anticonvulsant pharmacology of voltage-gated na channels...

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European Journal of Neuroscience, Vol. 17, pp. 1–11, 2003 © Federation of European Neuroscience Societies

doi:10.1046/j.1460-9568.2003.02710.x

EJN 2710

Prod. Cont.: Dharmesh

Anticonvulsant pharmacology of voltage-gated Na+ channels in hippocampal neurons of control and chronically epileptic rats

Stefan Remy,1,2 Bernd W. Urban,2 Christian E. Elger1 and Heinz Beck1

1Department of Epileptology and2Department of Anaesthesiology, University of Bonn Medical Center, Siegmund-Freud Str. 25, 53125 Bonn, Germany

Keywords: lamotrigine, phenytoin, pilocarpine model, sodium channel, valproate

Abstract

Voltage-gated Na+ channels are a main target of many first-line anticonvulsant drugs and their mechanism of action has been exten-sively investigated in cell lines and native neurons. Nevertheless, it is unknown whether the efficacy of these drugs might be alteredfollowing chronic epileptogenesis. We have, therefore, analysed the effects of phenytoin (100 µM), lamotrigine (100 µM) and valproate(600 µM) on Na+ currents in dissociated rat hippocampal granule neurons in the pilocarpine model of chronic epilepsy. In control ani-mals, all three substances exhibited modest tonic blocking effects on Na+ channels in their resting state. These effects of phenytoinand lamotrigine were reduced (by 77 and 64%) in epileptic compared with control animals. Phenytoin and valproate caused a shift inthe voltage dependence of fast inactivation in a hyperpolarizing direction, while all three substances shifted the voltage dependenceof activation in a depolarizing direction. The anticonvulsant effects on Na+ channel voltage dependence proved to be similar in con-trol and epileptic animals. The time course of fast recovery from inactivation was potently slowed by lamotrigine and phenytoin incontrol animals, while valproate had no effect. Interestingly, the effects of phenytoin on fast recovery from inactivation were signifi-cantly reduced in chronic epilepsy. Taken together, these results reveal that different anticonvulsant drugs may exert a distinct pat-tern of effects on native Na+ channels. Furthermore, the reduction of phenytoin and, to a less pronounced extent, lamotrigine effectsin chronic epilepsy raises the possibility that reduced pharmacosensitivity of Na+ channels may contribute to the development ofdrug resistance.

Introduction

A large number of structurally diverse antiepileptic drugs have beendeveloped which lower the propensity of CNS structures to generatehigh-frequency discharges. Putative mechanisms of action have beenidenti$ed for some of these substances, including effects on voltage-dependent ion channels, neurotransmitter receptors and enzymes(Rogawski & Porter, 1990; Bialer et al. 2001). Some of the most com-monly administered substances in temporal lobe epilepsy, such asphenytoin (PHT), carbamazepine (CBZ) and lamotrigine (LTG),potently inhibit voltage-gated Na+ channels with comparatively smalleffects being known on other ion channels or receptors at clinicallyrelevant concentrations (Ragsdale & Avoli, 1998).

Voltage-gated Na+ channels are transmembrane proteins composedof one of several pore-forming (a) subunits and accessory b subunits.They are closed at resting membrane potential but are rapidly acti-vated by depolarization, giving rise to Na+ inward currents. TheseNa+ currents decrease rapidly towards baseline levels as the Na+

channels undergo inactivation during prolonged depolarization. Fol-lowing inactivation, Na+ channels require repolarization in order toreturn to the resting state. The transition between these sets of func-tional states occurs within a time scale of milliseconds. These rapid

kinetics are essential for sustaining fast action potentials as well asrapid trains of action potentials.

A large number of studies have addressed the mechanism by whichanticonvulsant or local anaesthetic compounds inhibit voltage-depen-dent Na+ currents. Two distinct effects can be observed on Na+ chan-nels in their resting state: a block of the peak Na+ current amplitudeand a shift of the steady-state inactivation curve in a hyperpolarizingdirection resulting in voltage dependence of the blocking effects.These effects have been termed tonic inhibition (Ragsdale & Avoli,1998). More importantly, anticonvulsants and local anaestheticsinhibit Na+ currents in an activity- or use-dependent manner, i.e.blocking effects are more pronounced when the cell membrane isdepolarized. This effect probably results from preferential binding ofthese drugs to the inactivated state of the channel (Willow et al., 1985;Schwarz & Grigat, 1989; for review see Ragsdale & Avoli, 1998).Activity-dependent block of Na+ channels by anticonvulsants isthought to be the most important factor in their potent inhibition ofhigh-frequency neuronal discharges.

