Profound alterations in the intrinsic excitability of cerebellar Purkinje neurons following neurotoxin 3-acetylpyridine (3-AP)-induced ataxia in rat: new insights into the role of small conductance K+ channels.
ABSTRACT Alterations in the intrinsic properties of Purkinje cells (PCs) may contribute to the abnormal motor performance observed in ataxic rats. To investigate whether such changes in the intrinsic neuronal excitability could be attributed to the role of Ca(2+)-activated K(+) channels (K(Ca)), whole cell current clamp recordings were made from PCs in cerebellar slices of control and ataxic rats. 3-AP induced profound alterations in the intrinsic properties of PCs, as evidenced by a significant increase in both the membrane input resistance and the initial discharge frequency, along with the disruption of the firing regularity. In control PCs, the blockade of small conductance K(Ca) channels by UCL1684 resulted in a significant increase in the membrane input resistance, action potential (AP) half-width, time to peak of the AP and initial discharge frequency. SK channel blockade also significantly decreased the neuronal discharge regularity, the peak amplitude of the AP, the amplitude of the afterhyperpolarization and the spike frequency adaptation ratio. In contrast, in ataxic rats, both the firing regularity and the initial firing frequency were significantly increased by the blockade of SK channels. In conclusion, ataxia may arise from alterations in the functional contribution of SK channels, to the intrinsic properties of PCs.
PHYSIOLOGICAL RESEARCH • ISSN 0862-8408 (print) • ISSN 1802-9973 (online)
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Physiol. Res. 60: 355-365, 2011
Profound Alterations in the Intrinsic Excitability of Cerebellar
Purkinje Neurons Following Neurotoxin 3-Acetylpyridine (3-AP)-
Induced Ataxia in Rat: New Insights Into the Role of Small
Conductance K+ Channels
M. KAFFASHIAN1,2, M. SHABANI3, I. GOUDARZI4, G. BEHZADI1, A. ZALI3,
1Neuroscience Research Center and Department of Physiology, Medical School, Shahid Beheshti
University of Medical Sciences, Evin, Tehran, Iran, 2Department of Physiology, Faculty of
Medicine, Ilam University of Medical Sciences, Ilam, Iran, 3Neuroscience Research Center and
Physiology Research Center, Kerman University of Medical Sciences, Kerman, Iran, 4Department
of Biology, Damghan University of Basic Sciences, Damghan, Iran,5Department of Neurosurgery,
Shohada Hospital, Shahid Beheshti University of Medical Sciences, Tehran, Iran
Received May 17, 2010
Accepted September 17, 2010
On-line November 29, 2010
Alterations in the intrinsic properties of Purkinje cells (PCs) may
contribute to the abnormal motor performance observed in ataxic
rats. To investigate whether such changes in the intrinsic
neuronal excitability could be attributed to the role of
Ca2+-activated K+ channels (KCa), whole cell current clamp
recordings were made from PCs in cerebellar slices of control and
ataxic rats. 3-AP induced profound alterations in the intrinsic
properties of PCs, as evidenced by a significant increase in both
the membrane input resistance and the initial discharge
frequency, along with the disruption of the firing regularity. In
control PCs, the blockade of small conductance KCa channels by
UCL1684 resulted in a significant increase in the membrane input
resistance, action potential (AP) half-width, time to peak of the
AP and initial discharge frequency. SK channel blockade also
significantly decreased the neuronal discharge regularity, the
peak amplitude of the AP, the amplitude of the after-
hyperpolarization and the spike frequency adaptation ratio. In
contrast, in ataxic rats, both the firing regularity and the initial
firing frequency were significantly increased by the blockade of
SK channels. In conclusion, ataxia may arise from alterations in
the functional contribution of SK channels, to the intrinsic
properties of PCs.
