Contentslistsavailableatwww.sciencedirect.com
Epilepsy
Research
j o u r n al ho me p ag e :w w w . e l s e v i e r . c o m / l o c a t e / e p i l e p s y r e s
Seizures
triggered
by
pentylenetetrazol
in
marmosets
made
chronically
epileptic
with
pilocarpine
show
greater
refractoriness
to
treatment
Josy
Carolina
C.
Pontes
a,
Thiago
Z.
Lima
b,
Claudio
M.
Queiroz
c,
Simone
M.
Cinini
a,
Miriam
M.
Blanco
a,
Luiz
E.
Mello
a,∗aDepartamentodeFisiologia,UniversidadeFederaldeSãoPaulo,RuaPedrodeToledo669,3andar,SãoPaulo,SP04039-032,Brazil bHospitalIsraelitaAlbertEinstein,AvenidaAlbertEinstein,627,SãoPaulo,SP05652-000,Brazil
cBrainInstitute,UniversidadeFederaldoRioGrandedoNorte,AvenidaNascimentodeCastro,2155,Natal,RN59056-450,Brazil
a
r
t
i
c
l
e
i
n
f
o
Articlehistory:Received12March2016
Receivedinrevisedform29May2016 Accepted25June2016
Availableonline27June2016 Keywords: Epilepsy Pharmacoresistance Pentylenetetrazole Pilocarpine Anti-epilepticdrugs Animalmodel
a
b
s
t
r
a
c
t
Theefficiencyofmostofthenewantiepilepticdrugs(AEDs)onclinicaltrialsstillfallsshortthesuccess reportedinpre-clinicalstudies,possiblybecausethevalidityoftheanimalmodelsisinsufficienttofully representthehumanpathology.ToimprovethetranslationalvaluefortestingAEDs,weproposethe useofnon-humanprimates.Here,wesuggestthattriggeringlimbicseizureswithlowdosesofPTZin pilocarpine-treatedmarmosetsmightprovideamoreeffectivebasisforthedevelopmentofAED. Mar-mosetswithepilepticbackgroundweremoresusceptibletoseizuresinducedbyPTZ,whichwereatleast 3timeslongerandmoresevere(about6timesgreaterfrequencyofgeneralizedseizures)incomparison tonaïvepeers.Accordingly,PTZ-inducedseizureswereremarkablylessattenuatedbyAEDsin epilep-ticthannaïvemarmosets.Whilephenobarbital(40mg/kg)virtuallyabolishedseizuresregardlessofthe animal’sbackground,carbamazepine(120mg/kg)andvalproicacid(400mg/kg)couldnotprevent PTZ-inducedseizuresinepilepticanimalswiththesameefficiencyasobservedinnaïvepeers.VPAwasless effectiveregardingthedurationofindividualseizuresinepilepticanimals,asassessedinECoG(p=0.05). SimilarlyfollowingCBZtreatment,thebehavioralmanifestationofgeneralizedseizureslastedlongerin epileptic(p<0.05),whichwerealsomorefrequentthaninthenaïvegroup(p<0.05).Asexpected, epilep-ticmarmosetsexperiencingstrongerseizuresshowedmoreNPY-andFosB-immunostainedneurons inanumberofbrainareasassociatedwiththegenerationandspreadoflimbicseizures.Ourresults sug-gestthatPTZinducedseizuresoveranalreadyexistingepilepticbackgroundconstitutesareliableand controllablemeanforthescreeningofnewAEDs.
©2016ElsevierB.V.Allrightsreserved.
1. Introduction
Epilepsy is a common neurological disorder that affects at least50millionpeopleworldwide(LoscherandSchmidt,2002). Despiterecentdevelopmentsofanti-epilepticdrugsandinnovative approachesintheepilepsytreatment(Loscheretal.,2013)30%of patientswithtemporallobeepilepsy(TLE)stillareunabletohave
Abbreviations: AEDs,antiepilepticdrugs;CBZ,carbamazepine;ECoG, electro-corticogram;LFP,localfieldpotential;PB,phenobarbital;PFA,paraformaldehyde; PSD,powerspectrumdensity;PTZ,pentylenetetrazole;SE,statusepilepticus;SNr, substantianigraparsreticulate;SRF,serumresponsefactor;SRSs,spontaneous recurrentseizures;TLE,temporallobeepilepsy;VPA,valproicacid.
∗ Correspondingauthor.
E-mailaddress:lemello@unifesp.br(L.E.Mello).
theirseizurescontrolledasaconsequenceofpharmacoresistance (McKeownandMcNamara,2001)andalsodonotmeetthecriteria forsurgicalintervention(Centenoetal.,2006).Inthemorerecent years,asafirstchoiceapproachforseizurecontrol,pharmacology hasnotbroughtintoclinicalpracticethesamesuccessesreportedin pre-clinicaltrials(LoscherandSchmidt,2002).Onepossiblereason forsuchfailurereliesonthefactthatmostAEDsaretestedin ani-malmodelsthatnotcompletelymimichumanTLEandaremostly basedinacute,chemically-orelectrically-evokedseizures(Bialer andWhite2010;LoscherandSchmidt,2011;WhiteandLoscher, 2014).
In contrast, one of the key hallmarks of epilepsy,including TLE,istheoccurrenceofunpredicted,spontaneous(notevoked) andrecurrentseizures(SRSs). Inanimal modelsofTLE, sponta-neousseizuresareoftenobservedinthechronicphasethatensues
http://dx.doi.org/10.1016/j.eplepsyres.2016.06.012 0920-1211/©2016ElsevierB.V.Allrightsreserved.
injury,encompassinghippocampus,entorhinalandpiriform cor-tices,amygdala,thalamicandhypothalamicnuclei,claustrumand bednucleusofstriaterminalis(Covolanetal.,2000;Melloetal., 1993).Thiscontrastssignificantlywiththemorerestricteddamage seeninhumanTLEpatients(Engel,1996).Theinjectionof pilo-carpineinmarmosets(Perez-Mendesetal.,2011)hasrecentlybeen shown toresult in morerestricted neurodegeneration, ressem-blingthehumanpathology,withneuronaldamageandabnormal plasticitymainlyrestrictedtothelimbicsystem,particularlythe hippocampus(Perez-Mendesetal.,2011).Hereweexaminedthe effectofcarbamazepine(CBZ),phenobarbital(PB)andvalproicacid (VPA),threeclinically relevant and widelyusedAED, ina non-humanprimatemodelwherepredictableacutelyelicitedseizures (bymeansofPTZ)weregeneratedinanepilepticbrain(yielded epilepticbymeansofSEinductionwithpilocarpine).We aimed atprovidingananimalmodelthatwouldmorecloselymimicthe humanconditionandprovidetheeaseofacutelyinducedseizures.
