35-Pregnanolone glutamate, a use-dependent NMDA antagonist, reversed spatial learning deficit in an animal model of schizophrenia

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35-Pregnanolone glutamate, a use-dependent NMDA antagonist, reversed spatial learning deficit in an animal model of schizophrenia
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  (This is a sample cover image for this issue. The actual cover is not yet available at this time.) This article appeared in a journal published by Elsevier. The attachedcopy is furnished to the author for internal non-commercial researchand education use, including for instruction at the authors institutionand sharing with colleagues.Other uses, including reproduction and distribution, or selling orlicensing copies, or posting to personal, institutional or third partywebsites are prohibited.In most cases authors are permitted to post their version of thearticle (e.g. in Word or Tex form) to their personal website orinstitutional repository. Authors requiring further informationregarding Elsevier’s archiving and manuscript policies areencouraged to visit:http://www.elsevier.com/copyright  Author's personal copy BehaviouralBrainResearch 235 (2012) 82–88 ContentslistsavailableatSciVerseScienceDirect Behavioural   Brain   Research  journalhome   page:www.elsevier.com/locate/bbr Research   report 3  5  -Pregnanolone   glutamate,   a   use-dependent   NMDA   antagonist,   reversedspatial   learning   deficit   in   an   animal   model   of    schizophrenia Karel   Vales a ,   Lukas   Rambousek a , b , ∗ ,   Kristina   Holubova a ,    Jan   Svoboda a ,   Vera   Bubenikova-Valesova c ,Hana   Chodounska d ,   Ladislav   Vyklicky a ,   Ales   Stuchlik a a InstituteofPhysiology,AcademyofSciencesoftheCzechRepublic,CzechRepublic  b InstituteofChemicalTechnology,CzechRepublic  c PraguePsychiatricCenter,CzechRepublic  d InstituteofOrganicChemistryandBiochemistry,AcademyofSciencesoftheCzechRepublic,CzechRepublic  h   i   g   h   l   i   g   h   t   s   Wehave   developed   novel   neuroactive   steroid   pregnanolone   glutamate   (3  5  -P-Glu).   3  5  -P-Gluacts   as   ause-dependent   NMDA   receptor   antagonist.   3  5  -P-Glureversed   spatial   learning   deficit   in   an   animal   model   of    schizophrenia.   3  5  -P-Glu   did   not   influence   spatial   learning   of    intact   animals.   3  5  -P-Glumay   represent   procognitive   drug   without   side   effects. a   r   t   i   c   l   e   i   n   f   o  Articlehistory: Received7June2012Receivedinrevisedform9July2012Accepted12July2012 Available online xxx Keywords: Schizophrenia-likebehaviorMK-801Use-dependentNMDAantagonistAnxietyPregnanoloneglutamateCarouselmaze a   b   s   t   r   a   c   t Neuroactive   steroids   modulate   receptors   for   neurotransmitters   in   the   brain   and   thus   might   be   effica-cious   in   the   treatment   of    various   diseases   of    the   central   nervous   system   such   as   schizophrenia.   Wehave   designed   and   synthetized   anovel   use-dependent   NMDA   receptor   antagonist   3  5  -pregnanoloneglutamate   (3  5  -P-Glu).   In   this   study,   we   evaluate   procognitive   properties   of    3  5  -P-Glu   in   an   ani-mal   model   of    schizophrenia   induced   bysystemic   application   of    MK-801.   The   procognitive   propertieswere   evaluated   using   active   place   avoidance   on   a   rotating   arena   (Carousel   maze).   We   evaluated   effectsof3  5  -P-Glu   onthe   avoidance,   onlocomotor   activity,   and   anxiety.   3  5  -P-Glu   alone   altered   neitherspatial   learning   nor   locomotor   activity   in   control   animals.   