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Vol. 61, Issue 4, 921-927, April 2002
Anticonvulsant Drug Development Program, Department of Pharmacology and Toxicology, University of Utah, Salt Lake City, Utah
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Abstract |
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 Top
 Abstract
 Introduction
Materials and Methods
Results
Discussion
References
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The whole-cell patch-clamp technique was used to examine the effects of
retigabine, a novel anticonvulsant drug, on the electroresponsive properties of individual neurons as well as on neurotransmission between monosynaptically connected pairs of cultured mouse cortical neurons. Consistent with its known action on potassium channels, retigabine significantly hyperpolarized the resting membrane potentials of the neurons, decreased input resistance, and decreased the number of
action potentials generated by direct current injection. In addition,
retigabine potentiated inhibitory postsynaptic currents (IPSCs)
mediated by activation of 
-aminobutyric acidA
(GABAA) receptors. IPSC peak amplitude, 90-to-10% decay
time, weighted decay time constant, slow decay time constant, and,
consequently, the total charge transfer were all significantly enhanced
by retigabine in a dose-dependent manner. This effect was limited to
IPSCs; retigabine had no significant effect on excitatory postsynaptic currents (EPSCs) mediated by activation of
non-N-methyl-D-aspartate ionotropic
glutamate receptors. A form of short-term presynaptic plasticity,
paired-pulse depression, was not altered by retigabine, suggesting that
its effect on IPSCs is primarily postsynaptic. Consistent with the
hypothesis that retigabine increases inhibitory neurotransmission via a
direct action on the GABAA receptor, the peak amplitudes,
90-to-10% decay times, and total charge transfer of spontaneous
miniature IPSCs were also significantly increased. Therefore,
retigabine potently reduces excitability in neural circuits via a
synergistic combination of mechanisms.
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Introduction |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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The
novel anticonvulsant drug retigabine [D-23129;
N-(2-amino-4-(4-fluorobenzylamino)phenyl)carbamic acid ethyl
ester] has been found to effectively reduce or block seizure activity
in a wide variety of animal models of epilepsy (Dailey et al., 1995
; Rostock et al., 1996; Tober et al., 1996). Retigabine is structurally different and has a higher protective index than many of the commonly prescribed anticonvulsants (Rostock et al., 1996). Of particular interest is the recent finding that a primary mechanism of action of
retigabine is the enhanced activation of heteromeric potassium channels
composed of the KCNQ2 and KCNQ3 subunits (Main et al., 2000; Rundfeldt
and Netzer, 2000b; Wickenden et al., 2000). It has been demonstrated
that the channels formed by the KCNQ2/Q3 subunits underlie a neuronal
potassium current commonly referred to as the M current (Wang et al.,
1998; Shapiro et al., 2000). The M current is critical in determining
resting membrane potential and neuronal excitability in many brain
regions because of its sustained activation at potentials below the
threshold for action potential generation (Marrion, 1997). Consistent
with its ability to augment M channel currents, retigabine has been
found to effectively hyperpolarize and reduce action potential
generation in projection neurons located in layers II and III of the
entorhinal cortex (Hetka et al., 1999).
Previous work suggests that the mechanism of action of retigabine is
not restricted to potassium channels. Experiments in the hippocampal
slice preparation suggest that retigabine can increase synthesis of the
inhibitory neurotransmitter -aminobutyric acid (GABA) (Kapetanovic
et al., 1995). In addition, work by Rundfeldt and Netzer (2000a)
demonstrated that, in cultured rat cortical neurons, retigabine
potentiates Cl
currents induced by
subsaturating concentrations of exogenously applied GABA. However,
these findings contrast with those in the entorhinal cortex brain slice
preparation, where retigabine was found to have no effect on any
parameters of GABAA receptor-mediated IPSCs and
inhibitory postsynaptic potentials (Hetka et al., 1999). One obstacle
faced by Hetka et al. (1999) was that to reverse the effects of
retigabine on resting membrane potential and action potential
generation, the drug could only be superfused into brain slices for 2 min. Thus, the concentration of retigabine at the synapse was most
probably lower than that of the superfusion media; consequently, it is
not yet known whether retigabine, at concentrations comparable with
effective dose plasma levels [estimated to be between 3 and 10 µM,
(Tober et al., 1996)], modifies synaptic transmission. The present set
of experiments was therefore conducted in a simple in vitro model
system to circumvent these obstacles and directly determine the effects
of retigabine on excitatory and inhibitory synaptic transmission.
