ContentslistsavailableatScienceDirect
Behavioural
Brain
Research
j o ur na l h o me p a g e :w w w . e l s e v i e r . c o m / l o c a t e / b b r
Short
communication
Object
recognition
impairment
and
rescue
by
a
dopamine
D2
antagonist
in
hyperdopaminergic
mice
Arthur
S.C.
Franc¸
a
a,
Larissa
Muratori
b,
George
Carlos
Nascimento
c,
Catia
Mendes
Pereira
d,
Sidarta
Ribeiro
a,
Bruno
Lobão-Soares
e,∗aBrainInstitute,FederalUniversityofRioGrandedoNorte,RN59056-450,Brazil
bBiochemistryDepartment,FederalUniversityofRioGrandedoNorte,Brazil
cMedicalEngineeringDepartment,FederalUniversityofRioGrandedoNorte,Brazil
dEdmond&LilySafraInternationalInstituteofNeuroscienceofNatal,Brazil
eBiophysicsandPharmacologyDepartment,FederalUniversityofRioGrandedoNorte,RN59078-970,Brazil
h
i
g
h
l
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g
h
t
s
•Heterozygousmicefordopaminetransporter(DAT+/−)exhibithigherlevelsofsynapticdopamine.
•HereweconfirmedthatD2antagonismcaninterfereinobjectrecognition.
•WeobservedinDAT+/−anaturalphenotypeofimpairednovelobjectmemoryrecognition.
•Theinjectionofhaloperidolat0.05mgbeforeobjectexpositionrestoredobjectrecognition.
•ThiseffectcouldbeexplainedbyrestoringD2activitytooptimallevels,actingonmemoryacquisition.
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Articlehistory:
Received4September2015
Receivedinrevisedform29March2016
Accepted4April2016
Availableonline6April2016
Keywords: DAT-KO Heterozygous Haloperidol Dopamine Objectrecognition
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Genetically-modifiedmicewithoutthedopaminetransporter(DAT)arehyperdopaminergic,andserve
asmodelsforstudiesofaddiction,maniaandhyperactivedisorders.Hereweinvestigatedthecapacity
forobjectrecognitioninmildlyhyperdopaminergicmiceheterozygousforDAT(DAT+/−),with
synap-ticdopaminergiclevelssituatedbetweenthoseshownbyDAT−/−homozygousandwild-type(WT)
mice.WeusedaclassicaldopamineD2antagonist,haloperidol,tomodulatethelevelsofdopaminergic
transmissioninadose-dependentmanner,beforeorafterexploringnovelobjects.Incomparisonwith
WTmice,DAT+/−miceshowedadeficitinobjectrecognitionuponsubsequenttesting24hlater.This
deficitwascompensatedbyasingle0.05mg/kghaloperidolinjection30minbeforetraining.Inallmice,
a0.3mg/kghaloperidolinjectedimmediatelyaftertrainingimpairedobjectrecognition.Theresults
indi-catethatamildenhancementofdopaminergiclevelscanbedetrimentaltoobjectrecognition,andthat
thisdeficitcanberescuedbyalowdoseofaD2dopaminereceptorantagonist.Thissuggeststhatnovel
objectrecognitionisoptimalatintermediatelevelsofD2receptoractivity.
©2016ElsevierB.V.Allrightsreserved.
Dopamine(DA)isaneurotransmitterrelatedtocomplex
behav-iors,suchas:rewardperception,socialinteraction[1,2],andisalso
linkedtomemoryconsolidationbothinhumansandrodents[3].
AlterationsinDAsynapticregulationarerelatedtoalarge
vari-etyofmentaldiseases,suchasschizophrenia,hyperactivity,mood
disorders,andParkinsondisease[4,5].
∗ Correspondingauthor.
E-mailaddresses:brunolobaosoares@gmail.com,
brunolobaosoares@hotmail.com(B.Lobão-Soares).
DAhasmanyreceptorsubtypes,buttheyarebasicallydivided
inD1andD2families[3].DA,mainlythroughD1receptors,elicits
theonsetofthelatephaseoflongtermpotentiationinthe
hip-pocampus[6],controlplasticity-inducedproteinsynthesis[6],and
enhancethepersistenceofhippocampus-dependentmemories[7].
The involvement of both dopamine receptors families with
learningandmemoryiswidelyreportedforworkingmemory[3],
spatiallearning[3,6],aversivememory[7],reward-related
learn-ing[8]and cognitiveflexibility[9].In particular,impairmentin
objectrecognitionhasbeeninducedbyD2activitysuppressiondue
tohaloperidolIPinjection[10],byD1activitysuppressiontrough
http://dx.doi.org/10.1016/j.bbr.2016.04.009
SKF81297microinfusionintheprefrontalcortex[12].