• Intrinsic excitability
3-acetylpyridine • Purkinje neurons • Small conductance
Ca2+-activated K+ channels
M. Janahmadi, Neuroscience Research Centre and Department of
Physiology, Medical School, Shahid Beheshti University of Medical
Sciences, Evin, Tehran, Iran. PO. Box 19615-1178. E-mail:
email@example.com or Janahmadi@sbmu.ac.ir
Dysfunction or degeneration of Purkinje cells,
the key neuronal cells of the cerebellar circuitry, causes
ataxia. In our previous study, we showed that neurotoxin
3-acetylpyridine (3-AP) induced cerebellar ataxia, which
was associated with motor incoordination and alterations
in the morphological and intrinsic electrophysiological
characteristics of Purkinje cells (Janahmadi et al. 2009).
However, the cellular mechanisms underlying these
changes have not yet been fully determined. Intrinsic
neuronal excitability, which refers to the ability of a
neuron to fire action potentials in the absence of synaptic
356 Kaffashian et al.
inputs, could be directly attributed to the biochemical and
electrophysiological properties of intrinsic membrane
channels (Schulz 2006, Russo et al. 2008), so that
alterations in the function of these channels may lead to
plasticity in neuronal excitability and in neural circuits to
which they belong. Among the wide range of ion
channels, potassium channels, which are the largest and
most diverse group of ion channels, play a main
regulatory role in excitable cells. Consequently,
malfunction of these channels can result in several
neurological disorders, including ataxia. In animal
models of ataxia, Sausbier et al. (2004) reported that
Ca2+-activated K+ channel deficiency causes dysfunction
of Purkinje cells (PCs) and thereby results in cerebellar
ataxia. At the cellular level, they showed that cerebellar
Purkinje cells from mice lacking big conductance K+
channels display a dramatic reduction in spontaneous
activity. A remarkable reduction in the amplitude of post-
stimulus afterhyperpolarization (AHP), which is linked to
Ca2+-activated K+ conductance, was also recently
reported in a rat model of ataxia (Janahmadi et al. 2009).
In many neurons of the central nervous system, including
PCs, both large conductance K+ (BK) and small
conductance K+ (SK) channels are found (Shepard and
Bunney 1991, Sah 1996, Shah and Haylett 2000,
Cingolani et al. 2002, Faber and Sah 2002, Edgerton and
Reinhart 2003, Haghdoost-Yazdi et al. 2007, Maingret et
al. 2008), where they play an important role in the
regulation of neuronal excitability. Although the
physiological roles of calcium-dependent potassium
channels have been well documented, their functional
contribution to the pathophysiology of neurological
diseases (e.g. ataxia) is still poorly understood. To assess
the possible functional contribution of SK channels to the
altered intrinsic electrical properties of Purkinje neurons
in a rat model of cerebellar ataxia induced by
3-acetylpyridine (3-AP) (Janahmadi et al. 2009), the
effect of UCL 1684, a potent SK channel blocker, was
examined. 3-AP, which is a neurotoxin and a niacinamide
receptor antagonist, it is known to be an effective agent
that selectively destroys inferior olive neurons, the major
source of the climbing fibres innervating the cerebellar
Purkinje neurons (Balaban 1985, Torres-Aleman et al.
1996, Caddy and Vozeh 1997, Seoane et al. 2005). As a
result, the cerebellar cortex loses its climbing fibre input,
and the rats become ataxic (Llinás et al. 1975). It is well-
documented that the climbing input to a Purkinje neuron
is essential for normal cerebellar function because it
controls the intrinsic properties of the output of PCs
(Cerminara and Rawson 2004, McKay et al. 2007).
