Proepileptic phenotype of SV2A-deficient mice is

associated with reduced anticonvulsant

efficacy of levetiracetamRafal M. Kaminski, Michel Gillard, Karine Leclercq, Etienne Hanon, Genevieve Lorent,

Donald Dassesse, Alain Matagne, and Henrik Klitgaard

UCB Pharma S.A., CNS Research, B-1420 Braine-l’Alleud, Belgium

SUMMARY

Purpose: Synaptic vesicle protein 2A (SV2A) con-

stitutes a distinct binding site for an antiepileptic

drug levetiracetam (Keppra). In the present study

we characterized SV2A (+/)) heterozygous mice

in several seizure models and tested if the anticon-

vulsant efficacy of levetiracetam is reduced in

these mice.

Methods: Seizure thresholds of male SV2A (+/))

mice and their wild-type littermates were assessed

in pilocarpine (i.p.), kainic acid (s.c.), pentylenetet-

razol (i.v.), 6-Hz and maximal electroshock

models. Kindling development was compared in

amygdala and corneal kindling models. Ex vivo

binding of levetiracetam to SV2A was also

performed.

Results: Long-term electroencephalography (EEG)

monitoring and behavioral observations of SV2A

(+/)) mice did not reveal any spontaneous seizure

activity. However, a reduced seizure threshold of

SV2A (+/)) mice was observed in pilocarpine,

kainic acid, pentylenetetrazol, and 6-Hz models,

but not in maximal electroshock seizure model.

Accelerated epileptogenesis development was

also demonstrated in amygdala and corneal kind-

ling models. Anticonvulsant efficacy of levetirace-

tam, defined as its ability to increase seizure

threshold for 6 Hz electrical stimulation, was sig-

nificantly reduced (approx. 50%) in the SV2A (+/))

mice, consistently with reduced binding to SV2A

in these mice. In contrast, valproate produced the

same anticonvulsant effect in both SV2A (+/+) and

SV2A (+/)) mice.

Discussion: The present results evidence that

SV2A is involved in mediation of the in vivo anti-

convulsant activity of levetiracetam, in accor-

dance with its previously proposed mechanism of

action. Furthermore, the present data also indi-

cate that even partial SV2A deficiency may lead to

increased seizure vulnerability and accelerated

epileptogenesis.

KEY WORDS: Levetiracetam, SV2A, Epilepsy,

Antiepileptic drugs, Animal models.

The discovery that synaptic vesicle protein 2A (SV2A)is the binding site for the antiepileptic drug (AED) leveti-racetam (Keppra; UCB Pharma S.A., Braine-l’Alleud,Belgium) stimulated renewed interest in this protein(Lynch et al., 2004). In addition to being widely recog-nized as a presynaptic marker (Bajjalieh et al., 1993,1994), SV2A became an important target for the discoveryof novel antiepileptic therapies (Lynch et al., 2004;

Klitgaard & Verdru, 2007; Rogawski, 2008). In fact,brivaracetam, a novel high-affinity SV2A ligandwith inhibitory action on voltage-gated Na+ channels(Matagne et al., 2008) is currently in late stages of clinicaldevelopment for epilepsy.

The function of SV2A, ubiquitously expressed in thebrain, is far from being completely elucidated (Sudhof,2004). Most information about the role of SV2A in neuro-nal excitability comes from studies performed in knock-out mice (Crowder et al., 1999; Janz et al., 1999; Xu &Bajjalieh, 2001; Custer et al., 2006). It appears that SV2Ais not crucial for vesicle biogenesis or synaptic function,but modulates exocytosis of transmitter-containing vesi-cles (Crowder et al., 1999; Xu & Bajjalieh, 2001). Micelacking SV2A are characterized by a decrease in the

Accepted January 30, 2009; Early View publication April 19, 2009.Addresss correspondence to Rafal Kaminski, M.D., Ph.D., Head of

Epilepsy Pharmacology, UCB Pharma S.A., Chemin du Foriest, R9,B-1420 Braine-l’Alleud, Belgium. E-mail: rafal.kaminski@ucb.com

Wiley Periodicals, Inc.ª 2009 International League Against Epilepsy

Epilepsia, 50(7):1729–1740, 2009doi: 10.1111/j.1528-1167.2009.02089.x

FULL-LENGTH ORIGINAL RESEARCH

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calcium-dependent exocytotic burst, which is a measureof the availability of neurotransmitter vesicles ready torelease their content (Xu & Bajjalieh, 2001). Moreover,lack of SV2A results in decreased action potential–depen-dent neurotransmission, whereas action potential–inde-pendent neurotransmission remains normal (Crowderet al., 1999; Janz et al., 1999).

