The medial dorsal thalamic nucleus and the medial prefrontal cortex of the rat function together to support associative recognition and recency but not item recognition.

2013·0.64 MB·English

by  Cross, Laura

Research
The medial dorsal thalamic nucleus and the medial
prefrontal cortex of the rat function together
to support associative recognition and recency
but not item recognition
Laura Cross,1,3 Malcolm W. Brown,1 John P. Aggleton,2 and E. Clea Warburton1,4
1SchoolofPhysiologyandPharmacology,UniversityofBristol,Bristol,AvonBS81TD,UnitedKingdom;2SchoolofPsychology,
CardiffUniversity,CardiffCA103AT,UnitedKingdom
Inhumansrecognitionmemorydeficits,atypicalfeatureofdiencephalicamnesia,havebeententativelylinkedtomediodor-
salthalamicnucleus(MD)damage.AnimalstudieshaveoccasionallyinvestigatedtheroleoftheMDinsingle-itemrecogni-
tion,buthavenotsystematicallyanalyzeditsinvolvementinotherrecognitionmemoryprocesses.InExperiment1ratswith
bilateralexcitotoxiclesionsintheMDorthemedialprefrontalcortex(mPFC)weretestedintasksthatassessedsingle-item
recognition (novel object preference), associative recognition memory (object-in-place), and recency discrimination
(recencymemorytask).Experiment2examinedthefunctionalimportanceoftheinteractionsbetweentheMDandmPFC
using disconnection techniques. Unilateral excitotoxic lesions were placed in both the MD and the mPFC in either the
same(MD+mPFCIpsi)oroppositehemispheres(MD+mPFCContragroup).BilaterallesionsintheMDormPFCimpaired
object-in-placeandrecencymemorytasks,buthadnoeffectonnovelobjectpreference.InExperiment2theMD+mPFC
Contragroupwassignificantlyimpairedintheobject-in-placeandrecencymemorytaskscomparedwiththeMD+mPFC
Ipsigroup,butnovelobjectpreferencewasintact.Thus,connectionsbetweentheMDandmPFCarecriticalforrecognition
memorywhenthediscriminationsinvolveassociativeorrecencyinformation.However,therodentMDisnotnecessaryfor
single-itemrecognitionmemory.
Recognition memory, the ability to judge the prior occurrence mationconcerningpriorstimulusoccurrence(Fahyetal.1993;
ofastimulus,involvesmultipleprocessesandrequiresmultiple XiangandBrown1998,2004).
brainregions.Researchintohumanamnesiashowsthatthemedi- TheimportanceoftheratMDforsingle-itemrecognitionis,
al diencephalon is critical for normal recognition memory, al- however,farlessclear-cut(Mumbyetal.1993;Parkeretal.1997).
though neuropsychological studies have largely failed to link Thisuncertaintymayreflectdifferencesinthelearningdemands
thecontributionsofparticulardiencephalicsitestoaspectsofrec- ofthevariousstudies(Aggletonetal.2011).Thefirstgoalwas,
ognition(Aggletonetal.2011).Theprincipalreasonisthatthe therefore,toreexaminetheimportanceoftheratMDforitemrec-
pathology in human clinical cases almost invariably involves ognitionusingspontaneoustasksthatminimizeprocedurallearn-
multiplediencephalicnuclei. ing. The second goal was to extend the classes of recognition
Onediencephalicsitepotentiallylinkedtohumanrecogni- problemusedtoexaminetheratMD.Inadditiontosingle-item
tionmemoryisthemediodorsalthalamicnucleus(MD)(Victor recognition(testedusingthenovelobjectrecognitiontask[Fig.
et al. 1971; Victor 1987; Parkin et al. 1994; Isaac et al. 1998). 1A]), the present study examined object spatial memory (Fig.
Thislinkismoreevidentinanimallesionexperimentswithmon- 1B)associativerecognition(distinguishingbetweenmultiplefa-
keys,whichhaveconsistentlyindicatedarolefortheMDinitem miliaritemsintheiroriginalornovelconfigurationassessedusing
recognition (Aggleton and Mishkin 1983b; Zola-Morgan and anobject-in-placetask[Fig.1C])andrecencydiscriminations(as-
Squire 1985; Parker et al. 1997). From such findings, Aggleton sessedusingarecencyrecognitiontask[Fig.1D]).Thechoiceof
andBrown(1999)proposedamodelofitemrecognitionmemory taskwaspromptedbythefindingthatmPFClesionsinratsspare
thatincorporatestheMDwithinalargerneuralnetworkcentered single-itemrecognition,butdisruptassociativerecognitionand
on the perirhinal cortex and involving the prefrontal cortex recencyjudgments(Barkeretal.2007).Furthermore,inviewof
(AggletonandBrown1999).Thismodelissupportedbytheana- the dense, reciprocal interconnections between the MD and
tomical connections from the perirhinal cortex to the MD mPFC (Krettek and Price 1977; Groenewegen 1988; Ray and
(Russchenetal.1987;Saundersetal.2005)andtheobservation Price 1992, 1993; Taber et al. 2004) it might be predicted that
that the primate perirhinal cortex, medial prefrontal cortex theMDfunctionsinconcertwiththeprefrontalcortextoenable
(mPFC),andMDcontainneuronalpopulationsthatsignalinfor- associative recognition and recency judgments selectively. This
predictionissupportedindirectlybysomeoftheparalleleffects
3Present address: Department of Biochemistry & Physiology, of MD lesions and prefrontal lesions on memory in humans
UniversityofSurrey,Guildford,SurreyGU27TE,UnitedKingdom (Milner and Petrides 1984; Sandson et al. 1991; Van der Werf
4Correspondingauthor etal.2000),monkeys(ParkerandGaffan1998),andrats(Hunt
[email protected]
andAggleton1998;DiasandAggleton2000),butremainstobe
Articleisonlineathttp://www.learnmem.org/cgi/doi/10.1101/lm.028266.112.
FreelyavailableonlinethroughtheLearning&MemoryOpenAccessoption. testeddirectly.Thus,thethirdgoalofthepresentstudywasto
20:41–50#2013ColdSpringHarborLaboratoryPress 41 Learning&Memory
ISSN1549-5485/13;www.learnmem.org
MDthalamusandrecognitionmemory
A Novel object preference malshad someunilateral damagetothe central lateralnucleus
andpartialdamagetothecentralmedialnucleus.Forsixofthean-
Sample
imals,minordamage(,10%)wasincurredunilaterallyintheven-
tralanteriorlateralnucleusandanterodorsalnucleus.Fouranimals
had extensive damage to the paraventricular nucleus and five
hadbilateraldamagetotheposteriorparataenialnucleus.Three
animalsshowedminorunilateral(,10%)damagetothedentate
gyrus(DG)andoneanimalsustainedminordamagetotheDGbi-
B Object Location laterally.Importantly,theMDwastheonlycommonsiteoftissue
Sample Test lossacrossthecases.Diagrammaticrepresentationsofthecases
withthelargestandsmallestlesionsareshowninFigure2B.