Fourteen Wistar rats (3 to 4 weeks old) were
used in this study. The rats were divided into two groups
consisted of 8 rats in control group and 6 rats in 3-AP-
treated group. All animals (were housed at 22 °C and
maintained on a 12:12 h light/dark cycle with free access
to food and water. All procedures for the maintenance
and the use of the experimental animals were approved
by the Institutional Ethics Committee (IEC) at the
University of Shahid Beheshti Medical Sciences. To
induce ataxia, rats were given a single i.p. 65 mg/kg dose
of 3-acetylpyridine (Sigma-Aldrich)
physiological saline (Janahmadi et al. 2009). Our
previous results showed that there was no significant
difference between the behavioral responses in control
(untreated) and vehicle
(Janahmadi et al. 2009, Goudarzi et al. 2010). Therefore,
statistical comparison was performed between control
(untreated) and 3-AP-treated groups. The dose of
neurotoxin was chosen on the basis of previous studies
demonstrating that it caused a severe motor impairment
and induced cerebellar ataxia (Janahmadi et al. 2009).
Next, the electrophysiological recordings were done at
day 0 (pre-test) and one day after ataxia induction.
Animals were deeply anesthetized by inhalation
of ether and then decapitated. The rat brains were rapidly
removed and the cerebellar vermis was then dissected and
placed in ice-cold artificial cerebrospinal fluid (ACSF)
containing (in mM): 124 NaCl, 5 KCl, 1.2 KH2PO4,
1.3 MgSO4, 2.4 CaCl2, 26 NaHCO3 and 10 glucose,
bubbled continuously with carbogen gas (95 % oxygen
and 5 % carbon dioxide) to adjust the pH to 7.4.
Parasagittal slices (300 µm thick) were cut from the
vermis of rats using a vibroslicer (752M, Campden
Instruments Ltd, UK). The slices were incubated at 36 °C
for >30 min and then stored at room temperature.
Whole cell patch current clamp recording
After recovery (>1 h) at 22-25 °C, a single slice
was transferred to a submerged chamber mounted on the
stage of an upright microscope (Olympus; BX 51W) and
continuously superfused with oxygenated ACSF. The
flow rate was kept at 1-2 ml/min using a peristaltic pump
(Hugo Sachs Electronik, Ismatec, Germany). To study the
Alteration in SK Channel Function Causes Cerebellar Ataxia 357
intrinsic firing characteristics of Purkinje neurons, 1 mM
kynurenic acid and 100 µM picrotoxin, the blockers of
ionotropic glutamate (Stone 1993) and GABA (Yoon et
al. 1993) receptors, respectively, were added to the
recording ACSF. Whole cell patch clamp recordings
using a Multiclamp 700B amplifier (Molecular Devices,
Axon Instruments, Foster City, CA) in the current clamp
mode were performed on the cerebellar Purkinje cells and
were digitized with a Digidata computer interface (Axon
Instruments). Neurons were visually identified by their
shape and location using infrared differential interface
contrast (IR-DIC) video microscopy with a 60x water
immersion objective. The images were detected with an
IR-sensitive CCD camera (Hamamatsu, ORSA, Japan)
and displayed on a monitor.
Spontaneous activity was monitored with a
whole cell patch pipette (resistance 3-6 MΩ) pulled from
borosilicate glass using a PC-10 puller (Narishige, Japan)
and filled with a solution containing (in mM): 135
potassium methyl sulphate (KMeSO4), 10 KCl,
10 HEPES, 1 MgCl2, 2 Na2ATP, and 0.4 Na2GTP (pH
7.2, adjusted with KOH; 290 mOsm). After establishment
of the GΩ seal, the whole cell configuration was achieved
simply by the application of a brief suction to break
through the membrane. Cells with a seal < 1 GΩ before
the rupture of the membrane were discarded, and the test
seal function was constantly monitored throughout the
recording to ensure that the seal was stable. In addition,
the series resistance (typically <15 MΩ) was checked for
stability during the experiments. The signals were filtered
at 10 kHz and sampled at 20 kHz using Clampex 9
software (Axon Instruments).
