larger role (Shao et al. 1999a). When I BK was blocked
(red lines), the spike was broadened, causing the other
voltage-gated K+ currents (I DR , I A and I M ) to become
more strongly activated. Nevertheless, the first spike
was considerably broadened and the fAHP suppressed.
Together, these effects caused a stronger inactivation of
I Na and, in particular, a far less complete recovery from
inactivation of I Na between the spikes (see I Na , I-state).
This, in turn, caused a slight attenuation of the peak I Na
during the second to fourth spikes (I Na trace). Therefore,
the peak amplitude and rise slope of these spikes were
also reduced (phase plot, Fig. 8B), in agreement with
our experimental results. Thus, the amplitude of the
2nd AP was reduced from 69.4 ± 2.9 to 63.9 ± 3.5 mV
(n = 6, P < 0.01), whereas the 1st AP amplitude remained
unchanged (82.0 ± 2.5 and 81.3 ± 2.9 mV, before and after
IbTX, respectively, n = 6, P > 0.05, data not shown) (see
also Fig. 4Ac and Bb). The incomplete recovery from
inactivation of I Na raised the spike threshold slightly
(arrows in Fig. 8B), which contributed to prolonging the
interspike intervals, i.e. reducing the firing rate. However,
the strongest effect on firing rate was mediated via the
delayed rectifier K+ current, I DR . Note that I DR is by far
the largest current during the interspike intervals at higher
frequencies. I DR represents a current whose molecular
identity is not exactly known, although Kv2 subunits
are probably involved (Du et al. 2000), and a complete
kinetic description is lacking. Therefore, in our previous
IM
IsAHP
2nd Spike
450 Threshold
300
150
0
−150
−60 −40 −20 0
Vm (mV)
20
40
Figure 8. Computer simulations of a spike train and the
response of various ionic currents, while varying the BK channel
density
A, somatic responses to a current pulse (50 ms, 0.4 nA). The
membrane potential prior to the pulse was held at −60 mV by steady
current. The simulations were repeated for 3 different BK channel
densities: 0 (red), 130 (black), and 600 pS μm−2 (dashed black). The
first four spikes in the train are shown. Note that the delayed rectifier
potassium current (IDR ) increases considerably during the interspike
intervals when the spikes are broadened as a consequence of a
reduced BK channel density (0 pS μm−2 , red). Also, INa does not
recover completely from inactivation when the spikes get broader (INa ,
I-state). B, phase plot of the second spike. Note the increased threshold
and reduced rise slope and spike peak when IBK is blocked (red
arrow).
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872
N. Gu and others
(Vervaeke et al. 2006b) and present model, I DR is based
on a combination of voltage-clamp (e.g. Du et al. 2000)
and current-clamp data (e.g. Storm, 1987a; Shao et al.
1999a; see also Vervaeke et al. 2006b). Our model predicts
that the spike broadening caused by blocking I BK strongly
increases I DR , thus increasing refractoriness after the spike
and decreasing the firing frequency in the upper frequency
range. The spike broadening also caused an increased
activation of I M and I sAHP, but these currents are too small
(compared with I BK , I Na and I DR ) to have any substantial
effects on the initial high-frequency firing rate (Fig. 8A,
lower two rows).
When the I BK density was increased to 600 pS μm−2
(dotted lines in Fig. 8), the spikes became narrower and,
hence, I DR was substantially reduced, not only the I DR peak
during the spike, but also the I DR tail current between
the spikes. In parallel, I Na inactivation was reduced, and
recovery from inactivation more rapid. Together, these
mechanisms produced a briefer refractory period and,
hence, higher discharge frequencies.
Thus, our simulations provide an explanation for
how BK channels facilitate high-frequency firing: the
BK channels do this mainly by ensuring rapid spike
repolarization and narrow spikes, which prevent strong
activation of I DR and promote rapid recovery from I Na
inactivation after each spike.
The BK current generates early spike frequency
adaptation
Several lines of evidence indicate that the BK current
in CA1 pyramidal cells is transient, i.e. it decays within
less than 20 ms during sustained depolarization (Zbicz
& Weight, 1985; Storm, 1990; Shao et al. 1999a,b;
Halvorsrud et al. 1999; Gu et al. 2006; Vervaeke et al.
2006a). Therefore the current was dubbed ‘I CT ’, i.e.
transient Ca2+ -activated K-type current (Storm, 1990).
This inactivation contributes to frequency-dependent
spike broadening and the rapid decay of the fAHP during
a high-frequency discharge (Shao et al. 1999a).
We reasoned that the inactivation of the BK current
will also necessarily influence spike frequency and, in
particular, spike frequency adaptation. As outlined above,
the spike frequency is reduced when I BK inactivates,
due to enhanced I DR activation and Na+ channel
inactivation causing enhanced refractoriness. Therefore,
the inactivation of I BK during the initial spikes in a highfrequency train should cause spike frequency adaptation.
In order to test this hypothesis, we performed slice
experiments (Fig. 9A–C) as well as computer simulations
(Fig. 9D–F). To standardize the conditions, in both
experiments and simulations, the cells were held at
−60 mV and current steps were injected to keep the
instantaneous spike frequency of the first interval (1st ISI)
at about 100 Hz (Fig. 9Aa and Da).
J Physiol 580.3
In the slice experiments, BK channel blockade by bath
application of 100 nm IbTX, caused both a slowing of the
initial discharge frequency, as we saw before (Figs 2 and 4),
and also a reduction in the spike frequency adaptation. In
order to compare adaptation in spike trains with the same
initial frequency, a current step with stronger intensity was
injected into the cell, giving a frequency of ∼100 Hz for
the 1st ISI also after addition of IbTX (dashed lines in
Fig. 9A). In this situation, the frequency of the next spikes
(2nd and later ISIs) was clearly increased after adding
IbTX compared with the control condition, indicating
that BK channel blockade strongly reduced the early spike
frequency adaptation (Fig. 9Ab and Db). Similar results
were obtained in all the six CA1 pyramidal cells tested.
The data from six experiments are summarized in Fig. 9B
and C (∗∗ P < 0.01; NS, P > 0.05). Similar results were also
obtained while holding the cells at −70 mV (n = 6, see
above; data not shown).
In the computer simulations we used a similar protocol
and got similar results (Fig. 9D–F). Thus, the early
spike frequency adaptation was strongly reduced when
I BK was omitted from the model (Fig. 9Db) compared
with when I BK was included (Fig. 9Da). This is in close
qualitative agreement with the experimental results from
IbTX application (Fig. 9A–C), although there are minor
quantitative differences (at least compared with the
example shown in Fig. 9A), e.g. in the current needed
to obtain ∼100 Hz for the 1st ISI, the spike amplitude,
late spike frequencies, and the exact shape of the ISI and
frequency plots (Fig. 9E and F). No attempt was made
to tune the model in order to reduce these differences;
the simulation results shown in Fig. 9D–F are the ones
that were first obtained when the model was used for this
purpose. Thus, the modelling results represent a prediction
of the experimental results shown in Fig. 9A–C, rather than
just a post hoc explanation of them.
Our prediction was also confirmed by earlier
experiments and modelling. Thus, in Fig. 8A, the third ISI
is virtually identical with BK current (black, continuous
line: 130 pS μm−2 ) and without (red line), whereas the first
ISI is about 30% longer when the BK current is missing,
indicating reduced adaptation. A similar effect was seen
experimentally in response to IbTX (Fig. 2B). Figure 8A
also shows that the activation of I DR increased strongly
following the 2nd spike compared with the 1st spike with
normal BK current, whereas there was much less increase
in I DR in the simulation without BK channels (red line).