Experiment2:IpsilateralMD+mPFClesions
TwoanimalswereexcludedastherewasnodamageintheMD.In
theremaining10casesalltheanimalshadnear-completeunilat-
C Object-in-place erallesionsintheMDandmPFC.ThuswithinthemPFCtherewas
Sample Test extensivedamagetotheprelimbicandtheinfralimbiccortices,al-
thoughoneanimalshowedposterioranddeeplayersparingofthe
PL.Inonecasetheprimarymotorcortexshowedminordamage
(,10%)andinanothertherewasevidenceofveryminordamage
tothecorpuscallosum.Inhalfthecasestherewasminordam-
ageinthecingulatecortex,althoughthisdamagewasconfined
totheborderofthePL.Alltheanimalshadextensiveunilateral
D Recency Memory damageintheMD.Oneanimalhadsomeminorsparinginthelat-
eralMD,whiletwohadadditional lateraldamageaffecting the
Sample 1 Sample 2 Test dorsomedialpartofthelateraldorsalthalamus;inonecasethis
damageextendedintotheventrolateralpartofthelateraldorsal
thalamusandthemedialrostralpartofthelateralposteriorthal-
amus(largestlesioninFigure3).Sevenanimalsshowedasmall
amount of unilateral damage in the DG and anterior dorsal
Figure 1. Diagram of the four object recognition memory tasks. (A)
Novel object recognition task. (B) Object location task. (C) Object-
in-placetask.(D)Recencytask.
usedisconnectionproceduresthatexaminetheinteractionofMD
andmPFC.
Results
Histology
Experiment1:BilateralmPFClesions
AlltheanimalshadbilateraldamageinthemPFC.Allbuttwoan-
imalshadcompletelesionsofboththeprelimbiccortex(PL)and
theinfralimbic(IL)cortex.Thetwootheranimalshadsparingin
thedeeplayers.Alltheanimalsalsohadminorcingulatecortex
damagearoundtheborderwiththePL.Threeanimalshadasmall
amountofdamagetothemedialorbitalcortexandventralorbital
cortex.Twoanimalshadlesionsthatextendedtoaminordegree
intothesecondarymotorcortex,andoneanimalhadsustained
damageinthedorsalpeduncularcortex.Diagrammaticrepresen-
tationsofthecaseswiththelargestandsmallestlesionsareshown
inFigure2A.
Experiment1:BilateralMDlesions
Figure 2. Diagrammatic reconstructions showing the cases with the
Fouranimalswereexcluded,threeduetoincompletedamageto largest(gray)andsmallest(black)lesionsinthemPFC(A)andMD(B)
theMD,particularlyinonehemisphere,andoneastherewasaddi- lesiongroups.Thenumberscorrespondtotheapproximatepositionrel-
ative to bregma (Swanson 1998). (AC) Anterior cingulate cortex; (AV)
tionalbilateraldamagetoanteriorthalamicnucleiwithsomespar-
anteroventral thalamic nucleus; (AMD) anteromedial thalamic nucleus;
ing ofthe MD.Inthe remainingeightcasestherewasbilateral
(ILA)infralimbiccortex;(LD)lateraldorsalthalamicnucleus;(LP)lateral
damagetotheMD;however,allshowedaminoramount(,10%) posteriorthalamicnucleus; (MD) mediodorsal thalamic nucleus; (ORB)
ofsparinginthelateralextremesofonehemisphere.Alltheani- orbitalfrontalcortex;(PL)prelimbiccortex;(PT)parataenialnucleus.
www.learnmem.org 42 Learning&Memory
MDthalamusandrecognitionmemory
Novelobjectrecognitiontask
Recognition during the test phase. Figure 5, A and B, shows the
performance of all three groups (MD, mPFC, and sham) in the
testphasefollowingretentiondelaysofeither5min(Fig.5A)or
3h (Fig. 5B). Two-way ANOVA with the lesion and delay as
factors showed that therewas no significant main effect of the
lesion (F ¼0.5, P.0.1), no significant effect of the delay
(2,57)
(F ¼0.9, P.0.1), and no significant lesion-by-delay
(1,57)
interaction(F ¼1.8,P.0.1).Furtheranalysesrevealedthat
(2,57)
allgroupsshowedsignificantdiscriminationbetweennoveland
familiarobjectsacrossbothdelays(5-minsham:t ¼4.2,P,
(11)
0.001;MD:t ¼6.3,P,0.001;mPFC:t ¼4.1,P,0.01;3-h
(7) (11)
sham: t ¼5.7, P,0.001; MD: t ¼3.5, P,0.001; mPFC:
(11) (7)
t ¼5.8,P,0.001).
(11)
ThesedataindicatethatbilaterallesionsinneithertheMD
northemPFCaffectobjectrecognitionmemory.
Exploration in sample and test phases. Analysis of the total amount of
exploration completed in the sample phase or test phase
revealed no effect of the lesion in either phase at either delay
(all F’s,2.0). The data are presented in Table 1. In addition,
therewasnodifferenceintheamountofexplorationcompleted
in the test phase following a 5-min or 3-h delay F ¼3.64,
(1,28)
P.0.05.
Objectlocationtask
Figure 3. Diagrammatic reconstructions showing the cases with the
Recognitionduringthetestphase.Theperformanceofallthegroupsinthe
largest (gray) and smallest (black) lesions in the mPFC-MD Ipsi lesion
objectlocationtaskisshowninFigure5,CandD.Atthe5-min
group.Thenumberscorrespondtotheapproximatepositionrelativeto
bregma(Swanson1998).(AC)anteriorcingulatecortex;(AV)anteroven-
tralthalamicnucleus;(AMD)anteromedialthalamicnucleus;(ILA)infra-
limbiccortex;(LD)lateraldorsalthalamicnucleus;(LP)lateralposterior
thalamic nucleus; (MD) mediodorsal thalamic nucleus; (ORB) orbital
frontalcortex;(PL)prelimbiccortex;(PT)parataenialnucleus.
thalamusatthesiteoftheMDinjection.Diagrammaticrepresen-
tationsofthecaseswiththelargestandsmallestlesionsareshown
inFigure3.