The membrane properties and action potential
parameters were measured, including the firing
regularity, action potential half-width, time to peak, and
afterhyperpolarization (AHP) amplitude. In the current
clamp mode, the instantaneous firing frequency and spike
frequency adaptation (SFA) ratio were also measured.
The regularity of the firing was assessed using the
coefficient of variation (CV) of the interspike intervals,
which was calculated as the ratio of the standard
deviation to the mean. The action potential half-width
was the time difference between the rising and falling
phase of an action potential, measured at 50 % of the
amplitude of the spike. The instantaneous frequency was
calculated as the inverse of the first interspike interval for
the trains of action potential that were elicited by the
injection of depolarizing current pulses (1600 ms
duration, 0.1 nA and 0.5 nA). The SFA ratio is equal to
Finitial/Ffinal, where Finitial is the first instantaneous
frequency, calculated from the first interspike interval,
and Ffinal is the final instantaneous frequency, calculated
from the last interspike interval (Venance and Glowinski
2003). The amplitude of the AHP was measured from the
baseline (–60 mV) to the peak of the AHP. The resting
input resistance was measured from the slope of a linear
fit of the current-voltage curve of current-clamp
recordings. The action potential properties were measured
and compared while holding the cell membrane potential
at –60 mV.
All chemicals and drugs were obtained from
Sigma (UK). Drugs were stored in a stock from which
working solutions were prepared freshly every day. The
doses of the drugs were chosen based on preliminary
experiments using values from the literature (Daniel et al.
2004, Loewenstein et al. 2005, Moldavan et al. 2006).
Data are presented as the mean ± S.E.M.
Statistical comparisons were carried out using Student’s
t test or a two-way analysis of variance (ANOVA)
followed by a Tukey HSD post-hoc test. A p≤0.05 was
3-acetylpyridine-induced profound alterations in the
intrinsic membrane properties of Purkinje neurons
The present study examined how the intrinsic
properties of Purkinje neurons were affected by 3-AP, a
neurotoxin. Whole cell current clamp recordings were
made from a total of 45 cerebellar Purkinje neurons in
14 rats; 25 neurons were recorded from eight control rats
and 20 neurons were recorded from six rats treated with
In the control conditions, Purkinje cells
displayed a regular firing pattern (Fig. 1Ai) with a mean
input resistance of 73.35±0.45 MΩ (Fig. 2A) and mean
coefficient of variation (CV) of the interspike interval of
0.07±0.008 (n=25; Fig. 2B). In contrast, Purkinje neurons
from 3-AP-treated rats discharged with an irregular
pattern of activity, as evidenced by a significant increase
in the CV (0.18±0.03, n=15; Figs 1Bi and 2B), and
exhibited much larger input resistance (81.51±3.96 MΩ,
n=20, P<0.01; Fig. 2A). The duration of the action
potential at half the maximal amplitude was shortened
significantly (from 0.72±0.03 ms to 0.54±0.001 ms,
P<0.05; Fig. 2C), and the time to peak was significantly
358 Kaffashian et al.
increased (0.53±0.02 ms in control rats and 0.65±0.05 ms
in 3-AP-treated rats, p<0.05; Fig. 2C) by 3-AP treatment.
However, treatment did not affect the peak amplitude of
the action potentials (52.16±3.2 mV in control and
50.61±2.7 mV in ataxic groups; Fig. 2D) but did
significantly reduce the AHP amplitude (–5.82±0.25 mV
and –4.5±0.2 mV in control and ataxic groups,
respectively, p<0.01; Figs 1C and 2D).