Morespecifically,thelack ofD2receptorswasassociatedto
odordiscriminationinmice[13].TheimpairmentofD2activity
wasrelatedtosleepregulationandmemoryconsolidation,through
thedown-regulationof plasticityfactorsand reduction ofrapid
eyemovement(REM)sleepamount[10].TheantagonismofD2
receptorsalsoledtoelectrophysiologicalchanges duringobject
exploration[14].Furthermore,micelackingD2receptorsdoshow
memoryconsolidation deficits[15].Ontheotherhand,animals
withhighlevelsofsynapticDA,namelyknockoutmicefortheDA
transporter(DAT-KO),showimpairmentintheMorriswatermaze
task[9],and alsoimpairmentofspatial memoryin theY-maze
[16].Theseanimalspresented,however,lessimmobilitytimein
theforcedswimmingtask[17],andexhibitedanincreasein
loco-motion,reversedbyD2receptorblocking[17].
DAT-KOmicewereinitiallygenerated tostudytheinfluence
of hyperdopaminergia in physiological and behavioral
parame-ters,and inresponsetodopaminergicdrugadministration[13].
DAT—heterozygous (definedhereas DAT+/−)mice,expressing
onlyonecopyoftheDATgene,werealsoinvestigated[18].
Clear-anceofdopaminereleasedinthesynapsetakesthricemoretime
inDAT+/−micethaninwild-type(WT)mice[18].
Yet,inadditiontoneurochemicalalterationsleadingtoamild
DAincreaseatthesynapticlevel,onlyahandfulofstudieshave
describedmemoryalterationsinDAT+/−mice[19,20].Previous
studiesrevealedthatDAT+/−miceshowimpairmentinpattern
completioninapartialcueenvironment[19],andexhibitdecreased
anxiety-relatedbehaviors[20].ThemildhyperdopaminergicDAT
+/− micepresent biochemical changes [18] related tomemory
impairmentthatcouldbereversedbyD2downregulation[19].
ThepresentworkaimedtoinvestigatetherelationbetweenD2
activityandnovelobjectrecognitioninmildhyperdopaminergic
DAT+/−mice.Tothatend,weinvestigatedDAT+/−micetrained
intheobjectrecognition(OR)task,withassessmentatbasallevels
ofdopaminetransmissionaswellasundertheinfluenceof
differ-entdosesofhaloperidol,(0.05and0.3mg/Kg)beforeorafterthe
trainingsessionofthetask.
Atotalof75adult(2–5months)malemicewereused,
com-prising 39 WT (C57Bl-6 strain) and 36 heterozygous (DAT+/−
strain, on C57Bl/6J background). The animals were housed in
cages (2–4 animals/cage), under a 12h/12h light/dark cycle,
with lights on at 07:00, and food and water ad libitum.
Ani-malswere daily handledfor 5minfor 10 sessions prior tothe
experiments,in order todecrease stressresponses WTand KO
littermatesweregeneratedformC57BL/6J-129/SvJhybridDAT
het-erozygotes as previously described [18]. Mice were genotyped
byPCRusingsenseWT(5-CCCGTCTACCCATGAGTAAAA-3),sense
KO(5-TGACCGCTTCCTCGTGC-3)andacommonantisenseprimer
(5-CTCCACCTTCCTAGCACTAAC-3).Theproceduresappliedinthe
studyfollowedguidelinesoftheNationalInstitutesofHealthand
wereapprovedbytheEdmondandLilySafraInternational
Insti-tuteofNeuroscienceofNatalEthicsCommittee(protocolnumber
08/2010).
AnimalsweresubmittedtoanORtask,basedonthenovelty
explorationtendencyofrodents.Thetaskwasperformedina
cir-culararena(50cmdiameterand30cmhigh)inaroomwithdim
andwell-spread light,soastoavoid producingshadows inthe
apparatus.Animalswerenaïve totheapparatuswhen exposed.
We employed 6different objectspresented over 2 consecutive
days(twosessions);4 objects(A–D)werepresentedduringthe
initialexplorationsession(Firstsession,10min);and2unfamiliar
objects(EandF)replaced2familiarobjects(CandD)duringthe
secondsession,24hlater(testingsession,10min).Inorderto
eval-uatememoryrecognition,anobjectpreferenceratiowascalculated
(timespentwithEandF/AandBobjects).Noticethatiftheanimal
novelobjects[14,15](ratioEandF/AandB>1),iftheyhad
impair-mentinORtheywillexploresimilarlythefamiliarandthenovel
objects(ratioEandF/AandB=1).
Based onthe D2influence onlearning and memory [8–10],
weusedhaloperidoltoinduceORimpairment.Atthetimepoint
of 30min before exploration (B.E.) of the object, as well as
immediatelyafterexploration(I.A.E.),animalswereinjectedwith
haloperidol(0.3mg/Kgand0.05mg/Kg)orvehicle,andwerethen
allowedtobehavefreelyintheirhomecagesuntilthesecond
explo-rationsession.Inordertodifferentiatewhetherapossiblememory
deficitcouldbeduetomemoryacquisition,orspecificallyrelated
tothememoryconsolidationphase,weperformedinjectionsof
haloperidolatlowdosebothbeforeandafterobjectexploration.