2. Methods
2.1. Animals
Hereweused35 adultmarmosets(Callithrixjacchus)ofboth genders (2–8 years-old; 250–400g) from the primate facility of the Federal University of São Paulo (authorization number 36120-1).Eachexperimentalgroupwascomposedbyequal pro-portionofmalesandfemales(50%–50%).Atthebeginningofthe experiments, animals were individually housed in wired cages (50cm×50cm×50cm)undercontrolledconditions.Room tem-perature was kept stable (25±2◦C) and the light-dark cycle followeda12-hperiodicitywithlightsonat7:00am.Cage environ-mentwasenrichedwithbranchesacrossthecage,coloredwooden swingsandaplasticboxnest.Thedietwasbasedonchow sup-plemented withfresh fruitsand vegetables. Allprotocolswere consistentwithARRIVEguidelinesandwerecarriedoutaccording totheNationalInstitutesofHealthguideforthecareanduseof Lab-oratoryanimals.ProtocolsalsocompliedwithBrazilianlegislation andwerepreviouslyapprovedbytheAnimalCareandUse Com-mitteeoftheFederalUniversityofSãoPaulo(authorizationnumber 0147/10).Substantialeffortsweremadetoreducethenumberof experimentalanimalsused.
2.2. Electrodeimplantationandelectrocorticographyanalysis Aftersedationwithdiazepam(1mg/kg;i.m.)and anesthesia withketamine10%(10mg/kg;i.m.)andxylazine2%(0.5mg/kg; i.m.),we placeda bipolarelectrode (TA11CTAF40;DataSciences International,St.Paul,MN) over themotorcortex andthe asso-ciatedWiFitransmitterwasattachedtotheabdominalwall. All animalsweretreatedwithabroadspectrumveterinarianantibiotic (pentabiotic®0.1mL/kg;i.m.–acombinationofvariousformsof penicillinandstreptomycin)andflunixinmeglumineasanalgesic (Banamine®,1mg/kg;i.m.).
SEwasinducedasdescribedelsewhere(Perez-Mendesetal., 2011).In brief,scopolaminemethylbromide(1mg/kg,i.p.)was followed20minlaterbypilocarpine(250mg/kgi.p.) administra-tionandthereafterseizureswerehalted10minafterSEonsetwith diazepam(1.25mg/kg,i.p.)toreducemortality.Fourteenofthe20 pilocarpine-injectedmarmosetsdevelopedSEandwerepooledas theepilepticgroup.Theremainingsixanimals,thatdidnotdevelop SE(hencenon-epileptic),wereomittedindatapresentationand discussionforthesakeofclarity.Forsimplicitypilocarpine-induced epilepticanimalsarefurtherreferredtoasmerelyepileptic mar-mosets.Despiteintensivecare,threeanimalsdiedbecauseofSE. Fifteennaïveanimals(naïvegroup)weresubjecttothesame pro-cedures,exceptthatpilocarpinewasreplacedbyanequalvolume ofsalinesolution.
2.4. Ictalandnon-ictalbehaviors
We quantifiedthefrequencyand duration ofnatural behav-iors(StevensonandPoole1976)at30minlongepochs(starting approximatelyat8:00am)previoustothefirstdrugtesting ses-sion.Thisassessment comprisedlocomotionand scentmarking events;remaininginthenestboxandremainingstillinspecific quadrantsofthecage;movingaroundthecage;foodexamination andmastication;scratching;andgrooming.Asimilarassessment wasalsoperformedforictal orpre-ictalbehaviors,suchas fre-quencyoftongueandmouthautomatisms, mouthcleaningand headshakes,aswellasrepetitiveorofacialmovements,andintense salivation(thelatterbeingalsoalikelyconsequenceofperipheral cholinergicactivationbypilocarpine).Atlast,thebehavioral mani-festationofseizureswasevaluatedforsixhoursafterPTZinjection andseizureswerecharacterizedaccordingtoseizureseverityas adaptedfromtheRacinescale(Bachiegaetal.,2008).Inthiscontext, typeIIIandIV/Vseizuresarepresentedaspartialandgeneralized events,respectively.Thebehaviorsofthemarmosetswereacquired andcategorizedbymeansofTheObserverXT8.0software.
2.5. Drugtesting
DrugsweretestedthreemonthsafterTLEinductiontoallowfull recoveryofthemarmosets.AsillustratedinFig.1,animalswere subjectedtofivesessionsofdrugtesting, inwhichacompound wasgivenorally(gavage)precedingtheacuteseizureinductionby PTZ(40mg/kg,i.p.).Sessionsstartedapproximatelyat8:00amand thetestedcompoundwasgiven30minlater.Forthefirstandfifth drugtestingsessionsthecompoundwasinvariablysaline,whereas phenobarbital (PB, 40mg/kg), carbamazepine (CBZ, 120mg/kg) andvalproicacid(VPA,400mg/kg)wererandomlysortedinthe remainingsessions.ThedosageforPTZ,PBandCBZ administra-tionwerepreviouslydefinedforthecurrentexperiments(Bachiega etal.,2008),whereasthedoseofVPAwasbasedonprevious expe-riencewithrat(Blancoetal.,2009).Thetimeintervalbetweeneach AEDinjectionandPTZadministrationreflecteddifferencesin phar-macokineticsofeachdifferentAED:20minforCBZand30minfor VPA(LemosandCavalheiro,1995;Patsalos,2005),PBandsaline. Drugtestingsessionsweresegregatedbyaweekwashoutand
dur-Fig.1. Experimentaldesign.Animalswereimplantedwithtelemetry-associatedelectrodesoverthemotorcortexandweretheninjectedwitheithersalineorpilocarpine. PilocarpinecouldleadtotheonsetofSE(statusepilepticus),inwhichcaseanimalscomprisedthegroupEPI(epileptic),ornot(NEPIgroup;whichwereomittedindata presentationanddiscussion).Threemonthslatertestingof3antiepilepticdrugsagainstpentylenetetrazol(PTZ)seizureswascarriedoutfor5weeks.Forfirstandfifth sessionsthecompoundwasinvariablysaline,whereasvalproicacid(VPA),phenobarbital(PB)andcarbamazepine(CBZ)weresortedinremainingsessions.Afterlasttesting session,thebrainoftheseanimalswerefixedandprocessedforhistologyandimmunohistochemistry.
ingeachsessionanimalswerecontinuouslyrecordedbyvideoand ECoG.