Next, the evoked firing characteristics of PCs
were investigated. With a membrane potential of
–60 mV, trains of action potentials were elicited in both
the control and ataxic conditions when the weak (0.1 nA)
and strong (0.5 nA) depolarizing current pulses (1600 ms
duration) were injected. The reciprocal of the interspike
interval (1/first ISI) between the first two action
potentials was used to compare the initial instantaneous
firing frequency of PCs in response to the pulses of
depolarizing current. In comparison to the control
condition, treatment with 3-AP caused a significant
increase in the initial discharge frequency during a train
of action potentials that was evoked by injecting a strong
depolarizing pulse (0.5 nA, p<0.001, n=15, Fig. 3A). The
value of this parameter in control rats was 95.42±5.17 Hz
and 135.89±6.09 Hz in the ataxic group. However, there
was no significant difference between the control and
3-AP-treated groups in response to weak (0.1 nA)
depolarizing current injections (Fig. 3A).
In addition, to further quantify the effect of 3-AP
treatment on the responses evoked by depolarizing
current injections, the spike frequency adaptation ratio
was calculated. In ataxic rats, the mean SFA ratio
calculated for the trains of action potentials evoked by
0.5 nA current steps was significantly lower (40 %,
P<0.05) than in control groups (from 2.18±0.33 to
1.35±0.07; Fig. 3B), and therefore, the frequency of the
action potential discharge was increased in Purkinje
neurons from the ataxic group (Fig. 3C).
Contribution of small conductance K+ channels to
intrinsic properties of cerebellar Purkinje neurons:
a comparison between ataxic and control conditions
To determine the functional role of SK channels
and their contribution to the intrinsic properties of PCs,
the effect of UCL1684, a high-affinity blocker of SK
channels (SK1-3), was assessed both in the control and
ataxic conditions. In control Purkinje cells, a bath
application of UCL1684 (60 nM) caused the firing
pattern to become irregular (Fig. 1Aii), and a marked
reduction in the firing regularity was observed when the
firing coefficient of variation (a useful parameter to
describe the firing regularity) of PCs was compared to the
control conditions. This difference was evidenced by a
significant increase in the CV (0.23±0.05, p<0.001;
Fig. 1. Profound changes in the intrinsic firing properties of Purkinje neurons were induced in an animal model of ataxia by injection
with 3-AP. A(i), somatic whole cell patch clamp recording of spontaneous intrinsic firing under the control condition and A(ii) in the
presence of UCL 1684 (60 nM). The regularity of firing is shown on an expanded time scale (lower traces). B(i) intrinsic spontaneous
firing pattern of PCs recorded from 3-AP-treated rats and B(ii) following the application of UCL 1684 (60 nM). The change in firing
precision is shown on an expanded time scale (lower traces). (C-E) Superimposed traces from the control, control+UCL1684, 3-AP and
Alteration in SK Channel Function Causes Cerebellar Ataxia 359
Fig. 2. UCL 1684 differentially influences the electrophysiological
properties of PCs from normal and ataxic rats. (A) Effects of 3-AP
treatment and SK channel blockade on membrane input
resistance. (B) Summary data of the mean coefficient of variation
of spontaneous action potentials under the control conditions and
the 3-AP and UCL 1684 treatments. The effects of 3-AP
treatment and SK channel blockade by UCL 1684 (60 nM) (C) on
the half width and the time to peak of the action potential and
(D) on the AHP amplitude and the peak amplitude of action
potentials. *, **, ***, significantly different (P<0.05, P<0.01,
P<0.001) from control; +, ++, significantly different (P<0.05,
P<0.01) from the 3-AP-treated group.
A blockade of the SK channels also increased
the membrane input resistance (77.03±1.47 MΩ, P<0.05;
Fig. 2A). The firing activity in the Purkinje neurons from
ataxic rats became very regular (Fig. 1Bii), and the
coefficient of variation of the interspike interval was
significantly decreased (0.09±0.01, p<0.05; Fig. 2B);
however, the mean membrane resistance remained
unchanged in the presence of UCL 1684. Alterations in
the parameters of the action potentials upon application
of UCL 1684 were also explored. In the control
conditions, UCL 1684 significantly increased the time to
peak of the action potentials (0.77±0.04 ms, P<0.001,
Fig. 2C) and the half width of the action potentials
(0.87±0.02 ms, p<0.01; Fig. 2C) but significantly
decreased both the peak amplitude of the action potentials
(35.41±3.4 mV, p<0.001) and the AHP amplitude
(–4.41±0.1 mV, p<0.001; Fig. 2D). However, the
blockade of the SK channels by UCL 1684 neither
significantly changed the time to peak nor affected the
peak amplitude of the action potentials in the ataxic rats
(Figs 2C and D), but it caused a significant increase in the
duration of the action potentials (0.79±0.002 ms, p<0.05;
Fig. 2C) and a significant reduction in the AHP amplitude
(–3.29±0.17 mV, p<0.01; Fig. 2D).