In addition, Fig. 8A also shows that the inactivation of I Na
(I Na I-state) increased following the 3rd spike compared
with the 1st spike with normal BK current (black line),
whereas there is no such increase without BK channels (red
line; actually the I-state here was slightly reduced). Thus,
it seems that because the BK current facilitates spiking but
inactivates rapidly, it contributes to early spike frequency
adaptation.
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BK channels enhance high-frequency firing and adaptation
J Physiol 580.3
BK current inactivation underlies early spike
frequency adaptation
Figure 10A outlines the normal, inactivating I BK model,
with its closed (C), open (O), and inactivated (I) states.
With such an inactivating I BK in the model, injection
of a current pulse evoked a high-frequency spike train
with prominent spike frequency adaptation (Fig. 10Ca,
black upper trace). Here, the fAHP amplitude rapidly
decayed, because of rapid inactivation of I BK (Fig. 10Ca,
black middle trace). In contrast, in the non-inactivating I BK
model, the same current pulse produced a train of spikes
that showed much weaker spike frequency adaptation (red
plots in Fig. 10Ca and Cb), and each spike was followed by
a prominent fAHP (Fig. 10Ca, red upper trace; arrows).
In order to test more ‘directly’ our idea that rapid
inactivation of the BK current is essential for the
contribution of this current to early spike frequency
adaptation, we selectively removed the inactivation in
the model. By comparing a model with the normal,
inactivating I BK and a model in which the inactivation
mechanism of I BK was removed (Shao et al. 1999a), we
could address this issue in a direct way that was not
experimentally feasible (Fig. 10).
Experiment
Aa
Computer model
Da
1st - 3rd ISI
873
1st - 3rd ISI
Control
Control
20 mV
10 ms
-60mV
20 mV
-60mV
10 ms
1 nA
1 nA
1st - 3rd ISI
1st - 3rd ISI
b
b
IbTX
No IBK
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-60mV
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E
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Frequency (Hz)
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ISI (ms)
50
Frequency (Hz)
ISI (ms)
B
1 nA
1
2
3
ISI number
4
1
2
3
ISI number
Figure 9. Experiments and computer modelling showed reduced spike-frequency adaptation after BK
channel blockade
Aa, sample trace showing an early part of a spike train in response to a brief current injection. Before the pulse, the
cell was held at −60 mV by DC current injection. Note that in normal medium (Control), the cell showed strong
spike-frequency adaptation. Ab, after bath-application of 100 nM IbTX, a current step with stronger intensity was
injected to the cell in order to achieve the same firing frequency of the 1st ISI as before IbTX (vertical dashed lines).
Note that the 2nd and 3rd interspike intervals (ISIs) were clearly reduced compared with the control condition.
B and C, summary plots showing reduced spike-frequency adaptation after BK channel blockade by 100 nM IbTX:
B, the 2nd and 3rd ISIs were clearly reduced by IbTX when the 1st ISI was kept constant (NS: n = 6, P > 0.05; ∗∗ :
n = 6, P < 0.01). C, spike frequencies of the 2nd and 3rd ISIs were clearly increased by IbTX when the frequency
of the 1st ISI was kept constant (NS: n = 6, P > 0.05; ∗∗ : n = 6, P < 0.01). D–F, results from computer simulations
of the experiments shown in panels A–C, respectively.
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4
874
N. Gu and others
In this case, I BK showed no attenuation during the spike
train (Fig. 10Ca, red middle trace). This result indicates
that rapid inactivation of the BK current is essential for its
contribution to early spike frequency adaptation.
By giving a series of current steps of different intensities,
we also plotted the f–I curves (Fig. 10B). Removal of
I BK inactivation had essentially no effect on the 1st
ISI (Fig. 10B, left panel; note that the red and black
plots overlap). In contrast, there was a clear increase of
the average frequency of the first four spikes at high
stimulus current intensities (Fig. 10B, right panel). This
C
O
I
Ca
illustrates, again, that removing I BK inactivation did not
only increase excitability but also reduced the spike
frequency adaptation.
Next, we explored how I BK inactivation can affect
neuronal excitability during a burst of spikes in response
to excitatory synaptic input. Such spike bursts are typical
responses of CA1 pyramidal neurons (see e.g. Ranck,
1973; Harris et al. 2001). A synaptic conductance was
inserted at the apical dendrite of the model, 300 μm from
the soma, and the maximal conductance was adjusted
to evoke a burst of spikes at the soma (Fig. 10D).
Non inactivating IBK
200
160
120
80
Control IBK
40
0
0.0
ISI-1-3 Frequency (Hz)
IBK model
1st ISI Frequency (Hz)
B
A
J Physiol 580.3
Non inactivating IBK
200
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Current (nA)
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Current (nA)
IBK
Frequency (Hz)
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Vm
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Non inactivating IBK
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D
Control IBK
80
2
3
ISI no.
4
Control IBK
Non–inactivating IBK
slower recovery of INaT inactivation
20mV
10ms
20mV
10ms
Figure 10. Removing the inactivation of I BK in the model reduced the adaptation and increased the
excitability
A, diagram of the IBK Markov model states; C, closed state; O, open state; I, inactivated state. B, the rate of the I→C
transition was increased sufficiently to fully prevent cumulative IBK inactivation, thus producing a ‘non-inactivating’
BK current (red plot) (see Methods). Left panel: f–I relation of the first interspike interval was not changed by
removing IBK inactivation. Right panel: f–I relation for the first 4 spikes (average frequency) was increased by
removing IBK inactivation. Ca, top trace: spike train in response to a depolarizing current pulse for IBK with (black)
and without (red) inactivation (scale bar, 30 mV). Note the fast afterhyperpolarization (fAHP, arrows) following
every spike when inactivation was removed. Middle trace: IBK during the spike train (scale bars: 5 ms and 3 nA).
Cb, plot of adaptation based on the simulations shown in Ca. D, in the model, a synapse on the apical dendrite
(left cartoon) was activated strongly so that the EPSP evoked a burst of spikes at the soma. Top panels: a series of
somatic voltage responses of the model; the recovery rate of INa inactivation was reduced stepwise from fast (left
panels) to slow (right panels; see Methods). Bottom panels: same as above, but without inactivation of IBK .
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J Physiol 580.3
BK channels enhance high-frequency firing and adaptation
During such high-frequency bursts, recovery from I NaT
inactivation is also expected to be an important factor.
To explore the role also of this factor, we also varied
the latter parameter. Figure 10D shows four cases, with
different rates of recovery from I NaT inactivation (left, fast;
right, slow recovery). For each case we determined the
effect of removing I BK inactivation. With the fastest I NaT
inactivation recovery (Fig. 10D, left panel) removing I BK
inactivation reduced the spike frequency adaptation, and
increased the number of spikes in the burst. When the
I NaT inactivation recovery was slower, the removal of I BK
inactivation had stronger effects (Fig. 10D, middle traces).
However, when the recovery of I NaT inactivation was made
very slow (Fig. 10D, right traces), the I BK effect became less
significant. Thus, BK channel inactivation can contribute
strongly to the early spike frequency adaptation that is
characteristic for the bursting of hippocampal pyramidal
neurons in vivo (Ranck, 1973; Harris et al. 2001).