2.6. Histologicalprocessingandimmunohistochemistry
Euthanasiawasperformedoneweekafterthefifthsessionof drugtesting(inwhichallanimalsreceivedsalinefollowedbyPTZ). Theoneweekwashoutperiodwaslikelyenoughtoproteolytically eliminateanyfluctuationinFosB’slevelsthatmighthavebeen causedbyPTZoneweekbefore,hencepreventingthebiasfrom previoussessionsofdrugtestingoverthequantificationof FosB-expressingcells. For this, marmosets were deeply anesthetized (thiopental,30mg/Kg,intraperitoneal)andperfusedthroughthe heartwith200mLphosphatebuffer,pH7.4followedby400mL4% paraformaldehyde(PFA)in0.01MPBS,pH7.2.Next,brainswere sequentiallyimmersedinPFAand30%(w/v)sucrosesolutionfor 48heach.Atlast,30mthickbraincoronalsectionswerecutona cryostat.
Immunohistochemistrywascarriedoutaspreviouslydescribed (Hoffmanetal.,2008).Inbrief,primaryantibodiesweretitratedin PBS(1:15,000forneuropeptidey,NPY–Sigma-Aldrich,#N9528; and1:2000forFosB–Sigma-Aldrich,#AV32519)andtriton X-100,whereasbiotinylatedsecondaryantibodieswereusedinthe presenceof0.5%(v/v)goatserum.Next,horseradishperoxidase wascoupledtoantibodies’biotinbymeansofABCelitekitandthe colorimetricreactionwasbasedontheoxidationof diaminobenzi-dinefor20min.
2.7. Cellcounting
Cellcountingfromdifferentlabelingproceduresfollowed sim-ilarsamplingofplates,whosequantificationwasadaptedtothe abundanceofeachevent.Inallcases,thecellularcontentofthe frontalcortexwascomputedastheaverageof8plates(from 1 to13.8mmanteriortobregma),whereastemporalandentorhinal cortexcomprised6plates(from4.5to10mmanteriortobregma). Similarly,thecellcountinginCA1,CA3andhilusofthe hippocam-pusencompassed4plates(frombregma+3to−3mm).Estimation oftheneuronaldamagein thehippocampusofmarmosetswas basedonNissl-stainedsections.Tothisend,weacquiredimages fromsampledplatesat40xmagnificationandonlystained neu-ronssuperposedby10×10gridwerecomputed.Neuronswere identifiedbasedonmorphologicalcriteria.ToquantifyNPYand FosB-positiveneuronswesummedeveryimmunostainedcells presentinimagesfromsampledplates at10× and40× magni-fication,respectively.Forallquantification,theneuronalcontent wascomputedasanaverageofbothhemispheresandexpressed ascells/mm2.
2.8. Statisticalanalysis
Data, which fit the normal distribution (according to Kolmogorov-Smirnov test) was analyzed by T-test or ANOVA, otherwiseweusedtheWilcoxontestorKruskalWallis.In para-metricanalysis,thehypothesistestwasoftenfollowedbypost-hoc Tukey-KramerorFisher’sLSDtoconfirmeffectsanddeependata interpretation.Multipleregressionanalysiswasalsoexecutedto correlatetheamountofimmunostainedcells withictalactivity as recorded by ECoG. Finally, Chi-squared was computed to test differences regarding seizure incidence between different conditions treated withthe same AED. For all analysis, it was considered ␣=0.05. In addition to p>0.05, a statistical power higherthan0.7wasrequiredtodiscardeffects,otherwiseanalysis wasconsideredinconclusive.Forclarity,thestatisticalpowerwas omittedin statisticpresentation,butinconclusiveanalysiswere locallyindicated.
Natural behaviors were quantified in the first basal period, whereasictalbehaviors(aswellastheinfluenceoftheepileptic conditiononictalprofile)wereassessedafterthefirstdrug test-ingsession.EachAEDwasassumedtosuppressseizures,whenthe ensuingictalactivitywassignificantlylessthanthataftersaline treatment(thisanalysisispresentedinSupportinginformation). Theinfluenceoftheepilepticneuralbackgroundontheefficacy ofAEDswastestedastheeffectofexperimentalconditionwhen eachAEDwasanalyzedindividually.Thebehavioralanalysis con-sideredthesummationofseizureincidenceanddurationovera sixhoursperiodfollowingPTZadministration.Inthesameway, theanalysisofseizuresrecordedbyECoGprovidedtheduration ofseizuresasthesummationofictaleventsalong6uninterrupted hoursfollowingPTZadministration.
TheanalysisofNPY-andFosB-expressingneuronsalsotook intoaccounttwo additionalgroups:naïveandepilepticanimals thathadnotbeensubjecttosessionsofdrugtesting.Thisapproach wasaimedtodiscardapotentialinfluenceoftheAEDs(despite aoneweekwashout)overNPYandFosBexpression. Notwith-standing,somegroupswereomittedindatapresentationforthe sakeofclarity.
3. Results
3.1. Pilocarpine-treatedanimalsarecharacterizedbyanaltered “epileptic”background
Inrestingawakenedanimals,theLFPofepilepticmarmosets wasoftendominatedbyoscillationsaround3Hz,whereas peri-odswithfasteroscillations(8–12Hz)weremorefrequentinnaïve counterparts (Fig. 2A). As a result, when longer periods were
Fig.2.Pilocarpineleadstoanatomicalandfunctionalchanges.A)FifteensecondsofLFPacquiredinarestingawakeanimal.Thetaoscillationwasabundantinnaïveanimals’ LFP(blue),whereasadeltaoscillationprevailedintheepochsfromepilepticmarmosets(red).B)Themeannormalizedpowerspectrumdensity(PSD)fromonehourof restingawakeinnaïve(blue)andepileptic(red)groups.Dashedlinesillustratethestandarderrorsofthemean(SEM).Theinsetintheupperrightcornerhighlightsthemean powerintegratedindeltaandthetarange.ErrorbarsrepresentSEM,*indicatesp<0.05and#indicatesp<0.000001,bothfromt-test.C-D)Timespentscratchingandthe timeanimalswereshelteredinsidethenestbox,respectively,bothonbasalcondition,inwhichanimalsweredevoidofictalactivityandwerenotmanipulated.Errorbars representSEMand*indicatesp<0.05,accordingtoTukey-Kramer.E)RepresentativeimagesfromNisslstainingofthehilusofthedentategyrusfromanaïveanimal(E1) andanepilepticmarmoset(E2).InE3,themeanneuronaldensityinthehilusfromnaïveandepilepticgroups.ErrorbarsillustrateSEMand*indicatesp<0.05fromt-test.