Inthe   model   animals,   3  5  -P-Glu   reversedthe   MK-801-induced   cognitive   deficit   without   reducing   hyperlocomotion.   The   highest   dose   of    3  5  -P-Glu   also   showed   anxiolytic   properties.   Taken   together,   3  5  -P-Glu   may   participate   in   the   restorationof    normal   brain   functioning   and   these   results   may   facilitate   the   development   of    newpromising   drugsimproving   cognitive   functioning   in   schizophrenia. © 2012 Elsevier B.V. All rights reserved. 1.Introduction Schizophreniaisachronicanddevastatingmentalillnessthataffects1%oftheworld’spopulation[1,2].Threemaintypesof symptomscharacterizethisdisorder:positivesymptoms(hal-lucinations,delusions),negativesymptoms(socialwithdrawal,anhedonia,emotionalblunting),anddeficitsincognitivefunc-tions.Cognitivesymptomshavenegativeimpactonapatient’s ∗ Correspondingauthorat:DepartmentofNeurophysiologyofMemory,Instituteof    Physiology,AcademyofSciencesoftheCzechRepublic,Videnska1083,Prague4,142   20,CzechRepublic.Tel.:+420241062713;fax:+420296442488. E-mailaddress: rambousek@biomed.cas.cz(L.Rambousek). well-being,worsenfunctionalmanifestationsofschizophreniasymptoms,andreducepatientcompliance.Littlesuccessofvariouspharmacologicalstrategiesutilizedtoimprovecognitivefunctionsinschizophrenicpatientsunderlinesthedifficultyofcognitivedeficitremediation[3].Schizophreniasymptomsareinducedinhealthyvolunteers[4–6]orexacerbatedinschizophrenicpatients[7–9]byapplica- tionofnon-competitiveN-methyl- d -aspartate(NMDA)receptorantagonists(e.g.Phencyclidine,ketamine,MK-801).Inaddition,theiracuteorrepeatedadministrationshavebeenestablishedasapharmacologicaltooltoproducepositiveandcognitiveschizophrenia-likeintermediatephenotypesinrodents[10–12].Despitenoanimalmodelfullymimicsschizophreniasymptoms,administrationofNMDAantagonistsinducemotordysfunction, 0166-4328/$–seefrontmatter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.bbr.2012.07.020  Author's personal copy 84  K.Valesetal./BehaviouralBrainResearch  235 (2012) 82–88  2.3.3.   Elevated-plusmaze(EPM) Thepossibleanxiolyticpropertiesof3  5  -P-Gluweretestedinanelevated-plusmaze.Theapparatusconsistedoftwoopenarms(45cm × 10cm)   crossedatrightangleswithtwoopposedarmsofthesamelength.Two   oftheopposedarmswereenclosedbywalls40cmhigh,exceptforthecentralplatformwherethearmscrossed.Thewholeapparatuswaselevated50cmabovethefloor.Atthebeginningof    eachexperimenttheratwas   placedonthecentralplatformfacingtheclosedarm.The   numberofopenarmentriesandtimespentinopenarmswas   recordedduringa   5-mintestsession.  2.3.4.22-kHzultrasonicvocalization(USV) We   alsoexaminedaneffectof3  5  -P-Gluonultrasoundvocalizationsfollow-ing   exposuretoinescapablefootshock.Theexperimentalsetupconsistedofashockchamber(32.2cm × 25.5cm × 25.5cm)withthefloormadeofstainlesssteelrods(4.8mmindiameter)spaced10.75mmapart.Theultrasonicvocalizationwas   mea-suredbyamini-3batdetector(Noldus,Wageningen,TheNetherlands)attachedtotheceilingofthechamber.Thedetectorwassettoregistertheultrasonicvocaliza-tionsat22kHz.Thisfrequencywasfoundusefulfordifferentiatingfearandanxietyin   rats[36].Anaudiofilterreceiveddirectoutputfromabatdetectorandforwardedit   tothePC.Ashockdevicedelivered1-mAshocks.Onthefirstday,theratswereindividuallyplacedintotheshockchamberandafter30sofhabituationtheratsreceived6inescapableelectricfootshocks(1   shock/min)of10sduration.24hlatertheprocedurewasrepeated;however,the   ratsreceivedonlyone10sshockforrecollection.Afterwards,theyreceivedtesteddrugand30min   lateranimalsweregivenintoshockchamberfor10min.Duringthistesttrial,noshockswereadministered.Ultrasonicandaudiblevocaliza-tionsemittedbyanimalduringthetesttrialwererecordedsimultaneouslybydataacquisitionsoftware(UltraVox2.0;Noldus,Wageningen,TheNetherlands).Ultra-Vox   softwareautomaticallymonitoredthenumberanddurationofultrasonicandaudiblevocalizationsofeachrat.  