The aim of this study was twofold. Firstly, we wished to character-ize the actions of different anticonvulsant drugs known to act prima-rily via inhibition of Na+ channels in native neurons. Secondly, wewished to ascertain whether the ef$cacy of these drugs might bereduced following induction of chronic epilepsy. Such a mechanismmay be important because subgroups of epilepsy patients (Aicardi &Shorvon, 1997) and chronically epileptic animals (Löscher et al.,

Correspondence: Dr Heinz Beck, 1Department of Epileptology, as above.E-mail: [email protected]

Received 28 February 2003, revised 11 April 2003, accepted 15 April 2003

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© 2003 Federation of European Neuroscience Societies, European Journal of Neuroscience, 17, 1–11

1993; Nissinen & Pitkänen, 2000) develop resistance to common anti-epileptic drugs that might rely on reduced pharmacosensitivity of ionchannels. We have, therefore, examined the effects of PHT, LTG andvalproate (VPA) on Na+ channels in acutely dissociated hippocampalgranule cells both in control rats and in the chronic pilocarpine modelof epilepsy.

Materials and methods

Pilocarpine model of epilepsy

Chronically epileptic rats were prepared according to established pro-tocols (Turski et al., 1983). Brie#y, male Wistar rats (150–200 g, age30± 2 days) were injected with a single high dose of the muscarinicagonist pilocarpine (340 mg/kg i.p.), which induced status epilepticusin most (∼80%) animals. Peripheral muscarinic effects were reducedby prior administration of methyl-scopolamine (1 mg/kg s.c. 30 minbefore injecting pilocarpine). Diazepam (0.1 mg/kg s.c.) was adminis-tered to all animals 2 h after the pilocarpine injection. It terminated theconvulsions in the responsive rats and sedated all animals. The surviv-ing (∼81%) animals were closely tended, hydrated and fed in the lab-oratory until they recovered from the acute insult. They were video-monitored for at least 4 h/day for up to 2 months for the developmentof spontaneous epileptic seizures. Within 2–6 weeks of this treatment,all rats used in this study developed a chronic epileptic condition,expressed in two to four video-monitored spontaneous ‘limbic’ sei-zures (characterized by chewing, head nodding, forelimb clonus, rear-ing and falling) per week. The time between development of seizuresand the end of the experiment, when the animals were killed, variedbetween 2 and 4 weeks. These animals were used as the experimentalepileptic group. The animals were killed at the age of 75± 5 days. Agroup of untreated, age-matched rats housed under identical condi-tions for the same period of time was used as the control group.

Preparation of acutely dissociated dentate granule cells

Prior to decapitation, rats were deeply anaesthetized with ether. Coro-nal slices (400µm) were prepared from the hippocampus of controland pilocarpine-treated rats with a vibratome (VT1000S; Leica,Germany) in ice-cold arti$cial cerebrospinal #uid containing (in mM):NaCl, 125; NaHCO3, 25; KCl, 3; NaH2PO4, 1.25; MgCl2, 1; CaCl2, 2and glucose, 20 (pH 7.4; osmolarity, 305 mosmol; 95% O2 and 5%CO2). Slices were rapidly transferred into an arti$cial cerebrospinal#uid-$lled storage chamber in which they could be stored for up to10 h at room temperature. After an equilibration period of 60 min the$rst slice was transferred to a tube with 5 mL saline containing (inmM): CH3SO3Na, 145; KCl, 3; MgCl2, 1; CaCl2, 0.5, HEPES, 10 andglucose, 15 (pH 7.4, adjusted with NaOH). Pronase (protease typeXIV, 2 mg/mL; Sigma) was added to the oxygenated medium (100%O2). After an incubation period of 15 min at 35°C, the slice waswashed in pronase-free saline of an identical composition. The dentategyrus was dissected and triturated with $re-polished glass pipettes ofdecreasing aperture. Where possible, the hilus was separated from theslice during trituration. The Petri dish containing the cell suspensionwas then mounted on the stage of an inverted microscope (Telaval;Zeiss, Jena, Germany). Dissociated cells were allowed to settle for5–10 min and were superfused with an extracellular solution contain-ing (in mM): CH3SO3Na, 120; TEA, 20; KCl, 3; BaCl2, 5; MgCl2, 1;HEPES, 10; 4-aminopyridine, 4; CdCl2, 0.03 and glucose, 10 (pH 7.4,adjusted with NaOH; osmolarity, 310 mosmol/L). Isolated cellsshowed a round or ovoid small soma with a single apical process rem-iniscent of dentate granule cell morphology in situ. These neuronscould be clearly discriminated from neuron types with multipolar

processes and a large soma presumed to be interneurons. Only neu-rons with a typical dentate granule cell morphology were included inthis study. All animal experiments were conducted in accordance withthe guidelines of the University of Bonn Animal Care Committee andwere approved by the board on proper use of experimental animals ofthe state Nordrhein-Westfalen.