875
logical conditions. To test this, we stimulated the
Schaffer collateral fibres by positioning a tungsten
stimulation electrode in the middle of stratum radiatum,
and recorded the somatic population spike response
with an extracellular recording pipette placed in the
CA1 pyramidal layer (Fig. 11A). The experiments were
performed in the presence of GABAA - and NMDAreceptor blockers and in slightly elevated [K+ ]o (4.0 mm),
in order to obtain a purely excitatory synaptic response
with multiple population spikes, which remained stable
because NMDA-receptor-dependent forms of synaptic
plasticity were blocked. The stimulation intensity was
increased until a single stimulus evoked an excitatory
postsynaptic potential (EPSP) that triggered three to five
population spikes, with an interval of ∼5 ms (∼200 Hz)
between the first two population spikes under control
conditions (Fig. 11B). This kind of response resembles
the natural response pattern of CA1 pyramidal neurons,
which typically fire two to six spikes at 150–200 Hz during a
burst event in rats during behaviour (Ranck, 1973; Harris
et al. 2001). Furthermore, an advantage of extracellular
field potential recording is that it, being non-invasive, does
not disturb the intracellular milieu of the cell. Thus, it
avoids artifacts that may afflict intracellular recordings,
such as the artificial leak conductance caused by sharp
BK channel blockade reduced the firing frequency
of synaptically evoked population spikes in stratum
pyramidale
Finally, we wished to test whether BK channels also
increased the firing frequency under more physio-
A
B
Extracellular
Recording
Stimulation
Control
IbTx
PS3
PS2
0.5 mV
PS1
PS3
20
D
E
PS2
10
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5
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Time (min)
Decay Slope (mV/ms)
0
Frequency (Hz)
PS time (ms)
200
15
2 ms
Normalized
Decay slope (mV/ms)
C
Rise slope (mV/ms)
Figure 11. Blocking BK channels with IbTX
reduced the firing frequency of population
spikes in striatum pyramidale
A, a tungsten electrode was positioned in the middle
of stratum radiatum to stimulate the Schaffer
collaterals (stimulation duration, 150 μs; stimulation
interval, 30 s). A field recording electrode was
positioned in stratum pyramidale. The perfusion
medium contained 4.0 mM K+ , bicucculine (15 μM)
and APV (100 μM). B, under these conditions, a
single stimulus (arrow) evoked 3 population spikes
(PS). The stimulation artifact is indicated by an arrow.
The amplitude of the stimulus current was adjusted
so the interval between the first two population
spikes was ∼5 ms (i.e. ∼200 Hz). Bath application of
100 nM IbTX reduced the frequency of the
population spikes. C, the average (n = 4) time course
of the IbTX effect on the PS latencies following the
stimulation artifact (t = 0). D, average time course of
the IbTX effect on the frequency of the first and
second PS interval (ISI1 and ISI2). E, average time
course of the normalized change in decay slope of
the three population spikes. F and G, summary data
of the IbTX effect on the rise slopes and decay slopes
of the three population spikes (n = 4, NS: P = not
significant, ∗ 0.01 < P < 0.05, ∗∗ P < 0.01).
0
10 20 30
Time (min)
Control
IbTx
**
3
3
3
*
2
2
2
1
1
1
0
0
0
PS1
*
PS2
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PS3
876
N. Gu and others
microelectrode impalement (as used in this study), or
washout effects of whole-cell recording.
Adding IbTX (100 nm) broadened the population spikes
and reduced the frequency of the burst (Fig. 11B, dashed
and dotted line). The time course of the effects of IbTX
on the population spike latencies from the stimulus (spike
timing) is plotted, for all slices tested, in Fig. 11C, together
with the population spike interval frequency (Fig. 11D),
and the decay slope of the population spikes (Fig. 11E).
Interestingly, IbTX decreased the decay slope of the first
three population spikes but the effect was clearly less on
the third than on the first spike (Fig. 11E). This indicates
that BK-channel-dependent spike repolarization is most
prominent for the first few spikes in a burst, thus causing
frequency-dependent spike broadening, in agreement with
previous results from our lab (Shao et al. 1999a), as well as
with Figs 2–4. Thus, given that CA1 pyramidal cells in the
behaving animal mostly fire short, high-frequency bursts
of spikes rather than long spike trains, BK channel kinetics
seem to be well adapted to allow the type of bursting that
is characteristic of these cells in vivo.
Discussion
Main results
By combining slice electrophysiology and computational
modelling, we have investigated the functional roles of
BK channels in regulation of high-frequency firing in rat
CA1 hippocampal pyramidal neurons. We found that BK
channels facilitate high-frequency discharge (> ∼100 Hz),
thus increasing the spike frequency and spike number in
response to strong depolarizing current. By causing rapid
spike repolarization and a fast afterhyperpolarization
(fAHP), the BK channels mediate their facilitating effect.
Thus, the short spike duration caused by I BK limits the
activation of other, slower K+ channels (in particular,
the delayed rectifier, I DR ) and limits the inactivation
of Na+ channels. In addition, the BK-dependent fAHP
may promote faster recovery from Na+ inactivation and
faster closure of slower K+ channels (I DR ). In contrast
to DR channels, regulation of M-, sAHP- or SK-channel
recruitment does not seem to contribute appreciably to
the facilitating effect of BK channels.
Since the BK current itself inactivates rapidly, its
facilitating effect diminishes during the initial part of a
spike train, thus producing a novel form of early spike
frequency adaptation. Furthermore, the function of BK
channels is highly frequency dependent. Thus, little or
no effect of BK channel blockade was observed at spike
frequencies below 40 Hz.
In addition to the intracellular recordings of
repetitive firing in response to somatic current injection,
extracellular field recordings demonstrated that BK
channel activity is also important for high-frequency
J Physiol 580.3
burst firing in response to excitatory synaptic input to
the distal dendrites (Fig. 11). The latter conclusion was
also supported by model simulations. Taken together, our
results strongly support the idea that BK channels play an
important role in facilitating early high-frequency firing
and fast adaptation in hippocampal pyramidal neurons,
thus promoting the type of bursting that is characteristic
of these cells in vivo.
Mechanisms whereby BK channels promote
high-frequency discharge and bursting
BK channels are known to play a major role in spike
repolarization and generation of the fAHP in hippocampal
pyramidal neurons (Storm, 1987a,b; Lancaster & Nicoll,
1987; Shao et al. 1999a; Hu et al. 2001) as well as in a
variety of other neurons (Adams et al. 1982; Schwindt et al.
1988; Sah & McLachlan, 1992; Vergara et al. 1998; Womack
& Khodakhah, 2002; Sah & Faber, 2002). Converging
evidence indicates that the ability of BK channels to open
significantly during a single action potential, i.e. within
about 1 ms or less, depends critically on both their close
co-localization (within 20–40 nm; Marrion & Tavalin,
1998; Berkefeld et al. 2006) with voltage-gated Ca2+
channels, and on the rapid intrinsic voltage-dependent
gating of the BK channels (Storm, 1987b; Lancaster &
Nicoll, 1987; Blatz & Magleby, 1987; Marty, 1989; Gola &
Crest, 1993; Marrion & Tavalin, 1998; Vergara et al. 1998;
Sah & Faber, 2002; Berkefeld et al. 2006).