2.4.Dataanalysisandstatisticalmethods Thedataarepresentedasgroupaverages ± standarderrorsofmean(SEM).ThedatawerestatisticallyanalyzedbyprogramSTATISTICA8.0(StatSoft,USA)witha   two-wayANOVA.MK-801treatment(twofactorlevels)and3  5  -P-Glutreat-ment(threetosixfactorlevels)servedasindependentfactors.Intestsassessingtheeffectsof3  5  -P-Glualone(USV,EPM),one-wayANOVAwasconducted.Subse-quentNewman–Keulsposthoctestsfollowedwhenappropriate.Thesignificancelevelwas   setat  p <0.05. 3.Results  3.1.PlaceavoidanceonCarouselmaze Weassessedtheeffectof3  5  -P-GluonspatialMK-801-inducedcognitivedeficitinatestofcognitivecoordinationandspatiallearning,theCarouselmaze.Two-wayANOVArevealedasignificantinteractionbetweentheeffectsof3  5  -P-GluandMK-801onthenumberofentrancesintothepunishedsector F  (6,75)=3.3,  p <0.01,reflectingU-shapeddose-dependentpositiveeffectof3  5  -P-GluonthenumberofentrancesinratswithMK-801injection.Furthermore,theanalysisrevealedsignificantmaineffectof3  5  -P-Glu F  (6,75)=4.16,  p <0.01butnotMK-801treat-ment F  (1,75)=2.04,  p =0.15.PosthoctestsshowedthattheMK-801didinducecognitivedeficit,butitwasamelioratedby3  5  -P-Gluatdoses0.001mg/kg,0.01mg/kg,0.1mg/kgor1mg/kg.3  5  -P-Gluat10mg/kg,theimprovementwasonlymoderate.Applicationof3  5  -P-GluwithoutMK-801ledtosimilardose-dependenteffectonthenumberofentrancesthatwasnot,however,statis-ticallysignificant(Fig.1A).TheapplicationofMK-801slightly,yetinsignificantlystimu-latedlocomotoractivityintheCarouselmaze F  (1,75)=2.94,  p =0.09(Fig.1B).Inaddition,two-wayANOVArevealedmaineffectof 3  5  -P-Glutreatment F  (6,75)=3.05,  p =0.01.However,thesourceoftheseeffectscouldnotbedeterminedbyNewman–Keulsposthoctests.Interactionsbetweenthetwofactorsdidnotreachsig-nificance F  (6,75)=1.1,  p >0.05  3.2.Open-fieldtest  ThetendencyofMK-801tostimulatelocomotoractivityinCarouselmazewasaccentuatedintheopen-fieldtest(Fig.2). Fig.1. Effectsof3  5  -P-GluandMK-801treatmentonplaceavoidanceandloco-motoractivityontheCarousel.Averagegroupvalues ± SEM.**  p <0.01comparedtosaline/salinegroup.  #  p <0.05, ##  p <0.01comparedtoMK-801/salinegroup.Numbersbelowthebarsdenotethenumbersofanimalsinparticulargroups.EffectofMK-801(0.1mg/kg)and3  5  -P-Glutreatmentwasassessedbyameasureofspatiallearning(numberofentrances,(A)andlocomotoractivity(B).Administrationof3  5  -P-Gludidnotalterspatiallearningatanydoseinnaiverats.MK-801inducedspatiallearningdeficit,whichwas   reversedby3  5  -P-Gluadministrationatdosesof0.001,0.01,0.1and1mg/kg.Locomotoractivitywas   notsignificantlyaffectedeitherbyadministrationofMK-801or   anydoseof3  5  -P-Glu. Fig.2. Effectsof3  5  -P-GluandMK-801treatmentonlocomotionintheopen-field   test.Averagegroupvalues ± SEM*  p <0.05maineffectofMK-801administration.Numbersbelowthebarsdenotethenumbersofanimalsinparticulargroups.Admin-istrationof3  5  -P-Gludidnotalterlocomotoractivityatanydoseinnaiverats.MK-801(0.1mg/kg)inducedhyperlocomotion,whichwasnotreversedbyadmin-istrationofanydoseof3  5  -P-Glu.  Author's personal copy K.Valesetal./BehaviouralBrainResearch  235 (2012) 82–88 85 Fig.3. Effectof3  5  -P-Glutreatmentonanxietyintheelevated-plusmaze.Totaltimespentinopenarms(A),andthenumberofentriesintoopenarms(B).Averagegroupvalues ± SEM*  p <0.05comparedtothecontrolgroup.3  5  -P-Gluapplicationtendedtoincreasethetotaltimespentinopenarms;however,with-out   statisticallysignificanteffect.3  5  -P-Gluatadoseof10mg/kgincreasedthenumberofentriesintoopenarms. Atwo-wayANOVA(MK-801 × 3  5  -P-Glu)foundasignificantmaineffectofMK-801 F  (1,54)=8.98,  p <0.01,butnot3  5  -P-Glu F  (2,54)=0.36,  p >0.05orinteractionbetweenthetwodrugs F  (2,54)=0.49,  p >0.05.Thus,3  5  -P-GludoesnotaffectMK-801-inducedhyperactivity(Fig.2).Importantly,itdoesnotaffectopen-fieldlocomotionwhenappliedaloneeither.  