Next, we examined whether UCL 1684 could
differentially affect the evoked firing responses of PCs in
the control and ataxic conditions. The application of UCL
1684 significantly increased the initial firing frequencies
in the trains of action potentials elicited by injection with
a strong depolarizing current pulse (0.5 nA) but not in
response to a weak (0.1 nA) current step, in both control
(160.86±20.1, p<0.05) or ataxic rats (159.24±8.03,
p<0.05; Fig. 3A).
Furthermore, UCL 1684 (60 nM) in Purkinje
neurons from control but not from ataxic rats
significantly reduced the spike frequency adaptation
(from 2.18±0.33 to 1.34±0.16, p<0.05), thereby
enhancing Purkinje neuronal excitability (Figs 3B-D).
The present study examined the functional
consequences of alterations in the intrinsic properties of
Purkinje neurons in an animal model of ataxia induced by
neurotoxin 3-AP. The electrophysiological findings
demonstrated that, in ataxic rats, plastic changes in the
intrinsic electrophysiological properties of Purkinje
neurons were produced. These changes were manifested
as an increase in the firing irregularity that was
accompanied by a significant increase in CVISI, a
decrease in the time to peak and a significant decrease in
the half width and the AHP amplitude. Both the decrease
in the firing precision and the AHP amplitude could be
attributed to the suppression of SK potassium channels
(Hallworth et al. 2003). This decrease was also associated
with a significant increase in the initial instantaneous
frequency and a decrease in the SFA ratio in response to
the strong depolarizing pulse. To further determine the
360 Kaffashian et al.
possible mechanism(s) underlying such alterations in the
intrinsic electrophysiological behavior of PCs, the effects
of SK channel blockade on the intrinsic properties of PCs
were investigated. The present work indicated that the
blockade of SK channels by UCL 1684 (60 nM)
produced effects in control Purkinje neurons that were
almost entirely distinct from those recorded in ataxic rats;
the blockade of these channels disrupted the precision of
the firing in control PCs, but it decreased the irregularity
of the firing pattern while restoring the precision of the
firing in PCs from ataxic rats, as evidenced by a
significant increase and decrease in CVs, respectively.
The action potential parameters and the evoked firing
characteristics of control PCs appeared to be more
profoundly affected by SK channel blockade than did
those in the ataxic condition.
Previous studies have demonstrated that PCs are
capable of firing intrinsically even in the absence of
synaptic inputs (Llinás and Sugimori 1980, Häusser and
Clark 1997, Raman and Bean 1999, Womack and
Khodakhah 2002). However, distinct synaptic inputs are
necessary for producing distinct neuronal output
responses. Purkinje neurons receive two excitatory inputs
from a climbing fibre and additional input from many
parallel fibres. Olivary climbing fibre discharge plays an
important role in regulating cerebellar function by
controlling the intrinsic properties of PCs (Cerminara and
Rawson 2004, McKay et al. 2007, Janahmadi et al.