Theinjectionof0.3mg/Kghaloperidolbeforetheexplorationphase
resultsinpartialdeficitofmovementthatimpairsobject
explo-ration [17,21].Therefore, we couldnot determinewhetherthe
decrease in object recognition was due to impaired
consolida-tion,ortoa deficitinlocomotoractivityduringtheexploration
phase. For this reason, we didnot investigateanimals injected
with0.3mg/Kghaloperidolbeforetheexploration.Incontrast,the
useof the0.3mg/Kgdoseaftertheexplorationcouldnotaffect
themobilityneitherduringtheacquisitionphasenorduringthe
test/evocationphase,andthereforewesetouttoinvestigatethis
condition.
Theparameterofobjectexplorationconsideredthetime
ani-malsspentwiththewhiskersorfrontpawsincontactwithone
oftheobjectsforatleast0.5s,witha0.5sasaminimum
inter-valbout.Ontestsession,bypresentinghalfofobjectsasnovelty,
animalsshouldspendmoretimewithnovelobjects,givingriseto
unequalexplorationtimebetweennovelandfamiliarobjects[22].
ByrecordingvideoswithaPanasoniccameraandAMcap9.21free
software,wedefinedthepreferenceratioastheexploration
dura-tionofnovelobjects(EandF)dividedbythetimespentexploring
theobjectsthatwerethesameasintraininginsessions(objectsA
andB).
First we measured the influence of haloperidol (0.3 and
0.05mg/Kg)intheWTmicestrainduringORtask(Fig.1).We
exe-cutedtwodifferentstatisticalanalyses;one-wayANOVAfollowed
byBonferronicorrectionwasperformedtoanalyzethedifference
amonggroupssubmittedtovehicleorhaloperidol.Wealso
per-formedthedifferencebetweenthegroup(vehicleorhaloperidol)
andtheratio=1(describedabove),ttestagainst1,tofindoutifthe
preferenceratiopresentedbyWTgroupswassignificantdifferent
ofthehypothesisofnon-recognitionofobjects.Toverifypossible
effectsofhaloperidolinthemotorandmotivationsystem[8,17]
wemeasuredthetotaldistancetraveledandthetotaltimespent
inobjectexploration.
2WeappliedShapiro-Wilknormalityteststoassessthe
dis-tributionofthedata.Forthecomparisonwithinstrains,inwhich
treatment is the only independent variable, we used one-way
ANOVAs of three dependent variables (Preference Ratio, Total
ExplorationTimeandTotalDistanceTraveled).Forthecomparison
betweenstrainsweusedaTwo-wayANOVAwithstrainand
treat-mentasindependentvariables,followedbyBonferronipost-hoc
tests.Thecomparisonagainstonewasperformedbyapairedt-test
(alpha=95%,alldatawithBonferronicorrection).Thedescriptive
statisticscomprisenormalitytestvalues(W),mean±SEM,F,p
val-uesanddegreesoffreedom(DF)
FirstweappliedtheShapiro-Wilknormalitytestswithalldata
set groups to verify if the data followed the normal
distribu-tionin order tochoosecorrectly parametricor non-parametric
tests.Wvalues for theWildTypegroups were:Vehicle 30min
B.E., W=0.94; Halo0,05mg/Kg 30min B.E. I.A.E. W=0.95; Halo
0,3mg/KgI.A.E,W=0.93;VehicleI.A.E.,W=0.97;Halo0,05mg/Kg
Fig.1.Behavioralandpharmacologicalprocedures.HandledC57BL-6andDAT+/−micewerepresentedto4novelobjectsatlightperiods(trainingsession).Haloperidol
orvehiclewasinjected30minbeforeoftheexploration(B.E.)orimmediatelyafterexploration(I.A.E.)ofnovelobjectsexploration.Onedayaftertraining,animalswere
exposedto2novelobjectsand2familiarobjects(testsession).
DAT +/− animals, Shapiro-Wilk normalitytest W values were:
Vehicle 30minB.E., W=0.89; Halo0,05mg/Kg 30min B.E. I.A.E.
W=0.81;Halo0,3mg/Kg I.A.E,W=0.90;Vehicle I.A.E.,W=0.86;
Halo0,05mg/KgI.A.E,W=0.77.(Shapiro-Wilkpsummaryvalues
p>0.05).Therefore,inthisweusedonlyparametricteststoanalyze
allcomparisonsFig.1.