The whole-cell patch-clamp technique was used to record from
monosynaptically connected pairs of cultured cortical neurons. Cultured
neurons provide rapid and reversible access of the compound to the
synapses. In addition, this experimental paradigm allows us to directly
distinguish presynaptic and postsynaptic effects of drugs (Wilcox and
Dichter, 1994), because it provides the resolution required to analyze
spontaneous miniature postsynaptic currents. We report here that
retigabine effectively reduces excitability by hyperpolarizing neurons
and by decreasing the input resistance at membrane potentials near
action potential threshold, thus decreasing the number of action
potentials that can be elicited by direct current injection. Moreover,
we have found that retigabine dose dependently and reversibly
potentiates IPSCs mediated by activation of GABAA
receptors. Thus, retigabine has multiple actions that serve to dampen
excitability in neural circuits; these actions may underlie the potent
anticonvulsant profile of this compound.
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Materials and Methods |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Tissue Culture.
Fetal mouse cortical neurons were maintained
in primary culture using standard techniques, and all animals were
treated in accordance with the National Institutes of Health
Guide for the Care and Use of Laboratory Animals. Briefly, embryonic
Swiss-Webster mice were removed from anesthetized mice (Charles River,
Wilmington, MA) on gestational day 18 and the brains were quickly
removed (Skeen et al., 1994). Cortical hemispheres were dissected,
gently chopped into small pieces, and incubated for 2 min in
Dulbecco's modified Eagle's medium (Sigma, St. Louis, MO) containing
0.25% trypsin. Horse serum (2.0 ml) (Invitrogen, Carlsbad, CA) was
added for 30 s and the mixture was then placed into a 15-ml
centrifuge tube and spun for 3 min at 1800 rpm. Most of the supernatant
was discarded and the brains were triturated 10 to 14 times and spun down once more for 2 min at 1500 rpm. The pellet was reconstituted in
Dulbecco's modified Eagle's medium supplemented with 10% horse serum, 3% glucose, and 2% L-glutamine. After trituration,
cells were filtered and plated on 12-mm coverslips (Carolina Biological Supply Co., Burlington, NC) coated with poly(L-lysine)
(Peninsula Labs, Belmont, CA) and maintained in a humidified incubator
at 37°C in 5% CO2. Cultures were treated for
24 h with Ara-C when glial cells became about 70% confluent. In
some cases, 24 h after plating, the media was replaced with a
high-potassium media, supplemented with 20 mM KCl, to enhance survival
(Wilcox et al., 1994). Three times per week, the culture media was
replaced with either the standard or high-potassium media.
Electrophysiological Recordings.
As described previously,
whole-cell patch-clamp recordings (Hamill et al., 1981) were obtained
from monosynaptically connected pairs of neurons maintained in culture
2 to 4 weeks (Wilcox et al., 1994; Wilcox and Dichter, 1994; Cummings
et al., 1996). Glass capillaries (World Precision Instruments, Inc.)
were pulled to 3 to 6 M
resistance using a micropipette electrode
puller (Sutter Instrument Co.). For all data acquisition, Axopatch 200 Series amplifiers and pClamp 8.0 software were used (Axon Instruments, Union City, CA). Signals were filtered at 5 kHz with the exception of
mIPSC currents, which were filtered at 1 kHz. Data were acquired at 10 kHz for offline analysis using CLAMPFIT 8.0, Axograph, and/or the Mini
Analysis Program (Synaptosoft, Decatur, GA).