Two key observations indicated that SV2A is the tar-get site for levetiracetam. First, levetiracetam binding isabsent in SV2A ()/)) mice (Lynch et al., 2004). Sec-ond, the anticonvulsant potency of SV2A ligands inseveral epilepsy models strongly correlates with theirSV2A binding affinity (Lynch et al., 2004; Kaminskiet al., 2008). However, direct evidence that the in vivoanticonvulsant activity of levetiracetam is mediated bySV2A is still lacking. Genetically engineered knockoutanimals, which lack a given drug target, are frequentlyused in pharmacology to provide essential evidenceconcerning involvement of this target in mediation ofan anticonvulsant effect of its respective ligand(Rudolph et al., 1999; Szot et al., 2004). Similar proofof concept evidence could have been obtained by test-ing levetiracetam in SV2A ()/)) homozygous mice.However, these animals have severe seizures startingvery early in their development and do not survivebeyond 2–3 weeks after birth, which precludes theiruse in pharmacologic in vivo experiments (Crowderet al., 1999; Janz et al., 1999). In the present study, wedecided to use SV2A (+/)) mice, which are deficientin the SV2A protein, but develop normally after birth.We demonstrate that SV2A (+/)) mice do not displayany overt epileptic phenotype, but rather show pro-epileptic traits such as decreased seizure thresholds andaccelerated kindling development. In fact, the pheno-type of SV2A (+/)) in a range of different experimen-tal seizure models appears as a mirror image of theprofile of levetiracetam in these models. We also dem-onstrate for the first time a functional involvementof SV2A in mediation of the anticonvulsant effect oflevetiracetam, which is reduced in SV2A (+/)) mice.Some of the present results were previously publishedin abstract form (Leclercq et al., 2006, 2007).

Materials and Methods

AnimalsMale SV2A (+/)) mice and their littermate controls,

age 8–12 weeks, weighing 25–30 g, were used in alltests. All mice originated from the same breeding colony(UCB, Braine-l’Alleud, Belgium). SV2A (+/)) micedevelop normally and have normal brain morphology(Fig. 2), but slight decreases in body weight (<10%) andtemperature (�0.5�C) were observed (Lamberty et al.,2009). The mice were housed in groups of 3–5 percage (or single housed for kindling/EEG recording

experiments) and kept on a 12/12 h light/dark cycle withlights on at 6:00 h, having free access to standard pelletchow and water. On the day of testing they were habitu-ated to the experimental room for at least 1 h. Exceptfor kindling experiments, each mouse was used onlyonce. Experimental groups consisted of at least eightanimals. All animal experiments were done according tothe Helsinki declaration and conducted in accordancewith the guidelines of the European Community Councildirective 86/609/EEC. A local ethical committeeapproved the experimental protocol.

DrugsLevetiracetam (UCB) and valproate (Sigma, Bornem,

Belgium) were dissolved in saline and injected i.p. 60 and15 min before the tests, respectively. Injection volume forboth drugs was 10 ml/kg. Treatment doses and pretreat-ment times were based on pilot dose–response experi-ments performed with naive NMRI mice and previouslypublished reports (Klitgaard et al., 1998).

Binding studiesMice were injected i.p. with either levetiracetam

(0.054–544 mg/kg) or saline. At 60 min after administra-tion, animals were sacrificed and their forebrains quicklyremoved and homogenized in 750 ll of ice-cold 50 mM

Tris–HCl buffer (pH 7.4) containing 250 mM sucrose.Samples were frozen in liquid nitrogen and stored at)80�C until further experimental procedures. Bindingstudies were performed essentially as described by Gillardet al. (2003) with minor modifications. In brief, 10 ll ofbrain homogenates (650–750 lg protein) were incubatedfor 90 min at 4�C in a 50 mM Tris–HCl (pH 7.4) buffercontaining 2 mM MgCl2 and 2 nM [3H]ucb 30889 (32 Ci/mmol; custom synthesis by GE Healthcare, United King-dom). Membrane-bound radioligand was recovered byrapid filtration (<10 s/sample) through GF/C glass fiberfilters presoaked in 0.1% polyethyleneimine. Filters andsamples were rinsed by 8 ml of ice-cold 50 mM Tris–HClbuffer (pH 7.4). Radioactivity trapped on dried filters wasmeasured by liquid scintillation. Nonspecific binding wasdefined as the residual radioactivity measured in the pres-ence of 1 mM levetiracetam. Each sample was tested induplicate. Binding curves were analyzed by nonlinearregression using GraphPad Prism 4.0 (GraphPad Soft-ware, San Diego, CA, U.S.A.) with a model describingligand binding to a homogenous population of bindingsites. Percentage of receptor occupancy (RO) was definedas:

ROð%Þ ¼ 100� 100� BD �NSBð ÞBS �NSBð Þ

� �� �;

where, BD and BS are the total binding measured in thepresence or absence of drug, respectively, and NSB is thenonspecific binding.