considered,thepowerofdeltaoscillationswasenhancedin epilep-tic marmosets followed by a reduction in the power of theta oscillations,as compared tocontrols (Fig. 2B; t-test, p=0.0149 andp<0.000001,respectively).Thecontrastbetweenthe epilep-ticandnaïveneuralbackgroundswasalsomanifestedinnatural behaviors.Thetimeanimalsspentscratchingwasinfluencedby theepilepticcondition(Fig.2C;ANOVA,F=4.39,p=0.03),which was further confirmed by longer scratching by epileptic mar-mosetsascomparedtothenaïvegroup(Tukey-Kramer,p<0.05). Similarly,thetime shelteredinside thenestboxalsodepended on the epileptic condition (Fig. 2D; ANOVA, F=5.48, p=0.02). Indeed naïve animals remained longer periods inside the nest box than theepileptic group(Tukey-Kramer, p<0.05). In addi-tion structural changes induced by pilocarpine contributed to differentiate epileptic marmosetsfrom naïve animals. Epileptic marmosetspresented extensive mossy fibersprouting and dis-persion of the granularlayer of the dentategyrus (Supporting information,Figs.S1andS2,respectively).Atlast,theabundance of neurons in the hilus of the dentate gyrus was reduced in epilepticmarmosetsascomparedtonaïvepeers(Fig.2E;t-test, p<0.02),suggestinghippocampalatrophy.Altogethertheseeffects confirmthatchangesinducedbypilocarpineconstitutively differ-entiatetheepilepticneuralbackgroundfromthenaïvecondition regardingvarioushistologicalaspects,brainrhythmsand behav-ior.
3.2. ExpressionofneuropeptideYisenhancedinepileptic marmosets
NPYisaninhibitoryneuromodulator,whoseexpressionis aug-mentedunderaberrantelectricalactivity,hencethisisconsidered as anendogenous anti-epilepticresponse. Accordingly, the fre-quencyofseizuresreflected theamountofNPY-positivecellsin thefrontalcortex(R=0.69,p=0.01). Wecouldnot identifyany influenceoftheepilepticconditiononthenumberofNPY-positive cellsinentorhinalcortexandCA3(ANOVA,p=0.43,forentorhinal cortex;p=0.12forCA3;inconclusiveduetoinsufficient statisti-calpower).Inthehilusofthehippocampus,theinfluenceofthe epilepticbackgroundwasnotconclusive(becauseinsufficient sta-tistical power; ANOVA,p=0.06). On the other hand, in frontal (Fig.3A)andtemporalcorticesaswellasinCA1ofhippocampus, thequantityofNPY-positivecellsdependedontheepileptic con-dition(ANOVA,p<0.001forfrontalcortex;p=0.015fortemporal cortex;andp=0.02forCA1),giventhatthesecellsweremore abun-dantinepilepticmarmosetsascomparedtonaïvepeers(Fig.3Band C;Tukey-Kramer,p=0.0002,p=0.015,p=0.019,respectively).
3.3. PTZ-elicitedseizuresweremorefrequentandlastlongerin alreadyepilepticanimals
IndividualPTZ-inducedseizuresfrombothnaïve(Fig.4A)and epilepticmarmosets(Fig.4B)showedasimilarECoGarchitecture, withthree stages. Aftera moderate beginning,as evaluatedby
Fig.3.QuantificationofNPY-positiveneurons.Animals,whosedataaredepictedinA,BandCwerenotsubjectedtoAEDdrugtesting,hencesparedfromtheinfluence ofPTZ.A-1)TheimageexemplifiestheimmunohistochemistryforNPYinfrontalcortexofanaïvemarmoset,whereasthatofanepilepticpeerisrepresentedinA-2. QuantifiedimmunostainedcellsarerepresentedasgroupmeaninBandCforneocortexandhippocampus,respectively.ErrorbarsillustrateSEMand*indicatesp<0.05 fromTukey-Kramer.
meansofpoweranalysis,seizuresevolvedtoasustainedperiod withhighpowercenteredaround10Hz.Thereafterthisdiminished infrequencytowardstheterminationphase.Seizureendingwas markedbyhighpowerslowoscillations(around2Hz),whose val-leyswereconcomitantwithfasteroscillations.Atlastapost-ictal depressionmarkedtheendofeachictalevent.Theseizure exem-plifiedinFig.4Bmaysuggestthatseizuresemergedwithfaster oscillationsinepilepticmarmosets,althoughthisfeaturewasless clearwhenseizuresareaveraged(datanotshown).
Theepilepticconditionalsodidnotaffectthelatencyforthe firstseizureafterPTZadministration(datanotshown).However, theepilepticconditioninfluencedthefrequencyanddurationin whichthesePTZ-inducedseizuresemerged, favoringthe occur-renceofseizures,whichemergedaslongersequencesofictalevents (classifiedasSE-likeseizures;Fig.4C;Kruskal-Wallis,p=0.05)in contrasttothescarcityof thesesequencesinnaïvemarmosets. Indeed,nonaïvemarmosetshowedtheseSE-likeseizuresafterPTZ administrationasopposedto55%oftheepilepticcohort(tableS1 inSupportinginformation;Chi-squared,p<0.05).Inaddition,the frequencyofthebehavioralmanifestationofseizures,dependedon theepilepticcondition(Fig.4D;ANOVA,F=6,40,p=0.01),giventhe incidenceofictaleventswasgreaterinepilepticmarmosetsas com-paredtonaïvecounterparts(Tukey-Kramer,p<0.05).Thishigher frequencywasalsoassociatedwithlongerbehavioralexpression of seizures in epileptic marmosets (Fig. 4D; ANOVA, p=0.03), whichwasfurtherconfirmedbypost-hocanalysis(Tukey-Kramer, p=0.02).Theepilepticconditionseemedtoworsen theseverity of theictal profileelicited by PTZ, given that seizure intensity wasconcentratedintypeIV/Vseizures(ANOVA,F=4.42,p=0.03 andTukey-Kramer,p<0.05forfrequency;ANOVA,F=4.27,p=0.04 andFisher’s LSD,p<0.05, for duration).In linewiththelonger behavioralexpressionofseizures,epilepticmarmosetshadlonger durationsofseizure-relatedorofacialmovementsascomparedto
thenaïvegroup(Fig.4D-3;Kruskal-Wallis,p=0.02).Vocalization, spasmsoflimbsandvisualdisorientationmayalsoaccountfor dif-ferencesinthebehavioralexpressionofseizuresbetweenepileptic andcontrolmarmosets,althoughtheseeffectswerenot statisti-callysignificant(Kruskal-Wallisp=0.07andWilcoxonp=0.06for thelatter).Giventhattheinfluenceoftheepilepticconditionover seizureselicitedbyPTZreliedmostlyonthefrequencyand dura-tionoftheseevents,thesubsequentinvestigationoftheeffectsof AEDswascenteredinthesetwoparameters.