Whole-cell patch-clamp recording

Patch pipettes with a resistance of 2.0–3.0 MΩ were pulled fromborosilicate glass capillaries (1.5 mm o.d., 1 mm i.d.; Science Prod-ucts, Hofheim, Germany) on a PP-830 puller (Narishige, Tokyo,Japan). Pipettes were $lled with an intracellular solution containing(in mM): Cs-methanesulphonate, 87.5; TEA, 20; MgCl2, 5; HEPES,10; BAPTA, 5; CaCl2, 0.5; adenosine-5′-triphosphate (Na+2-ATP), 10and guanosine-5′-triphosphate, 0.5 (pH adjusted to 7.40 with NaOH).The osmolarity was adjusted with sucrose to 300± 6 mosmol. Tight-seal whole-cell recordings were obtained at room temperature(21–24°C) according to Hamill et al. (1981). The seal resistance was>1 GΩ in all recordings and the series resistance was 4± 2 MΩmeasured by the dial settings for series resistance compensation onthe Axopatch 200A ampli$er (Axon Instruments, USA). Resistanceand capacitance compensation (between 85 and 95% each) were usedto improve the voltage control. The maximal residual voltage errorwas 2.3± 0.3 mV for the control group and 2.5± 0.4 mV for thechronically epileptic group. Voltage commands were delivered viathe Axopatch 200A ampli$er and the resulting current was recordedonline with the PCLAMP 8.0 acquisition and analysis program. Cur-rent signals were $ltered at 5 kHz (−3dB, four-pole low-pass Bessel$lter) and sampled at 20 kHz or more by an interface (DigiData1322A; Axon Instruments). Residual capacitance transients and leakconductances were subtracted using a P/4 protocol. Eperiments wereperformed on cells from at least $ve rats for each experimentalparadigm.

Drugs and application

Different concentrations of LTG (Glaxo Wellcome), PHT (Sigma) andVPA (Sigma) in the extracellular solution were applied with a superfu-sion pipette placed at a distance of 50–100µm from the cell body. TheLTG, PHT and VPA were dissolved in dimethyl sulphoxide and thendiluted into the bath solution to the desired concentrations for experi-ments (100, 100 and 600µM, respectively). Control recordingsshowed that the maximal concentration of dimethyl sulphoxide had nodetectable effects on the Na+ currents in acutely dissociated dentategranule neurons.

Voltage clamp paradigms, fitting and data analysis

Voltage-dependent steady-state activation and inactivation

The voltage dependence of activation and inactivation was determinedusing standard protocols. The conductance G(V) was calculatedaccording to

where VNa is the Na+ reversal potential, V the command potential andI(V) the peak current amplitude. G(V) was then $tted with the follow-ing Boltzmann equation:

Where Gmax is the maximum Na+ conductance, V50 is the voltagewhere G(V) is half of Gmax and km indicates the slope of the relation-ship between channel inactivation and membrane voltage.

( ) ( ) ( ) (1)/ NaG V I V V V= −

max 50 m( ) /1 exp[( ) / ] (2)G V G V V k= + −

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Anticonvulsant pharmacology of native Na+ 3


The magnitude of the window current resulting from the overlap inthe voltage-dependent activation and inactivation curves was esti-mated by multiplying the Boltzmann equations best describing theNa+ current voltage dependence of activation and inactivation, respec-tively (Ketelaars et al. 2001). The resultant value is an estimate of theprobability of the Na+ channel being in a noninactivating state that issuitable for comparison between different experimental groups.

Double-pulse experiments

A conditioning pulse from −80 to −10 mV was followed by a varyingrecovery period (1 ms−5 s) at −80 mV and a subsequent test pulse to−10 mV (5 ms). The duration of the conditioning pulse was 10 ms.Time constants of recovery τfast and τslow were extracted from the dou-ble exponential equation:

where I(t) is the current amplitude at timepoint t after onset of thevoltage command and Afast and Aslow are the respective amplitude con-tributions of the recovery time constants.

A nonlinear Levenberg–Marquardt algorithm was used for all $ts.Statistical comparison was performed with Student's unpaired t-test ata signi$cance level α of 0.05. All results are presented as the mean±SEM.

Results

We have examined the effects of the three antiepileptic drugs PHT,LTG and VPA on voltage-gated Na+ channels in hippocampal dentategranule neurons in detail. All analyses were performed both in controlrats and the chronic pilocarpine model of epilepsy (see Materials andmethods). As in a previous study, differences between the Na+ currentproperties in control and chronically epileptic animals were observedwith respect to the voltage-dependent activation and inactivationbehaviour (see Table 1).