Recording Solutions. For cell-pair and single-cell recording other than mIPSCs, the HEPES-buffered saline (HBS) extracellular recording solution contained 142 mM NaCl, 1.5 mM KCl, 10 mM HEPES, 1 mM MgCl2, 3 mM CaCl2, 10 mM glucose, and 20 mM sucrose. To determine the effect of retigabine on input resistance, tetrodotoxin (TTX; 500 nM) and CdCl2 (200 µM) were included in the bath. The pH was maintained at 7.34-7.36 and the osmolality ranged from 315 to 318 mOsm. The internal electrode solution contained 130 mM potassium gluconate, 10 mM KCl, 10 mM HEPES, 1 mM EGTA, 0.1 mM CaCl2, and 10 mM glucose. The pH and osmolality of the internal solution were maintained at 7.28 and 290 mOsm, respectively.
For mIPSC recording, the HBS external solution contained 142 mM NaCl, 5 mM KCl, 10 mM HEPES, 2 mM CaCl2, 2 mM MgCl2, and 10 mM glucose. The pH and osmolarity were the same as the above external solution. To block EPSCs and Na+ currents, 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) (10 µM) and TTX (500 nM) were added, respectively. The internal electrode solution contained 140 mM KCl, 1 mM EGTA, 10 mM HEPES, 0.1 mM CaCl2, and 10 mM glucose. The pH and osmolarity were maintained at 7.28 and 305 to 308 mOsM, respectively. Retigabine was supplied by ASTA Medica AG (Frankfurt, Germany). Stock solutions of 0.05 M were made in 50% dimethyl sulfoxide, frozen, and thawed as needed. The working concentration of dimethyl sulfoxide was
0.01%. Stocks of 0.01 M linopirdine were made in 10% HCl, frozen,
and thawed as needed. Linopirdine and all other buffer chemicals were
supplied by Sigma.
Data Analysis.
When determining the effects of retigabine on
electroresponsive properties of single cortical neurons, the whole-cell
current-clamp mode was used. Membrane potentials were maintained at
65 mV by direct current as needed. Currents ranging from 200 pA to
+325 pA, in increments of 25 pA, were injected into single neurons. Recordings were terminated if, in control HBS solution, current injections of 
50 pA did not produce two or more action potentials. The effects of retigabine on action potential firing, input resistance, and resting membrane potential were analyzed.
). A high external
divalent cation concentration was used in the external solution (3.0 mM
CaCl2 and 1.0 mM MgCl2) to
reduce the probability of spontaneous action potentials and activation
of any polysynaptic pathways (Wilcox et al., 1994). Because a low chloride containing internal solution was used, reversal potentials could be used to distinguish IPSCs from EPSCs. At 40 mV, IPSCs were
outward, whereas EPSCs were inward; in addition, IPSCs and EPSCs were
also distinguishable based on the duration of the PSC decay (Wilcox et
al., 1994). As previously demonstrated for rat hippocampal cultures
(Wilcox et al., 1994), IPSCs could be completely and reversibly blocked
with bicuculline (10 µM), a selective GABAA receptor antagonist, and EPSCs could be completely and reversibly blocked with the non-NMDA receptor antagonist CNQX (10 µM) (data not shown).
Using Clampfit 8.0 (Axon Instruments), the evoked PSC and mIPSC peak
amplitude, 90-to-10% decay time, decay time constant (
), total
charge transfer, and paired-pulse ratio were analyzed. EPSCs were
adequately fit with first-order exponential equations in both control
and retigabine solutions; IPSCs could be fit with first-order equations
in control solution, but second-order equations were almost always
required in the presence of retigabine. Decay phases of the currents
were fit with a double exponential equation of the form: I(t) = If × exp(t/f) + Is × exp(t/s), where
If is the amplitude of the fast component,
Is is the amplitude of the slow component, and
f and s are the fast
and slow time constants, respectively. Weighted time constants are
calculated using an equation of the form: w = [If / (If + Is)] × f + [Is / (If + Is)] × s (Rumbaugh and
Vicini, 1999). Spontaneous mIPSC peak amplitude and total charge
transfer were plotted on relative cumulative frequency histograms, and
the Kolmogorov-Smirnov test statistic was used to determine the
significance of retigabine's effects on mIPSCs. All values are
presented as mean ± S.E.M.