ContralateralMD+mPFClesions
Twoanimalswereexcluded.Oneanimalwasexcluded asthere
wasnodamageintheMDandonebecauseofextradamagein
the mammillothalamic tract region. In the remaining 10 cases
all the animals had unilateral lesions in the MD and mPFC in
thecontralateralhemispheres.Alltheanimalshadlargeunilateral
lesionsintheprelimbicandinfralimbiccortices,althoughonean-
imalshowedsparinginthe deeplayersof thePL. Twoanimals
showedminimal(,10%)damagetothemotorcortex.Inallcases,
the cingulate cortex was damaged by about 50% in the region
closest tothe PL.Inall casestherewasa largeunilateral lesion
intheMD.Theanimalsshowedasmalldegreeofsparingofeither
theanteriororposteriorpartsoftheMD,butneverofboth.Sixan-
imalsalsoshowedadegreeofunilateraldamagetotheDGandthe
anteriordorsalthalamicnucleusaroundtheareaoftheinjection
site.Sixanimalsalsohadminor(,10%)damagetotheintrame-
dullarylamina.Diagrammaticrepresentationsofthecaseswith
thelargestandsmallestlesionsareshowninFigure4.
Figure 4. Diagrammatic reconstructions showing the cases with the
Behavior: Experiment 1 largest (gray) and smallest (black) lesions in the mPFC–MD Contra
Afterhistologicalanalysis,thefinalgroupnumberswereasfol- lesiongroup.Thenumberscorrespondtotheapproximatepositionrela-
lows:sham,n¼12;mPFC,n¼12;MD,n¼8.Asstated,thosean- tivetobregma(Swanson1998).(AC)anteriorcingulatecortex;(AV)ante-
roventralthalamicnucleus;(AMD)anteromedialthalamicnucleus;(ILA)
imalsthat didnotcompletethe necessarylevels ofexploration
infralimbiccortex;(LD)lateraldorsalthalamicnucleus;(LP)lateralposte-
wereexcludedfromthefinalanalyses,asindicatedbyreducedde- riorthalamicnucleus;(MD)mediodorsalthalamicnucleus;(ORB)orbital
greesoffreedominthequotedstatisticaltests. frontalcortex;(PL)prelimbiccortex;(PT)parataenialnucleus.
www.learnmem.org 43 Learning&Memory
MDthalamusandrecognitionmemory
withthelesiongroupanddelayasfactorsrevealedasignificant
maineffectofthelesion(F ¼19.3,P,0.001),butnoeffect
(2,57)
ofthedelay(F ¼3.1, P.0.1)andnosignificantlesion-by-
(1,57)
delay interaction (F ¼1.5, P.0.1). Post hoc analyses
(2,57)
showedthattheperformancesoftheMDandmPFCgroupswere
significantlyworsethanthatoftheshamgroupfollowingbotha
5-min delay (MD, P,0.001; mPFC, P,0.05) and a 3-h delay
(MD, P,0.01; mPFC, P,0.05). The sham control group
displayed significant discrimination between the stationary
objects and the objects that had exchanged position following
both retention delays (5min: t ¼7.1, P,0.001; 3h: t ¼
(11) (11)
3.1,P,0.01),butthemPFCgroupdidnot(5min:mPFC:t ¼
(10)
1.3,P.0.1;3h:mPFC:t ¼0.7,P.0.1).TheMDgroupalso
(11)
didnotdiscriminateatthe5-mindelay(MD:t ¼1.3,P.0.1),
(7)
but at the 3-h delaythis group showed a significant preference
for the stationary objects compared with the objects that had
exchanged position (MD: t ¼ 22.7, P,0.05); hence the MD
(7)
lesionproducedonlypartialimpairmentatthe3-hdelay.
ThesedatasuggestthatboththeMDandmPFCarenecessary
fornormalobject-in-placeassociativememory.
Figure5. PerformanceofthebilateralMDandmPFClesiongroupsin
object recognitionandobject locationtaskswhentestedwitha5-min
or3-hdelay.(A)Objectrecognitionperformance ata5-min delay.(B) Exploration in sample and test phases. Analysis of the total amount of
Objectrecognitionperformanceata3-hdelay.(C)Objectlocationperfor-
explorationconductedinthesamplephaseortestphaserevealed
manceata5-mindelay.(D)Objectlocationperformanceata3-hdelay.
Shownforeachgroupisthemean(+SEM)discriminationratio(DR). noeffectofthelesionfollowingeitherdelay(allF’s,1.5).The
explorationdataareshowninTable1.
delay experiment one animal from each lesion group was Recencymemorytask
excludedduetoinsufficientlevelsofobjectexploration,andat
Recognitionduringthetestphase.Theperformancesofthesham,bilateral
the3-hdelaytwoMDanimalswereexcluded.ANOVAwiththe
MD,andmPFClesiongroupsintherecencymemorytaskareshown
lesion group and delay as factors revealed no significant main
in Figure 6C, and a statistical comparison of the mean
effect of the lesion (F ¼0.1, P.0.1) and of the delay
(2,51) discriminationratiosagainstzeroperformanceshowedthatonly
(F ¼3.4, P.0.1), and no significant lesion-by-delay
(1,51) theshamcontrolgroupsignificantlydiscriminatedbetweenthe
interaction(F ¼0.04,P.0.1).Furtheranalysisshowedthat
(2,51) ‘old’and‘recent’objects(sham:t ¼2.5,P,0.05),whilethe
all the lesion groups were able to discriminate between novel (11)
MD and mPFC groups did not (MD: t ¼0.5, P.0.1; mPFC:
andfamiliarlocationsfollowingadelayof5min(sham:t ¼ (7)
(10) t ¼0.2, P.0.1). Thus, while the ANOVA did not reveal any
5.2, P,0.001; MD: t ¼2.5, P,0.05; mPFC: t ¼5.5, P, (11)
(6) (10) significantgroupeffect(F ¼1.0,P.0.1),bothlesiongroups
0.001) or 3h (sham: t ¼3.9, P,0.01; MD: t ¼3.9, P, (2,29)
(10) (5) failedto showan ability to use recency information to effect a
0.01;mPFC:t ¼6.7,P,0.001).
(10) recognitionmemoryjudgment.
ThesedataindicatethatthebilaterallesionsintheMDand
mPFC had no effect on the performance of the object location
task. Exploration in sample and test phases. Analysis of the total amount of
exploration conducted in the sample phases or test phase
Exploration in sample and test phases. Analysis of the total amount of revealed no effect of the lesion (all F’s,1.5). The data are
exploration completed in the sample phase or test phase showninTable2.
revealed no effect of the lesion (all F’s,1.5). The data are
presentedinTable1. Behavior:Experiment 2
Following histological analysis the final numbers of animals in
Object-in-placetask the MD+mPFC Ipsi and MD+mPFC Contra lesion groups
Recognitionduringthetestphase.Figure6,AandB,showstheperformance were10ineachgroup.Asstated,animalsthatdidnotcomplete
ofthebilateralMDandmPFCgroupsintheobject-in-placetask thenecessarylevelsofexplorationwereexcludedfromthefinal
followinga5-min(Fig.6A)or3-hdelay(Fig.6B).OnemPFCwas analyses,asindicatedbyreduceddegreesoffreedominthequoted
excludedfromtheanalysisatthe5-mindelay.Two-wayANOVA statisticaltests.