We $rst determined the effects of all three substances on Na+

channels in their resting state. To this end, the holding potential wasset to −80 mV and Na+ currents were elicited with the voltage stepprotocol shown in the inset of Fig. 1A. We recorded Na+ currents

fast fast slow slow 3( ) [1 exp( )] [1 exp( )] (3)/ /I t A t A t Aτ τ= − − + − − +

Table 1. Comparison of functional properties of Na+ channels in the dentate granule neurons of control and chronically epileptic rats

Group

Activation curve Inactivation curve Recovery

V1/2 (mV) (n) SF (n) V1/2 (mV) (n) SF (n) τfast (ms) (n) Afast (%) (n) τslow (ms) (n) Aslow (%) (n)

Controls −24.2± 0.7 (8) 5.3± 0.2 (8) −50.7± 1.1 (11) 5.8± 0.4 (11) 7.0± 0.3 (12) 76± 1.0 (12) 496± 49 (12) 25± 1.0 (12)Epileptic −29.1± 0.6∗ (8) 5.1± 0.3 (8) −46.1± 1.0∗ (9) 5.5± 0.4 (9) 7.1± 0.5 (12) 75± 1.0 (12) 511± 36 (12) 26± 1.0 (12)

The Boltzmann $ts (eqn 2) of the activation and inactivation curves (n= number of rats) yielded the voltage of half-maximal activation/inactivation (V1/2) and theslope factor (SF). The time course of the recovery from inactivation was analysed with a double-exponential function (eqn 3) yielding τfast, τslow and the amplitudecontribution (∗P< 0.05, Student's unpaired t-test). In this analysis the maximal residual voltage errors did not exceed 1.5 mV.

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Fig. 1. Tonic inhibition of Na+ currents in dentate granule neurons from con-trol and epileptic rats. (A) Superimposed whole-cell current traces recordedunder control conditions, during the application of 100µM lamotrigine (LTG),100µM phenytoin (PHT) and 600µM valproate (VPA) (grey traces, see aster-isks) and after washout. Na+ currents were elicited by a 15-ms pulse to−10 mV from the −100 mV pre-pulse potential of 200 ms duration (see inset).The holding potential was −80 mV. (B) The percentage of the Na+ peak currentamplitude blocked following application of 100µM LTG (n= 5), 100µM PHT(n= 6) and 600µM VPA (n= 5) in control ( ) and chronically epileptic ( )rats (asterisks indicate signi$cant differences, P< 0.05, Student's unpaired t-test). The maximal amplitude was 3.4± 0.9 nA for the recordings obtainedfrom control rats and 5.1± 1.1 nA for the chronically epileptic rats.

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before, during (asterisks, Fig. 1B) and after application of 100µM

LTG, 100µM PHT and 600µM VPA. The LTG and PHT led to amodest but reversible reduction of the peak Na+ current amplitude inthe control group (LTG, 15.0± 4.8%; PHT, 21.7± 2.8%; VPA,1.6± 1.3%, n= 5–10) and the chronically epileptic group (LTG,3.5± 0.6%; PHT, 7.8± 4.0%; VPA, 1.5± 1.4%, n= 5–6). The mag-nitude of this tonic blocking effect was signi$cantly higher in con-trol animals compared with chronically epileptic animals for LTGand PHT (P< 0.05).

We next determined whether the effects of these anticonvulsantdrugs display voltage dependence. To this end, we varied the voltageof a conditioning pre-pulse preceding the test pulse as depicted in theinset of Fig. 2A. This brief conditioning pulse was suf$cient to revealdifferences in the voltage-dependent block by PHT, VPA and LTG.When blocking effects were quanti$ed at various conditioning pre-pulse potentials, the fraction of block induced by application of PHTand VPA increased steeply with more depolarized pre-pulses (Fig. 2A

and B, two rightmost panels). In contrast, the blocking effects of LTGwere less strongly voltage dependent (Fig. 2A and B, leftmost panels).The voltage-independent effects of LTG and PHT depicted in Fig. 1are also clearly apparent in Fig. 2B. It has to be noted that binding ofanticonvulsants to the inactivated state of Na+ channels is thought tobe slow. Thus, differences observed here may re#ect differences eitherin the time course of anticonvulsant binding to Na+ channels or differ-ences in voltage dependence of the block.

As voltage-dependent blocking effects, as shown in Fig. 2, areexpressed as a shift of the voltage-dependent inactivation curve in ahyperpolarized direction, we analysed the effect of PHT, LTG andVPA on voltage-dependent inactivation of Na+ channels. Voltage-dependent inactivation curves were constructed from recordingsobtained using voltage paradigms as used in Fig. 2B (see inset,Fig. 3A). Representative current recordings under control conditionsare shown in Fig. 3A. The application of PHT (Fig. 3C) appeared tocause a larger shift in the voltage-dependent inactivation curve than

Fig. 2. Voltage-dependent inhibition of Na+ currents in control and epileptic rats. (A) Superimposed recordings of Na+ currents in control neurons elicited by testpulses to −10 mV following 100 ms pre-pulse potentials of −60 mV (upper traces) or −100 mV (lower traces). The voltage protocol is depicted in the inset. Aster-isks indicate the current trace recorded following application of lamotrigine (LTG), phenytoin (PHT) or valproate (VPA), respectively. (B) Percentage of the peakNa+ current amplitude blocked by application of 100µM LTG (leftmost panel, n= 5–6), 100µM PHT (middle panel, n= 6–7) and 600µM VPA (rightmost panel,n= 5) at different pre-pulse potentials in granule neurons of control ( ) and chronically epileptic ( ) rats. The peak amplitude was 3.6± 0.8 nA for the recordingsobtained from control rats and 5.6± 1.0 nA for the chronically epileptic rats.