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ImmunocytochemistryMice were deeply anesthetized with sodium pentobar-

bital (60 mg/kg, i.p.) and transcardially perfused witha physiologic solution containing heparin (4,000 IU/L in0.9% saline) followed by 0.12 M phosphate buffered4% formaldehyde freshly prepared from paraformalde-hyde (VWR International, Fontenay-sous-Bois, France).Brains were removed and postfixed in the same fixativeovernight at 4�C. They were rinsed in several changesof water and then progressively dehydrated, embeddedin paraffin, cut into 5-lm sections, collected on posi-tively charged glass slides (DAKO, Glostrup, Denmark)and kept in serial order at room temperature until use.Following deparaffinization and rehydration, the sliceswere placed in citrate buffer (0.1 M; pH 6.0) and heatedby microwave (3 min at 750W then 2 times 5 min at350W) for antigen unmasking. The endogenous peroxid-ases activity was blocked in a peroxidase blocking solu-tion (DAKO) for 15 min. The nonspecific binding wasblocked in 0.5% cold-water-fish gelatin (Sigma Aldrich,Steinheim, Germany) diluted in Tris buffered saline(TBS; 0.05 mol/L Tris–HCl, 0.15 mol/L NaCl, 20%Tween, pH 7.6) for 15 min. Thereafter, sections wereincubated for 1 h at room temperature with anti-SV2Apolyclonal antiserum (ref SC-11936, Santa Cruz Inc.,Santa Cruz, CA, U.S.A.) diluted at 1/200 in 0.1% TBS-CWF-Gelatin (DAKO). The unbound primary antibodywas removed by rinsing with TBS (0.05 mol/L Tris–HCl, 0.15 mol/L NaCl, 20% Tween, pH 7.6). Goatgamma globulins were bounded with biotinylated anti-goat gamma globulin at 1/600 dilution in 0.1% TBS-CWF-Gelatin for 30 min (DAKO). Following TBSrinse, the binding signal was amplified by an incubationof avidin-biotin-peroxidase complex (Vector Laborato-ries, Burlingame, CA, U.S.A.) for 30 min. The immuno-reaction was visualized by using peroxidase substrate3,3¢ diaminobenzidine tetrahydrochloride (DAB sub-strate chromogene system, DAKO) for 10 min. Thereaction was stopped by TBS rinsing (0.05 mol/LTris–HCl, 0.15mol/L NaCl, 20% Tween, pH 7.6). Aftercounterstaining with Mayer hematoxylin and subsequentdehydration, the sections were cleared with xylene,coverslipped with DPX (Sigma Aldrich), and examinedand photographed in a microscope.

In addition to the specificity confirmation of theSV2A antiserum by the manufacturer, several controlexperiments were performed in order to validate theaccurate antigenic target recognition: (1) preabsorptionof antibody with 0.26 lg/ml of the correspondingantigenic peptide (SC-11936P; Santa Cruz Inc.) for60 min; (2) preincubation of primary antibody with0.26 lg/ml of irrelevant peptides of SV2B and SV2Cisoforms (SC-11941P and SC-11944P; Santa CruzInc.) for 60 min; (3) omission of the primary anti-body (replacement by TBS-CWF-Gelatin 0.1%); and

(4) check for lack of labeling on sections where noSV2A isoform is expressed (SV2A )/) mice).

Digitalized images with 256 gray levels were analyzedwith the public domain software Image J Program(National Institutes of Health, Bethesda, MD, U.S.A.).The SV2A expression was quantified at four standardtransverse sections, anteroposterior from bregma: +0.98,)2.06, )3.28, and )6.84 mm (Paxinos & Franklin, 2001).The intensity of the SV2A immunostaining was evaluatedby means of a relative optical density (ROD) value, whichwas obtained after transformation of mean gray valuesusing the formula ROD = log (256/mean gray value).Before measuring, on each section, an averaged opticaldensity of the background level was subtracted to obtaincorrected values.

Surgery and EEG recordingThe animals were anesthetized with medetomidine

hydrochloride (0.5 mg/kg, i.p., Domitor; Pfizer Louvain-la-Neuve, Belgium) and ketamine (50 mg/kg, i.p., Imal-gene; Merial, Lyon, France) for implantation of EEGelectrodes. For experiments involving EEG monitoring,the mice were implanted with a monopolar depth elec-trode into the CA1 region of the hippocampus (coordi-nates vs. bregma: )1.94 mm anteroposterior, )1.0 mmlateral, )1.25 mm depth). Three monopolar cortical elec-trodes, frontal left (+1.0 mm anteroposterior, )2.0 mmlateral) and occipital left/right: ()4.0 mm anteroposterior,+/)4 mm lateral) were also implanted. A ground electrodewas placed in the left prefrontal bone. EEG activity fromboth hippocampal and cortical electrodes was recordedwith a Model 15 Neurodata Amplifier System (GrassTechnologies, West Warwick, RI, U.S.A.) for a total of12 h (two 6-h sessions). For amygdala kindling experi-ments a bipolar depth electrode (0.4 mm diameter,0.2 mm distance between electrode tips) was implantedinto the basolateral amygdala ()1.82 mm anteroposterior,)2.5 mm lateral, )5 mm depth). In addition, a monopolarelectrode in the left occipital cortex ()4.0 mm anteropos-terior, )3 mm lateral) was also implanted. A ground elec-trode was placed in the left prefrontal bone. Cortical andamygdala activity was recorded with the same Grass EEGsystem before and after kindling stimulation. Accuracy ofelectrode implantation was verified postmortem andcompared against an anatomic atlas (Paxinos & Franklin,2001).