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Results |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Effects of Retigabine on the Electroresponsive Properties of
Neurons.
Initial experiments examined the effects of retigabine on
the electroresponsive properties of cultured cortical neurons.
Retigabine (1, 10, and 50 µM) significantly attenuated the ability of
prolonged current injection to elicit action potentials (Fig.
1A). During retigabine administration,
and especially at the highest concentrations, current amplitudes that
had previously elicited action potentials in control solution often
failed to generate action potentials (Fig. 1B). This effect was
completely and rapidly reversible. Interestingly, after washing the
drug out, it was common for the number of elicited action potentials to
surpass that of the initial control solution. The number of action
potentials elicited in both control and drug solutions varied from cell
to cell, but retigabine consistently reduced the number of action
potentials elicited. In addition, retigabine significantly
hyperpolarized the resting membrane potential of all cells dose
dependently (Fig. 2.). From a membrane
potential of 65 mV, neurons were consistently hyperpolarized by
2.4 ± 0.6 mV in 1 µM retigabine (n = 5),
5.4 ± 1.0 mV in 10 µM retigabine (n = 8); and
by 7.2 ± 1.0 mV in 50 µM retigabine (n = 16).
This hyperpolarization does not account entirely for action potential
attenuation, because a steady-state current was injected to return the
cells to the same membrane potential as during control (65 mV). Even
with this steady-state current injection, it was still necessary to
inject larger current amplitudes to elicit action potentials in the
presence of drug. To determine whether the hyperpolarization induced by
retigabine was caused by enhanced activation of the KCNQ2/3 channel,
the KCNQ2/3 antagonist linopirdine (10 µM) was coperfused with
retigabine (10 µM) (Rundfeldt and Netzer, 2000b; Wickenden et al.,
2000). Under these conditions, linopirdine completely blocked the
effect of retigabine on resting membrane potential (Fig. 2).
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+100 pA. Retigabine decreased input
resistance in the depolarized range, which is reflected as a decreased
slope.
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Effects of Retigabine on IPSCs and EPSCs.
While recording from
pairs of monosynaptically connected neurons, presynaptic cells were
recorded in current-clamp mode so that action potentials could be
evoked, and postsynaptic cells were voltage-clamped at 70 or 80 mV
while PSCs were elicited and recorded. All analyzed pair data were
acquired from 25 pairs of monosynaptically connected neurons: four
excitatory pairs and 21 inhibitory.
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values. Weighted values were then
calculated from the data as described previously (Rumbaugh and Vicini,
1999). All IPSC kinetic data are represented in Table 1. Kinetic data
in control conditions were pooled. To compare IPSCs across conditions,
only decays that could be adequately fit by second-order equations were
analyzed. Retigabine at 10 and 50 µM significantly enhanced the
percentage of the decay phase of the IPSC that was attributed to the
slow , and the overall weighted values were also significantly
increased (Fig. 4D; Table 1). In addition, the fast component was
significantly decreased in 50 µM retigabine (Table 1), although this
is most probably not caused by any direct action on factors
contributing to the fast component of decay, but instead by the
dramatic enhancement of the percentage of the decay that the slow encompassed after administration of 50 µM retigabine.
In contrast to the effects on IPSCs, all measured parameters of EPSCs,
including rise time, peak amplitude, 90-to-10% decay time, decay time
constant, and total charge transfer, were unaffected by retigabine (50 µM, n = 4) (Fig. 5,
A and B). First-order exponential equations adequately fit
EPSCs in control and retigabine conditions.