Table1. Meanexplorationtimes+SEMduringthesampleandtestphaseoftheobjectrecognition,objectlocation,andobject-in-place
tasksat5-minor3-hdelay
Objectrecognition Objectlocation Object-in-place
Sample(s) Test(s) Sample(s) Test(s) Sample(s) Test(s)
5min 3h 5min 3h 5min 3h 5min 3h 5min 3h 5min 3h
Sham 30.8+2.6 30.2+2.7 27.1+2.9 22.0+2.0 24.6+1.8 28.3+2.1 16.2+1.3 15.3+1.6 46.0+2.8 47.4+4.9 20.7+2.5 33.7+3.4
MD 32.0+2.1 29.7+3.4 24.7+4.1 17.8+2.0 27.3+2.7 29.1+2.3 22.3+1.6 16.8+2.6 44.7+3.7 48.0+4.5 20.9+4.3 33.1+3.8
mPFC 29.4+1.7 29.9+2.0 21.8+1.5 22.7+1.4 24.9+1.6 27.5+2.8 19.8+2.5 15.1+1.2 47.9+4.0 52.7+3.6 21.9+2.4 27.6+1.8
www.learnmem.org 44 Learning&Memory
MDthalamusandrecognitionmemory
object-in-place task. One animal from the MD+mPFC group
was excluded from the analyses due to insufficient levels of
exploration.One-wayANOVAconfirmedasignificantmaineffect
of lesion type (F ¼8.6, P,0.01). Subsequent ana-lyses
(1,18)
confirmed that only the MD+mPFC Ipsi lesion group showed
sig-nificantdiscriminationbetweentherearrangedandunmoved
objects in the test phase (MD+mPFC Ipsi: t ¼4.6, P,0.01;
(8)
MD+mPFC Contra: t ¼1.1, P.0.1). Thus contralateral
(9)
unilateral lesions in the MD and mPFC resulted in significant
impairmentsinobject-in-placeperformance.
Exploration in sample and test phases. Analysis of the total amount of
exploration conducted in the sample or test phase of the
object-in-placetaskrevealednosignificantdifferences(allF’s,
1.0).ThedataareshowninTable3.
Recencytask
Figure6. PerformanceofbilateralMDandmPFClesiongroupsinthe Recognition during the test phase. Figure 8B shows the performance of
object-in-place and temporal order memory tasks. (A) Object-in-place both lesion groups in the recency task. One-way ANOVA
performance following a 5-min delay. (B) Object-in-place performance showedasignificantmaineffectoflesiontype(F ¼4.8,P,
following a 3-h delay. (C) Temporal order memory performance. (∗) 0.05). Additional analyses revealed that only th(1e,1M8)D+mPFC
P,0.05, (∗∗) P,0.01, (∗∗∗) P,0.001. Shown for each group is the
Ipsi lesion group showed a significantly greater preference for
mean(+SEM)discriminationratio(DR).
the object previously seen in the first sample phase (MD+
mPFC Ipsi: t ¼3.6, P,0.01; MD+mPFC Contra: t ¼0.7,
(9) (9)
P.0.1). Contralateral unilateral lesions in the MD and mPFC
Novelobjectrecognitiontask
significantly impaired performance in the recency task
Recognition during the test phase. Figure 7, A and B, shows the
comparedwithanimalswithipsilaterallesions.
performance of the MD+mPFC Ipsi and MD+mPFC Contra
lesiongroupsintheobjectrecognitiontaskandshowsthatthe
performance of these two groups did not differ from one Exploration in sample and test phases. Analysis of the total amounts of
another. ANOVA with the lesion group and delay as factors explorationconductedintherecencytaskrevealednoeffectof
showed no significant main effect of lesion type (F ¼1.3, thelesionineitherthesamplephaseorthetestphase(allF’s,
(1,36)
P.0.1), of delay (F ¼0.3, P.0.1), and no lesion 1.0).ThedataareshowninTable4.
(1,36)
type-by-delay interaction (F ¼0.08, P.0.1). Additional
(1,36)
analyses revealed that both groups significantly discriminated Discussion
the novel from the familiar objects following delays of 5min
(MD+mPFCContra:t(9)¼4.1,P,0.01;MD+mPFCIpsi:t(9)¼ Thepresentstudyisthefirsttoexaminesystematicallythemne-
4.7,P,0.01),and3h(MD+mPFCContra:t(9)¼3.7,P,0.01; monic importance of the rodent MD in recognition memory
MD+mPFCIpsi:t(9)¼4.1,P,0.001). anditrevealedfourimportantfindings.First,theMDwasshown
nottobenecessaryforsingle-itemrecognition.Second,theMD
Exploration in sample and test phases. Analysis of the total amount of wasfoundtobecriticalforobject-in-placeassociativerecognition
explorationconductedinthesamplephaseortestphaseofthe andrecencyrecognition.Third,thisstudyclearlyshowedthatthe
novelobjectrecognitiontaskrevealednosignificantdifferences effectsofMDdamageonalltherecognitionmemorytaskstested
atanydelay(allF’s,2.0).ThedataareshowninTable3. in the current study using these measurements were indistin-
guishable from the effects of damage to the mPFC. The fourth
findingwasthatdisconnectionoftheMDandmPFCsignificantly
Objectlocationtask
impairedobject-in-placeandrecencyrecognitionmemory.Thus
Recognitionduringthetestphase. TheperformancesoftheMD+mPFC togethertheseresultsdemonstratethattheMDmaybeincorpo-
Ipsi and MD+mPFC Contra lesion groups in the object ratedwithinaneuralcircuitforrecognitionmemorywhenjudg-
locationtaskfollowinga3-hdelayareshowninFigure7C;the ments of prior occurrence involve a spatial/associational or
two lesion groups did not differ from one another. Analysis recencycomponent.