Amygdala kindlingAfter a recovery period (10 days postsurgery), the kind-

ling procedure began with determination of an afterdis-charge threshold (ADT). Monophasic pulses, 1-s trains of1-ms square-wave (50 Hz), were delivered via bipolarelectrode (see above). Stimulation was started at 10 lA(base-to-peak) and increased in 10-lA steps until an after-discharge (at least 5 s) was elicited. The lowest current

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intensity that triggered an afterdischarge was consideredas the ADT. Subsequently, the mice were kindled byonce-daily stimulations with a current of intensity equal tothe individual ADT values (1-s monophasic square-wavepulses). The behavioral severity of seizures was scoredaccording to Racine’s scale (Racine, 1972), where stage1 = facial twitches and chewing, stage 2 = head nodding,stage 3 = bilateral forelimb clonus, stage 4 = rearing withforelimbs clonus, and stage 5 = generalized clonus withrearing and falling.

Corneal kindlingKindling was induced by twice-daily stimulations with

a current intensity of 2 mA for 3 s delivered via cornealelectrodes connected to a stimulator (WITT IndustrieElektronik, Berlin, Germany) as described previously(Matagne & Klitgaard, 1998). Each day the stimulationswere separated by at least a 4-h interval. A drop of 0.4%oxybuprocaine hydrochloride (Unicaine, Thea, France)was placed on the eyes before stimulation. The behavioraleffects of kindling were assessed in each group of animalsaccording to Racine’s scale (see above).

Threshold for pilocarpine-, kainic acid-, and pentyl-enetetrazol (PTZ)-induced seizures

Increasing doses of pilocarpine (i.p.) and kainic acid(s.c.) (both from Sigma) were injected to determine theseizure thresholds expressed as a dose required to induceconvulsions in 50% of tested animals (CD50 value). Meth-ylscopolamine (1 mg/kg, i.p., Sigma) was injected 30 minbefore administration of pilocarpine to prevent peripheralcholinergic symptoms. Immediately after administrationof the convulsants mice were placed into individual cagesand observed for 60 min (pilocarpine) and 90 min (kainicacid). The presence of generalized clonic convulsions withloss of righting reflex was noted and CD50 values werecomputed. A fixed dose of kainic acid (16 mg/kg, s.c.)was injected in separate groups of SV2A (+/+) and SV2A(+/)) mice, and their EEG was monitored for 2 h precededby 30 min baseline recording.

Figure 1.

Results of binding experiments performed with a spe-

cific SV2A ligand [3H]ucb 30889 in SV2A (+/+) and

SV2A (+/)) mice. Scatchard plot (panel A) illustrates

relation between specific binding (B) and specific bind-

ing divided by free radioligand concentration (F).

Ex-vivo SV2A radioligand binding after intraperitoneal

(i.p.) administration of levetiracetam (panel B). Calcu-

lated occupancy of SV2A after i.p. administration of lev-

etiracetam (panel C). Data expressed as mean ± SEM,

n = 1–2 mice per dose.

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PTZ (i.v.) infusion was performed as previouslydescribed (Kaminski et al., 2005). Before infusion, micewere briefly restrained, and a 30-gauge needle attached toa 0.3-m long polyethylene tubing (PE-10) was inserted intothe lateral tail vein. The needle was gently secured to thetail with plastic tape. The tubing was connected to a syringemounted on infusion pump (Harvard Apparatus, Holliston,MA, U.S.A.). PTZ (5 mg/ml) (Sigma) was infused at therate of 0.25 ml/min in freely moving mice. The time fromthe start of the infusion to the onset of clonic convulsionswas recorded. The threshold doses of PTZ were calculatedaccording to the following formula: threshold dose [mg/kg] = (PTZ concentration [mg/ml] · infusion rate [ml/s] ·infusion duration [s] · 1,000)/weight of mouse [g]).

Threshold for maximal electroshock (MES) and 6-Hzseizures

MES was induced by electrical stimulation (0.2 s,50 Hz) delivered via corneal electrodes connected to a

stimulator (WITT Industrie Elektronik). Tonic hind limbextension was the seizure endpoint in this test (Klitgaardet al., 1998). 6-Hz seizures were also induced by cornealstimulation, but with different current parameters(0.2 ms-duration monopolar pulses at 6 Hz for 3 s) deliv-ered by a stimulator (ECT Unit 57800; Ugo Basile,Comerio, Italy). Behavioral immobility of at least 7 sduration was the seizure endpoint in this test (Kaminskiet al., 2004). In each test, separate groups of animalswere challenged with varying current intensities, whichallowed determination of a CS50 value (i.e., currentrequired to induce seizures in 50% of animals). A dropof 0.4% oxybuprocaine hydrochloride (Unicaine) wasplaced on the eyes before electrical stimulation. Treat-ment doses selected for the experiments in the 6 Hzmodel were based on pilot dose–response studies per-formed with naive NMRI mice. In the present experi-ments, both levetiracetam and valproate were tested atdoses one step lower and one step higher (on log scale)

Figure 2.