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LTG and VPA (Fig. 3B and D, respectively). Fitting the datapointsshown in Fig. 3B–D with Boltzmann equations (solid lines superim-posed on the datapoints, see Materials and methods) allowed us tocompare shifts in the voltage of half-maximal inactivation quantita-tively (V1/2inact, Fig. 3E). In control animals, the application of PHT orVPA caused an average shift of V1/2inact in the hyperpolarizing direc-tion of 3.7± 0.4 (n= 5) and 1.7± 0.5 mV (n= 6). The effects of LTGon V1/2inact were negligible (0.7± 0.2 mV, n= 5). Similar shifts inV1/2inact were observed in chronically epileptic animals followingapplication of PHT (shift in V1/2inact: 2.9± 0.5 mV, n= 7), VPA(2.0± 0.7 mV, n= 5) and LTG (0.25± 0.4 mV, n= 6).

Next, we examined the effects of LTG, PHT and VPA on Na+ cur-rent voltage-dependent activation. Activation curves were generatedusing standard voltage protocols (see inset, Fig. 4A) and plotting thenormalized conductance at the peak of the Na+ current versus the testpulse potential. The datapoints were $tted with a Boltzmann function(see Materials and methods) in order to determine the voltage of half-

maximal activation (V1/2act). In control animals, V1/2act was modestlyshifted in a depolarizing direction by all three substances. The averageshift was 1.5± 0.5 mV for 100µM PHT, 4.1± 1.1 mV for 100µM LTGand 4.6± 1.3 mV for 600µM VPA (n= 5, 6 and 5, respectively,p< 0.05). The average shifts in V1/2act were not signi$cantly differentin the pilocarpine model of chronic epilepsy compared with controlanimals (PHT, 1.5± 0.6 mV; LTG, 2.1± 1.0 mV; VPA, 4.1± 1.5 mV,n= 7, 6 and 5, respectively). The average shifts in V1/2act in control andepileptic animals are summarized in Fig. 4E.

The analysis of voltage-dependent activation and inactivationallowed us to determine the magnitude of the window current result-ing from the overlap between the voltage-dependent activation andinactivation curves. The amplitude of the window current was largerin chronically epileptic animals, as suggested by previous studies(Ketelaars et al. 2001; Remy et al. 2002). Amongst the three anticon-vulsant drugs tested, the amplitude of the window current was moststrongly affected by VPA (44 and 30% in control and epileptic

Fig. 3. Effects of lamotrigine (LTG), phenytoin (PHT) and valproate (VPA) on the inactivation of Na+ channels. (A) Representative recordings of Na+ currents in acontrol neuron elicited by the voltage step family displayed in the inset. (B–D) Voltage-dependent inactivation of Na+ currents recorded in individual neurons undercontrol conditions ( ), during the application of 100µM LTG, 100µM PHT and 600µM VPA () and after washout ( ). Peak Na+ current amplitudes were normal-ized and plotted versus the pre-pulse potential [see inset in A]. Solid lines represent Boltzmann functions $tted to the data points. (E) Average shift of the midpointof the inactivation (V1/2Inact) caused by LTG (n= 5), PHT (n= 6) and VPA (n= 5) in neurons of control ( ) and chronically epileptic ( ) rats (n= 6,7 and 5, respec-tively). ACSF, Arti$cial cerebrospinal #uid.

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animals, respectively, Fig. 5C). Smaller effects were observed for PHT(29 and 31%, Fig. 5B) and LTG (17 and 17%, Fig. 5A).

The slowing of the recovery from fast inactivation is considered tobe one main mechanism by which anticonvulsant drugs reduce theavailability of Na+ channels during high-frequency depolarizations.To characterize the effects of PHT, LTG and VPA on recovery fromfast inactivation, we have used double-pulse experiments with inter-pulse intervals varying from 1 ms to 5 s (see Fig. 6, inset). Representa-tive experiments are depicted under control conditions (Fig. 6A–C,upper panels), following application of the anticonvulsant drugs (mid-dle panels) and after washout (lower panels).

Clearly, both LTG and PHT potently slowed the time course ofrecovery from fast inactivation both in control animals (Figs 6 and7A, two leftmost panels) and in the chronic pilocarpine model of epi-lepsy (Figs 6 and 7B, two leftmost panels). In marked contrast, appli-cation of VPA did not appear to alter the fast recovery from

inactivation in either experimental group (Figs 6, and 7A and B,rightmost panels).