3.4. Seizuresinepilepticanimalsengagedalargerneuronal networkthanseizuresinnaïveanimals
FosBis alongerstableindirectmarkerof neuronal activa-tion,hencesuitabletoidentifyneuronalpopulationsengagedin seizureprecipitation.Underthepresentconditions,weobserved thatseizureinductionbyPTZalteredthenumberofFosB-positive cells,inamannerthatwasspecifictotheepilepticcondition,inall analyzedbrainareas(Fig.5BandC;ANOVA,p<0.001forfrontal, temporalandentorhinalcortices,CA3andCA1;p=0.03forthehilus ofthehippocampus).Thiseffectisconfirmedbythegreater abun-danceofimmunostainedcellsinepilepticmarmosetsascompared tonaïveanimals(Tukey-Kramer,allvaluesofp≤0.05).Epileptic marmosetspresentedahighernumberofFosB-positivecellsin theneocortexirrespectiveofictalactivityinducedbyPTZ(datanot shown).Yetthedifferences,inthisbrainarea,ascomparedtonaive animals,islargelyincreasedasaresultofPTZseizureinduction, giventhatonlyepilepticmarmosetshadafurtherincreaseinthe abundanceofFosB-immunostainedcellsafterPTZ.Ontheother handthenumberofFosB-expressingneuronsintheCA1andCA3 ofthehippocampussurpassedthatofnaïvepeersonlyfollowing acuteseizureinductionbyPTZ(datanotshown).Altogetherthese
Fig.4. TheinfluenceofchronicepilepticinductionbypilocarpineoveracuteseizureevocationbyPTZ.A)Upperpanel:13minofLFPfromanaïvemarmosetstartingatPTZ administration,showingoneseizure,whichisindicatedwithanarrowheadandisexpandedinthemiddlepanel,withcorrespondingPSDplottedbellow(lowerpanel).B) SimilaranalysisasshowninAbutnowforanepilepticmarmoset.C)Theboxplotillustratesmedian,quartiles,minimumandmaximumvaluesforthedurationofchains ofseizures(i.e.,seizuresnotinterruptedeitherbypost-ictaldepressionorbehavioralmanifestationofconsciousness.#indicatesp≤0.05accordingtoKruskal-Wallis,D-E) BehavioralevaluationofseizureselicitedbyPTZduringsixhoursfollowingPTZadministration.Seizureswerefirstcategorized,accordingtoseverityfollowinganadapted Racinescale:seizuretypesIIIandIV/Varepresentedaspartialandgeneralizedevents,respectively.SeizurefrequencyisplottedinDanddurationispresentedinE.Colorful barsillustrategroupmeans,whereaserrorbarsrepresenttheSEM.*indicatep<0.05accordingtoTukey-Kramer.F)Durationoforofacialmovementsaccompanyingseizures, representedasmedian,quartiles,minimumandmaximumvalues.#indicatesp≤0.05accordingtoKruskal-Wallis.
Fig.5. QuantificationofFosB-positiveneurons.ThedatadepictedinA,BandCwerecollectedfollowingthelastdrugtestingsession,inwhichsalineprecededPTZ,one weekafterlastAEDtreatment.A-1)TheimageexemplifiestheimmunohistochemistryforFosBinthefrontalcortexofanaïvemarmoset,whereasthatofanepilepticpeer isrepresentedinA-2.TheimmunostainedcellswherequantifiedandarerepresentedasgroupmeaninBandC,forneocortexandhippocampus,respectively.Errorbars illustrateSEMand*indicatesp<0.05fromTukey-Kramer.
evidencesindicatethatseizureinductionbyPTZengagesalarger neuronalnetworkinanimalswithanepilepticbackground.
3.5. EfficacyofAEDswaslessinepilepticanimals
Ingeneral,epilepticanimalswerelessresponsivethannaïve animalstotheanticonvulsanteffectsofthetesteddrugs.The excep-tionwasPB,whichwasequallyeffectiveonthecontrolofevery evaluatedictalparameterinbothepilepticmarmosetsandnaïve peers(Fig.6B-1–B-4).Incontrast,VPAwaslesseffectiveinepileptic animals,regardingthedurationofindividualseizures,asassessed inECoG(Fig.4A-3;Kruskal-Wallis,p=0.05).
TheeffectofCBZonthedurationofbothisolatedandSE-like seizuresalsodependedontheepilepticbackground(Fig.6C-3and C-4;Kruskal-Wallis,p=0.02andp=0.03,respectively).Similarly theepilepticconditioninfluencedtheeffectofCBZonthe behav-ioralmanifestationofthemoreseveregeneralizedseizures(type IV/V; Duration: Fig.6A-3;ANOVA, F=5.85, p=0.01. Frequency:
Fig.6B-3;ANOVA,F=4.07,p=0.04).SeizuresuppressionbyCBZ wasless evident in epileptic marmosets.As a result, following CBZtreatment,thebehavioralmanifestationoftypeIV/Vseizures lastedlongerinepilepticthaninthenaïvegroup(Fig.6B-3; Tukey-Kramer,p<0.05)andtype IV/Vseizuresweremorefrequent in epilepticmarmosetsthaninnaïveanimals(Fig.6A-3;Fisher’sLSD, p<0.05).The effectofCBZ onseizurefrequency alsodepended ontheepilepticconditionwhenallbehavioralictalobservations wereconsidered,regardlessofseizureseverity(Fig.6A-3;ANOVA, F=6.07,p=0.01),giventhatthefrequencyofalltypesofbehavioral seizureswasgreaterinepilepticthaninthenaïvegroup(Fisher’s LSD,p<0.05).
4. Discussion
Hereweobservedthat,intheabsenceofAEDs,theacuteseizures evokedbyPTZwereaccentuatedinepilepticmarmosets.In gen-eral, all AEDs suppressed seizures, even though withdifferent degreesofsuccess. PBwastheonlyevaluatedAEDtomarkedly abolishsevereseizureseveninepilepticanimals.Inturntheseizure blockingeffectof CBZandVPA weremostly restrictedtonaïve marmosets.Moreover,theepilepticconditionwasassociatedwith differentnaturalandictal behaviors.Theseepilepticmarmosets alsopresentedgreaternumbersofNPY-positivecellsinfrontaland temporalcorticesaswellasinCA1ofhippocampus.Inaddition,the abundanceofFosB-positivecellswasgreaterinepileptic mar-mosetsinbrainareasassociatedwithgenesisandpropagationof seizures.Altogetherthesefindingssubstantiatethe hyperexcitabil-ityconferredbytheepilepticbackground,overwhichtherewasa diminishedefficacyofclassicalAEDs.
Theepilepticconditionwasassociatedwithlongertime scratch-ing,a naturalbehavior relatedtoanxietyin marmosets(Barros etal.,2000).Accordingly,theanxiolyticslorazepam(Schinoetal., 1991),midazolam(Maestripierietal.,1991)anddiazepam(Cilia andPiper,1997)havebeenshowntoreducescratchingin anxi-etymodelsin nonhumanprimates.Also,theshortertimespent inthenestboxbyepilepticanimalsmayreflectenhanced anxi-ety,amooddisorder,which affectstwiceasmanypersonswith epilepsyasinthegeneralpopulationanddeterioratesthelife qual-ityofthesepatients(Beyenburgetal.,2005).Indeedithasbeen suggestedthatincreasedlocomotoractivityinmarmosets(Barros etal.,2000),mightbeseenasindicativeofanxiety.