SV2A immunostaining of coronal brain sections of SV2A (+/+) (first row) and SV2A (+/)) mice (second row). Brain

slices of SV2A (+/)) mice show strong reduction in the labeling of SV2A in comparison to slices taken from the

wild-type littermates. Schematic drawing (bottom left corner) illustrates the four brain levels (dashed lines) chosen

for quantitative analysis. These four levels correspond to +0.98, )2.06, )3.28, and )6.84 mm anteroposterior from

bregma. Bar charts (bottom right corner) illustrate expression of SV2A (mean ± SD) in relative optical density

(ROD) measures at the four selected brain levels. *p < 0.05; **p < 0.01; ***p < 0.001 as compared to the wild-type

littermates (Student’s t-test, n = 6 mice per genotype).

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than their respective ED50 values against 6 Hz in naiveNMRI mice (data not shown).

Data analysis and statisticsDose- or current-response experiments allowed deter-

mination of seizure thresholds in the SV2A (+/+) and(+/)) mice, which were expressed as CD50 or CS50 inmg/kg or mA, respectively. These values were determinedand statistically compared after nonlinear fitting of thedose- or current-responses curves with GraphPad Prism4.0 (GraphPad Software) according to a previouslydescribed method (Likhodii et al., 2003). The NS50 values,defined as the number of electrical stimulations necessaryto induce kindling in 50% of animals, were also deter-mined and compared with GraphPad Prism 4.0 softwareafter nonlinear curve fitting. Average ROD values, ADTvalues, and threshold doses of PTZ were comparedusing Student’s t-test. The 95% confidence intervals ofindividual CS50 values obtained in the 6-Hz model afterpretreatment with two different doses of levetiracetam orvalproate were transformed into SEM. Subsequently, theDCS50s = [CS50 (DRUG) - CS50 (SALINE)] for each mousegenotype were calculated and compared statistically withStudent’s t-test according to the method described byPorreca et al. (1990).

Results

Binding experiments performed in brain membranesconfirmed that the Bmax value (4 pmol/mg) for a selectiveSV2A radioligand, [3H] ucb 30889, is reduced by 50% inSV2A (+/)) when compared to the Bmax value (8.8 pmol/mg) obtained with their wild-type littermates (Fig. 1A).However, the radioligand dissociation constant (Kd) wascomparable between SV2A (+/+) and (+/)) mice, 52 and69 nM, respectively. These observations were further cor-roborated by ex vivo binding experiments, which alsorevealed that SV2A (+/)) mice have half of the proteinavailable for binding as compared to their wild-type litter-mates (Fig. 1B). Injection of levetiracetam to both SV2A(+/+) and SV2A (+/)) mice produced similar displace-ment of ex vivo radioligand binding and the slopes of dis-placement curves were not statistically different (Fig. 1B).Ex vivo SV2A occupancy calculated from these curveswas almost identical in both genotypes (Fig. 1C). Conse-quently, the IC50 values of levetiracetam calculated fromthe ex vivo binding curves were also similar in both SV2A(+/+) and SV2A (+/)) mice, 14.0 and 13.7 mg/kg, respec-tively.

SV2A (+/)) mice are characterized by reduced expres-sion of SV2A protein as illustrated by immunocytochem-istry studies (Fig. 2). Quantitative analysis of the RODlabeling performed in forebrain, midbrain, and hindbrainsections of SV2A (+/)) mice revealed consistent anduniform signal decrease, which indicates homogenous

reduction in the protein expression throughout the brain(Fig. 2).

Amygdala stimulation revealed that an afterdischargecan be elicited in SV2A (+/)) mice by lower intensity cur-rent than that required for SV2A (+/+) mice. Therefore,SV2A (+/)) mice are characterized by significantlyreduced ADT in comparison to their wild-type littermates(Table 1). Furthermore, the first kindling stimulationalready produced generalized seizures (stage 5) in 2 of 10SV2A (+/)) mice, whereas the first stage 5 seizure wasobserved in SV2A (+/+) mice in only one animal after sixstimulations (Fig. 3A). All SV2A-deficient mice deve-loped stage 5 seizures after 12 stimulations, whereas 20stimulations were required for their wild-type littermatesto reach that state (Fig. 3A). Statistical comparison of thenumber of stimulations predicted to induce stage 5 sei-zures in 50% of animals (NS50) revealed significant differ-ence between the genotypes, confirming faster kindlingdevelopment in SV2A (+/)) mice (Fig. 3B). Consistently,the average seizure score was also higher in the SV2A(+/)) mice during kindling (Fig. 3C), but there were nodifferences in the afterdischarge duration (Fig. 3D).Accelerated epileptogenesis was also observed in cornealkindling experiments, where all SV2A (+/)) mice devel-oped stage 5 seizures after 10 days of stimulation, whereasSV2A (+/+) mice required 22 days of stimulation(Fig. 3E). Consequently, the NS50 value was also signifi-cantly lower in SV2A (+/)) mice (Fig. 3F).