WeappliedANOVAtocomparethepreferenceratioamongWT
groups.ANOVAcomparisonofpreferenceratiorevealeda
signif-icantdifferenceamonggroups(F=11.74,df=4,38andp<0.0001)
(Fig.2A).TheBonferroni’spost-hocshowedsignificantpreference
ratiodecreasesofhaloperidol0.3mg/Kgincomparisontotheother
groups(seedetailsinTable1).Thehigherdoseofhaloperidolwas
theonlytreatmentthatledtoimpairmentinOR,supportedbythe
t-testagainst1,t=1.74,p=0.6.Theothergroupsshowed
signif-icantpreferenceratiohigherthan1:VehicleI.A.E.(t=4.57df=6,
p=0.019);Haloperidol0.05mg/KgI.A.E.(t=4.14df=7,p=0.021);
Vehicle30minB.E.(t=11.13df=7,p<0.0001);and Haloperidol
0.05mg/Kg30minB.E.(t=11.05df=7,p<0.0001).
Thesecondpartofthestudytestedifthemilddisbalanceofthe
DAlevelsinDAT+/−micestrainhadinfluenceinORtask.Therefore,
weanalyzedtheeffectofhaloperidol(0.3and0.05mg/Kg)during
ORtask.
Weappliedone-wayANOVAtocompareallthegroupsofDAT
+/−(Fig.2B).TheANOVAcomparisonofpreferenceratiorevealed
a significant difference among groups (F=12.44, df=4,35 and
p<0.0001).Bonferroniposthocrevealedthatthetreatmentwith
haloperidol(0.05mg/Kg)injected30minbeforeexplorationledto
ahigherpreferenceratioincomparisontotheothergroups(see
detailsinTable1).Whenweperformedthecomparisonto1,DAT
+/−withhaloperidol0.05mg/Kg30minB.E.wastheonlygroupto
presentdifference(t=5.621,df=6,p=0.007),demonstratingtobe
theonlygroupwithapreferenceratiocompatiblewithnovelOR.
Theothercomparisonsbetween1andDAT+/−groupsrevealed
nodifference:VehicleI.A.E.(t=0.73 df=6,p>0.99); Haloperidol
0.05mg/Kg I.A.E.(t=0.39,df=7,p>0.99);Haloperidol0.3mg/Kg
I.A.E.(t=0.26 df=6, p>0.99); and Vehicle30minB.E. (t=2.095,
df=6,p=0.4).
WealsomeasuredtheinfluenceofhaloperidolinWTorDAT+/−
byusingthestrainsandhaloperidoldosageasindependent
vari-ables.TheTwo-wayANOVA(Strain:F (1,65)=21.78,p<0.0001;
Treatment:F(4,65)=41.65,p<0.0001;Interaction:F(4,65)=4.39,
p=0.072)revealedthatbothtreatmentandstrainhasinfluencein
thepreference ratiooftheanimals(Fig.2C).Finally,weapplied
the Bonferroni post hocto be able to compare where in each
treatmentboth strainshavedifferent preferenceratios.
Bonfer-roniposthocshowedinhaloperidol0.3mg/KgI.A.E.groupsimilar
resultsconsideringWTandDAT+/−preferenceratios(p>0.05).
Both strains presented ratios similar to 1 indicating that both
strainshaveimpairmentinOR.Haloperidolat0.05mg/Kg30min
B.E.alsoinduceda similarpreferenceratioconsideringthetwo
strains(p>0.5).Nevertheless,inthiscasebothstrainsrevealeda
preferenceratiohigherthan1indicatingthattheyareableto
rec-ognizenovelobjects.RegardingthecomparisonofWTandDAT
Preference Ratio
Preference Ratio
Preference Ratio
A - Haloperidol Effects on WT Mice
B - Haloperidol Effects on DAT +/- Mice
C - WT vs DAT +/- Mice
Fig.2. Haloperidoldifferentially modulatesmemoryconsolidation inWTand
DAT+/−miceinanovelobjectrecognitiontask.A—Effectoftwodifferenthaloperidol
(halo)doses(0.05and0.3mg/Kg)wheninjectedimmediatelyafterexposition(I.A.E.)
and30minbeforeexposition(B.E.)inwildtype(WT)micenovelobjectpreference
ratio(unfamiliar/familiarobjectexplorationduration),usedasmemory
consolida-tion(M.C.)parameter.ComparisonthroughANOVArevealedthatinjectionofhalo
0.3mg/KgimmediatelyafterexplorationledtoadecreaseonM.Cwhencompared
tocontrolgroup(p<0.01)andothergroups.Also,thedoseof0.05mg/Kginjected
30minbeforetheexploration(B.E.)revealedanassociationwithincreasednovel
objectexplorationwhencomparedtoothergroupsexceptingitscontrolvehicle
group(p<0.001).B—EffectofdifferenthalodoseswheninjectedI.A.E.and30min
B.E.inheterozygousmiceinnovelobjectpreferenceratio.ANOVArevealedthat
injectionofhalo0.05mg/KgB.E.increasedpreferenceratiowhencomparedtoall
othergroups(p<0.01).C—DAT+/−andWTmicedisclosedifferentpreferenceratios
andspecificresponsestohaloinjection.WTandDAT+/−grouppairswerecompared
ineachtreatmentusingStudent’sTtest.DAT+/−groupsrevealednonovelty
recog-nitioninvehicleandhalo0.3and0.05I.A.E.,aswellasvehicleB.E.(ratioscloseto
1.0),anddecreasedratioscomparedtoWTinbothvehicletreatments(I.A.EandB.E.)
