Effects of microinjections of apomorphine and haloperidol into the inferior colliculus on prepulse inhibition of the acoustic startle reflex in rat

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NeuroscienceLetters509 (2012) 60–63

ContentslistsavailableatSciVerseScienceDirect

Neuroscience

Letters

jo u rn a l h om epa ge :w w w . e l s e v i e r . c o m / l o c a t e / n e u l e t

Effects

of

microinjections

of

apomorphine

and

haloperidol

into

the

inferior

colliculus

on

prepulse

inhibition

of

the

acoustic

startle

reflex

in

rat

Susan

Satake, Karen

Yamada,

Liana

Lins

Melo,

Regina

Barbosa

Silva

∗

LaboratóriodePsicologiaExperimental,DepartamentodeBiociências,UniversidadeFederaldeSãoPaulo(Unifesp),Av.D.AnaCosta,95,Santos(SP)11060-001,Brazil

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received24September2011 Receivedinrevisedform 20December2011 Accepted23December2011

Keywords: Apomorphine Haloperidol Inferiorcolliculus Prepulseinhibition Rat

Schizophrenia

a

b

s

t

r

a

c

t

Prepulseinhibition(PPI)isthereductioninthestartleresponsecausedbyalowintensitynon-startling

stimulus(prepulse)whichispresentedshortlybeforethestartlestimulusandisanoperationalmeasure

ofsensorimotorgating.PPIisimpairedinschizophreniapatientsandinratswithcentraldopamine

(DA)activation.Theinferiorcolliculus(IC)isacriticalpartoftheauditorypathwaymediatingacoustic

PPI.TheactivationoftheICbytheacousticprepulsereducesstartlemagnitude.Theaimofthisstudy

wastoelucidatetheroleofDAtransmissionoftheIConthedevelopmentofacousticPPI.Bilateral

microinjectionsofapomorphine(9.0␮g/0.5␮L),anagonistofD2 receptors,intotheICdisruptedPPI

whilemicroinjectionsofhaloperidol(0.5␮g/0.5␮L),anantagonistofD2receptors,intothisstructuredid

notalterPPI.Theseresultssuggestthatdopamine-mediatedmechanismsoftheICareinvolvedinthe

expressionofPPIinrodents.

1. Introduction

Theacousticstartleresponseisevokedbyasuddenandloud acoustic stimulus and is expressed as a rapid contraction of the facial and skeletal muscles. The magnitude of theacoustic startleresponsecanbereducedbyarelativelyweaksound (pre-pulse) presented immediatelybeforethestartle-eliciting sound [8,11,19,21,25].Thisphenomenonhasbeentermedprepulse inhi-bition(PPI)oftheacousticstartleresponse(ASR),andmayreflect the functioning of a pre attention filtering system protecting the brain from sensory overload [11,14]. Deficits in PPI have been observed in several neuropsychiatric disorders, including schizophrenia[11],obsessive-compulsivedisorder[23], Hunting-ton’sdisease[24]andTourettesyndrome[4].Thesedeficitsmay belinkedtoimpairmentsinsensorimotorgatingamechanismthat enablesnormalindividualstosuppressor“gate”irrelevantor inter-feringinformationinsensory,cognitiveandmotordomains,which allowsthehierarchicalorganizationofthemostrelevant informa-tion[7,11,14].PPIprovidesanimportantoperationalmeasureof sensorimotor gating.Thisjustifiedthe studyof PPIfor a better understandingofthepathophysiologyofschizophreniaandaidin thedevelopmentofnewtherapies.

∗Correspondingauthorat:DepartamentodeBiociências,UniversidadeFederal deSãoPaulo(Unifesp),Av.D.AnaCosta,95,11060-001Santos(SP),Brazil. Tel.:+551332218058;fax:+551332232592.

E-mailaddress:newstein@hotmail.com(R.BarbosaSilva).

Previousstudiesindicated thattheprimaryneuralpathways mediatingPPIisinthebrainstemandthattheinferiorcolliculus (IC)wascrucial[14,22].ThecentralnucleusoftheICreceives audi-toryinput,whichisrelayedtotheexternalnucleusoftheICbefore goingtothemiddlelayersofthesuperiorcolliculus(SC).Inturn,the SCsendsbilateralprojectionstothepedunculopontinetegmental nucleus(PPTg)[1,8,10].Thetransientactivationofthesemidbrain nucleibytheprepulseisconvertedintolong-lastinginhibitionof thegiantneuronsofthecaudalpontinereticularnucleus(PnC)thus reducingstartleresponse[8].LargelesionsoftheICeliminatedthe inhibitionofacousticstartlebyauditorybutnotbyvisualprepulses [8,13,20].ElectricalstimulationoftheICbeforetheacoustic star-tlestimulusattenuatedPPIinratswithoutmajoreffectsonstartle amplitude[14,22].Therefore,theICisacriticalpartoftheauditory pathwaymediatingacousticPPI[8,10].