The fast spike repolarization caused by the joint action
of BK channels and purely voltage-gated K+ channels, has
two main effects: (1) it limits the inactivation of the transient, spike-generating Na+ current (I NaT ), whose degree
of inactivation is important for determining the spike
threshold and therefore the timing of the next spike; and
(2) it limits the activation of slower voltage-dependent K+
conductances that are also activated by the spike. Because
of their slow deactivation, the latter are particularly
important for determining the interspike intervals. As the
M-, SK- and sAHP-currents are slow, important for spike
frequency regulation (Madison & Nicoll, 1984; Storm,
1989; Storm, 1990; Pedarzani & Storm, 1993; Gu et al. 2005;
Peters et al. 2005), and would be more strongly activated
by broadened spikes caused by BK channel blockade,
they might be suspected to contribute importantly to the
slowing of the discharge caused by BK channel blockers.
However, when we tested this idea experimentally, by
blocking M-, SK- and sAHP-channels, none of these
slow K+ conductances seemed to be important for the
BK-dependent facilitation of high-frequency discharge.
Our computer model yielded the same result: although
both I sAHP and I M had substantial effects on sustained
low-frequency firing (in agreement with Madison & Nicoll,
1984; Pedarzani & Storm, 1993; Gu et al. 2005; Peters et al.
2005), their effects on the initial high-frequency firing
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J Physiol 580.3
BK channels enhance high-frequency firing and adaptation
was small, and they contributed essentially nothing to the
BK-dependent facilitation (Fig. 6).
Instead, our simulations predict that the delayed
rectifier, I DR , is the main K+ current contributing to the
BK-dependent facilitation of high-frequency firing during
brief spike trains or bursts. The main reasons for the
dominant role of I DR are evident from Fig. 8: the amount
of I DR that is activated by a spike (3–4 nA) is far larger
than I sAHP and I M (0.003–0.6 nA), and the tail current
of I DR after each spike is sufficiently slow and large to
strongly delay the subsequent spike. The A-current, I A ,
is about as large as I DR during the first spike in the train
(∼3 nA), but because of the rapid deactivation kinetics of
I A , it decays almost to zero well before the next spike, and
has therefore little influence on spike timing or frequency
in this situation.
These model predictions are consistent with all our
experimental data so far, including the lack of effect of
SK channel blockade. (Recall that SK channels were not
included in this model because they do not seem to
contribute measurably to normal repetitive firing (Gu
et al. 2005) and we lack sufficiently detailed data for
CA1 pyramidal cells.) However, the precise role of I DR
must await further experimental testing. In contrast,
the role of Na+ inactivation is supported by convergent
evidence. Thus, the elevation of the spike threshold and
reduced dV /dt of the spike upstroke (following BK channel
blockade) are both consistent with this mechanism.
In addition to the consequences of BK-dependent rapid
spike repolarization discussed above, BK channels also
influence the initial discharge frequency by generating
the fAHP. On one hand, the fAHP should contribute
to refractoriness by hyperpolarizing the cell away from
the spike threshold immediately after the spike. On the
other hand, the fAHP is expected to promote both rapid
recovery from Na+ inactivation and rapid closure of
voltage-dependent K+ conductances (in particular I DR )
between the spikes. The relative importance of the two
latter factors depends sensitively on the exact voltage- and
time-dependences of these two processes, and must await
more detailed analysis to be quantitatively determined.
However, our experimental results as well as our model
simulations indicate that the facilitating effects of BK
channels dominate over fAHP-mediated refractoriness, at
least for the spike frequency range explored here. Thus,
the combined effects of spike duration-induced facilitation
(in particular, via reductions in I DR tail current and Na+
inactivation) and fAHP-induced facilitation (via speeding
of I DR closure and recovery from Na+ inactivation) are
evidently larger than the relatively modest hyperpolarizing
effect of the fAHP away from threshold.
The explanation outlined above is in accordance with
all our electrophysiological and modelling results. In
particular, it explains why application of a BK channel
blocker strongly reduced the early firing rate (Figs 1 and
877
3). In contrast, BK channel blockade had no detectable
effect on discharge frequency or the number of spikes in
response to a weak depolarizing current, yielding spike
frequencies less than 50 Hz (Figs 1 and 2). Our modelling
indicated that the lack of BK channel effects at low
discharge frequencies was caused by two factors: (1) a
nearly complete recovery from Na+ channel inactivation
between the spikes – even after IbTX-broadened spikes –
when the frequency is so low; and (2) a minimal effect of
the increased activation of DR channels by the broadened
spikes, because the DR channels have time to close between
the spikes at such low frequencies.
Which properties enable BK channels to facilitate
high-frequency firing?
Which properties make a K+ channel type primarily
inhibitory or excitatory/facilitating? Presumably, this is not
only determined by the properties of the K+ channel alone,
but by the entire biophysical context in which it operates:
the properties and distribution of various ion channels
within the cell, as well as its passive membrane properties
and geometry. Thus, K+ channels of the same type may
play different roles in different neuron types, in different
parts of the same neuron, and in different situations.
Nevertheless, it seems that some general properties are
needed to enable K+ channels to facilitate high-frequency
firing, and that these rules must also apply to BK channels
in CA1 pyramidal cells: (1) the channels should have
sufficiently rapid activation kinetics to turn on during a
spike; (2) they should produce a large outward current
contributing substantially to spike repolarization and/or
fast AHP (thus preventing strong Ca2+ influx, strong
activation of slower K+ currents, and strong Na+ channel
inactivation); (3) they should have deactivation kinetics
that are neither too fast nor too slow: i.e. the channels
should stay open long enough to ensure rapid, full spike
repolarization and a deep, fast AHP (thus promoting rapid
voltage-dependent closure of the slower K+ currents and
rapid recovery from Na+ channel inactivation); but on
the other hand the BK channels should close before the
earliest time at which the next spike may occur, in order
to avoid delaying it. These requirements place quite strict
boundaries on optimal BK channel kinetics. Since an
action potential in CA1 pyramidal cells lasts ∼1–2 ms, the
BK channels should activate substantially in ∼1 ms and
since these cells can fire at rates up to ∼250 Hz, i.e. with
∼4 ms intervals, and the spike lasts ∼1 ms, the BK channels
should deactivate nearly completely in < 3 ms, but not
much faster, in order to obtain a powerful repolarization
and fAHP. Interestingly, Lien & Jonas (2003) have given
a nice illustration of this point by showing directly, using
dynamic clamp, that the analogous role of Kv3 current
in interneurons depends on the deactivation kinetics in
exactly this way.
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N. Gu and others
BK current inactivation and a novel form of spike
frequency adaptation
As previously described, several lines of evidence indicate
that the BK current in CA1 pyramidal cells is transient,
i.e. it decays within a few milliseconds during sustained
depolarization (Storm, 1990; Halvorsrud et al. 1999; Shao
et al. 1999a; Vervaeke et al. 2006a). Therefore, the current
was dubbed ‘I CT ’, i.e. transient Ca2+ -activated K+ current,
by Storm (1990) and Shao et al. (1999a). Our group
has previously also shown how this inactivation produces
frequency-dependent spike broadening and rapid decay of
the fAHP during a high-frequency discharge (Shao et al.
1999a).
The inactivation of the BK current will also necessarily
influence excitability and spike frequency, in particular
spike frequency adaptation. As outlined above, the spike
frequency is reduced when I BK disappears, due to enhanced
I DR activation and Na+ channel inactivation causing
enhanced refractoriness. Therefore, the inactivation of I BK
during the initial spikes in a high-frequency train should
contribute to spike frequency adaptation. This logical,
qualitative prediction was confirmed by our modelling as
well as our experiments. For example, in Fig. 8A, the third
ISI is virtually identical with BK current (black, continuous
line: 130 pS μm−2 ) and without (red line), whereas the
first ISI is about 30% longer when the BK current is
missing, indicating reduced adaptation. A similar effect
was seen experimentally in response to IbTX (Fig. 2B).