Injection of pilocarpine produced a dose-dependentincrease in the occurrence of generalized convulsions in

Table 1. Seizure thresholds of SV2A (+/+)

and SV2A (+/–) mice in different

experimental models

Seizure model

Genotype

SV2A (+/+) SV2A (+/–)

Afterdischarge threshold

(ADT in lA)

80.0 (64.5–95.5) 63.0 (43.9–82.1)*

Pilocarpine

(CD50 in mg/kg)

335.4 (317.6–354.2) 248.2 (235.3–261.8)*

Kainic acid

(CD50 in mg/kg)

34.9 (34.3–35.5) 30.1 (29.6–30.7)*

PTZ (i.v.)

(THD in mg/kg)

61.3 (54.0–68.5) 48.5 (43.3–53.8)*

6 Hz (CS50 in mA) 19.4 (18.9–19.9) 14.0 (8.7–22.5)*

MES (CS50 in mA) 14.6 (13.7–15.6) 14.2 (13.5–14.9)

ADT, afterdischarge threshold, that is, the minimal current

required to elicit an afterdischarge in the EEG; CD50, a dose

required to induce generalized convulsions in 50% of tested

animals; THD, threshold dose required to elicit generalized

clonic convulsions in all animals; CS50, a current required to

induce convulsions in 50% of animals; 95% confidence intervals

in parentheses; i.v., intravenous infusion.

*p < 0.05, Student’s t-test.

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both genotypes of SV2A mice. However, pilocarpine’sdose-response curve obtained after testing of SV2A (+/))mice was shifted to the left in comparison to the oneobtained with SV2A (+/+) mice (Fig. 4A). This resulted ina significantly lower CD50 value, indicating increased vul-nerability of SV2A (+/)) animals to pilocarpine-inducedseizures (Table 1). Similar results, that is, a leftward shiftof the dose-response and lower CD50 values for the induc-tion of generalized seizures, were observed in SV2A (+/))mice after kainic acid–induced convulsions (Fig. 4B;Table 1). The threshold dose of PTZ required to induceclonic convulsions was also significantly reduced inSV2A (+/)) mice (Table 1). These mice also displayedincreased sensitivity to 6-Hz seizures, illustrated by a left-ward shift in the response to stimulation current (Fig. 4C)and a decreased CS50 value (Table 1). This result was inaccordance with lower seizure thresholds of SV2A (+/))mice observed in other models, that is, amygdala kindling,pilocarpine, kainate, and PTZ. However, in striking con-trast to those models, the threshold for MES remainedunchanged in the SV2A (+/)) mice (Fig. 4D, Table 1).

Although EEG monitoring (12 h total) failed todetect any epileptiform activity in naive SV2A (+/))

mice, these animals displayed much more robust andprolonged epileptic bursts after kainic acid challenge(Fig. 5). In fact, SV2A (+/)) mice continued to dis-play ictal EEG activity at 60–120 min after kainicacid injection, whereas only interictal spikes and sharpwaves were visible in the EEG of SV2A (+/+) miceduring this period (Fig. 5).

In the 6-Hz model, levetiracetam (17 and 170 mg/kg) administered to SV2A (+/+) mice produced dose-dependent rightward shifts in responses to stimulationcurrent (Fig. 6A), congruent with elevations of theseizure threshold (Fig. 6C). In the SV2A (+/)) mice,the lower dose of levetiracetam (17 mg/kg) also pro-duced rightward shift in current response (Fig. 6B) andan elevation of seizure threshold that was comparablewith SV2A (+/+) mice (Fig. 6C). However, the higherdose of levetiracetam (170 mg/kg) produced a muchless pronounced shift in current response (Fig. 6B) anda significantly smaller (�50% less) elevation in seizurethreshold of SV2A (+/)) mice (Fig. 6C). In contrast tolevetiracetam, two doses of valproate (124 and 166 mg/kg) produced comparable elevations of seizure thresh-old in both genotypes of SV2A mice (Fig. 6C).

Figure 3.

Development of amygdala and

corneal kindling in SV2A (+/+)

and SV2A (+/)) mice. Panels A

and E illustrate the percentage

of mice responding with stage 5

seizure (Racine, 1972) after daily

electrical stimulations during

amygdala or corneal kindling,

respectively. Panels C and D

illustrate average seizure score

and afterdischarge duration

during amygdala kindling,

respectively. The NS50 values

with 95% confidence intervals,

defined as the number of

electrical stimulations necessary

to induce amygdala and corneal

kindling in 50% of animals (panels

B and F, respectively), were

determined after nonlinear

curve fitting and compared

statistically (*p < 0.05, Student’s

t-test; n = 9–11 mice per

group).

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Discussion

Binding experiments in SV2A (+/+) wild-type andSV2A (+/)) heterozygous mice indicated that the affinityof SV2A ligands remained unaltered and the relativeex-vivo SV2A occupancy curves for levetiracetam werecompletely overlapping. However, SV2A (+/)) mice

displayed 50% less sites available for binding of leveti-racetam, consistent with its proposed mechanism of action(Lynch et al., 2004).

It has been reported that SV2A ()/)) homozygous micedevelop severe spontaneous seizure phenotype, withlethality occurring within 2–3 weeks of postnatal develop-ment (Crowder et al., 1999; Janz et al., 1999). The SV2A

Figure 4.

