Epilepsia, 43(7):685–690, 2002 Blackwell Publishing, Inc. © International League Against Epilepsy Neocortical Potassium Currents Are Enhanced by the Antiepileptic Drug Lamotrigine *†Cristina Zona, *Virginia Tancredi, †Patrizia Longone, *Giovanna D’Arcangelo, ‡Margherita D’Antuono, ‡Mario Manfredi, and ‡§Massimo Avoli *Dipartimento di Neuroscienze, Università degli Studi di Roma “Tor Vergata,” and †IRCCS Santa Lucia, Roma; ‡IRCCS Neuromed, Pozzilli (Isernia), Italy; and §Montreal Neurological Institute Departments of Neurology and Neurosurgery, McGill University, Montréal, Québec, Canada Summary: Purpose: We used field-potential recordings in slices of rat cerebral cortex along with whole-cell patch recordings from rat neocortical cells in culture to test the hypothesis that the antiepileptic drug (AED) lamotrigine (LTG) modulates K+-mediated, hyperpolarizing currents. Methods: Extracellular field-potential recordings were performed in neocortical slices obtained from Wistar rats aged 25–50 days. Rat neocortical neurons in culture were subjected to the whole-cell mode of voltage clamping under experimental conditions designed to study voltage-gated K+ currents. Results: In the in vitro slice preparation, LTG (100–400 ␮M) reduced and/or abolished epileptiform discharges induced by 4-aminopyridine (4AP, 100 ␮M; n ⳱ 10), at doses that were significantly higher than those required to affect epileptiform activity recorded in Mg2+-free medium (n ⳱ 8). We also dis- covered that in cultured cortical cells, LTG (100–500 ␮M; n ⳱ 13) increased a transient, 4AP-sensitive, outward current elicited by depolarizing commands in medium containing voltagegated Ca2+ and Na+ channel antagonists. Moreover, we did not observe any change in a late, tetraethylammonium-sensitive outward current. Conclusions: Our data indicate that LTG, in addition to the well-known reduction of voltage-gated Na+ currents, potentiates 4AP-sensitive, K+-mediated hyperpolarizing conductances in cortical neurons. This mechanism of action contributes to the anticonvulsant effects exerted by LTG in experimental models of epileptiform discharge, and presumably in clinical practice. Key Words: Lamotrigine—K+ currents—Cortical cells— Epileptiform discharges—4-Aminopyridine. Antiepileptic drugs (AEDs) exert their effects by interacting with cellular mechanisms that include reduction of voltage-gated Na+ channels and/or glutamatergic excitation, as well as enhancement of ␥-aminobutyric acid (GABA)-mediated inhibition (1,2). It is unclear whether AEDs also modulate K+-mediated, hyperpolarizing conductances. K+ channels contribute to the regulation of brain excitability and thus may play an active role in controlling epileptic synchronization (3). In line with this view, K+ channel blockers readily induce epileptiform discharges in the in vivo and in the in vitro preparations (4–6). In addition, mice with induced deletions of various K+ channel genes exhibit seizures (7,8). We reported some years ago that therapeutic concentrations of the AED carbamazepine (CBZ) potentiate outward, voltage-dependent K+ currents in rat neocortical cells (9). However, with the exception of an earlier study describing a benzodiazepine (BZD)-induced enhancement of the Ca2+-dependent K+-mediated afterhyperpolarization (10) or an increase in late K+ currents during treatment with valproic acid (VPA) (11), little evidence has been provided regarding the ability of AEDs to act on this therapeutic target. This mechanism is even more relevant to the treatment of epileptic seizures, as mutations in K+ channel genes have been reported in some epileptic syndromes (12). While testing the effects of some newer AEDs on epileptiform discharges recorded in vitro during different pharmacologic manipulations, we noticed that the doses of lamotrigine (LTG) required to reduce the epileptiform activity induced by the K+ channel blocker 4-aminopyridine (4AP) were larger than those capable of inhibiting the activity disclosed by applying Mg 2+-free medium. This evidence suggests that LTG, in addition to the well-documented state-dependent block of voltagegated Na+ channels (13,14), may control neuronal excitation by modulating K + -mediated hyperpolarizing conductances. To test this hypothesis, we used fieldpotential recordings from slices of rat cerebral cortex as Accepted March 9, 2002. Address correspondence and reprint requests to Dr. M. Avoli at 3801 University St., Montréal, Québec, H3A 2B4 Canada. E-mail: massimo.avoli@mcgill.ca 685 686 C. ZONA ET AL. well as whole-cell voltage-clamping techniques in cortical