PPIdeficitssimilartothoseseeninschizophreniapatientscan beinducedin ratsbysystemic administrationof adirect oran indirectdopaminergic(DA)agonist,N-methyl-d_-aspartate_(NMDA) glutamatergicantagonist, anddirect or anindirectserotonergic (5HT)agonist[11].Moretypically,deficitsinPPIareinducedby theadministrationofadopamineagonist,suchasapomorphine, withattenuationoftheapomorphine-induceddeficitrepresenting a meansbywhich potentialantipsychoticagents canbe evalu-ated[11].Apomorphinehasbeenreportedtobeafullagonistof D2receptors[11].ThedisruptioninPPIcausedbyDAagonistsis reversedbythetypicalantipsychotichaloperidol,adrugwithhigh D2receptor-bindingaffinity.Althoughapomorphineand haloperi-dolare‘D2-prefering’drugs,theyalsohavearelativelyhighD1

Open access under the Elsevier OA license.

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S.Satakeetal./NeuroscienceLetters509 (2012) 60–63 61

affinity.Haloperidolisconsideredaclassicalantypsichoticacting primarilyasDAantagonists.ThepresenceofD2receptorsinthe IChasbeenreportedusingtechniquessuchas autoradiography afterlabelingwithhighlyselectiveligands,insituhybridization, andnorthernblotanalysis[5,16,26].

Thus,thepurposeofthepresentstudywastoelucidatetherole ofdopaminergictransmissionintheIContheexpressionofacoustic PPI.Forthatweinvestigatedwhetherbilateralmicroinjectionsof apomorphineorhaloperidolintotheICwouldaffectPPI.

2. Materialsandmethods

2.1. Animals

Atotal of29 naïvemale Wistarratsprovided byCEDEME – FederalUniversityofSaoPauloweighing250–300gatthe begin-ningoftheexperimentswereusedforallexperiments.Theywere housedinindividualPlexiglas-wallcagesina12:12dark/lightcycle (lightsonat07:00am)understandardconditionsina tempera-ture(22±1◦_C)_and_humidity₍₅₅_±_5%)_controlled_room_with_food

andwatergivenadlibitumfortheextentofthestudy.The exper-imentswereconductedduringthelight phaseof thelight/dark cycle,between12:00and18:00h.Theexperimentsreportedinthis studywereperformedincompliancewiththerecommendationsof SBNeC(BrazilianSocietyforNeuroscienceandBehavior),whichare basedontheUSNationalInstitutesofHealthGuideforCareand UseofLaboratoryAnimals(PublicationNo.85-23,revised1985) andwereapprovedbytheethicscommitteeoftheFederal Univer-sityofSaoPaulo(801/09).Alleffortsweremadetominimizethe numberofanimalsusedandtheirsuffering.

2.2. Drugs

Apomorphinehydrochlorideataconcentrationof9.0␮g/0.5␮L

(Sigma,USA)wasdissolvedindistilledwater[2,16].Haloperidol (Janssen,Belgium)waspreparedfrom5mg ampoules,in which thedrugispresentin1mLofvehiclesolutioncontaining6mg lac-ticacid.Thissolutionwassubsequentlydilutedwithphysiological salinetoobtaintherequiredconcentrationof0.5␮g/0.5␮L[16].

Thesedoseswerebasedonpreviousstudies[16].Theinjections weredonebilaterally.Drugsolutionswerefreshlypreparedbefore administration.Distilledwater orphysiological salineservedas vehiclecontrolforICmicroinjections.

2.3. Startlechambers

Twocommercialstartlechambersdevices(InsightEquipment, Brazil)wereusedsimultaneouslytorecordtheamplitudeofthe acousticstartleresponseinthePPItest.Theequipmentconsistedof awire-meshcage(16.5cm×5.1cm×7.6cm)whichwasconnected toa stabilimeter(responseplatform,36.5cm×11.5cm×4.5cm) withfourthumbnail-screws,insideaventilated,sound-attenuated chamber(48cm×48cm×45cm).Noiseburstswerepresentedvia

ahigh-frequencyloudspeakerlocated24cmfromthewire-mesh cage.Thestartlereactionoftheratwithinthewire-meshcage gen-eratedapressureonthestabilimeter,andsignalswereamplified, digitizedandanalyzedbythesoftwareofthestartlemeasurement system(InsightEquipment,Brazil),andinterfaceassembly,which also digitized, and recorded stabilimeter readings. Calibrations were performed weekly to maintain accurate acoustic stimuli presentationsandensureequivalentsensitivitiesoftheresponse platformsoverthetestsessions.Animalbehaviorwasrecordedby aninfraredcamera(SafetyView)locatedbehindthestabilimeter, allowingthe discrimination of all possible behaviors,with the

signalbeingrelayedtoavideoandamonitorinanotherroomvia

aclosedcircuit.