3.3.Elevated-plusmaze(EPM)andultrasonicvocalization(USV) Thesebehavioralparadigmswereusedtotestwhether3  5  -P-Gluadministrationaloneinfluencesanxiety-relatedbehavior.Ratstreatedwith3  5  -P-Gluatadoseof10mg/kgmadesignificantlymoreentriesintoopenarmsintheelevatedplusmazetest F  (3,28)=3.06,  p <0.05,butthetimespentinopenarmswasnotsig-nificantlyalteredby3  5  -P-Glutreatment F  (3,28)=2.23,  p =0.11(Fig.3).Furthermore,3  5  -P-Glutreatedratstended(butinsignif-icantly)tovocalizelessfrequently F  (1,33)=2.27,  p =0.1andwithshorterdurationsoftheiremittedsounds F  (1,33)=2.19,  p =0.11duringatestsessionofultrasoundvocalizationtest(Fig.4). 4.Discussion Inthisstudy,wedemonstrateprocognitiveeffectsof3  5  -P-GluinananimalmodelofschizophreniabythesystemicadministrationofMK-801(dizocilpine)atadoseof0.1mg/kg.ThisdoseofMK-801wasselectedbecauseitelicitsacognitivedeficitintheCarouselmazewithoutlocomotoractivitysensitiza-tion[12].HigherdosesofMK-801(suchas0.2mg/kgor0.3mg/kg)severelyaffectmotoractivityconfoundingthecognitiveeffectsintheCarouselmaze[12,31].Thenumberofentrancesintotheto-be-avoidedsectorintheCarouselmazewassignificantlydecreasedafterapplicationof0.001mg/kg,0.01mg/kg,1mg/kgand0.1mg/kgof3  5  -P-GluandMK-801(comparedtoMK-801alone;Fig.1A).Thelowest(0.0001mg/kg)andthehighestdose(10mg/kg)of 3  5  -P-Glufailedtoproduceprocognitiveeffect(Fig.1A)inthismodel.TheseresultssuggestaU-shapeddose-dependenteffect Fig.4. Effectof3  5  -P-Glutreatmentonultrasonicvocalization.Numberofvocalizations(A)andtotaldurationofvocalization(B).Averagegroupvalues ± SEM.3  5  -P-Glutendedtodecreasethevocalizations;however,withoutstatisticallysignificanteffect. of3  5  -P-Glu.Thiseffectmightbeduetothebindingof3  5  -P-GlutoGABA A  receptor(unpublishedresults).Itisconceivablethatthelowestdose(0.0001mg/kg)wasnotsufficienttoinhibitNMDAreceptorsandnormalizecognitivefunctions.Incontrast,thehighestdose(10mg/kg)mighthaveactivatedGABA A  recep-tors,preventingthe“stabilization”effectontheexcitatorysystem,whichwas   observedatthedoserangeof0.001–1mg/kg.WehaveobservedasimilarU-shapedeffectinourpreviousstudy[29],where3  5  -P-Gluapplied30min   afterNMDAlesionshowedneu-roprotectiveeffectatdosesof0.1mg/kgand1mg/kgbutnotat10mg/kg.Asnotedabove,higherdosesof3  5  -P-Glumay   inter-actwithGABAergicneurotransmissionandthusmodulategeneraleffectsof3  5  -P-Glu.Thisassumptionissupportedbythefindingthatthehighdose(10mg/kg)of3  5  -P-GluproducedasignificantanxiolyticeffectinEPMtest.Animportantgoalwastotestthepossibilitythatadministra-tionof3  5  -P-Glu,whichhasanNMDAreceptor-antagonisticaction,induceshyperlocomotionandcognitivedeficitinnaive,healthyrats(notethatsucheffectsaretypicalformanyNMDAreceptorantagonists;forreviewsee[38].Recentreportsindi-catethatneuroactivesteroidsmay   beefficientinthetreatmentof variousbraindisordersandpathologicalstatessuchasschizophre-nia,neurodegenerationandpain[29,39–41].   On   theotherhand,administrationofNMDAantagonistssuchasketamine,phencycli-dine,ordizocilpine(MK-801)inducesseveremotorandcognitiveimpairments[4–6]andotherundesirableside-effectsoftenlead-ingtotheexpressionofpsychoticsymptoms.Theseside-effectsmakenon-competitiveantagonistsunacceptableforclinicalusedespitetheirsignificantneuroprotectivepotential[42].However,thesedrugsprovedtobeextremelyusefulinpreclinicalresearchforinductionofschizophrenia-likebehaviorsinanimalmodels,especiallyinrodents[10–12],   totestplausibilityofthehypogluta-matergichypothesisofschizophrenia[43].Ourresultsdemonstratethatadministrationof3  5  -P-Glualonecausedneithersignifi-cantalternationsinlocomotoractivity,norcognitivedeficitinthe
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