revealednoeffectoflesiontype(F(1,18)¼0.7,P.0.1)andboth MDdamagehasbeenshowntoimpairdelayednonmatch-
groups showed significant discrimination between novel and ing-to-sample(DNMS)tasksinprimates(AggletonandMishkin
familiarlocations(MD+mPFCIpsi:t(9)¼6.8,P,0.001;MD+ 1983a; Zola-Morgan and Squire 1985); however, the results for
mPFCContra:t(9)¼3.0,P,0.01). ratsappeartobelessconsistent(Aggletonetal.2011).Testsusing
Exploration in sample and test phases. Analyses of the total amount of
exploration in the sample or test phases of the object location Table2. Meanexplorationtimes+SEMduringsamplephases
andtestphaseofthetemporalordertask
task revealed no significant differences (all F’s,1.0). The data
areshowninTable3. Firstsample Secondsample Test
exploration exploration exploration
Object-in-placetask Sham 35.3+2.9 36.4+2.9 23.5+2.2
MD 32.6+2.4 34.9+3.1 20.8+2.7
Recognitionduringthetestphase.Figure8Ashowstheperformancesofthe mPFC 38.8+3.0 40.3+4.1 24.6+2.3
MD+mPFC Ipsi and MD+mPFC Contra lesion groups in the
www.learnmem.org 45 Learning&Memory
MDthalamusandrecognitionmemory
portanceofthemPFCincertainrecognitionmemoryprocesses,it
wasimportanttocomparedirectlytheroleoftheMDwiththatof
themPFCindifferentformsofrecognitionmemoryotherthan
single-itemobjectrecognition, i.e.,objectlocationrecognition,
object-in-place, and recency memory. Bilateral lesions in both
theMDandmPFCsignificantlyimpairedobject-in-placeandre-
cencymemoryperformance,buthadnoapparenteffectonob-
jectlocationmemory.Theobjectlocationtaskassessestherat’s
abilitytorecognizethatanobjectisinapreviouslyunoccupied
place, but does not require identification of which object goes
where, and therefore does not require associative recognition.
Whileregionsimportantforspatiallearning,e.g.,thehippocam-
pusandfornix,arerequiredforthistask(EnnaceurandAggleton
1997; Barkerand Warburton2011b), the intactperformance of
theratswithMDandwithmPFClesionsisconsistentwithagrow-
ingnumberofstudiesthatreportalackofspatialmemorydeficit
inratsfollowingMD(Neaveetal.1993;HuntandAggleton1998;
Mitchell and Dalrymple-Alford 2005; Dolleman-van der Weel
Figure7. PerformanceoftheMD+mPFCIpsiandMD+mPFCContra etal.2009)ormPFClesions(Barkeretal.2007).
lesiongroupsinobjectrecognitionandobjectlocationtasksfollowinga
Three animals in the mPFC group had a small amount of
5-mindelayor3-hdelay.(A)Objectrecognitionperformancefollowing
damage in the medial and ventral orbital cortex. Inspection of
a 5-min delay. (B) Object recognition performance following a 3-h
delay.(C)Objectlocationperformancefollowinga5-mindelay.Shown their individual discrimination ratios revealed that the pattern
foreachgroupisthemean(+SEM)discriminationratio(DR). ofperformanceoftheseanimalswasnodifferenttothatofthe
groupasawhole.Thustheseanimalsshowednodeficitsintheob-
jectrecognitionorobjectlocationtasks,but,consistentwiththe
reinforcedDNMShavefoundthatMDlesionsinratsimpairthe remainderofthemPFClesiongroup,theyshowedpoorerdiscrim-
learning of task contingencies (Hunt and Aggleton 1991; ination in the object-in-place and temporal order tasks. While
Mumbyetal.1993),butrecognitionperformanceoverrelatively withasmallgroup(n¼3)onecannotdrawfirmconclusions,it
shortdelaysappearsnormal,followingtaskmastery.Thesingle doesnotappearfromthepatternofperformanceintheseanimals
previousstudy to usespontaneous objectrecognition(Mitchell thatdamagetotheorbitalfrontalcortexcontributestothemne-
andDalrymple-Alford2005)reportednoeffectofMDlesionsafter monicdeficitsseen.
theone(2-h)retentiondelayexamined. ThedemonstrationthatbilaterallesionsinMDhavenoef-
Inlightofthecontradictoryevidenceconcerningtheimpor- fectonanobjectlocationtaskallowsustoexcludethepossibility
tanceoftheMDinsingle-itemrecognition,thefindingsfromthe thattheobserveddeficitsinobject-in-placeandrecencymemory
presentstudy,i.e.,thatbilateralMDlesionshavenoeffectonnov- maybetheresultoftheslightdamagetotheDGthatoccurredin
elobjectrecognitionafterashortorlongretentiondelayareofim- someoftheanimals(unilateraldamageinthreecases,bilateral
portanceastheysuggestthatthedeficitspreviouslyseeninrats damage in one case). Further, as MD lesions had no effect on
whenusingrewardedDNMS(HuntandAggleton1991;Mumby single-item recognition or object location, the object-in-place
etal.1993)mayreflecttheeffectsofMDdamageonaspectsof andrecencyrecognitionmemorydeficitsarenottheresultofim-
taskperformanceotherthanrecognition.Onepossiblecandidate pairedobjectidentificationorobjectfamiliaritydiscrimination.
isrewardassociationlearning,aprocesscriticalforDNMStaskac- Astheobject-in-placetaskrequiresthesubjecttoencodefourdif-
quisition,andindeedpreviousstudiesshowthatrewardlearning ferentobjectsitmaybeconsideredtobeamoredifficulttaskthan
iscompromisedbyMDdamage(Corbitetal. 2003). Numerous theobjectrecognitiontask.Thusinthepresentstudythesample
studieshaveshowntheperirhinalcortextobecriticalforobject timeintheobject-in-placetaskisgreaterthanthatintheobject
recognition memory (Ennaceur et al. 1996; Ennaceur and recognitiontasktoenablethesubjectstospendlongerexploring
Aggleton1997;Busseyetal.2000;Barkeretal.2007)andinlight andencodingtheobjects.Ifweconsidertheperformanceofthe
ofthepresentnullresultitisnoteworthythatanatomicalstudies shamgroupinbothtasksitisclearthatthereisnodifferencein
haveshownlittleevidenceofdirectconnectionsbetweentheMD the levels of discrimination, indicating that the sham group
andperirhinalcortexintherat(RayandPrice1992).Thisarrange- doesnotfindtheobject-in-placetaskmoredifficult.Theabsence
mentappearsdifferenttothatintheratbrainwheredirectperirhi- ofanydifferencesinperformancesupportstheconclusionthat
nal projections to the MD have been traced (Aggleton and theimpairmentsinobject-in-placerecognitionmemoryfollow-
Mishkin1983a;Russchenetal.1987). ing lesions in the MD and the mPFC reflect the importance of
There are strong anatomical connections between the MD thesebrainregionsselectivelyinassociativerecognitionmemory,
andthemPFCand,asourpreviousstudiesdemonstratedtheim- andnotthedifferencesintaskdifficulty.