andinhalo0.05mg/kgI.A.E.TherewasnodifferenceconcerningWTandDAT+/−
inhalo0.05mg/kgB.I.*p<0.05;**p<0.01;***p<0.001.
+/−groups:vehicleI.A.E.,haloperidol0.05mg/KgI.A.E.andvehicle
30minB.E.revealedadifferenceinthepreferenceratio(seedetails
inTable1).Amongthosethreetreatments,WTanimalspresented
StatisticalsummaryofthesignificantresultsinthecomparisonsamongWTandDAT
+/−.ANOVAcomparisonsofpreferenceratiowereappliedamongWTandDAT+/−
separately.Two-wayANOVAcomparisonswereappliedtocomparetheWTandDAT
+/−groupsfollowedbyBonferronipost-hoctests.Comparisonswereperformed
consideringtwoindependentvariables:treatmentandstrainline.
Wild-TypeMice ANOVA
F PValue DF
11.74 0.0001 (4.38)
Bonferroni
Groups Mean±SEM;N PValue
VehicleI.A.E.xHalo0.3mg/KgI.A.E. 2.28±0.28;N=7 P<0.01 1.23±0.13;N=8
Halo0.05mg/KgB.E.xHalo0.05mg/KgI.A.E 2.73±0.15;N=8 P<0.001 1.66±0.16;N=8
VehicleB.E.xHalo0.3mg/KgI.A.E. 2.20±0.10;N=8 P<0.01 1.23±0.13;N=8
Halo0.05mg/KgB.E.xHalo0.3mg/KgI.A.E 2.73±0.15;N=8 P<0.001 1.23±0.13;N=8 DAT+/-Mice ANOVA F PValue DF 12.44 0.0001 (4.35) Bonferroni
Groups Mean±SEM;N PValue
Halo0.05mg/KgB.E.xVehicleI.A.E 2.25±0.22;N=7 P<0.001 1.07±0.10;N=7
Halo0.05mg/KgB.E.xHalo 0.05mg/KgI.A.E
2.25±0.22;N=7 P<0.001 0.96±0.10;N=8
Halo0.05mg/KgB.E.xHalo 0.3mg/KgI.A.E
2.25±0.22;N=7 P<0.001 0.97±0.12;N=7
Halo0.05mg/KgB.E.xVehicleB.E 2.25±0.22;N=7 P<0.01 1.38±0.18;N=7
Wild-TypevsDAT+/-Mice
Two-WayANOVA
Sourceofvariation F PValue DF
Interaction 4.39 0.0720 (4.65)
Strain 21.78 0.0001 (1.65)
Treatment 41.65 0.0001 (4.65)
Bonferroni
Groups Mean±SEM;N PValue
VehicleI.A.E(WTvsHz) 2.28±0.28;N=7 P<0.01 1.07±0.10;N=7 Halo0.05mg/KgI.A.E.(WTvsHz) 1.66±0.16;N=8 P<0.05 0.96±0.10;N=8 VehicleB.E 2.20±0.10;N=8 P<0.01 (WTvsHz) 1.38±0.18;N=7
TotestwhethertheinvolvementofD2receptorsinthe
motiva-tionalandmotorsystemsplayedaroleduringtheexplorationof
objectsinthetestsession,wemeasuredthetotalexplorationtime
andthetotal distancetraveled.WeappliedANOVAtocompare
thetotal explorationtime among WTgroups. ANOVA
compari-sonoftotal explorationrevealeda significantdifferenceamong
groups(F=17.59,df=4,39andp<0.0001),butthecomparisonof
thetotaldistancetraveledintheWTgroupsrevealedno
signifi-cantdifference(F=2.372,df=4,39andp=0.0712).Weappliedthe
samecomparisontotheDAT+/−groups.Thecomparisonoftotal
explorationtimerevealednodifference(F=1.805,df=4,35 and
p=0.153).Whenwecomparedthetotaldistancetraveled,a
signif-icantdifferencewasdetected(F=4.684,df=4,35andp=0.0045).
Finally,weappliedtheTwo-WayANOVAtocomparebothstrains.