Figure 8A also shows that the inactivation of I Na (I Na
I-state) increased following the 3rd spike compared with
the firsts spike with normal BK current (black line),
whereas there is no such increase without BK channels
(red line; actually the I-state here is slightly reduced). Thus,
because the BK current facilitates spiking but inactivates
rapidly, it contributes to early spike frequency adaptation.
Interestingly, this mechanism of I BK -dependent spike
frequency adaptation is almost exactly the opposite to
the ‘classical’ mechanism of K+ channel-dependent spike
frequency adaptation, which has been described for,
e.g. the ‘Ks’ current in molluscan neurons (Partridge &
Stevens, 1976), the M- and SK-currents in sympathetic
neurons (Adams et al. 1986), the M- and sAHP-currents in
hippocampal and neocortical neurons (Madison & Nicoll,
1984; Storm, 1989; Gu et al. 2005; Peters et al. 2005). In all
the latter cases, the adaptation results from a cumulative
build-up of a slowly activating K+ conductance, causing a
progressive reduction in excitability and spike frequency,
often in the form of a true feed-back regulation of spike
frequency (Partridge & Stevens, 1976; Storm, 1990; Hille,
2001). In contrast, in the I BK -dependent spike frequency
adaptation, the K+ conductance that is the primary cause
of the adaptation, g BK , enhances high-frequency firing
rather than slowing it down, and this K+ conductance
is decaying rather than building up. Only secondarily,
J Physiol 580.3
as a consequence of g BK inactivation and the resulting
spike broadening, is there an increased recruitment of
other, slower K+ conductances (mainly g DR ), but this
occurs by an increase in K+ conductance activation
by each (broadened) spike, rather than the cumulative
build-up of slowly deactivating K+ conductances, which
characterizes the classical cases of K+ current-dependent
adaptation, e.g. I Ks , I M , I Ks , I SK /I AHP , and I sAHP (Partridge
& Stevens, 1976; Adams et al. 1986; Madison & Nicoll,
1984; Storm, 1990; Gu et al. 2005). Thus, I BK -dependent
adaptation is due to a selective K+ conductance-dependent
facilitation of the early spikes rather than a selective
K+ conductance-dependent reduction of the later spike
frequency. To our knowledge, this type of adaptation
mechanism has not previously been described.
Comparison with previous results
In view of the widespread idea that K+ channels are
mainly inhibitory, serving to limit excitation (e.g. Connor
& Stevens, 1971; Partridge & Stevens, 1976; Madison
& Nicoll, 1982; Madison & Nicoll, 1984; Storm, 1990;
Hille, 2001), it is perhaps not surprising that previous
reports mention only the inhibitory role of BK channels
(Lancaster & Nicoll, 1987; Alger & Williamson, 1988;
Williamson & Alger, 1990; Storm, 1990), and the same
limitation applies to both early and recent reviews of
BK channels and their functions (Blatz & Magleby, 1987;
Marty, 1989; Sah & McLachlan, 1992; Vergara et al. 1998;
Salkoff et al. 2006). Thus, based on the report that BK
channel blockade increased the initial discharge frequency
and reduced burst duration in CA1 pyramidal cells, it was
previously suggested that the effect of BK channels is to
reduce the spike frequency and excitability (Lancaster &
Nicoll, 1987; Alger & Williamson, 1988; Williamson &
Alger, 1990). In apparent agreement, subsequent studies
suggested that BK channels contribute significantly to
the medium AHP following a spike burst, in particular
when the cell was depolarized (Williamson & Alger, 1990).
Other evidence also suggested that these channels may
generate a small mAHP component (in current clamp)
and a corresponding tail current (in voltage clamp) under
artificial conditions (when several other K+ currents had
been suppressed by 40 μm carbachol, etc.; Lancaster &
Adams, 1986; Storm, 1989). These data were interpreted as
support for an I BK component of the mAHP and early spike
frequency adaptation (Lancaster & Nicoll, 1987; Storm,
1990; Williamson & Alger, 1990).
As discussed above, our data clearly support the
conclusion that I BK contributes to early spike frequency
adaptation, although the mechanism we find is very
different from the one proposed originally. However,
our observation that both the selective (IbTX) and nonselective (TEA) BK channel blockers consistently inhibited
the high-frequency discharge (Figs 1–3), indicates that the
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J Physiol 580.3
BK channels enhance high-frequency firing and adaptation
dominating effect of BK channels in CA1 pyramidal cells
is facilitating. This result does not, however, preclude that
I BK may contribute a small component to the mAHP.
(However, unpublished data suggest that BK channels do
not contribute appreciably to the mAHP. This is the theme
of a separate study; N. Gu & J. F. Storm, in preparation).
Although there are slight differences in the experimental
conditions between this study and those of Lancaster
& Nicoll (1987) and Williamson & Alger (1990), they
all used essentially the same approach (sharp electrode
intracellular recordings from CA1 pyramidal in rat
hippocampal slices at 20–31◦ C, etc.). Why, then, did
Lancaster & Nicoll (1987) find a reduced spike frequency
after blocking the BK channels whereas we consistently
found the opposite?
Two factors may help to explain the apparent
discrepancy. (1) We observed the strongest effects of
IbTX at frequencies higher than ∼50 Hz (Fig. 5), whereas
Lancaster & Nicoll (1987) limited their tests of BK channel
blockade (by ChTX or TEA) to spike frequencies of ∼50 Hz
or less (see their Figs 3 and 8), and ChTX was tested
in only two cells. Therefore, a facilitating effect of I BK
might have been overlooked. (2) Their and other early
studies relied on channel blockers and manipulations
(TEA, ChTX, Mn2+ , Cd2+ , Ca2+ -free medium) that are not
selective for BK channels, because selective blockers were
not available at the time (Lancaster & Nicoll, 1987; Storm,
1989; Williamson & Alger, 1990). Thus, the effects might
have been partly due to other channels than BK. However,
this explanation may not apply to ChTX (Lancaster &
Nicoll, 1987; Williamson & Alger, 1990), since no other
ChTX-sensitive channels than BK have been shown to exist
in rat CA1 pyramidal cells. Thus, the ChTX-sensitive Kv1.3
channels do not appear to be significantly expressed in this
cell type (Beckh & Pongs, 1990).
The early results of Williamson & Alger (1990) and
Storm (1989) may have been influenced by other factors.
(i) As was common at that time, KCl-filled microelectrodes, which cause artificially high intracellular Cl−
concentration, were used to achieve good voltage-clamp
recordings (Storm, 1989; Williamson & Alger, 1990). Since
both muscarinic agonists and high [Cl− ]i may affect
multiple channel types (Akaike & Sadoshima, 1989; Zhang
et al. 1994), the results obtained under those conditions
are probably less representative of the physiological state
than the present results, which were obtained with highly
selective channel blockers and intracellular media that are
likely to cause minimal perturbations (Zhang et al. 1994).
(ii) Since a selective M-channel blocker was unavailable
until XE991 was introduced in 1998 (Wang et al. 1998), a
high dose of the cholinergic agonist carbachol (40 μm) was
used to first eliminate I M (and I sAHP ), in order to study the
putative BK channel contribution in relative isolation (see
Fig. 4 in Storm, 1989). However, this strong cholinergic
stimulation may also have affected the BK channels or the
879
Ca2+ transients that activate them. Therefore, the results
from those previous studies do not really contradict the
results of our present study. In conclusion, the available
evidence seems to support the conclusion that BK channels
facilitate high-frequency discharge in CA1 pyramidal cells
under normal conditions.