Table3. Meanexplorationtimes+SEMduringsamplephasesandtestphaseoftheobjectrecognition,objectlocation,and
object-in-placetasks
Objectrecognition Objectlocation Object-in-place
Sample(s) Test(s) Sample(s) Test(s) Sample(s) Test(s)
5min 3h 5min 3h
MD+mPFCContra 39.0+1.4 39.2+1.7 34.3+3.4 39.6+4.8 44.7+3.9 29.8+3.8 62.1+5.2 28.0+2.8
MD+mPFCIpsi 37.5+1.4 36.0+1.7 32.0+3.4 41.6+4.8 46.2+3.9 31.4+3.8 59.6+5.5 32.5+2.9
www.learnmem.org 46 Learning&Memory
MDthalamusandrecognitionmemory
object-in-placeandrecencytaskswassignificantlyabovechance.
Therefore,damagetobothregionswithinthesamehemisphere
appearedtospareperformance,afindingconsistentwithnumer-
ousotherstudiesthatemployadisconnectiontechniquetoestab-
lishregionalinterdependencies(Florescoetal.1997;Barkeretal.
2007;BarkerandWarburton2011b).Theseresultsthereforesug-
gestthattheconjointnecessityfortheMDandmPFCforsuccess-
ful recognition memory is due to the fact that these regions
operatewithinanintegratedneuralcircuit.
Asthepresentstudyusedpermanentlesionstoestablishthe
importanceofMDandmPFCinrecognitionmemory,wecannot
Figure8. PerformanceoftheMD+mPFCIpsiandMD+mPFCContra identifywhetherthecontributionsoftheMDandmPFCareatthe
lesion groups in object-in-place and temporal order memory tasks. (A) sameoratdifferentstagesofrecognitionmemory.Wehaveprevi-
Object-in-place task performance. (B) Temporal order memory perfor-
mance task. (∗) P,0.05, (∗∗) P,0.01. Shown for each group is the ouslyshownthatthemPFCiscriticalforboththeacquisitionand
mean(+SEM)discriminationratio(DR). retrievalofobject-in-placememoryandthatitsrolemaybeinthe
integrationofobjectandspatialinformationorfortheretrievalof
object–spatialinformationthroughanengagementwiththehip-
pocampus (Barker et al. 2007; Barker and Warburton 2011b).
One previousstudyalso foundthatlesionsin the MDim- ConcerningtheMD,onepossibilityisthatthisregionactsasa
paired a spontaneous recency memory task (Mitchell and critical relay between the medial temporal lobe and the mPFC,
Laiacona 1998), however the present study has extended upon though such an explanation goes against present views of the
thisresultbysystematicallyexploringtheroleoftheMDinrecog- roleofthalamicnuclei(Sherman2007;Tsanovetal.2011)and
nitionmemory,anddirectlycomparingthisrolewiththatofthe leavesuncertainwhythemanydirectconnectionslinkingtempo-
mPFCfordifferenttypesofinformation(singleitem,spatialloca- ralandfrontalregionsarenotsufficient.Ithasalsobeensuggested
tion,object-in-place,andrecency)usingidenticalapparatusand thatablationoftheMDinprimatesproduceshypoactivityinthe
stimulustypesandsimilarretentiondelays.Further,theorderin mPFC(ParkerandGaffan1998)and,therefore,thedeficitsinrats
whichtheanimalsweretestedinthebatteryoftasksinterleaved withMDlesionsmightbeascribedtosuchafrontaldysfunction.
thenovelobjectrecognition,objectlocation,andobject-in-place Inthepresentstudy,histologicalanalysisofthemPFCinanimals
tasks,andthusitisunlikelythattaskordercanaccountfortheob- withabilateralMDlesionshowednoindicationofcellloss,yet
serveddeficits.Thisstudythereforedemonstratesforthefirsttime previous studies have shown disruptive effects in regions distal
thattheMDisselectivelyrequiredforbothassociativeandrecen- toalesionsite,evenwhensuchregionsappearnormalbystan-
cyrecognitionmemoryinrats. dardhistologicalmeasures(VanGroenetal.1993;Gardenetal.
Inlightoftheseresults,theAggletonandBrown(1999)mod- 2009), and thus further study of potential diaschesis in the
elofrecognitionmemory,whichplacedtheMDsolelywithina mPFCisrequired.
neural circuit for item familiarity through an interaction with Itispossiblethatduringrecognitionmemoryperformance
theperirhinalcortex,requiresadegreeofreevaluation.Inarecent theMDiscriticalfornon-mnemoniccognitiveprocessessuchas
reviewAggletonetal.(2011)proposedanewmodelofthalamic behavioral flexibility (Block et al. 2007). If this were the case
contributionstorecognitionmemory,themulti-effectmultinu- then during associative or recency recognition memory tasks,
clei(MEMN)model.ThismodelassertsthattheMDcancontrib- theMD–mPFCconnectionmightbenecessarytodirectongoing
utetobothfamiliarityandrecollectiveprocesseseitherdirectly behaviortoward,forexample,thenovelobject–placeconfigura-
viaaninteractionwiththeprefrontalcortexorindirectlyasare- tion. Alternatively, the MD may be required for acquisition of
sultofcorticaldiaschesis(Aggletonetal.2011).Thismodelissup- newinformationorforretrieval.Inprimatesthemagnocellular
portedbythecurrentfindingsregardingassociativerecognition, MD has previously been shown to be necessary for acquisition
alongwithmorerecentclinicalresults(Pergolaetal.2012)that butnotforretrievalofpreoperativelylearntobject-in-placedis-
pointtocontributionsfromtheparvicellularMDforrecollective criminations(MitchellandGaffan2008).However,assuchtasks
aspectsofrecognition.Atthesametime,theredoesappeartobe are quite distinct from those used here, an examination of the
apotentialincreaseintheimportanceoftheMDforfamiliarity- separate roles of the rat MD in acquisition or retrieval is now
basedobjectrecognitioninmonkeysversusrats,potentiallyre- warranted.