2009). Inactivating or chemically destroying the inferior
olive climbing fibre system has been shown to result in
marked modifications in the spike firing behavior of
Purkinje cells (Colin et al. 1981, Montarolo et al. 1982,
Cerminara and Rawson 2004, Janahmadi et al. 2009). In
agreement with previously published data, here we
observed a marked alteration in the intrinsic excitability
of Purkinje cells from ataxic rats. Neuronal intrinsic
excitability plays a critical role in the transition of
synaptic inputs to the particular output function; hence,
alterations in the intrinsic properties of neuronal cells
may profoundly affect the functioning neuronal circuits.
We have recently demonstrated that abolition of the
inferior olivary climbing fibre using neurotoxin 3-AP
caused changes in the intrinsic firing pattern of PCs,
which was associated with a decrease in the precision of
firing, and that pretreatment with combined riluzole and
3-AP restored the normal intrinsic properties (Janahmadi
et al. 2009), thereby improving the motor performance of
rats. It was assumed that the neuroprotective action of
riluzole was due to the opening of intrinsic SK channels.
It has also been previously shown that the precision of
pacemaking in PCs is maintained mainly by KCa channels
(Womack and Khodakhah 2003, Womack et al. 2004);
thus, the reduction of KCa channel activity may be the
main cause of firing irregularity in the PCs of ataxic mice
Fig. 3. Effects of 3-AP treatment and UCL 1684 application on evoked firing responses of Purkinje neurons. (A) Histogram showing the
first instantaneous firing frequency (1/first ISI) for low and high frequency discharges evoked by weak (0.1 nA) and strong (0.5 nA)
depolarizing current pulses (1600 ms), both in the control and ataxic conditions before and after UCL 1684 treatments. (B) Comparison
of the spike frequency adaptation (SFA) ratio before and after a bath application of UCL 1684 (60 nM) in the control and ataxic
conditions. (C and D) Representative traces illustrating the differences in action potential firing recorded from Purkinje neurons in
control and ataxic groups when elicited by depolarizing current injections (0.1 nA, left and 0.5 nA, right) before and after SK channel
blockade by UCL1684. *, **, ***, significantly different (p<0.05, p<0.01, p<0.001) from controls; +, significant difference (p<0.05)
from 3-AP treated (ataxic) groups.
Alteration in SK Channel Function Causes Cerebellar Ataxia 361
(Sausbier et al. 2004, Walter et al. 2006). Nevertheless,
the functional contribution of K+ channels, particularly
SK channels, to the intrinsic electrophysiological
properties of PCs in the ataxic condition has not yet been
A marked alteration in the first instantaneous
firing frequency, and thus the interspike interval, in
response to the strong current pulse (0.5 nA), but not a
weak step, following 3-AP treatment indicates that SK
channels, which are important for controlling early spike
frequency adaptation, have
changes. Therefore, the effects of potent SK channels
blockers on the intrinsic electrophysiological properties
of PCs in control and ataxic conditions were assessed.
Possible ionic mechanisms underlying plastic changes in
the intrinsic properties of PCs following 3-AP-induced
Ataxia is a neurological disease characterized by
a lack of balance and coordination; the decreased
neuronal firing precision may be an underlying
mechanism (Sausbier et al. 2004, Shakkottai et al. 2004,
Walter et al. 2006). To determine the functional role of
SK channels in firing irregularity, the effects of UCL
1684, a potent SK blocker (Dunn 1999), on intrinsic
electrophysiological properties of PCs were compared
between control and ataxic conditions. The blockade of
SK channels induced firing irregularity, changed the
action potential shape, suppressed the AHP amplitude,
increased both the half width and the firing frequency and
decreased the SFA ratio in the control condition.
Blockade of these channels also produced similar effects
on the half width, the AHP amplitude and the initial
firing frequency of PCs in the ataxic group. However, in
ataxic rats, the blockade of SK channels led to an increase
in the firing precision but left the SFA ratio unchanged.