Characterization of seizure

vulnerability of SV2A (+/+) and

SV2A (+/)) mice. Dose-respon-

ses for seizures induced by

pilocarpine (panel A) and kainic

acid (panel B). Current-respon-

ses for seizures induced by

6-Hz electrical stimulation

(panel C) and maximal electro-

shock (panel D). Each data

point represents percentage of

animals (n ‡ 8) responding with

seizures. Experimental points in

each panel were used for

nonlinear curve-fitting with

sigmoidal dose- or current-

response model.

Epilepsia ILAE

Figure 5.

Cortical electroencephalography (EEG) recorded with a monopolar depth electrode in the CA1 region of

hippocampus in SV2A (+/+) (left panel) and SV2A (+/)) (right panel) mice before (baseline) and at different times

after kainic acid (16 mg/kg, s.c.) injection.

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(+/)) mice appeared indistinguishable from wildtype lit-termates and did not display any convulsions (Janz et al.,1999). Consequently, our EEG monitoring experimentsdid not reveal any epileptiform activity in these animals.However, based on the data from SV2A ()/)) mice, a pro-epileptic phenotype of SV2A heterozygotes was antici-pated and demonstrated in the present study. Remarkably,a recent report revealed reduced expression of SV2A inhippocampal samples obtained from both epileptic ratsand patients with temporal lobe epilepsy (van Vliet et al.,2008). Therefore, even partial reduction of SV2A expres-sion leads to increased seizure susceptibility and acceler-ated epileptogenesis.

The proepileptic phenotype of SV2A (+/)) mice wasobserved in kindling, pilocarpine, kainate, pentylenetetra-zol (i.v.), and 6 Hz models, but not in the MES model.Interestingly, this profile matches very well with theprotective activity of levetiracetam in those models(Table 2). In fact, levetiracetam has been demonstrated toinhibit the development of amygdala kindling and sug-gested to have antiepileptogenic effects (Lçscher et al.,1998; Husum et al., 2004; Margineanu et al., 2008). Thesedata appear to be substantiated by the present resultsindicating accelerated amygdala kindling development inSV2A-deficient mice. In order to rule out that such a fasterkindling acquisition was a function of reduced ADT, and

Figure 6.

Current-responses for seizures induced by 6-Hz electrical stimulation after treatment with levetiracetam (LEV) in

SV2A (+/+) mice (panel A) and SV2A (+/)) mice (panel B). Points representing the percentage of animals (n ‡ 8 per

group) responding with seizures were used for sigmoidal curve-fitting. CS50 value represents a current required to

induce convulsions in 50% of animals. Panel C illustrates the DCS50s = [CS50 (DRUG) - CS50 (SALINE)] ± SEM calculated

after treatment with two different doses of LEV or valproate (VPA). Values were compared statistically with

Student’s t-test.

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thus of increased seizure susceptibility, both SV2A (+/+)and SV2A (+/)) mice were stimulated with a current thatwas equal to the individual ADT of each mouse. In addi-tion, a similar but even more pronounced phenotype wasobserved in the corneal kindling model. It should be notedthat both levetiracetam and brivaracetam delay kindlingdevelopment in this model (Matagne & Klitgaard, 1998;Matagne et al., 2008). Finally, levetiracetam affordsprotection against generalized pilocarpine- and kainicacid–induced seizures (Klitgaard et al., 1998). Althoughlevetiracetam is not active against seizures induced by su-pramaximal doses of PTZ s.c. (Klitgaard et al., 1998), itdoes elevate the threshold for seizures induced by PTZ i.v.(Lçscher & Hçnack, 1993). Interestingly, SV2A (+/))mice are significantly more susceptible to all these chemo-convulsants and require much lower doses than their wild-type littermates to induce generalized convulsions. Instriking contrast to the above-described observations, theSV2A (+/)) mice did not differ from their wild-type litter-mates when tested in the MES model; and levetiracetam isnot active against MES seizures (Klitgaard et al., 1998)(Table 2). It is important to emphasize that different brainregions appear to be recruited depending on seizure-inducing stimulus or model (Barton et al., 2001; Eellset al., 2004). Brainstem regions are involved in mediationof tonic convulsions, whereas forebrain structures are acti-vated during forelimb clonus (Eells et al., 2004). Theexpression of SV2A protein appears to be uniformlydecreased throughout the forebrain, midbrain, and hind-brain of SV2A (+/)) mice (Fig. 2), and on this basis it isdifficult to explain why there is no difference in their vul-nerability to MES. However, it could be hypothesized thatthe function of SV2A protein in response to seizure-induc-

ing stimuli that activate forelimb structures, particularlylimbic system, is more important than in response toseizure activity involving hindbrain structures asdemonstrated during MES (Browning & Nelson, 1986).