flecting species differences in afferents to the MD (Russchen Thepresentstudyaddstoagrowingbodyofevidencethatfor
etal.1987)andthegreaterinfluenceofprefrontalareasforobject associativeandrecencyrecognitionmemory,informationneces-
recognitionmemoryinprimates(Aggletonetal.2011). saryforsuccessfulperformanceisheldinmultiplebrainregions,
TotestthefirstofthepossibilitiesraisedbytheMEMNmod- includingtheperirhinalcortex,hippocampus,andMD.Further,
el,i.e.,thattheinvolvementoftheMDinrecognitionmemoryis itappearsthateachoftheseregionsisrequiredtointeractwith
through an interaction with the prefrontal cortex, we explored themPFC(Hannessonetal.2004;Barkeretal.2007;Barkerand
the functional importance of an interaction between these re- Warburton2011b).Takentogethertheseresultsshowthatduring
gionsusingadisconnectionanalysis.Inanimalswithcombined
unilateral MD and mPFC lesions in opposite hemispheres
(MD+mPFCContragroup),performanceintheobject-in-place Table4. Meanexplorationtimes+SEMduringsamplephases
and recency tasks was significantly impaired compared with andtestphaseofthetemporalordermemorytask
that of animals with combined ipsilateral lesions (MD+mPFC
Ipsigroup).HeretheMD+mPFCIpsigroupservedascontrols, Firstsample Secondsample Test
exploration exploration exploration
as these animals sustained the same amount of damage, albeit
inthesamehemisphere,asanimalsintheMD+mPFCContra MD+mPFC 53.0+2.2 36.7+3.0 31.3+2.1
Contra
group.Whileitisimpossibletoruleoutentirelythepossibility
MD+mPFC 50.8+4.5 39.3+2.9 32.8+3.0
thattheipsilaterallesionsproducedsomesmalleffectonperfor-
Ipsi
mance,thediscriminationoftheMD+mPFCIpsigroupinthe
www.learnmem.org 47 Learning&Memory
MDthalamusandrecognitionmemory
complex recognition memory tasks in which the processing of line. The scalp was further anesthetized using lidocaine, cut,
itemsrequiresacontextual(placeortime)tag,theMDappears andretracted.Aftercraniotomy,excitotoxiclesionstothetarget
tohaveakeyrolewithinthisneuralnetwork,Further,theresults regionsweremadeusingN-methyl-D-aspartate(NMDA)dissolved
inphosphatebuffer(PB),injectedthrougha1-mLHamiltonsy-
indicatethatthenatureofto-be-remembereddetermineswhich
ringeatthefollowingcoordinatesrelativetobregma:mPFC,ante-
brainregionsbecomeengaged.
rior–posterior (AP) +2.7mm, mediolateral (ML) +0.7mm,
Recognitionmemoryperformancehasbeencarefullyexam- dorsoventral (DV) 24.5mm and 22.2mm, and AP +4.0mm,
inedinalimitednumberofclinicalcasesinwhichtheMDisdam- ML +0.7mm, DV 23.5mm and 22.0; MD, AP +3.7mm, ML
aged,andresultsindicateaninvolvementofthisthalamicnucleus +0.7mm,DV +4.6.ThecoordinatesforthemPFC lesionwere
in associative recognition (Aggleton et al. 2011; Pergola et al. calculated relative to bregma, while the coordinates for MD
2012).Ofparticularinterestisastudyofapatientwithaninfarct were calculated relative to ear-bar zero. For the mPFC lesion
involvingtheinferiorcapsulargenu,whichdisruptedtheconnec- each NMDA injection (0.09 M, 0.28 mL) was made gradually
tionbetweenthethalamus(includingtheMD)andtheprefrontal over 4min and the needle left in situ for a further 4min. For
theMDlesion,NMDA(0.12M,0.36mL)wasinjectedintoeach
cortex (Schnider et al. 1996). On testing, nonassociative recog-
sitegraduallyover5minandtheneedleleftinsituforafurther
nitionmemorywasfoundtobenormalwhileassociativeandtem-
5min.
poralorderrecognitionmemorywerebothsignificantlyimpaired
Oncesurgerywascompletedtheskinwassuturedandanan-
(Schnideretal.1996).Thepreciselocusofdamageresponsiblefor
tibiotic powder (Acramide) applied. All the animals received at
thememorydeficitsinthisstudyisunclearasinevitablythelesion least5mLofglucosesalinesubcutaneouslyandsystemicanalge-
involved other regions, including the anterior thalamic nuclei siaintramuscularly(0.05mLVetergesic)beforetheendofsurgery.
andthalamicconnectionswiththeamygdala.However,thedefi- Hypromelloseeyedropsweregivenatthebeginningandendof
citsinbothtemporalorderandcontextualrecognitionmemory surgery.Theanimalswereallowedtorecoverforatleast10dbe-
accordwellwiththeresultspresentedhere. forehabituationtothebehavioralarenacommenced.
Insummary,resultsfromthisstudydemonstratethattheMD
iscriticalforobject-in-placeandrecencymemoryperformance.
Histology
Furthermore,fortheserecognitionmemoryjudgments,aninter-
actionbetweentheMDandthemPFCisequallynecessaryasthe Oncompletionofthebehavioraltaskstheanimalsweresacrificed
bytranscardialperfusionwithPBfollowedby4%paraformalde-
integrity of the structures themselves. Our previous studies re-
hyde(PFA).Thebrainswerepost-fixedin4%PFAforaminimum
vealedaneuralcircuitinvolvingthehippocampus,perirhinalcor-
of24hfollowedby48hin30%sucroseinPB.Coronalsections
tex,andmPFCforbothassociativerecognitionandrecognition
(40mm)werecutonacryostatandthesectionsmounteddirectly
memory discriminations based on recency information (Barker ontogelatin-coatedslides,stainedusingcresylviolet,andcover-
etal.2007;BarkerandWarburton2011a).Theevidencenowsug- slippedusingDPXmountingmedium.Slideswerethenviewed
geststhattheMDisafurthercriticalcomponentofthiscircuit. underalightmicroscopeandtheextentoflesionsrecorded.
ToassesstheextentofthemPFClesionallsectionsalongthe
APaxisofeachratbrainbetween+4.7mmand+1.5mm(relative
Materials and Methods
tobregma)wereselectedandcomparedwiththoseinaratbrain
atlas(Swanson1998).ToassesstheextentoftheMDlesion,all
Subjects
sections along the AP axis of each rat brainbetween 21.5 and
Experiment1used36maleratsandExperiment2used24male 24.20(relativetobregma)wereselectedandcomparedwiththe
rats(DAstrain;BantinandKingman,Hull,UK),weighing220– ratbrainatlas(Swanson1998).