These findings suggest the differential contribution of SK
channels to the intrinsic excitability of PCs in normal and
ataxic conditions, possibly because of plastic alterations
in the intrinsic properties of the PCs that occurred in the
ataxic condition. Cerebellar Purkinje neurons express
Ca2+ -activated K+ channels on their soma and dendrites
(Gahwiler and Llano 1989, Knaus et al. 1996, Jacquin
and Gruol 1999, Cingolani et al. 2002), which are
activated by Ca2+ influx through P/Q type voltage-gated
calcium channels (Vergara et al. 1998, Womack et al.
2004). BK channels contribute to the electrophysiological
properties of PCs, including
repolarization and fast AHP that follows a single action
potential (Edgerton and Reinhart 2003, Womack and
Khodakhah 2003, 2004, McKay and Turner 2004). In
contrast, SK channels play a pivotal role in shaping the
neuronal firing pattern (Wolfart et al. 2001, Cloues and
Sather 2003) and in regulating spike frequency adaptation
(Yen et al. 1999, Pedarzani et al. 2005, Brosh et al. 2006,
Vatanparast and Janahmadi 2009) in many nerve cells,
including PCs. SK channels, which are voltage-
insensitive and blocked by apamin, a bee venom toxin
(Hallworth et al. 2003, Sah 1996), play a role in setting
the intrinsic firing frequency (Edgerton and Reinhart
2003) and contribute to a slow AHP current that may last
several seconds following bursts of action potentials
(Marrion and Tavalin 1998, Sotcker et al. 1999, Bowden
et al. 2001). These channels are active at membrane
potentials that are close to the cell resting potential in
mature PCs and participate in the regulation of neuronal
hyperexcitability and burst firing (Haghdoost et al. 2007).
Considering the suppressive effect of UCL 1684
on the AHP amplitude, an increase in the neuronal
excitability and a decrease in the regularity of firing were
expected in the control group; however, in the ataxic
condition, blockade of the SK channels decreased the
firing regularity and induced an irregular firing pattern in
the PCs. These results suggest that SK channels play an
important role in regulating the firing behavior: the
dysfunction of these channels potentially may contribute
to the disruption of the normal firing behavior of PCs
seen in ataxic rats. It is believed that the AHP enhances
the precision of firing (Deister et al. 2009); therefore, the
firing irregularity observed in the presence of UCL 1684
in control PCs could be due to the blockade of the SK
channel, which is responsible for the action potential
AHP. Apamin, a selective blocker of SK channels, has
also been reported to induce an irregular firing pattern in
dopaminergic neurons of the substantia nigra (Lovejoy et
al. 2001) and midbrain (Ji et al. 2009).
In addition, UCL 1684 significantly decreased
the SFA ratio in the control conditions but not in the
ataxic conditions, suggesting that SK channels play a
significant role in affecting early spike frequency
adaptation in normal PCs; this result may reflect the
down-regulation of SK channels in the ataxic condition.
Several mechanisms have been proposed to underlie early
SFA, including a slow activation of outward currents, a
slow reduction in inward currents, the summation of the
AHP, and a reduction in the availability of fast Na+
channels (Miles et al. 2005, Gu et al. 2007, Vatanparast
and Janahmadi 2009). Here a reduction in both the peak
362 Kaffashian et al.
amplitude of the action potential and the AHP amplitude
and an increase in the time of peak imply that a reduction
in Na+ channel availability and/or summation of the AHP
could be the most likely underlying mechanisms of SFA
in the control conditions, but not in the ataxic conditions.
However, further voltage clamp study is needed to
address this issue. There is also evidence supporting the
involvement of the AHP in the SFA phenomenon in other
cell types, where blockade of AHP conductance leads to
reductions in spike frequency adaptation (Madison and
In conclusion, the present data strongly support
the idea that cerebellar ataxia, induced by neurotoxin
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Conflict of Interest
There is no conflict of interest.
This work was sponsored by a grant (No. 85032/14) from
Iran National Science Foundation (INSF) and supported
by Deputy of Research, Shahid Beheshti Medical School.
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