2.4. Procedures

2.4.1. Surgery

The animals were anesthetized with sodium pentobarbital (45mg/kg,ip)andfixedinastereotaxicframe(DavidKopf,USA). Theupperincisorbarwasset3.3mmbelowtheinterauralline, suchthattheskullwashorizontalbetweenbregmaandlambda. A stainless steel guide cannula (o.d. 0.6mm, i.d. 0.4mm) was introduced vertically bilaterally, aimed at theIC using the fol-lowingcoordinates,withthebregmaservingasthereferencefor each plane: antero-posterior=−8.8mm; medio-lateral=1.5mm anddorso-ventral=3.5mm[17].Theguidecannulawasaffixedto theskullwithacrylicresinandtwostainlesssteelscrews.Astylette insideeachguidecannulapreventedobstruction.Allsubjectswere allowedaperiodof7daysofrecoveryaftersurgerywithadlibitum

accesstofoodandwater.

2.4.2. Baselinestartlesession

One weekafter surgery, all rats underwent a brief baseline startle/PPIsessionconsistingof120dBpulse-alonetrialsand pre-pulse+pulse trials in which a prepulse stimulus, 12dB above backgroundnoise,waspresented100msbeforetheonsetofthe 120dB pulse.After a 5minperiod of acclimationin the startle chamber,withaconstant backgroundnoise(65dB) that contin-uedthroughouttheremainderofthesession,atotalof24trials were presented in a pseudorandom order:18 presentations of a 40ms,120dBbroadbandburst and 6trials inwhich a 77dB, 20msburstprecededthe120dBburstby100ms.The experimen-talgroupswereestablishedbyusingthemeanstartleresponseto the120dBpulse-alonetrialsandthemean%PPIcalculatedfrom theprepulse+pulsetrials(seeformulainSection2.5),sothatall groupshadcomparablebaselinestartlereactivityandPPI reduc-inginter-groupvariability.Thedayafterthebaselinesession,drug testingtookplace[18].

2.4.3. Microinjections

Afterremovalofthestylette,microinjectionsweremadeusing thin dentalneedle stainlesssteelcannulae(Mizzy, o.d.0.3mm) introducedbilaterallythroughtheguidecannulaeuntiltheirlower endswere1mmbelowtheguidecannulae.Eachinfusioncannula wasconnectedtoa10␮LHamiltonsyringebypolyethylene

tub-ing,andavolumeof0.5␮Lofvehicleordrugsolutionwasdelivered

simultaneouslyintoeachICover1minbyaninfusionpump(Insight Equipment,Brazil).Theneedlewasleftinplaceforanadditional 1minafterinjection.Afterthat,thestylettewasreplaced.Therats wereplacedinthestartlechambersimmediatelyaftertheinjection.

2.4.4. TestingstartleandPPI

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beginningofthesessiontoachievearelativelystablelevelof star-tlereactivityforthereminderofthesession.Anotherfiveofthe pulse-alonetrials,whichwerealsonotincludedinthecalculation ofPPIvalues,werepresentedattheendofthetestsession,with theremainingtwelve120dBtrialspresentedinthemiddleofthe session.Anaverageof15s(rangingfrom7to23s)separated con-secutivetrials.Thetotaldurationofthesessionwasapproximately 20min[18].

2.5. Dataanalysis

2.5.1. StartleandPPI

Twomeasureswerecalculatedfromthesedataforeach ani-mal. First, the amount of PPI was calculated as a percentage scorefor each prepulse+pulsetrialtype:%PPI=100−{[(startle response for prepulse+pulse trial)/(startle response for pulse-alonetrial)]×100}.Second,startlemagnitudewascalculatedasthe averageresponsetoallofthepulse-alonetrials.PPIdatawere ana-lyzedwithtwo-factoranalysisofvariance(ANOVA)withtreatment asbetween-subjectsfactorandtrialtype(prepulseintensity)as repeatedmeasure(within-subjectsfactor).Startlemagnitudedata wereanalyzedwithone-factor(treatment)ANOVA.Posthoc anal-yseswerecarriedoutusingTukey’stest.Thealphalevelwassetat 0.05.