We found a significant interaction regarding total exploration
(Strain:F (1.65)=11.27,p=0.0013; Treatment: F(4, 65)=8.398,
parisonof total distancetraveleddidnot revealany significant
interaction(Strain:F (1,65)=27.30,p<0.0001; Treatment:F(4,
65)=6.746,p<0.0001;Interaction:F(4,65)=1.236,p=0.304).
ThepresentstudyshowsthatDAD2antagonisthaloperidolwas
abletoboth decreaseandincrease memoryformation,
depend-ingonthemousestrain,andonthetimeofinjection.Specifically,
wereplicatedWTmicedata,inwhichhaloperidol0.3mg/Kgafter
explorationledtoanimpairmentinamemorytaskrelatedtoOR
[10,14].We alsoshowedthat mildhyperdopaminergicDAT+/−
micehaveabasalimpairmentinthisparameter.Thereafter,inDAT
+/−mice,wedemonstratedthathaloinjectionbefore(0.05mg/kg),
butnotafterexploration(0.05or0.3mg/kg),wasabletorecover
ORcapacitytosimilarlevelsoftheirWTcorrespondents.
Dopamine D1 and D2 receptors are linked toattention and
memoryconsolidationandtobothmesolimbicaland
mesocorti-caldopaminergicpathways[3,6].Rossatoetal.reportedthatD1
familyreceptormodulatesthelong-termmemorystorage
persis-tenceintheinhibitoryavoidancetask[7],aswellD1participatein
thespatialmemoryprocess[23].Similarly,thesystemicinjection
andintra-accumbensofD2antagonistimpairspatialmemory.Both
D1andD2suppressionresultsinimpairmenttorecognizechanges
intheenvironment[24].
TheuseofDAT-KOmiceasamodeltostudythebehaviors
asso-ciatedwithdopamineiswidelydescribed.DAT-KOmiceexhibit
hyper locomotion [18] and this phenotypecan be reversed by
haloperidolinjection[17,20].DAT-KOanimalsalsoexhibita
pref-erence fortheborders of anopenfield [20], and adecrease of
immobilitytimeintheforcedswimmingtask[17].When
main-tainedinisolation,DAT-KOmiceexhibitincreaseinreactivityand
aggressionwhensubmittedtosocialinteractions[2].DAT-KOmice
alsopresentdeficitsinodorrecognition[13]andimpairmentinthe
normalwake-sleepcycle[25].UsingDAT-KO,Moriceetal.showed
thattheseanimalshadimpairmentinthewater-mazeparadigm,
andlong-termdepression[9].Incontrast,onlyafewstudieshave
beenpublishedonDAT+/−mice,amildmodelof
hyperdopaminer-gia.Inthismodel,amilddisbalanceofdopamineresultsinseveral
cognitivechanges[17,19,20].DAT+/−miceexhibitlessimmobility
timeduringtheforcedswimmingtest[17],highertimespentinthe
centerofanopenfield[20],andmoretimespentintheopenarms
incomparisontoWTmiceinaplusmaze[19]orzeromaze[20].In
addition,theseheterozygousmiceshowedanincreaseinthe
explo-rationofobjectsplacedinanopenfield[17],andinthefrequency
ofobjectexploration[20].DAT+/−miceshowedimpairedpattern
completioninanenvironmentwithfewspatialcues,whichcould
bereversedbyhaloperidol[19].However,noimpairmentofORhas
beendescribed[19,20].
InthepresentworkwefoundbasalimpairmentinORinthe
DAT+/−mice(withoutpharmacologicalinterference),differently
frompreviousstudies[19,20],butourORprotocolwasquite
dif-ferentfromtheprotocolusedbyLietal.Weused50%lesstimeof
objectexplorationsessionsandtwicethenumberofnovelobjects
thantheprotocolofthatstudy[19].Theincreaseintheamountof
novelobjects,combinedwithlesstimetoexplorethem,likelyled
tothedifferencesbetweenthepreviouslypublishedresultsand
ourown.In thePogorolevprotocol,animalswere submittedto
7consecutivedaysofnovelexploration.Theanimalshad3daily
sessionstoexplorethesameobjects.Inthefourthdaythe
place-mentoftheobjectswaschanged,andinthesixthdayoneofthe
objectswasreplaced[20].Mostlikely,anymildimpairmentofOR
inDAT+/−miceshouldbereversedbyseveralconsecutivecontacts
withthesameobjects.Similarlytoourresults,patterncompletion
impairmentwasreversedbyhaloperidolinjectioninDAT+/−mice.
[19].
Secondly,regardingexplorationandoverallmobility,notethat
study,relatedtomemoryinDAT+/− orWTmice,areprobably
notinfluencedbyanalterationofthesetwovariables.Asshown
intheresultsabove,theWT0.3mg/KgI.A.E.groupexhibitedthe
samelevelsoftotalexplorationseeninthevehicleI.A.Egroup,but
theyexploreddifferentlythenewandfamiliarobjects(seeFig.2A).