The effects of the different anticonvulsant drugs on recovery fromfast inactivation were quantitatively analysed by $tting the time courseof recovery with a double-exponential equation shown superimposedon the datapoints in Fig. 7A and B. In control animals, application ofPHT and LTG potently increased the fast time constant τfast (Fig. 8A;PHT, 2.3-fold from 6.41± 0.42 to 14.82± 1.89 ms, n= 8, P< 0.05;LTG, 1.9-fold from 6.22± 0.88 to 11.70± 0.60 ms, n= 11, P< 0.05).In addition, the amplitude contribution of the rapidly recovering com-ponent was signi$cantly decreased following application of PHT andLTG, while no changes were observed for VPA (Fig. 8C). In contrastto τfast, the slower recovery time constant τslow was unaltered followingapplication of any anticonvulsant drug (Fig. 8B). The amplitude con-tribution of this slower recovery component was increased followingthe application of PHT and LTG but not VPA (Fig. 8D).

Fig. 4. Effects of lamotrigine (LTG), phenytoin (PHT) and valproate (VPA) on the activation of Na+ channels. (A) Representative recording of Na+ currents in acontrol neuron elicited by the voltage protocol presented in the inset. (B–D) Voltage-dependent activation of Na+ currents recorded in three control neurons undercontrol conditions ( ), during the application of 100µM LTG, 100µM PHT and 600µM VPA () and after washout ( ). The conductances (see Materials and meth-ods) were normalized and plotted versus the test pulse voltage [see inset in (A)]. Solid lines represent Boltzmann functions $tted to the data points. (E) Average shiftof the midpoint of the activation curve (V1/2act) induced by application of LTG (n= 5), PHT (n= 6) and VPA (n= 5) in neurons of control ( ) and chronically epilep-tic ( ) rats (n= 6, 7 and 5). ACSF, Arti$cial cerebrospinal #uid.

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Fig. 5. Sensitivity of the window current resulting from the overlap of the voltage-dependent activation and inactivation curves to anticonvulsant drugs. The proba-bility of the Na+ channels being in a noninactivating state was estimated by multipliying the Boltzmann equations best describing Na+ current voltage dependenceof activation and inactivation. The resulting relations were plotted under control conditions (black lines) and following application of lamotrigine (LTG), phenytoin(PHT) and valproate (VPA), respectively (grey lines). Analyses are shown in an identical manner for control animals (A) and epileptic animals (B).

Fig. 6. The recovery from inactivation of the Na+ channel is slowed by lamotrigine (LTG) and phenytoin (PHT) but not by valproate (VPA). (A) Fast recovery frominactivation was analysed with double pulse experiments at a recovery potential of −80 mV, with various intervals (∆t) between a 10-ms conditioning pulse(−10 mV) and a 10-ms test pulse (see inset). Representative families of original traces recorded under control conditions [arti$cial cerebrospinal #uid (ACSF)], dur-ing application of 100µM LTG, 100µM PHT and 600µM VPA (drug) and following washout (wash) in three different dentate granule neurons of control rats.[Q22

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Qualitatively, PHT and LTG exerted similar effects in the pilo-carpine model of chronic epilepsy. Both PHT and LTG resulted in pro-nounced slowing of τfast. Both compounds also reduced the amplitudecontribution of the recovery process in pilocarpine-treated animals(Fig. 8C). Interestingly, when the effects of LTG and PHT were com-pared between control animals and chronically epileptic animals, PHTwas signi$cantly less effective in slowing in the pilocarpine model ofepilepsy. All other effects of PHT and LTG were unchanged by induc-tion of chronic experimental epilepsy. As surmised from inspection ofthe data in Figs 5, and 6C and F, application of 600µM VPA did notalter either τfast or τslow in dentate granule neurons isolated either fromcontrol or chronically epileptic animals (Fig. 8A and B).

Discussion

In this study, we have investigated the effects of three majoranticonvulsant drugs, PHT, LTG and VPA, on one of their main phar-macological targets, the voltage-gated Na+ channel, in native hippoc-ampal neurons. We have extended this analysis to consider whether theactivity of these substances is altered following chronic epileptogenesis.

Effects of lamotrigine, phenytoin and valproate in control rats

All three substances tested in this study modestly blocked the peakNa+ conductance elicited from hyperpolarized membrane potentials.