Theinductionofachronicepilepticconditionbypilocarpine turned marmosetsmore responsiveto PTZ, accentuating dura-tionandfrequencyespeciallyofmoresevereseizures(generalized, typeIV/V).Thiseffectwasrathersubtleinrats(underasimilar
Fig.6. TheefficacyofAEDs,accordingtotheepilepticcondition.RowsA,BandCrepresentparametersfromseizuresevokedbyPTZinanimalspreviouslytreatedwithvalproic acid,phenobarbitalandcarbamazepine,respectively.Columns1and2represent,respectively,thesummationoffrequencyanddurationofthebehavioralmanifestationof seizuresoversixhoursafterPTZadministration.Seizureswereclassifiedaspartialorgeneralizedevents,reflectingtypeIIIorIV/VofanadaptedRacinescale.Colorbars illustratethemeanofthegroup,whereaserrorbarsindicateSEM.+and*indicatesp≤0.05accordingtoFisher’sLSDandTukey-Kramer,respectively.Column3represents thesummationofthedurationofisolatedseizuresasrecordedinECoGalong6uninterruptedhoursafterPTZadministration.Theabsenceofpost-ictaldepressionand lackingbehavioralevidenceofconsciousnesscharacterizedthechainsofseizuresasSE-likeevents,whosedurationsarepresentedincolumn4asasum.Theboxplots(blue) illustratemedian,quartiles,minimumandmaximumvalues.#indicatesp≤0.05accordingtoKruskal-Wallis.
pilocarpine/PTZprotocol),whichpresentedsimilarprofileof par-tialandgeneralizedseizuresinbothepilepticandnaïverodents (Blancoetal.,2009).Importantly,thisenhancedictalprone con-dition might have undermined theefficacy of AEDs, given the suppressionofseizuresbyAEDswaslessinthechronicepileptic condition.Indeed,whereastheoccurrenceofseizureselicitedby PTZwascompletelyabolishedbyVPAinnaïvesubjects,thesame ictaleventstillpersistedin20%ofepilepticmarmosetsafterVPA. Thiswascomparabletoseizureinhibitioninspontaneously seiz-ingrats,whoseepileptiformactivitywastriggeredbyelectrical stimulationoftheamygdala(NissinenandPitkanen,2007).Yet,the presentexperimentaldesignaddstheconveniencetoconcentrate datacollection,giventheseizuremeasurementsaredefinedbythe experimenterupontheadministrationofPTZ.
Thus,under thechronicepilepticconditioninducedby pilo-carpine there is a failure of VPA in suppressing seizures. The mechanismunderlyingthispharmacoresistancemightberelated inparttoGABAergicneuronsfromsubstantianigrapars reticu-lata(Snr) (McNamaraetal.,1984),whose firingrateis reduced
by VPA. This decline, on pharmacoresistant amygdala-kindled rats (Tollner et al., 2011), is possibly due to reduced activity ofGABA-synthesizingenzymeglutamatedecarboxylase(Loscher andSchwark,1985).Thus,chronicepilepticanimalsseemtolack GABAergicinhibitioninSNrtomediateseizuresuppressionbyVPA. Similarly,underCBZtreatment,epilepticmarmosetsdeveloped longerSE-likeseizures,whichreflectedlonger,morefrequentand severebehavioralmanifestations(generalized,typeIV/Vseizures) thannaïvesubjects.CBZabolishedSE-likeseizuresinnaïve ani-mals,whereastheseeventswerelargelynotattenuatedinepileptic marmosets.Theseevidencesindicatethatepilepticinductionby pilocarpinealsoleadtopharmacoresistancetoCBZ.The mecha-nismofactionreportedforCBZreliesonthereductionofneuronal excitabilitythroughtheblockageofvoltage-gatedNa+ channels (CourtneyandEtter,1983).Indeed,thesechannelsbecome insen-sitivetoCBZinCA1neuronsofthehippocampusofkindledrats (VreugdenhilandWadman, 1999), andin humanpatientswith epilepsy(Vreugdenhiletal.,1998).Thusthepharmacoresistance toCBZshownheremightbeassociatedtothefadingofan
anti-epilepticmechanism causedbylongerictal activity,which only occursinchronicmodels.Thus,similarlytoVPA,theexpectedCBZ efficacy,basedondatafromacutemodels,doesnotshowupwhen thetreatmentisassessedinanepilepticbackground.
PBwasoncethemostwidelyusedfirst-lineantiepilepticdrug, butitsusehasdeclinedinwealthycountriesgivenanumberof sideeffects,chieflycognitiveandfunctionalimpairments(Elafros etal.,2014).Theadverse effectsof PBincludesedation, hyper-activity,impairedcognition,depressedmoodandaffect,anemia, hepatotoxicityandteratogenicity(KwanandBrodie,2004).Inour experimentalconditions,PBwasremarkablyefficientinthe over-allcontrolofseizuresregardlessoftheepilepticbackgroundover whichthedrugwasbeingtested.ThehigherefficacyofPBinthe presentconditionscontraststhemeta-analysis,inwhichPBis con-sideredashavingcomparableefficacytootherAEDs,suchasVPA (KwanandBrodie,2004).ThismightbeassociatedwithbothPTZ andPBactingonthesameGABA-Achannel.PBbindstotheGABA-A receptorinthesiteforbenzodiazepine,whichextendsthetimethe chloridechannelremainsopened(Twymanetal.,1989),whereas PTZblocksthechlorideinfluxbymeansofthebindingtothe picro-toxinsiteofthesameGABA-Achannel(Huangetal.,2001).Even thoughtheantagonismbetweenPTZandPBdoesnotrelyona competitionforthesamebindingsite,thepreviousPB adminis-trationmaypromptconformationalchanges,whichmightreduce theresponsivenessofGABA-AchanneltoPTZ.However,the effi-cacyofPBpresentedherecorroboratestheanticonvulsanteffect onsimilarcombinedpilocarpine-PTZinmice(Tollneretal.,2016) butcontrastswithitsnegligibleeffectinanequivalentmodelin rats(Blancoetal.,2009).Thisdivergencemightberelatedtothe P-glycoprotein,adrugeffluxtransporterlocatedintheblood-brain barrier(BBB).P-glycoproteinwasshowntoreducethe concentra-tionofPBinthebrain,henceleadingtolossofefficacy(Loscherand Potschka,2005)anditsexpressionis3foldshigherintheratbrain ascomparedtomarmosetsandhumans(Hoshietal.,2013).The comparable BBBpermeabilitybetween humansand marmosets (Hoshietal.,2013)seemstoconsistanotheradvantageto accu-ratelyforecasttheefficacyofAEDsinhumanswhenusingthese animalsfortesting.