Furthermore,weobservednodifferenceamonggroupscomparing
0.3mg/kgandtheirrespectivecontrolsinthemobilityparameter
(distancetraveled).Thesamebasicexplanationcanbeappliedto
theDAT+/−:thestatisticsindicatenodifferenceintotalexploration
timewhenwecompareDATwithvehicleandwithhalo0.05mg/kg
or0.5mg/kg,(andonlydifferencesbetweenI.A.EandB.E.
treat-ments,treatedinsequence)butthesegroupsexhibiteddifferential
explorationofthenewandfamiliarobjects(seeFig.2B).
Ausefulrationalein thiscase istoexplain theresultsasan
increaseinpotentiallystressfulstimulipresentedtotheDAT+/−
animals,sincemoreobjectswerepresentedandtheanimalshad
lesstimetobefamiliarizedwiththeenvironment.Thisincreasein
potentiallystressfulconditionsmaybesubtleforwildtypeanimals,
butnotforDAT+/−animalswithamilddopamineexcess.
Inagreementwiththishypothesis,ithasbeendemonstrated
thattheenhancedactivityregulationofDATreceptorinWTmice
occursasa synapticresponsetorestraintstress,and thiseffect
occurs even in haloperidol-treated animals [26]. Since DAT+/−
micehave less DATreceptors, theymight be more susceptible
tothis stress-associatedphenomenon.Despiteofthescarcityof
pharmacologicalandneuroanatomicaldatainDAT+/−mice,
DAT-KOlittermatemiceexhibita differentneurocircuitry[27]and a
non-dopaminergicacuteresponsetopsychostimulantdrugs[28].
FutureinvestigationoftheneurocircuitryofDAT+/−animalsshall
clarifywhataretheneuralcorrelatesofthedifferentialmodulation
ofitsrewardsystemimpliedbyourresults.
Interestingly,withregard tomotor parameters,weobserved
thatWTmicewithinjectionbeforeexplorationandDAT+/−mice
withB.E.injectionat0.05mg/Kgofhaloperidol,presented
locomo-tionalterationswhencomparedtobothcontrolandto0.3mg/kg
after exploration. The differences are more evident in DAT+/−
mice,whichpresented,antagonically,adecrease oftotal
explo-rationininjectionsB.E.aswellasanincreaseindistancetraveled
(0.05mg/kgB.E.).Thesefindings,togetherwithanincreaseinDAT
+/−(distancetraveled)at0.05mg/kgafterexposure,alsosuggest,
additionally,anincreasedsusceptibilityof motormodulationin
DAT-+/−animalsduetopreviousinjectionstress,andto
haloperi-dolactionatlowerdoses.
ObjectrecognitiondeficitsinDAT+/−were,infact,themain
findingsinthisstudy:thisfindingissupportedbyrelatedresearch.
Thereis involvement of D2 receptor activity in neuroplasticity
markersimpairmentassociated withORdeficits[10,14].A high
doseofhaloperidol(0.3mg/Kg)leadtoadecreaseinCAMKII,
ZIF-268and BDNF [10]after object exploration.In the samestudy
above,thehaloperidolinjectedanimalsthatpresentedimpairment
inORhadsignificantdecreaseintheREMsleepduration[10];they
alsorevealedastrongcorrelationbetweenREMsleepdecreaseand
ORimpairment[10].
Besidescorroboratingpreviousstudiesinthefield,datafrom
WTmicewereimportantforthecomparisonwithDAT+/−results
onmemoryimpairment.DAT+/−micedidnotreceivemajor
scien-tificattentionasdidtheirknockoutcounterparts,sincetheyonly
exhibitamildhyperdopaminergiaandexhibitlessphenotypic
dif-ferences[18].Nevertheless,inthepresentstudythesemiceshowed
aclearimpairmentinOR.Thesedataarerelevantsincethey
indi-catethatamildhyperdopaminergiacanbedetrimentalenoughto
causeattentionormemorydeficitsrelatedtonoveltyrecognition.
Itisimportanttonotethatthetwostrainsinvestigatedhere,
WTandDAT+/−,presentdifferentbasallevelsofdopamine[18].
Because of these different basal synaptic dopamine levels, we
expecteddifferentsensibilitytodopamineantagonistsinthesetwo
micestrains.AhighersensibilitywasobservedinDAT+/−miceata
lowhaloperidoldose(0.05mg/Kg,BEinjection),sincehaloperidol
revertedthenaturalORdeficitphenotype,andnotablyenhanced
object memoryformation in this strain. In contrast,in WT we
didnotdetectanydifferencebetweenhaloperidolandvehicleBE,
usingtheANOVAtest.In fact,inthiscasea trendwasrevealed
byStudent’sTtest(t=2.69,df=14andp=0.0176),indicatingthat
haloperidolBEata low0.05mg/kgdosecouldpossiblyenhance
objectrecognitioninWTmice.Weinterpretthesespecificstrain
variationsatlowhaloperidoldosesasdifferentsusceptibilitiestoa
minormodulationofdopaminereceptors.However,wheninjected
afterexploration,haloperidolatthesamelowdosedidnotincrease
ORinDAT+/−miceduringthetestsession.Together,bothresults
suggestthatthisimpairmentcouldmostprobablybeduetoDA
imbalance duringmemoryacquisitionin DAT+/− mice.