cells maintained in tissue culture. The findings reported here indicate that LTG potentiates a 4APsensitive, K + -mediated outward current in cortical neurons, and that this mechanism of action contributes to the ability of LTG to control epileptiform synchronization in neocortical networks. METHODS Experiments were performed in cerebral cortex slices obtained from 25- to 50-day-old Wistar rats or in cultured neocortical cells prepared from dissociated neurons of Wistar rat embryos at day 14. Details on the preparation of brain slices (15) or tissue cultures (9,16) are reported in previous studies from our laboratories. Neocortical slices were maintained in a tissue chamber where they lay in an interface between oxygenated (95% O2/5% CO2) artificial cerebral spinal fluid (aCSF) and humidified gas at 34°C (pH 7.4). aCSF composition was (in mM): NaCl, 124; KCl, 2; KH2PO4, 1.25; MgSO4, 2; CaCl2, 2; NaHCO3, 26; and glucose, 10. Synchronous epileptiform discharges were induced by 4AP (100 ␮M) addition to the aCSF or by omitting MgSO4 from the aCSF (so-called Mg2+-free medium). aCSF containing increasing concentrations of LTG (100–400 ␮M) was applied in a cumulative fashion for ∼30 min at each dose. The effects induced by the different concentrations of LTG were measured after 20 min of superfusion with any given dose of LTG. Extracellular field potential recordings were made at ∼800 ␮m from the pia with aCSFfilled electrodes (resistance, 2–8 M⍀). Signals were fed to a DC high-impedance amplifier and displayed on a Gould pen recorder. Neocortical cells maintained in culture were studied at room temperature in a medium containing the following (in mM): NaCl, 120; KCl, 3; CaCl2, 2; MgCl2, 2; glucose, 20; HEPES/NaOH, 10; buffered to pH 7.3. In addition, tetrodotoxin (1 ␮M) and Cd2+ (0.2 mM) were added to the medium to block Na+ and Ca2+ currents, respectively. Currents were recorded from the soma of these neurons 10–18 days old in culture, in the whole-cell configuration with patch electrodes that were filled with a solution containing (in mM): KCl, 120; CaCl2, 0.24; EGTA, 5; glucose, 30; HEPES, 10; and buffered to pH 7.3 with KOH. The resistance of the filled electrode was ∼3–4 M⍀. 4AP (2 mM), tetraethylammonium (TEA, 10 mM), and different solutions of LTG were applied by gravity with small tubes (<1 mm diameter) placed near the patched cell. The response to a given drug concentration was evident in <1 s after the tap controlling the drug perfusion was opened. Chemicals used for both slice and tissue culture experiments were acquired from Sigma. Our study is based on data obtained from 18 neocorEpilepsia, Vol. 43, No. 7, 2002 tical slices analyzed with field-potential recordings and 21 neocortical cells patched in tissue culture. Throughout this article, measurements are expressed as mean ± SD, and n indicates the number of slices or neocortical cells used for any given pharmacologic protocol. Statistical analysis of the data obtained under control conditions and during any experimental manipulation was performed with paired or unpaired Student’s t tests as well as with analysis of variance (ANOVA). Data were considered significantly different at p < 0.01. RESULTS First we established the effects induced by LTG on epileptiform discharges recorded in neocortical slices during application of medium containing 4AP (17) or of medium from which Mg2+ had been omitted (18). In both cases, field-potential recordings showed the occurrence of prolonged epileptiform discharges with features that closely approximated the electrographic activity that accompanies a seizure. These synchronous events will hereafter be termed ictal discharges. In particular, 4APinduced ictal discharges had a duration of 22.4 ± 10.6 s (n ⳱ 10) and occurred at intervals of 42.6 ± 19.0 s (n ⳱ 9; Fig. 1A, in Control), whereas ictal events induced by Mg2+-free medium lasted 19.7 ± 9.8 s (n ⳱ 8) and repeated at intervals of 25.5 ± 16.6 s (n ⳱ 8; Fig. 1B, Control). During 4AP application, we could also record interictal-like events that consisted of negative-going field potentials occurring between the ictal discharges (Fig. 1A, Control, arrows). Interictal activity during application of Mg2+-free medium was rarely seen in the experiments included in this study. Bath application of 100–400 ␮M LTG caused a reduction of the duration and of the rate of occurrence of both 4AP-induced and Mg2+-free–induced ictal discharges (Fig. 1A and B). However, the effects induced by LTG were consistently more pronounced when tested on Mg2+-free–induced epileptiform