290gpriortotesting.Alltheanimalswerehousedingroupsof
four, under a 12-h light/dark cycle (light phase, 18.00 to 6.00)
withadlibitumaccesstofoodandwater.Behavioraltestingwas Apparatus
conductedduringthedarkphaseofthiscycle.Allanimalproce-
Behavioral testing took place in a wooden open-topped arena
dureswereperformedinaccordancewithUKAnimalsScientific
(50-cm high, 95-cm wide, and 100-cm long), with gray walls
ProceduresAct(1986)andassociatedguidelines.Alleffortswere
andexternalblackcurtainstoaheightof1.5mtorestrictdistal
madetominimizeanysufferingandthenumberofanimalsused.
cues.Thefloorwascoveredinsawdust,whichservedtoconceal
the identical green Duplo mats (15cm×15cm), onto which
Surgery the objects were attached to prevent them being displaced. In
alltheexperimentsthesawdustwascleanedbetweeneachani-
Experiment1 mal.Explorationwasmonitoredusinganoverheadcameraand
recordedontovideotape.Theamountofobjectexplorationwas
Rats received bilateral excitotoxic lesions in the mPFC or MD.
determined using in-house counting software on a computer
Controlanimalsreceivedshamsurgery;halfthecontrolanimals
withintheroomwhich,inresponsetoakeypressfromtheexper-
received sham mPFC lesions, and the other half received sham
imenter(onekeyfortheleftobject,onekeyfortherightobject),
MDsurgeries.Fortheshamsurgeriestheanimalsunderwentthe
recordedtheamountofexplorationcompletedwithin20-sectime
samesurgicalproceduresasthetwolesiongroupswiththeexcep-
bins.ObjectswereconstructedfromDuploandvariedincolorand
tionthatnoexcitotoxinwasinjectedoncetheneedlehadbeen sizefrom9×8×7cmto25×15×10cm.Newobjectswereused
lowered(n¼12forallgroups).
foreveryexperiment.
Experiment2
Behavioral procedures
AlltheratsreceivedcombinedunilaterallesionsintheMDand
the mPFC. In one group these lesions were placed in the same
hemisphere (MD+mPFC Ipsi group), while in the other group Habituation
these lesions were placed in contralateral hemispheres (MD+ Priortotesting,alltheanimalswerehandledforaweekandhabit-
mPFCContragroup)(n¼12forbothgroups).Thesideofdamage uatedtotheemptyarenaforfourdays.Forthefirsttwoofthese
(i.e.,leftorrighthemisphere),wascounterbalancedwithineach dayseachcageofanimals(fourrats)washabituatedtothearena
group. togetherfor15min.Forthenexttwosessions,eachratwashabit-
Beforesurgeryalltheratswereanesthetized(isoflurane:in- uated individually for 5min in the emptyarena. Animals were
duction 4%; maintenance 2%–4%) and placed in a stereotaxic alsohabituatedindividuallyfora5-minperiodpriortothetasks
frame with the incisor bar set at +5mm above the interaural in which the appearance of the arena had been altered from
www.learnmem.org 48 Learning&Memory
MDthalamusandrecognitionmemory
that of the standard arena (i.e., for the object location and across the rats. A representation of the procedure is shown in
object-in-placetasks). Figure1D.
Behavioral measuresand statistical analyses
Familiaritydiscrimination(anovelobjectrecognitiontask)
Explorationoftheobjects,definedastheanimalorientatingits
Inthesamplephase,theanimalswereintroducedtoanarenacon-
nosetowardtheobjectatadistanceof ,1cmfromtheobject,
tainingtwoidenticalcopiesofoneobject.Animalswereallowed
was measured with the experimenter blind to the lesion status
toexplorethetwocopiesfreelyuntiltheyhadcompletedatotal
oftheanimal.Anyotherbehavior,suchaslookingaroundwhile
of40secofobjectexploration,orhadspent4mininthearena.
sittingonorrestingagainsttheobjectorusingtheobjecttorear
Animalswereremovedfromthearenaandplacedintheirhome
whilelookingaroundthearena,wasnotconsideredasexplora-
cagewithinthetestingroom,foradelayperiod(5minor3h)dur-
tion. Animals were excluded from the analysis on the basis of
ingwhichtimethearenaandobjectswerecleaned.Followingthe
lowexplorationlevels(,15secinthesamplephase).Adiscrimi-
delay,animalswereplacedbackintothearena,whichnowcon-
nationratiowasgeneratedforeachanimal.Thediscriminationra-
tainedanothercopyoftheobjectseeninthesamplephaseand
tiowascalculatedasthetimespentbyeachanimalexploringthe
anovelobject.Theanimalwasallowedtoexploretheobjectsfree-
novelobjectminusthatexploringthefamiliarobject,dividedby
lyfor3minandwasthenreturnedtoitshomecage.Theobject
thetotaltimespentexploringbothobjects.Inthecaseoftheob-
actingasthesampleobjectandthepositionoftheobjectswere
jectrecognitiontask,thenovelobjectwasanobjectthathadnev-
counterbalancedacrosstheanimals.Arepresentationofthepro-
er been previously encountered, while for the object-in-place
cedureisshowninFigure1A.
and object location tasks the novel stimulus was considered to
betheobjectthathadaltereditspositioninthearenafromthat
inthesamplephase.Inthecaseofthetemporalordertask,the
Spatialdiscrimination(objectlocationtask)
‘novel’stimulus wasthe object encountered in the firstsample
Theobjectlocationtaskwasconductedinasimilarwaytotheob- phase. Group comparisons used ANOVA followed by post-hoc
jectrecognitiontask;however,twoofthecurtainssurrounding Newman–Keulstests.Additionalanalysisexaminedwhetherin-
thearenawereremovedtoallowtherattoviewextra-mazecues dividual groups had discriminated between the objects using a
andoneofthearenawallswaspaintedadifferentcolortoprovide one-sample t-test (two-tailed) where significance was assumed
an intra-maze cue. During the sample phase, animals were al- whenP,0.05.
lowedtoexploretwoidenticalcopiesofanobjectfor4minbefore Therecognitionmemoryexperimentswereconductedinthe
beingremovedfromthearenaforthedelayperiod(5-minor3-h followingorder:Objectrecognitionwitha3-hdelay,objectloca-
delay in Experiment 1 and 3-h delay only in Experiment 2). tionwitha5-mindelay,object-in-placewitha5-mindelay,object
Followingthedelay,theanimalswereplacedbackintothearena, locationwitha3-hdelay,recency,object-in-placewitha3-hde-
whichnowcontainedtworeplicasoftheobjectsfromthesample lay, and object recognition with a 5-min delay. The animals
phase.Oneobjectwasreplacedinthelocationpreviouslyoccu- were run in this order to minimize the effects of task orderon
pied bya sample-phase object,but the otherobject was placed performance.
inanewlocationwithinthearena.Objectexplorationwasrecord-
edfor3min.Thepositionofthemovedobjectwascounterbal-
ancedacrossrats.Arepresentationoftheprocedureisshownin Acknowledgments
Figure1B.
This research was funded by a project grant from the
Biotechnology and Biological Sciences Research Council (grant
no. BB100310X1) and the Wellcome Trust. We thank Jane
Associativerecognitionmemory(object-in-placetask)
RobbinsforassistancewiththehistologyandLucyWilliamsfor
Object-in-place testing was conducted in an arena identical to conductinganumberofpreliminarystudies.
thatusedfortheobjectlocationtask.Inthistask,fourdifferent
objectswereplacedinthefourcornersofthearenaandduring
thesamplephasetheanimalswereallowedtoexplorethesefour References
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