Conse-quently,sincetheacquisitionphaseseemstobedirectlyaffected,
nofurtherpharmacologicalinterventionaftermemoryacquisition
couldexertanyeffectonORinDAT+/−mice,asweobservedwith
haloperidolinjectionsafterobjectexploration.
Interestingly,thesamedrugthatcausedmemorydeficitsafter
explorationinWTmicealsocausedanincreaseinthisparameterin
DAT+/−mice,whenusedbeforeexplorationandatalowerdose.An
invertedU-shapeactivity,inwhichoptimalactivitylevelsenhance
performance, is proposedfor DAD1 receptor activation,and is
relatedtogoal-orientedtasks,memoryconsolidationand
atten-tion[29,30].Inthisapproachmodel,bothloworhighDAreceptor
activationreducestheperformanceintheseactivities,andmedium
optimalactivationproducesthebestperformances.Aninteresting
studyconductedinhumansindicatestheinvertedU-shaped,D-2
receptor-basedneuroplasticityisalsoobserved[31].
SinceweusedadrugthatismainlyaD2receptorantagonist,
theresultscanindicatethatD2activationlevelsmayalsoinduce
aninvertedU-shapeperformanceinmicerelatedtoOR.TheD2
receptoractionmaymodulateORbothatacquisitionand
consoli-dationphases.Weinferthatinthepresentstudy,bothhighandlow
D2DAreceptoractivationlevelsledtoobjectmemoryimpairment.
ThehighD2activationcouldberelativetoDAT+/−atbasal
perfor-mance,whichwasrevertedbyhaloperidolinpre-exploration(at
0.05mg\kg).ThelowactivationcasecouldbethatofWT,impaired
byhaloperidolinjectionafterexploration(at0.3mg\kg).
These resultsindicate that optimalD2 activitylevelscan be
pharmacologically induced,andcan potentiallybeconsidereda
therapeutictargetfordopamine-relateddysfunctions.Indeed,the
useofD2/D3familyantagonistshasbeendescribedforreducingthe
impairmentofmemorydeficitsinducedbyamnesiccompounds
inrodents[32].ThisisalsosupportedbythetrendforOR
facili-tationinWTinjectedwith0.05mg/kghaloperidol.Altogether,the
resultsindicatethatD2familyantagonistsinlowdosescanproduce
memoryenhancement.
In summary,we reportherean ORdeficit inDAT +/− mice.
Importantly,injectionoftheD2DAantagonistbeforeobject
explo-rationrevertedthisphenotype.Theseresultsarecomplementedby
anhaloperidol-induceddecreaseofORinWTmicewheninjected
afterthetrainingsession.ThisindicatesthatD2DAreceptorscan
modulateORtasksbyactingbothinattentionalprocessesduring
acquisition,andduringsubsequentmemoryconsolidation.More
studiescouldbeperformedexaminingotherphenotypic
charac-teristicsofDAT+/− mice,inordertoassociatethesebehavioral
deficitstomolecularandelectrophysiologicalparameters.
Further-more, additionalpharmacologicalstudies involvingDAagonists
andantagonistsfordifferentreceptorsareinordertobuildamore
comprehensiveunderstandingofD2regulationofattentionand
None.Theauthorsdeclarenocompetinginterests.
Acknowledgments
We thank Marc Caron and Miguel Nicolelis for making the
DAT+/− mice available; Valéria Arboes for animal care. We
thank to the listo f grants: Grant (1) FAPERN-PPP (Dr.
Lobão-Soares:2013–2014). Grant(2) CNPQ-Universal;Grantnumber:
484408/2013-5(Dr.Lobão-Soares).Grant(3)CNPqUniversal;Grant
number:481351/2011-6(Dr.Ribeiro).Grant(4)FINEPGrant
num-ber01.06.1092.00(Dr.Ribeiro).Grant(5)FAPERN/CNPqPronem.
Grant Number: 003/2011 (Dr. Ribeiro). Grant (6) FAPESP
Cen-ter for NeuromathematicsGrant number: #2013/07699-08(Dr.
Ribeiro).Grant(7)UFRN.Grantnumber00001/2010(Dr.Ribeiro).
(8)ConselhoNacionaldeDesenvolvimentoCientíficoeTecnológico
(CNPQ)Researchproductivity306604/2012-4.
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