The key observation in the present study was that leveti-racetam displayed reduced anticonvulsant efficacy inSV2A-deficient mice, which was illustrated by its failureto produce the same degree of increase in the threshold for6-Hz seizures as in the wild-type animals. In contrast,valproate, which has SV2A-unrelated mechanism ofaction (Noyer et al., 1995; Lçscher, 1999), produced thesame magnitude of threshold increase in both genotypes.Levetiracetam and valproate, as well as several otherAEDs, have been reported to display protective efficacyagainst 6-Hz seizures (Barton et al., 2001). Interestingly,only those two AEDs maintained their efficacy against 6-Hz seizures elicited by higher stimulation current(44 mA). At this current intensity, the 6-Hz model becameresistant to other AEDs (Barton et al., 2001). Therefore,for the present study we decided to use valproate as a ref-erence compound for comparison with levetiracetam. Fur-thermore, Barton et al. (2001) demonstrated that thepotency of levetiracetam and valproate decreased signifi-cantly as the stimulation current was increased. Conse-quently, we could not simply determine the ED50 valuesagainst 6 Hz seizures for these compounds in SV2A (+/+)and SV2A (+/)) mice, because such comparison would bebiased by the difference in seizure thresholds between thetwo genotypes of mice. The ED50 values are generally cal-culated after stimulation with a fixed current, which in thiscase would be a confounding factor, given the differencesin 6-Hz seizure vulnerability of these mice. As a conse-quence, we decided to use an unbiased approach andascertain whether the same doses of levetiracetam and val-proate would produce the same magnitude of thresholdincrease for 6-Hz seizures in both SV2A (+/+) and SV2A(+/)) mice. Remarkably, this was true only in the case ofvalproate. A low dose of valproate produced an increasein the threshold for 6-Hz seizures, which was comparablebetween SV2A (+/+) and SV2A (+/)) mice. A higher doseof valproate afforded further increase of the threshold,which again was almost identical in the two genotypes.Similarly, a low dose of levetiracetam produced compara-ble increases of the seizure threshold in both genotypes.However, in contrast to valproate, a higher dose of leveti-racetam failed to provide an additional threshold increasein the SV2A (+/)) mice, whereas the threshold was furtherincreased in SV2A (+/+) mice. In fact, a higher dose oflevetiracetam produced a 50% higher threshold increasein wild-type mice compared to the increase obtained inSV2 (+/)) mice. It is important to remember that SV2A(+/)) mice still express 50% of the SV2A protein, andperhaps even limited binding of levetiracetam may affordthe same degree of protection observed after a lowerdose of the drug. Furthermore, SV2A (+/)) mice have a

Table 2. Phenotype of SV2A (+/)) mice and

anticonvulsant profile of levetiracetam in

seizure models

Epilepsy model

SV2A phenotype

difference

Protective activity of

levetiracetam

Amygdala kindling

Afterdischarge threshold Yes Yesa,b

Kindling development Yes Yesa,b

Corneal kindling Yes Yesc

Pilocarpine Yes Yesd

Kainic acid Yes Yesd

PTZ (iv) Yes Yese

6 Hz Yes Yesf

MES No Nod

MES, maximal electroshock; PTZ, pentylenetetrazol.aLoscher et al., 1998.bHusum et al., 2004.cMatagne & Klitgaard, 1998.dKlitgaard et al., 1998.eLoscher & Honack, 1993.fBarton et al., 2001.

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significantly reduced threshold for 6-Hz seizures, and atlower stimulation currents it might have been somewhateasier to elevate the threshold with the same dose of leveti-racetam. The difference in the effects of levetiracetam onseizure threshold became significant only at the dose thatoccupied nearly all of the SV2A binding sites in bothgenotypes, but since SV2A (+/)) have 50% less sitesavailable for levetiracetam binding, the degree of seizureprotection was also reduced by approximately 50%.

Proepileptic phenotype of SV2A-deficient micetogether with reduced anticonvulsant efficacy of leveti-racetam in these mice suggests that it may act as a positivemodulator of SV2A. This, however, still needs to be dem-onstrated at the molecular level by additional mechanisticstudies. Finally, the function of SV2A also remains to befully elucidated. Although these results strongly suggest apivotal involvement of SV2A in the mediation of theanticonvulsant action of levetiracetam, several othermechanisms may also contribute to the protective effectsof the drug (Niespodziany et al., 2001; Rigo et al., 2002;Angehagen et al., 2003; Madeja et al., 2003; Nagarkattiet al., 2008).

The present study provides the first direct evidencethat SV2A is involved in mediation of the anticonvul-sant activity of levetiracetam in vivo. Furthermore, ourdata also indicate that even partial SV2A deficiencymay lead to increased seizure vulnerability and acceler-ated epileptogenesis. Taken together these data stronglysuggest that SV2A constitutes the primary mechanismfor the antiepileptic effect of levetiracetam.

Acknowledgments

We are very grateful to Dr. Donald Dassesse for well-organized man-agement of the breeding colony of SV2A transgenic animals and toMr. Michel Neveux for excellent technical expertise and help withEEG/kindling experiments. Skilful technical assistance of Mrs. ColetteChaussee, Mrs. Fabienne Coddens, Mrs. Marie-Christine Tordeur andMrs. Zita Povegliano is kindly acknowledged.

We confirm that we have read the Journal’s position on issues involved inethical publication and affirm that this report is consistent with thoseguidelines. All the authors are full-time employees of UCB Pharma S.A.,the manufacturer of levetiracetam (Keppra�).

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