Comparison with other cell types
Although K+ channels are often portrayed as inhibitory, it
has been known for decades that fast spike repolarization
caused by K+ currents is important for high-frequency
firing (Hodgkin & Huxley, 1952; Hille, 2001). More
recently, it has been described how channels of the Kv3
family (mainly Kv3.1 and Kv3.2), due to their unusually fast
activating and deactivating properties, support sustained
high-frequency spike trains, by producing brief spikes and
fast deep afterhyperpolariztions (AHPs) (Rudy et al. 1999).
For example, blockade of Kv3 channels in interneurons
produced cumulative Na+ channel inactivation, causing
slowing of the firing rate (Erisir et al. 1999; Lien & Jonas,
2003). In the light of these studies, it is not surprising that
BK channels, by causing relatively fast spike repolarization
and fAHP in pyramidal cells, can facilitate high-frequency
firing. Since BK channels contribute to spike repolarization
and fAHPs in many neurons (Adams et al. 1982; Storm,
1987a; Lancaster & Nicoll, 1987; Schwindt et al. 1988;
Womack & Khodakhah, 2002), it is likely that their
facilitating effect is a widespread phenomenon. This is
supported by a recent paper, which appeared while this
study was in progress (Brenner et al. 2005). It reports
that BK channels in mice lacking BK β 4 -subunits promote
repetitive firing in dentate granule cells by narrowing the
spike, thus reducing the duration of the AHP. The authors
also suggest that this effect, which resembles the one we
report here, is a likely cause of the temporal lobe seizures
of this mouse strain (Brenner et al. 2005).
Clinical implications
Recently, a familial form of human epilepsy and
paroxysmal dyskinesia was found to be associated with
a gain-of-function mutation in the BK channel α-subunit
(Du et al. 2005). The mutation caused a ∼4-fold increase
in the Ca2+ sensitivity of the channel, leading to a larger
and faster BK current. It was speculated that this might
cause increased brain excitability because of (1) faster
spike repolarization and reduced Na+ inactivation, or
(2) hyperpolarization-induced bursting as in absence
seizures, or (3) hyperpolarization-induced activation of
the h-current (Du et al. 2005). In view of the results
presented here and those of Brenner et al. (2005), it seems
likely that the first of these mechanisms is important
for this form of epilepsy in humans. The results of Du
et al. (2005) provide a fascinating example of how the
BK channel-induced facilitation mechanisms described in
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N. Gu and others
this study might be of functional and clinical importance.
In addition to these mechanisms, reduced excitability of
GABAergic neurons (Zhang & McBain, 1995) or effects at
GABAergic synapses (Goldberg et al. 2005) might underlie
the epilepsy caused by the gain-of-function BK channel
mutation (Du et al. 2005).
References
Adams PR, Constanti A, Brown DA & Clark RB (1982).
Intracellular Ca2+ activates a fast voltage-sensitive K+
current in vertebrate sympathetic neurones. Nature 296,
746–749.
Adams PR, Jones SW, Pennefather P, Brown DA, Koch C &
Lancaster B (1986). Slow synaptic transmission in frog
sympathetic ganglia. J Exp Biol 124, 259–285.
Akaike N & Sadoshima J (1989). Caffeine affects four different
ionic currents in the bull-frog sympathetic neurone. J Physiol
412, 221–244.
Alger BE & Williamson A (1988). A transient calciumdependent potassium component of the epileptiform burst
after-hyperpolarization in rat hippocampus. J Physiol 399,
191–205.
Beckh S & Pongs O (1990). Members of the RCK potassium
channel family are differentially expressed in the rat nervous
system. EMBO J 9, 777–782.
Berkefeld H, Sailer CA, Bildl W, Rohde V, Thumfart JO, Eble S,
Klugbauer N, Reisinger E, Bischofberger J, Oliver D, Knaus
HG, Schulte U & Fakler B (2006). BKCa-Cav channel
complexes mediate rapid and localized Ca2+ -activated K+
signaling. Science 314, 615–620.
Blatz AL & Magleby KL (1987). Calcium-activated potassium
channels. Trends Neurosci 10, 463–467.
Borg-Graham L (1987). Modelling the somatic electrical
behaviour of hippocampal pyramidal neurons. Master’s
Thesis, Massachusetts Institute of Technology, Cambridge,
MA, USA (also appears at MIT AI laboratory Technical
report 1161) Ref Type: Thesis/Dissertation.
Borg-Graham L (1999). Interpretations of data and
mechanisms for hippocampal pyramidal cell models. In
Cerebral Cortex, Vol. 12: Cortical Models, ed. Jones EG,
Ulinski PS & Peter A. Plenum Press, New York.
Borg-Graham LJ (2000). Additional efficient computation of
branched nerve equations: adaptive time step and ideal
voltage clamp. J Comput Neurosci 8, 209–226.
Brenner R, Chen QH, Vilaythong A, Toney GM, Noebels JL &
Aldrich RW (2005). BK channel β4 subunit reduces dentate
gyrus excitability and protects against temporal lobe
seizures. Nat Neurosci 8, 1752–1759.
Brown DA & Adams PR (1980). Muscarinic suppression of a
novel voltage-sensitive K+ current in a vertebrate neurone.
Nature 283, 673–676.
Browne DL, Gancher ST, Nutt JG, Brunt ER, Smith EA, Kramer
P & Litt M (1994). Episodic ataxia/myokymia syndrome is
associated with point mutations in the human potassium
channel gene, KCNA1. Nat Genet 8, 136–140.
Cai X, Liang CW, Muralidharan S, Kao JP, Tang CM &
Thompson SM (2004). Unique roles of SK and Kv4.2
potassium channels in dendritic integration. Neuron 44,
351–364.
J Physiol 580.3
Connor JA & Stevens CF (1971). Prediction of repetitive firing
behaviour from voltage clamp data on an isolated neurone
soma. J Physiol 213, 31–53.
Du J, Haak LL, Phillips-Tansey E, Russell JT & McBain CJ
(2000). Frequency-dependent regulation of rat hippocampal
somato-dendritic excitability by the K+ channel subunit
Kv2.1. J Physiol 522, 19–31.
Du W, Bautista JF, Yang H, ez-Sampedro A, You SA, Wang L,
Kotagal P, Luders HO, Shi J, Cui J, Richerson GB & Wang
QK (2005). Calcium-sensitive potassium channelopathy in
human epilepsy and paroxysmal movement disorder. Nat
Genet 37, 733–738.
Erisir A, Lau D, Rudy B & Leonard CS (1999). Function of
specific K+ channels in sustained high-frequency firing of
fast-spiking neocortical interneurons. J Neurophysiol 82,
2476–2489.
Galvez A, Gimenez-Gallego G, Reuben JP, Roy-Contancin L,
Feigenbaum P, Kaczorowski GJ & Garcia ML (1990).
Purification and characterization of a unique, potent,
peptidyl probe for the high conductance calcium-activated
potassium channel from venom of the scorpion Buthus
tamulus. J Biol Chem 265, 11083–11090.
Gho M & Ganetzky B (1992). Analysis of repolarization of
presynaptic motor terminals in Drosophila larvae using
potassium-channel-blocking drugs and mutations. J Exp Biol
170, 93–111.
Gola M & Crest M (1993). Colocalization of active KCa
channels and Ca2+ channels within Ca2+ domains in helix
neurons. Neuron 10, 689–699.