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Effects of Retigabine on Short-Term Plasticity.
Evoking two
PSCs with a short interstimulus interval of less than 4 s often
leads to a decreased amplitude of the second PSC (Deisz and Prince,
1989; Mott et al., 1993; Wilcox and Dichter, 1994; Cummings et al.,
1996); this presynaptic phenomenon is referred to as paired-pulse
depression (PPD). It is thought that changes in the degree of PPD are
caused by presynaptic mechanisms related to the release of
neurotransmitter (del Castillo and Katz, 1954; Otis and Mody, 1992;
Wilcox and Dichter, 1994). PPD elicited with interstimulus intervals of
300 ms at both excitatory (n = 4) and inhibitory
synapses (n = 16) was not effected by retigabine at any
concentration (Fig. 6, B and C). This
suggests that the observed effects of retigabine on IPSC amplitude and
decay time constant are caused primarily by a direct postsynaptic
action on the GABAA receptor rather than a
presynaptic alteration of neurotransmitter release.
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Effects of Retigabine on mIPSCs.
To test the hypothesis that
the effects of retigabine on IPSCs were mediated by a direct action on
postsynaptic GABAA receptors, we analyzed mIPSCs
in the presence of retigabine (50 µM). Single cells were
voltage-clamped at 70 mV in the presence of TTX and CNQX to block
Na+ channels and excitatory non-NMDA receptors,
respectively. The effects of retigabine (50 µM) on peak amplitude,
90-to-10% decay time, and total charge of mIPSCs were examined
(n = 5 cells). Cumulative probability histograms were
generated from the mIPSC data obtained, and the Kolmogorov-Smirnov test
statistic was used to determine the significance of the effects of
retigabine. Although retigabine significantly potentiated mIPSC peak
amplitude in only three of the five cells recorded, (Fig.
7C), both the 90-to-10% decay times and
total charge transfer were substantially and significantly increased in
all five cells (Fig. 7, D and E). The 10-to-90% rise time was also
significantly increased in two of five cells (data not shown). The
effects of retigabine on mIPSCs closely paralleled those on evoked
IPSCs, confirming that its effect on IPSCs is caused primarily by a
postsynaptic action at GABAA receptors.
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Discussion |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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The experiments presented here investigated the effects of the novel anticonvulsant retigabine on cortical neurons maintained in culture. Retigabine was found to modulate several electroresponsive properties of all neurons tested and to significantly enhance inhibitory neurotransmission. Specifically, in all neurons examined, retigabine hyperpolarized the resting membrane potential, reduced input resistance at membrane potentials depolarized from resting membrane potential, decreased action potential generation, and significantly enhanced IPSCs through a direct action on postsynaptic GABAA receptors. Therefore, retigabine can dampen excitability in neural circuits not only by inhibiting action potential generation in many types of neurons but also by enhancing inhibition at GABAergic synapses.
The direct actions of retigabine on individual neurons that we describe
in our culture system are similar to what has been described in the
entorhinal cortex brain slice preparation and in rat sympathetic
neurons in culture (Hetka et al., 1999; Tatulian et al., 2001).
Retigabine was found to decrease the input resistance of neurons at
potentials depolarized from the resting membrane potential, thereby
making it necessary to inject more depolarizing current to reach
threshold for action potential generation. This outward rectification
effectively reduced the number of action potentials that could be
elicited via direct current injection. This action has been attributed
to the ability of retigabine to enhance flow through the M-channel, a
potassium channel that is thought to be a heteromultimer composed of
KCNQ2/Q3 potassium channel subunits (Hetka et al., 1999; Main et al.,
2000; Rundfeldt and Netzer, 2000b; Wickenden et al., 2000; Tatulian et
al., 2001). This is of particular interest with regard to the treatment
of epilepsy because it has been demonstrated that compounds that reduce
current flow through the M-channel are potent convulsants (Rogawski,
2000). In addition, loss of function mutations in the KCNQ2/Q3 channel
underlie the epilepsy syndrome known as benign familial neonatal
convulsions (Biervert et al., 1998; Charlier et al., 1998; Singh et
al., 1998). Therefore, mechanisms that enhance current flow through
this channel, such as that which occurs in the case of retigabine, are
likely to reduce excitability and should prove useful in the treatment
of seizure disorders.