This tonic inhibition was also observed, to a different extent, in studiesof Na+ channel pharmacology in expression systems (Lang et al.,1993; Xie et al. 2001) and other types of native neurons (Kuo & Lu,1997). The inhibition by PHT and VPA was voltage dependent, withstronger blocking effects being observed when depolarizing pre-pulses were applied prior to eliciting Na+ currents. The voltage depen-dence of PHT and VPA effects was expressed as a shift of the inactiva-tion curve towards more hyperpolarized potentials, similar to previousstudies (Willow et al., 1985; Schwarz & Grigat, 1989; Ragsdale et al.,1991; Lang et al., 1993; Vreugdenhil & Wadman, 1999). In our hands,LTG failed to shift V1/2inact signi$cantly in hippocampal granule neu-rons, even though it does so potently at the same concentration(100µM) in mouse neuroblastoma cells (Lang et al., 1993) or cerebel-lar granule cells (Zona & Avoli, 1997). The discrepancies betweendifferent studies may be explained, in part, by differences in the volt-age step protocols. We and others (Xie et al., 1995; Vreugdenhil &Wadman, 1999) have employed brief pre-pulses to induce inactivationin order to examine fast inactivation processes. Many previous studieshave used much longer pre-pulses (5–9 s) to induce inactivation (Kuo& Bean, 1994). As binding of anticonvulsants to the inactivated stateof the Na+ channel is thought to occur in a time range of seconds,pharmacological effects would be expected to be saturated, and thusmore pronounced, with long pre-pulses. Indeed, selective effects ofLTG on slow but not fast inactivation processes have been demon-strated (Xie et al., 1995).

Fig. 7. Recovery from inactivation of the Na+ channel is slowed by lamotrigine (LTG) and phenytoin (PHT) but not by valproate (VPA). (A and B) Normalized cur-rent amplitudes of the recovery from fast inactivation in the dentate granule neurons of control (A) and chronic epileptic (B) rats were plotted against the interpulseduration on a logarithmic time scale. Fast recovery from inactivation was analysed with double pulse experiments at a recovery potential of −80 mV, with variousintervals (∆t) between a 10-ms conditioning pulse (−10 mV) and a 10-ms test pulse (see inset). The recovery process is displayed under control conditions ( ), dur-ing application of 100µM LTG (n= 10–11), 100µM PHT (n= 7–8) and 600µM VPA (n= 6–7) () and after washout ( ). The data were best described by a dou-ble-exponential equation (eqn 3, see Materials and methods) shown superimposed on the data points. ACSF, Arti$cial cerebrospinal #uid.

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Most anticonvulsant drugs acting on Na+ channels have beenshown to display an activity dependence of blocking effects, i.e.blocking effects are enhanced when neurons are repetitively depolar-ized at higher frequencies. This activity dependence is expressed as aslowing of recovery from fast Na+ channel inactivation (Schwarz &Grigat, 1989). In our hands, both LTG and PHT potently slowed therecovery from fast inactivation, as described previously in cell lines(Lang et al., 1993) and dissociated CA1 neurons (Kuo & Lu, 1997).The VPA did not exert any effects on the recovery from inactivation inrat dentate granule neurons, consistent with the lack of use-dependentblock of the human brain IIA Na+ channel by VPA (Xie et al. 2001).

In contrast to these $ndings, slowing of fast recovery from inactiva-tion by VPA has been described in other preparations (Vreugdenhilet al., 1998; Vreugdenhil & Wadman, 1999), possibly due to differen-tial VPA sensitivity of Na+ channel isoforms.

Altered anticonvulsant effects in the pilocarpine model of chronic epilepsy

In this study, we observed differences between control and epilepticanimals with respect to the tonic inhibition of Na+ channel amplitudefor PHT and LTG (77 and 64% reduction of blocking effects in thepilocarpine model, respectively) without changes in the voltagedependence of block. In addition, the slowing of the fast recoveryfrom inactivation by PHT was less marked in epileptic animals (35%reduction in the effects on τfast). All other effects of anticonvulsantdrugs were similar in control and epileptic animals.

What would the expected consequences of these changes be forthe ef$cacy of PHT or LTG in the inhibition of epileptiform activ-ity? With regard to tonic block of Na+ channels, impaired ef$cacy ofPHT and LTG would mean that these substances no longer reducethe number of Na+ channels available at resting potential. This mayresult in reduced effects of these substances on the properties of indi-vidual action potentials, such as rise time, threshold and amplitude(Selzer, 1984; Colbert & Johnston, 1996). The reduction of PHTeffects on the recovery from inactivation may be more relevant withregard to high-frequency neuronal activity because slowing of Na+

channel recovery from inactivation is thought to underlie activity- orfrequency-dependent blocking effects. Thus, reduced effects onrecovery from inactivation would be expected to render PHT lessef$cient in blocking high-frequency neuronal discharges (McLean &Macdonald, 1983). This loss of PHT ef$cacy in chronically epilepticanimals would become more and more noticeable with higher dis-charge frequencies. Such use-dependent effects have been shown tooccur at lower concentrations of PHT than those necessary to alterthe morphology of single action potentials (McLean & Macdonald,1983).