Goldberg EM, Watanabe S, Chang SY, Joho RH, Huang ZJ,
Leonard CS & Rudy B (2005). Specific functions of
synaptically localized potassium channels in synaptic
transmission at the neocortical GABAergic fast-spiking cell
synapse. J Neurosci 25, 5230–5235.
Graham LJ (2004). The Surf-Hippo Neuron Simulation
System, v3.5a Ref Type: Computer Program http://www.
neurophys.biomedicale.univ-paris5.fr/∼graham/surf-hippo.
html
Gu N, Vervaeke K, Hu H & Storm JF (2005). Kv7/KCNQ/M and
HCN/h, but not KCa2/SK channels, contribute to the
somatic medium after-hyperpolarization and excitability
control in CA1 hippocampal pyramidal cells. J Physiol 566,
689–715.
Gu N, Vervaeke K & Storm JF (2006). Large-conductance
calcium-activated K+ channels (BK) channels promote
high-frequency firing in rat CA1 hippocampal pyramidal
neurons. FENS Forum Abstracts 3, A189.7.
Gustafsson B, Galvan M, Grafe P & Wigstrom H (1982). A
transient outward current in a mammalian central
neurone blocked by 4-aminopyridine. Nature 299,
252–254.
Hadley JK, Noda M, Selyanko AA, Wood IC, Abogadie FC &
Brown DA (2000). Differential tetraethylammonium
sensitivity of KCNQ1-4 potassium channels. Br J Pharmacol
129, 413–415.
Halvorsrud R, Shao LR, Bouskila Y, Ramakers GM & Storm JF
(1999). Evidence that BK-type Ca2+ -dependent K+ channels
contribute to frequency-dependent action potential
broadening in rat CA1 hippocampal pyramidal cells. Abstr
Soc Neurosci 25, 453.
C 2007 The Authors. Journal compilation C 2007 The Physiological Society
Downloaded from J Physiol (jp.physoc.org) at UNIV OF TEXAS-AUSTIN on February 24, 2010
J Physiol 580.3
BK channels enhance high-frequency firing and adaptation
Harris KD, Hirase H, Leinekugel X, Henze DA & Buzsaki G
(2001). Temporal interaction between single spikes and
complex spike bursts in hippocampal pyramidal cells.
Neuron 32, 141–149.
Hille B (2001). Ion Channels of Excitable Membranes, 3rd edn.
Sinauer Associates, Inc., Sunderland, MA, USA.
Hines M (1984). Efficient computation of branched nerve
equations. Int J Biomed Comput 15, 69–76.
Hodgkin AL & Huxley AF (1952). Currents carried by sodium
and potassium ions through the membrane of the giant axon
of Loligo. J Physiol 116, 449–472.
Hoffman DA, Magee JC, Colbert CM & Johnston D (1997). K+
channel regulation of signal propagation in dendrites of
hippocampal pyramidal neurons. Nature 387, 869–875.
Hotson JR & Prince DA (1981). Penicillin- and barium-induced
epileptiform bursting in hippocampal neurons: actions on
Ca2+ and K+ potentials. Ann Neurol 10, 11–17.
Hu H, Shao LR, Chavoshy S, Gu N, Trieb M, Behrens R, Laake
P, Pongs O, Knaus HG, Ottersen OP & Storm JF (2001).
Presynaptic Ca2+ -activated K+ channels in glutamatergic
hippocampal terminals and their role in spike repolarization
and regulation of transmitter release. J Neurosci 21,
9585–9597.
Hu H, Vervaeke K & Storm JF (2002). Two forms of electrical
resonance at theta frequencies, generated by M-current,
h-current and persistent Na+ current in rat hippocampal
pyramidal cells. J Physiol 545, 783–805.
Jin W, Sugaya A, Tsuda T, Ohguchi H & Sugaya E (2000).
Relationship between large conductance calcium-activated
potassium channel and bursting activity. Brain Res 860,
21–28.
Johnston D, Hoffman DA, Magee JC, Poolos NP, Watanabe S,
Colbert CM & Migliore M (2000). Dendritic potassium
channels in hippocampal pyramidal neurons. J Physiol 525,
75–81.
Lancaster B & Adams PR (1986). Calcium-dependent current
generating the afterhyperpolarization of hippocampal
neurons. J Neurophysiol 55, 1268–1282.
Lancaster B & Nicoll RA (1987). Properties of two calciumactivated hyperpolarizations in rat hippocampal neurones.
J Physiol 389, 187–203.
Lien CC & Jonas P (2003). Kv3 potassium conductance is
necessary and kinetically optimized for high-frequency
action potential generation in hippocampal interneurons.
J Neurosci 23, 2058–2068.
Madison DV & Nicoll RA (1982). Noradrenaline blocks
accommodation of pyramidal cell discharge in the
hippocampus. Nature 299, 636–638.
Madison DV & Nicoll RA (1984). Control of the repetitive
discharge of rat CA 1 pyramidal neurones in vitro. J Physiol
354, 319–331.
Marrion NV & Tavalin SJ (1998). Selective activation of
Ca2+ -activated K+ channels by co-localized Ca2+
channels in hippocampal neurons. Nature 395,
900–905.
Marty A (1989). The physiological role of calcium-dependent
channels. Trends Neurosci 12, 420–424.
Nelson AB, Krispel CM, du Sekirnjak C & LS (2003).
Long-lasting increases in intrinsic excitability triggered by
inhibition. Neuron 40, 609–620.
881
Ngo-Anh TJ, Bloodgood BL, Lin M, Sabatini BL, Maylie J &
Adelman JP (2005). SK channels and NMDA receptors form
a Ca2+ -mediated feedback loop in dendritic spines. Nat
Neurosci 8, 642–649.
Partridge LD & Stevens CF (1976). A mechanism for spike
frequency adaptation. J Physiol 256, 315–332.
Pedarzani P & Storm JF (1993). PKA mediates the effects of
monoamine transmitters on the K+ current underlying the
slow spike frequency adaptation in hippocampal neurons.
Neuron 11, 1023–1035.
Peters HC, Hu H, Pongs O, Storm JF & Isbrandt D (2005).
Conditional transgenic suppression of M channels in mouse
brain reveals functions in neuronal excitability, resonance
and behavior. Nat Neurosci 8, 51–60.
Rall W, Burke RE, Holmes WR, Jack JJ, Redman SJ & Segev I
(1992). Matching dendritic neuron models to experimental
data. Physiol Rev 72, S159–S186.
Ramakers GM & Storm JF (2002). A postsynaptic transient K+
current modulated by arachidonic acid regulates synaptic
integration and threshold for LTP induction in hippocampal
pyramidal cells. Proc Natl Acad Sci U S A 99,
10144–10149.
Ranck JB Jr (1973). Studies on single neurons in dorsal
hippocampal formation and septum in unrestrained rats. I.
Behavioral correlates and firing repertoires. Exp Neurol 41,
461–531.
Rudy B, Chow A, Lau D, Amarillo Y, Ozaita A, Saganich M,
Moreno H, Nadal MS, Hernandez-Pineda R,
Hernandez-Cruz A, Erisir A, Leonard C & Vega-Saenz de ME
(1999). Contributions of Kv3 channels to neuronal
excitability. Ann N Y Acad Sci 868, 304–343.
Rudy B & Mcbain CJ (2001). Kv3 channels: voltage-gated K+
channels designed for high-frequency repetitive firing.
Trends Neurosci 24, 517–526.