Our experiments also demonstrate that retigabine, at physiologically
relevant concentrations, can significantly increase
GABAA receptor-mediated IPSCs via a direct action
on these receptors. The experiments on IPSCs, spontaneous mIPSCs, and
baseline noise support the hypothesis that retigabine, like
diethyl-lactam, is most effective under conditions in which the synapse
is not saturated by GABA (Hill et al., 1998; Perrais and Ropert, 1999;
Hajos et al., 2000; Leao et al., 2000). Thus, the most robust effect of retigabine on IPSCs occurs during the decay phase of the IPSC, when
concentrations of GABA in the synapse are diminishing. Single-channel experiments should be performed to determine the precise mechanism of
action of retigabine on the GABAA receptor-gated
ion channel complex.
Previous efforts to study the effects of retigabine on
GABAA receptors have produced conflicting
reports. Although in cultured rat cortical neurons, retigabine
concentrations of 10 µM and above resulted in potentiation of
currents evoked by the exogenous application of GABA (Rundfeldt and
Netzer, 2000a), 100 µM retigabine had no significant effect on the
peak amplitudes of evoked IPSCs in rat entorhinal cortex brain slice
preparations (Hetka et al., 1999). It is not currently understood why
the brain slice experiments did not demonstrate any effects of
retigabine on IPSCs. However, this discrepancy could be attributed to a
variety of differences in the experimental protocols, such as an
insufficient concentration of retigabine at the synapses in the brain
slice experiments, the temperature at which experiments were performed
(Perrais and Ropert, 1999), and possible differences in the subunit
composition of postsynaptic GABAA receptors in
culture versus the brain slice.
The culture preparation used in these experiments is a useful tool for determining the effects of novel compounds on synaptic transmission in a simple neural circuit. Using this experimental paradigm, we have determined that retigabine, at concentrations comparable with therapeutically effective plasma concentrations, effectively decreases excitability in neural circuits by hyperpolarizing neurons, impeding action potential generation, and greatly enhancing current flow through GABAA receptors by prolonging IPSCs. Furthermore, these unique synergistic actions of retigabine provide a novel approach for the therapeutic management of seizure disorders.
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Acknowledgments |
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We thank Drs. Harold Wolf and H. Steve White for encouragement and support. In addition, we thank Cynthia Levinthal and Tim Pruess for tissue culture preparation and maintenance and David Daberkow with help on initial miniature IPSC experiments.
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Footnotes |
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Received June 27, 2001; Accepted January 15, 2002
This work was supported by National Institutes of Health contract N01-NS42311 and an ASPET Summer Fellowship to M.M.K.
Address correspondence to: Karen S. Wilcox, Ph.D., Anticonvulsant Drug Development Program, Department of Pharmacology and Toxicology, University of Utah, 20 S 2030 E Room 408, Salt Lake City, UT 84112. E-mail: kwilcox{at}deans.pharm.utah.edu
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Abbreviations |
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GABA, -aminobutyric acid;
IPSC, inhibitory postsynaptic current;
TTX, tetrodotoxin;
EPSC, excitatory
postsynaptic currents;
mIPSC, miniature inhibitory postsynaptic
current;
HBS, HEPES-buffered saline;
CNQX, 6-cyano-7-nitroquinoxaline-2,3-dione;
PSC, postsynaptic current;
PPD, paired-pulse depression.
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References |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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V
occurred for all current injections 
, the effect of 1 µM retigabine approached
statistical significance at 95% confidence, with p < 0.085.) D, for IPSC decay phases that were adequately fit with
second-order exponential equations, retigabine significantly enhanced
weighted 