What is the correlation between a loss of PHT effects on Na+ chan-nels and sensitivity to PHT in the intact animal? Despite the loss ofPHT effects on Na+ channels demonstrated in this study, PHT is quiteeffective in blocking chronic convulsive seizures in pilocarpine-treated rats (Leite & Cavalheiro, 1995). One reason for this discrep-ancy could be that, despite the loss of PHT ef$cacy in dentate granulecells, PHT is suf$ciently active in other brain regions involved in thegeneration of convulsive seizures. It is presently unknown whetherchronic epilepsy alters anticonvulsant ef$cacy in other brain regionsinvolved in ictogenesis and this will be an important subject for futureexperiments. In another study, the PHT sensitivity of both Na+ chan-nels and intact animals was examined in an animal model of pharma-coresistance (Löscher et al., 1993). In these experiments, PHTresistance was not associated with altered tonic block of Na+ channelsby PHT (Jeub et al. 2002) but recovery from Na+ channel inactivationand use-dependent blocking effects were not studied.

Nevertheless, several studies support the idea that loss of ion chan-nel drug sensitivity may occur in chronic epilepsy. For instance, a

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Fig. 8. The fast time constant of Na+ channel recovery is increased by lamot-rigine (LTG) and valproate (VPA), but is signi$cantly less affected by pheny-toin (PHT) in neurons of chronic epileptic rats. (A–D) The percentage ofchange of the fast and slow recovery time constants τfast (A) and τslow (B) andthe corresponding amplitudes Afast (C) and Aslow (D) caused by application of100µM LTG (n= 10–11), 100µM PHT (n= 7–8) and 600µM VPA (n= 6–7) indentate granule neurons of control ( ) and chronically epileptic ( ) rats. Aster-isks indicate signi$cant difference (P< 0.05, Student's unpaired t-test).

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10 S. Remy et al.


diminished effect of CBZ has been observed on the steady-state inacti-vation properties of Na+ channels in CA1 neurons from kindled ani-mals, but these effects were only transient (Vreugdenhil & Wadman,1999). Circumstantial evidence indicates that a similar loss of sensi-tivity in CA1 neurons may occur in human epilepsy (Vreugdenhilet al., 1998). In contrast to these comparatively modest effects, a com-plete loss of CBZ effects on the recovery from fast Na+ channel inac-tivation was observed in chronic experimental epilepsy and in humanCBZ-resistant patients (Remy et al. 2002). Interestingly, data fromepilepsy patients and the kindling model of epilepsy suggest that VPAeffects on Na+ channels are unaltered in seizure foci (Vreugdenhilet al., 1998; Vreugdenhil & Wadman, 1999), consistent with data pre-sented here demonstrating unaltered VPA sensitivity of Na+ channelsin the pilocarpine model.

What relation do these data have to the development of drug resis-tance, either in chronic human epilepsy (Aicardi & Shorvon, 1997) orin some animal models of epilepsy (Löscher et al., 1993)? Our data donot allow us to directly address this question, since we did not dis-criminate subgroups of pilocarpine-treated rats with and without resis-tance to antiepileptic drugs. Nevertheless, our present and previousresults suggest a scenario in which the use-dependent block of Na+

channels by CBZ is virtually completely lost in chronic epilepsy(Remy et al. 2002) while PHT effects are quantitatively reduced (thisstudy). At the very least, this suggests that, for PHT, LTG and VPA,other mechanisms may also contribute to the development of drugresistance. These may conceivably consist of decreased sensitivity ofother targets of these drugs. In addition to altered properties of drugtargets, a further intriguing possibility would be that the properties oftransporters that regulate intraparenchymal drug concentrations arealtered in chronic epilepsy. Indeed, multiple drug transporters havebeen shown to be up-regulated, both in animal models of epilepsy andin human epilepsy (Tishler et al., 1995; Sisodiya et al., 1999, 2001,2002a,b; Dombrowski et al. 2001). These multidrug transportersef$ciently transport different anticonvulsants including PHT, LTG andVPA (see Löscher & Potschka 2002 for review).

Taken together, our and previously published results suggest that itwill be necessary to dissect the relative importance of altered drug tar-gets versus altered regulation of intraparenchymal drug concentrationsfor each anticonvulsant drug in order to understand what relation thesetwo candidate mechanisms may have to the development of drugresistance.

Acknowledgements

This research was supported by the graduate programme 246 of the DeutscheForschungsgemeinschaft ‘Pathogenese von Krankheiten des Nervensystems’, aUniversity of Bonn Medical Center grant ‘BONFOR’, the SFB-TR3 and theGerman–Israel collaborative research programme of the MOS and the BMBF/DLR.

Abbreviations

CBZ, carbamazepine; LTG, lamotrigine; PHT, phenytoin; VPA, valproic acid.

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Journal: EJNArticle : 2710

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