discharges. Accordingly, by increasing the doses of LTG, we found that the median inhibitory concentration (IC50) for the reduction induced by this drug on both duration and rate of occurrence of Mg2+-free–induced ictal discharges was significantly lower (140 and 192 ␮M for the duration and rate of occurrence, respectively) than with 4AP-induced ictal events (370 and 367 ␮M). Moreover, maximal concentrations of ltg (i.e., 400 ␮M) abolished the ictal discharges in all but one slice perfused with Mg2+-free medium (n ⳱ 8). In contrast, blockade of ictal activity by a similar concentration of LTG was seen only in four of 10 slices treated with 4AP. These findings indicate, therefore, that the use of 4AP, which is a K+ channel blocker, may interfere with some mechanisms of LTG action that are linked with the modulation of K+ outward currents. LAMOTRIGINE POTENTIATES K+ CURRENTS 687 FIG. 1. Effects induced by lamotrigine on the epileptiform activity induced in neocortical slices by bath application of 4-aminopyridine (4AP; 100 µM) or medium from which Mg2+ had been omitted. A: Ictal discharges induced by 4AP are reduced in duration and rate of occurrence by bath application of increasing concentrations of lamotrigine (LTG). Arrows, interictal-like events. B: Ictal discharges recorded during application of Mg2+-free medium. Also in this experiment, LTG reduces the duration and the rate of occurrence of the epileptiform discharges, but these effects are more pronounced than with 4AP-induced discharges. C, D: Dose–response curves of the effects induced by increasing doses of LTG on the epileptiform discharges induced by 4AP and Mg2+-free medium in six and eight experiments, respectively. These results are expressed as percentage of the inhibition exerted by the different doses of LTG on the epileptiform patterns recorded under control conditions. Next we analyzed the effects induced by LTG on outward K+ currents generated by neocortical cells in culture during depolarizing pulses (–20, 10, and 30 mV) delivered from a holding potential of –80mV. These experiments were carried out in medium containing tetrodotoxin and Cd2+ to abolish voltage-gated Na+ and Ca2+ currents, respectively. As reported in a previous study from our laboratories (16), this protocol elicited voltagedependent, outward currents that increased rapidly (within 3–7 ms) to a peak and then decayed to a steady level (Fig. 2A, Control). Moreover, the fast peak of the current response was largely inactivated by bringing the holding potential to values less negative than –50mV; this procedure revealed a delayed steady outward current (not shown) (16). Hence neocortical cells studied under control conditions generated outward responses charac- terized by two components with different kinetics and voltage dependence (16). As illustrated in Fig. 2, application of LTG (100–500 ␮M) caused an increase in the amplitude of the voltagegated outward responses generated by rat neocortical cells (n ⳱ 13). However, these effects were consistently more pronounced when measured at the time of the early peak of the response (Fig. 2B and C). The enhancement of voltage-gated outward currents induced by LTG was dose dependent and characterized by an EC50 of 276 ␮M (Fig. 2D). Outward responses recorded in rat neocortical cells during blockade of voltage-gated Na+ and Ca2+ channels are contributed by a fast, transient 4AP-sensitive current that is inactivated at holding potentials less negative than –50mV, and a late persistent current that is blocked by Epilepsia, Vol. 43, No. 7, 2002 688 C. ZONA ET AL. FIG. 2. Effects induced by lamotrigine (LTG) on the outward K+ currents generated by neocortical cells in culture during depolarizing commands. A: Outward responses induced by three depolarizing commands (from –80 mV to –20, 10, and 30 mV) under control conditions and during application of 200 µM LTG. Note that LTG increases the peak amplitude of the three outward currents. B, C: Plots of the amplitudes of the outward responses elicited by different commands from a holding potential of –80 mV before and during application of LTG. The amplitude of the outward currents were measured at the peak of the response (B, ∼20 ms after the onset of the command) and at the end (C). In both panels, diamond is control and square is LTG. D: Dose–response curves of the percentage increase in the peak of the outward currents induced by LTG. The EC50 was 276 µM, and the command potential, 10 mV. TEA and not influenced by changing the holding potential (16). Hence, we established whether LTG was capable of modulating