Rutecki PA, Lebeda FJ & Johnston D (1987). 4-Aminopyridine
produces epileptiform activity in hippocampus and
enhances synaptic excitation and inhibition. J Neurophysiol
57, 1911–1924.
Sah P & Faber ES (2002). Channels underlying neuronal
calcium-activated potassium currents. Prog Neurobiol 66,
345–353.
Sah P & McLachlan EM (1992). Potassium currents
contributing to action potential repolarization and the
afterhyperpolarization in rat vagal motoneurons.
J Neurophysiol 68, 1834–1841.
Salkoff L, Butler A, Ferreira G, Santi C & Wei A (2006).
High-conductance potassium channels of the SLO family.
Nat Rev Neurosci 7, 921–931.
Sausbier M, Hu H, Arntz C, Feil S, Kamm S, Adelsberger H
et al. (2004). Cerebellar ataxia and Purkinje cell dysfunction
caused by Ca2+ -activated K+ channel deficiency. Proc Natl
Acad Sci U S A 101, 9474–9478.
Schwindt PC, Spain WJ, Foehring RC, Stafstrom CE, Chubb
MC & Crill WE (1988). Multiple potassium conductances
and their functions in neurons from cat sensorimotor cortex
in vitro. J Neurophysiol 59, 424–449.
Shao LR, Halvorsrud R, Borg-Graham L & Storm JF (1999a).
The role of BK-type Ca2+ -dependent K+ channels in spike
broadening during repetitive firing in rat hippocampal
pyramidal cells. J Physiol 521, 135–146.
C 2007 The Authors. Journal compilation C 2007 The Physiological Society
Downloaded from J Physiol (jp.physoc.org) at UNIV OF TEXAS-AUSTIN on February 24, 2010
882
N. Gu and others
Shao LR, Halvorsrud R, Bouskila Y, Ramakers GM,
Borg-Graham L & Storm JF (1999b). Evidence that BK-type
Ca2+ -dependent K+ channels contribute to frequencydependent action potential broadening in rat CA1
hippocampal pyramidal cells. Physiologist 42, A-21.
Smart SL, Lopantsev V, Zhang CL, Robbins CA, Wang H, Chiu
SY, Schwartzkroin PA, Messing A & Tempel BL (1998).
Deletion of the KV 1.1 potassium channel causes epilepsy in
mice. Neuron 20, 809–819.
Steinlein OK (2001). Genes and mutations in idiopathic
epilepsy. Am J Med Genet 106, 139–145.
Stocker M, Krause M & Pedarzani P (1999). An apaminsensitive Ca2+ -activated K+ current in hippocampal
pyramidal neurons. Proc Natl Acad Sci U S A 96, 4662–4667.
Storm JF (1987a). Action potential repolarization and a fast
after-hyperpolarization in rat hippocampal pyramidal cells.
J Physiol 385, 733–759.
Storm JF (1987b). Intracellular injection of a Ca2+ chelator
inhibits spike repolarization in hippocampal neurons. Brain
Res 435, 387–392.
Storm JF (1988). Temporal integration by a slowly inactivating
K+ current in hippocampal neurons. Nature 336, 379–381.
Storm JF (1989). An after-hyperpolarization of medium
duration in rat hippocampal pyramidal cells. J Physiol 409,
171–190.
Storm JF (1990). Potassium currents in hippocampal
pyramidal cells. Prog Brain Res 83, 161–187.
Storm JF, Sartorius T, Gu N, Sausbier M, Sausbier U & Ruth P
(2006). Alterations in cortical EEG activity, neuronal
excitability and sleep-wake behavior in BK channel-deficient
mice. Abstr Soc Neurosci 627.9, D23.
Stuart G & Spruston N (1998). Determinants of voltage
attenuation in neocortical pyramidal neuron dendrites.
J Neurosci 18, 3501–3510.
Swensen AM & Bean BP (2003). Ionic mechanisms of burst
firing in dissociated Purkinje neurons. J Neurosci 23,
9650–9663.
Takahashi T (1990). Membrane currents in visually identified
motoneurones of neonatal rat spinal cord. J Physiol 423,
27–46.
Vergara C, Latorre R, Marrion NV & Adelman JP (1998).
Calcium-activated potassium channels. Curr Opin Neurobiol
8, 321–329.
Vervaeke K, Gu N & Storm JF (2006a). Large conductance
calcium activated K+ (BK) channels promote highfrequency firing in rat CA1 hippocampal pyramidal neurons.
Abstr Soc Neurosci 627.7, D21.
Vervaeke K, Hu H, Graham LJ & Storm JF (2006b). Contrasting
effects of the persistent Na+ current on neuronal excitability
and spike timing. Neuron 49, 257–270.
Wang HS, Pan Z, Shi W, Brown BS, Wymore RS, Cohen IS,
Dixon JE & Mckinnon D (1998). KCNQ2 and KCNQ3
potassium channel subunits: molecular correlates of the
M-channel. Science 282, 1890–1893.
J Physiol 580.3
Williamson A & Alger BE (1990). Characterization of an early
afterhyperpolarization after a brief train of action potentials
in rat hippocampal neurons in vitro. J Neurophysiol 63,
72–81.
Womack MD & Khodakhah K (2002). Characterization of large
conductance Ca2+ -activated K+ channels in cerebellar
Purkinje neurons. Eur J Neurosci 16, 1214–1222.
Zbicz KL & Weight FF (1985). Transient voltage and calciumdependent outward currents in hippocampal CA3 pyramidal
neurons. J Neurophysiol 53, 1038–1058.
Zhang XF, Gopalakrishnan M & Shieh CC (2003). Modulation
of action potential firing by iberiotoxin and NS1619 in rat
dorsal root ganglion neurons. Neuroscience 122, 1003–1011.
Zhang L & McBain CJ (1995). Potassium conductances
underlying repolarization and after-hyperpolarization in rat
CA1 hippocampal interneurones. J Physiol 488, 661–672.
Zhang L, Weiner JL, Valiante TA, Velumian AA, Watson PL,
Jahromi SS, Schertzer S, Pennefather P & Carlen PL (1994).
Whole-cell recording of the Ca2+ -dependent slow
afterhyperpolarization in hippocampal neurones: effects of
internally applied anions. Pflugers Arch 426, 247–253.
Acknowledgements
We thank Lyle Graham, university Rene Descartes, Paris, for
the continuous development and support of the Surf-Hippo
simulator. This study was supported by the Norwegian Research
Council (N.F.R.) via the MH, FUGE, EMBIO and Norwegian
Centre of Excellence programmes. G.N. performed all the
intracellular current-clamp experiments included in this study
(including those presented in Figs 1–4, 5A and B, 7 and 9A–C);
K.V. performed the computer modelling (illustrated in Figs 5C
and D, 6, 8, 9D–F and 10) and extracellular field potential
recording illustrated in Fig. 11. J.F.S. performed pilot intracellular current-clamp experiments. The manuscript was mainly
written by G.N. and J.F.S.
Supplemental material
The online version of this paper can be accessed at:
DOI: 10.1113/jphysiol.2006.126367
http://jp.physoc.org/cgi/content/full/jphysiol.2006.
126367/DC1
and contains supplemental material entitled: Supplemental
computational methods (reprinted from Neuron 49, 257–270.
Vervaeke K, Hu H, Graham LJ & Storm JF (2006). Contrasting
effects of the persistent Na+ current on neuronal excitability and
spike timing, with permission from Elsevier).
This material can also be found as part of the full-text
HTML version available from http://www.blackwellsynergy.com
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