3. Results

Histologicalanalysisrevealedthatthetipsofthecannulaewere situatedinside thecentralorcorticaldorsalnuclei oftheIC,as showninFig.1.Notallsitesofinjectionarerepresentedbecause ofseveraloverlaps.Theeffectsoflocaladministrationof apomor-phineorhaloperidolintotheIConPPIandstartlemagnitudeare depictedinFig.2AandB,respectively.Two-wayANOVArevealeda significantmaineffectoftreatment[F(2,26)=9.0;P<0.01].Posthoc

analysisshowedthatthePPIresponsewasdisruptedin apomor-phinetreatedratsacrossallprepulseintensitiescomparedtothe vehicleandhaloperidol-treatedgroups.Therewasnomaineffect ofthefactorofprepulseintensity[F(2,52)=2.30;P=0.068],andno treatment×prepulseintensityinteractionwasobserved(P>0.05). Therewasnodifferencebetweenvehicleandhaloperidol-treated groups(P>0.05).One-wayANOVAofstartlemagnitudeonpulse alonetrialsdidnotrevealasignificanteffectoftreatmentbetween apomorphine and haloperidol-treated groups compared to the vehicle[F(2,28)=1.16; P=0.206].Since there wasno difference betweenthevehiclegroupstheyweregroupedtogether.

4. Discussion

The present experiment tested the effects of intracollicular administrationofapomorphineorhaloperidolonPPIandthe mag-nitudeofthestartle.Theresultsdemonstratethatapomorphine disrupted PPI atall three prepulses intensities, while haloperi-doldidnotaffectPPIsuggestingthatdopaminergicmechanisms intheICmaybeatleastpartiallyresponsibleforthemediation ofthiseffect.Thesefindingsareconsistentwithreportsshowing thatstimulation,notblockade,ofD2-familyreceptorsbysystemic administration of direct DA receptor agonists such as apomor-phinesubstantiallyorcompletelydisruptsPPIinrats[3,11,15,25]. InaccordancewiththeseresultsMeloetal.[16]showedthatlatent inhibition(LI),amodelfortheinformationfilteringthatunderlies attentionalprocesseswasdisruptedbyapomorphine microinjec-tionintheICwhilemicroinjectionofhaloperidolintothisstructure didnotinterferewithLI.

AlthoughitiswellestablishedthatlesionsoftheICdisruptPPIof acousticstartle[8,13,20]itwasnotpreviouslyknownwhetherDA

Fig.1. Locationofcannulaplacements(blackdots)intheIConcross-sectionsfrom thePaxinosandWatson[17]atlas.Figuresrepresenttheatlascoordinatesin mil-limeters,posteriortobregma.Notallsitesofmicroinjectionarerepresentedbecause ofseveraldotsoverlaps.

neurotransmissionwithintheICaffectthisresponse.Thepresent study showed that apomorphine infused into the IC decreased PPI.Therefore, theseresultssuggest thatDA neurotransmission not only acts at PPI-modulating forebrain circuits (e.g. ventral tegmentalarea,nucleusaccumbens,ventralpallidum,and pedun-culopontinetegmentalnucleus)[12]butalsodirectlyatlowerlevel system,inthebasiccircuitry,mediatingPPI.

Inthisstudy,thelackofamaineffectofprepulseintensitywas surprising.Typicallywegetmoreinhibitionwithincreasing pre-pulseintensities.Nevertheless,despitetheabsenceofamaineffect ofprepulseintensity,apomorphinewasshowntodisruptPPIatall prepulseintensities.

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Fig.2.Mean+S.E.M.percentofprepulseinhibition(A)andaveragestartle ampli-tude(B)inmalesWistarratsfollowingbilateralmicroinjectionsofapomorphine (9.0␮g/0.5␮L)andhaloperidol(0.5␮g/0.5␮L)intotheinferiorcolliculus.N=11 vehiclegroup;N=10apomorphinegroup;N=8haloperidolgroup.*P<0.01 com-paredtovehicleandhaloperidol.

Thus,deficitsinPPIinducedbyapomorphinemicroinjectioninto theICarenotduetoalterationsinbaselinestartlereactivity.

Insummary,thepresentfindingssuggeststhatdopaminergic neurotransmissionoftheICcanbeinvolved inthemediationof PPIinrodents. Apomorphinedisrupted PPIwhenmicroinjected directlytotheIC.Incontrast,microinjectionofhaloperidolinto thisstructuredidnotseemtointerferewithPPI.

TheseresultssuggestthatactivationorblockadeofD2-family receptorsoftheICdifferentiallyaffectsthisresponse.

Contributors

SusanSatakeandKarenYamadaareundergraduatestudents of the Universidade Federal de São Paulo. They have materi-allyparticipatedintheresearch,andweresupportedbyFAPESP (proc.09/07347-0and09/07278-9).Dr.Melo,andDr.BarbosaSilva weretheprofessorsintheUniversidadeFederaldeSãoPaulo.They haveparticipatedinthearticlepreparation.Thisworkwas spon-soredbyFAPESP(proc.09/05703-4).Allco-authorshaveseenand agreewiththecontentsofthemanuscriptandthereisnofinancial interesttoreport.

Acknowledgments

This work was sponsored by FAPESP (proc. 09/05703-4). S.Y. Satakeand K.Y.Yamada were supported by FAPESP (proc. 09/07347-0and09/07278-9).

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