The increased number of NPY-positive cells in frontal and temporalcorticesandCA1ofthehippocampusofanimalsmade chronicallyepilepticbypilocarpinecorroboratessimilarfindingsin othermodels(Husumetal.,2000;VezzaniandSperk,2004).Indeed NPYisaninhibitoryneuromodulator,whosesynthesisisprompted byseizures(VezzaniandSperk,2004),andisthusconsideredaspart ofanendogenousantiepilepticmechanism(Richichietal.,2004). Accordingly,hereNPYexpressionincreasedinthetemporalcortex ofnaïve marmosetsas a consequenceof repetitivePTZ admin-istration,inherent tothe drugtesting protocol.In contrast,the expressionofNPYintheepilepticanimalswasindifferenttoPTZ,as thoughtheepilepticconditionhadalreadysaturatedthis endoge-nousanti-epilepticsystem.Inanyeventtheover-expressionofNPY inepilepticascomparedtonaïveanimalsisaclearindicationofthe alteredbackgroundneuronalnetworkofepilepticanimals.
Themarmosetsmadechronicallyepilepticbypilocarpine pre-sentedmoreFosB-positivecellsinCA1,CA3andthehilusofthe dentategyrus,corroboratingtheincreasedexpressionfoundinthe hippocampusofratswithSRSsinducedbykainicacid(Bingetal., 1997).IncreasedFosBexpressioninthefrontalcortexwasalready showntofollowrepeatedelectroconvulsiveseizures(Hiroietal., 1998).Herewefoundthatasimilarincrementalsotookplacein temporalandentorhinalcorticesofmarmosetsasaconsequence ofthechronicepilepticconditionduetopilocarpine,hence gen-eralizingtheenhancedFosBexpressiontootherareasofseizure propagation.FosBexpressionisconditionedtotheactivationof serumresponsefactor(SRF)inresponsetorepetitivestimulation (Morrisetal.,2000).Accordingly,SRFwasfoundtobeincreased
afterepilepticinductionbypilocarpine(Nadler,2003).FosB dif-fers from theremaining productsof the immediateearly gene fosBbecausetheC-terminustruncation,whichrenderstoFosB increasedstability: 10hin culture (Ulery et al.,2006).Yet,the turnoverofFosBcanbereducedbyphosphorylationinthebrain. AstheresultFosBlevelsoriginatedfromfivedaysofincreased expressionmaypersistforupto10days (Ulery-Reynolds etal., 2009).ThusitseemsprudenttoassumethatFosBinducedby acuteevents,suchasAEDsandPTZadministrationineachsessionof drugtesting,wouldnotpersistafteraweekwashout.Inthissense, thenumberofcellsexpressingFosBmightlesslikelyreflectthe influenceofprevioussessionsofdrugscreening,andmightreflect theoutcomeofacuteseizureinductionbyPTZ.Inaddition,a pos-siblebiasfromacumulativeeffectofthedrugtestingsessionsis ratherunlikely,givenallictalparametersremainedunchanged fol-lowingthelastadministrationofPTZascomparedtothefirstone (e.g.,therewasnokindlingeffect).Therefore,thegreaterexpression ofFosBinepilepticascomparedtonaïveanimalsindicatesthat genesisandpropagationofseizureinducedbyPTZlikely appro-priatesamuchlargerneuronalnetworkintheformer.Thisresult mighthelptoexplaintheenhancedexpressionofPTZseizuresin theepilepticanimals,overwhichAEDswerelessefficient.
Supported by histological evidence, here we show that the greaterseizurepropensityseeninepilepticanimalswasalso asso-ciatedtoadiminishedeffectivenessofAEDsintheseanimals.We forwardthatinductionofPTZseizuresinmarmosetsmade chroni-callyepilepticbypilocarpinemightconstitutearelevantmeansfor investigatingpotentialnewAED.
Conflictofinterest
WethankFundac¸ãodeAmparoàPesquisadoEstadodeSão Paulo(FAPESP),Brazil,andConselhoNacionaldeDesenvolvimento CientíficoeTecnológico(CNPQ),Brazil,forfinancialsupport.Every stepofthisworkisdevoidofinfluencebyfundingsources. More-over,authorsconfirmtheabsenceofconflictsofinterest.
Acknowledgements
WeacknowledgeEvaldoCaralJuniorandFernandadePaulafor technicalassistance.
AppendixA. Supplementarydata
Supplementarydataassociatedwiththisarticlecanbefound,in theonlineversion,athttp://dx.doi.org/10.1016/j.eplepsyres.2016. 06.012.
References
Bachiega,J.C.,Blanco,M.M.,Perez-Mendes,P.,Cinini,S.M.,Covolan,L.,Mello,L.E., 2008.Behavioralcharacterizationofpentylenetetrazol-inducedseizuresinthe marmoset.EpilepsyBehav.13,70–76.
Barros,M.,Boere,V.,Huston,J.P.,Tomaz,C.,2000.Measuringfearandanxietyin themarmoset(Callithrixpenicillata)withanovelpredatorconfrontation model:effectsofdiazepam.Behav.BrainRes.108,205–211.
Beyenburg,S.,Mitchell,A.J.,Schmidt,D.,Elger,C.E.,Reuber,M.,2005.Anxietyin patientswithepilepsy:systematicreviewandsuggestionsforclinical management.EpilepsyBehav.7,161–171.
Bialer,M.,White,H.S.,2010.Keyfactorsinthediscoveryanddevelopmentofnew antiepilepticdrugs.Nat.Rev.DrugDiscov.9,68–82.
Bing,G.,Wilson,B.,Hudson,P.,Jin,L.,Feng,Z.,Zhang,W.,etal.,1997.Asingledose ofkainicacidelevatesthelevelsofenkephalinsandactivatorprotein-1 transcriptionfactorsinthehippocampusforupto1year.Proc.Natl.Acad.Sci. U.S.A.94,9422–9427.
Blanco,M.M.,dosSantosJr.,J.G.,Perez-Mendes,P.,Kohek,S.R.,Cavarsan,C.F., Hummel,M.,etal.,2009.Assessmentofseizuresusceptibilityinpilocarpine epilepticandnonepilepticWistarratsandofseizurereinductionwith pentylenetetrazoleandelectroshockmodels.Epilepsia50,824–831.
Hiroi,N.,Marek,G.J.,Brown,J.R.,Ye,H.,Saudou,F.,Vaidya,V.A.,etal.,1998. EssentialroleofthefosBgeneinmolecular,cellular,andbehavioralactionsof chronicelectroconvulsiveseizures.J.Neurosci.18,6952–6962.