one or both of these two pharmacologically distinct, outward responses. To this end, we analyzed the effects of this AED on outward currents generated during application of either TEA (10 mM; n ⳱ 5) or 4AP (2 mM; n ⳱ 3). As illustrated in Fig. 3A, LTG enhanced the outward current generated in the presence of TEA, and this effect was reversible (not shown). In contrast, it did not modify the amplitude of steady outward responses recorded during application of 4AP (Fig. 3B). Effects induced by LTG on outward currents recorded in the presence of TEA or 4AP are summarized in Fig. 3C. DISCUSSION We have found that LTG can reduce epileptiform activity generated by rat neocortical slices treated with medium that either contains 4AP or from which Mg2+ is omitted. This evidence is well in line with the antiepileptic action exerted by this drug in clinical practice as Epilepsia, Vol. 43, No. 7, 2002 well as in animal models of epileptiform discharges in vivo (19–21). However, we have also noticed that the doses of LTG required to exert similar effects in these two models of in vitro epileptiform activity are larger when testing the ictal discharges induced by 4AP. A main mechanism of action for LTG resides in the well-documented state-dependent block of voltage-gated Na+ channels (13,14). This action, which is shared by several AEDs (1), leads to inhibition of sustained actionpotential firing that is the hallmark of neuronal networks participating in epileptiform synchronization. Sustained action-potential discharges are recorded during epileptiform discharges induced by 4AP or Mg2+-free medium. However, we have found that the doses of LTG required to reduce epileptiform discharges induced by Mg2+-free medium are lower than those capable of exerting similar effects on 4AP-induced epileptiform activity. This evidence leads us to consider the possibility that the different sensitivity of these two types of discharges to LTG is caused by an additional mechanism of action that is differently modulated in one of the two models. In particular, the possibility that 4AP could interfere with K+ LAMOTRIGINE POTENTIATES K+ CURRENTS 689 LTG concentrations that were similar to those capable of reducing voltage-gated Na+ channels in the same preparation (14). An increase of outward currents has been reported for CBZ in cultured neocortical cells (9). Interestingly, as for CBZ, the enhancement induced by LTG was associated with the modulation of the early outward response that is blocked by 4AP. Outward currents play a fundamental role in controlling neuronal excitability and thus may limit the intensity of firing, thereby reducing the duration of epileptiform discharges. Hence, the effects of LTG reported here may be relevant for its anticonvulsant action. In addition, recent evidence indicates that some epileptic syndromes occurring in both animals and humans are caused by mutations in K+ channel genes (12). In conclusion, we documented the existence of an additional target for the AED lamotrigine. Moreover, we linked this mechanism of action, which is exerted on intrinsic neocortical cell properties, to the ability of LTG to reduce epileptiform synchronization in vitro. We propose that this mechanism may contribute to the antiepileptic effects of LTG in vivo, as well as to the regulation of abnormal K+-channel behavior that may accompany several types of neuropsychiatric disorders. Acknowledgment: This work was supported by the Canadian Institutes of Health Research (grant MT-8109) and the Savoy Foundation. M.D. was a fellow of the Fragile X Research Foundation of Canada. We thank Ms. T. Papadopoulos for secretarial assistance. REFERENCES FIG. 3. Lamotrigine (LTG) potentiates the outward responses generated by rat neocortical cells during application of tetraethylammonium (TEA), but not those elicited in the presence of 4AP. A, B: Raw data obtained by applying LTG (200 µM) during application of 10 mM TEA (A) or 2 mM 4AP (B). Note that LTG enhances the outward current generated in the presence of TEA, but it does not modify the amplitude of the steady outward responses recorded during application of 4AP. C: Plots of the effects induced by LTG (200 µ M ) on the outward currents generated during application of TEA or 4AP (command potential, 10 mV). outward currents was attractive, given the well-known ability of this convulsant to block K+ currents. Such a mechanism was considered for LTG in a study in which this drug was tested on the intrinsic excitability of rat CA1 pyramidal cells (22). 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