Hoffman,G.E.,Le,W.W.,Sita,L.V.,2008.Theimportanceoftitratingantibodiesfor immunocytochemicalmethods.Curr.Protoc.Neurosci.,http://dx.doi.org/10. 1002/0471142301.ns0212s45(Chapter2Unit2.12).
Hoshi,Y.,Uchida,Y.,Tachikawa,M.,Inoue,T.,Ohtsuki,S.,Terasaki,T.,2013. Quantitativeatlasofblood–brainbarriertransporters,receptors,andtight junctionproteinsinratsandcommonmarmoset.J.Pharm.Sci.102,3343–3355. Huang,R.Q.,Bell-Horner,C.L.,Dibas,M.I.,Covey,D.F.,Drewe,J.A.,Dillon,G.H.,2001. Pentylenetetrazole-inducedinhibitionofrecombinantgamma-aminobutyric acidtypeA(GABA(A))receptors:mechanismandsiteofaction.J.Pharmacol. Exp.Ther.298,986–995.
Husum,H.,Mikkelsen,J.D.,Hogg,S.,Mathe,A.A.,Mork,A.,2000.Involvementof hippocampalneuropeptideYinmediatingthechronicactionsoflithium, electroconvulsivestimulationandcitalopram.Neuropharmacology39, 1463–1473.
Kwan,P.,Brodie,M.J.,2004.Phenobarbitalforthetreatmentofepilepsyinthe21st century:acriticalreview.Epilepsia45,1141–1149.
Lemos,T.,Cavalheiro,E.A.,1995.Suppressionofpilocarpine-inducedstatus epilepticusandthelatedevelopmentofepilepsyinrats.Exp.BrainRes.102, 423–428.
Loscher,W.,Potschka,H.,2005.Drugresistanceinbraindiseasesandtheroleof drugeffluxtransporters.Nat.Rev.Neurosci.6,591–602.
Loscher,W.,Schmidt,D.,2002.Newhorizonsinthedevelopmentofantiepileptic drugs.EpilepsyRes.50,3–16.
Loscher,W.,Schmidt,D.,2011.Modernantiepilepticdrugdevelopmenthasfailed todeliver:waysoutofthecurrentdilemma.Epilepsia52,657–678. Loscher,W.,Schwark,W.S.,1985.EvidenceforimpairedGABAergicactivityinthe
substantianigraofamygdaloidkindledrats.BrainRes.339,146–150. Loscher,W.,Klitgaard,H.,Twyman,R.E.,Schmidt,D.,2013.Newavenuesfor
anti-epilepticdrugdiscoveryanddevelopment.Nat.Rev.DrugDiscov.12, 757–776.
Maestripieri,D.,Martel,F.L.,Nevison,C.M.,Simpson,M.J.,Keverne,E.B.,1991. Anxietyinrhesusmonkeyinfantsinrelationtointeractionswiththeirmother andothersocialcompanions.Dev.Psychobiol.24,571–581.
McKeown,M.J.,McNamara,J.O.,2001.Whendoepilepticseizuresreallybegin? Neuron30,1–3.
McNamara,J.O.,Galloway,M.T.,Rigsbee,L.C.,Shin,C.,1984.Evidenceimplicating substantianigrainregulationofkindledseizurethreshold.J.Neurosci.4, 2410–2417.
Patsalos,P.N.,2005.Propertiesofantiepilepticdrugsinthetreatmentofidiopathic generalizedepilepsies.Epilepsia46(Suppl.9),140–148.
Perez-Mendes,P.,Blanco,M.M.,Calcagnotto,M.E.,Cinini,S.M.,Bachiega,J.,Papoti, D.,etal.,2011.Modelingepileptogenesisandtemporallobeepilepsyina non-humanprimate.EpilepsyRes.96,45–57.
Richichi,C.,Lin,E.J.,Stefanin,D.,Colella,D.,Ravizza,T.,Grignaschi,G.,etal.,2004. Anticonvulsantandantiepileptogeniceffectsmediatedbyadeno-associated virusvectorneuropeptideYexpressionintherathippocampus.J.Neurosci.24, 3051–3059.
Schino,G.,Troisi,A.,Perretta,G.,Monaco,V.,1991.Measuringanxietyin nonhumanprimates:effectoflorazepamonmacaquescratching.Pharmacol. Biochem.Behav.38,889–891.
Stevenson,M.F.,Poole,T.B.,1976.Anethogramofthecommonmarmoset(Calithrix jacchusjacchus):generalbehaviouralrepertoire.Anim.Behav.24,428–451. Tollner,K.,Twele,F.,Loscher,W.,2016.Evaluationofthepentylenetetrazole
seizurethresholdtestinepilepticmiceassurrogatemodelfordrugtesting againstpharmacoresistantseizures.EpilepsyBehav.57,95–104.
Tollner,K.,Wolf,S.,Loscher,W.,Gernert,M.,2011.Theanticonvulsantresponseto valproateinkindledratsiscorrelatedwithitseffectonneuronalfiringinthe substantianigraparsreticulata:anewmechanismofpharmacoresistance.J. Neurosci.31,16423–16434.
Twyman,R.E.,Rogers,C.J.,Macdonald,R.L.,1989.Differentialregulationof gamma-aminobutyricacidreceptorchannelsbydiazepamandphenobarbital. Ann.Neurol.25,213–220.
Ulery,P.G.,Rudenko,G.,Nestler,E.J.,2006.RegulationofDeltaFosBstabilityby phosphorylation.J.Neurosci.26,5131–5142.
Ulery-Reynolds,P.G.,Castillo,M.A.,Vialou,V.,Russo,S.J.,Nestler,E.J.,2009. PhosphorylationofDeltaFosBmediatesitsstabilityinvivo.Neuroscience158, 369–372.
Vezzani,A.,Sperk,G.,2004.OverexpressionofNPYandY2receptorsinepileptic braintissue:anendogenousneuroprotectivemechanismintemporallobe epilepsy?Neuropeptides38,245–252.
Vreugdenhil,M.,vanVeelen,C.W.,vanRijen,P.C.,LopesdaSilva,F.H.,Wadman, W.J.,1998.Effectofvalproicacidonsodiumcurrentsincorticalneuronsfrom patientswithpharmaco-resistanttemporallobeepilepsy.EpilepsyRes.32, 309–320.
Vreugdenhil,M.,Wadman,W.J.,1999.ModulationofsodiumcurrentsinratCA1 neuronsbycarbamazepineandvalproateafterkindlingepileptogenesis. Epilepsia40,1512–1522.
White,H.S.,Loscher,W.,2014.Searchingfortheidealantiepileptogenicagentin experimentalmodels:singletreatmentversuscombinatorialtreatment strategies.Neurotherapeutics11,373–384.
