Edinburgh Research Explorer
Everyday memory: towards a translationally effective method ofmodeling the encoding, forgetting and enhancement of memory
Citation for published version:Nonaka, M, Fitzpatrick, R, Lapira, J, Wheeler, D, Spooner, PA, Corcoles-Parada, M, Munoz-Lopez, M, Tully,T, Peters, M & Morris, RGM 2017, 'Everyday memory: towards a translationally effective method ofmodeling the encoding, forgetting and enhancement of memory', European Journal of Neuroscience.https://doi.org/10.1111/ejn.13637
Digital Object Identifier (DOI):10.1111/ejn.13637
Link:Link to publication record in Edinburgh Research Explorer
Document Version:Peer reviewed version
Published In:European Journal of Neuroscience
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Download date: 04. Apr. 2020
1
Dr 47 – RGMM, MN - FINAL
ResearchReport/TechnicalSpotlight/NewMethod
Everydaymemory:towardsatranslationallyeffectivemethodof
modelingtheencoding,forgettingandenhancementofmemory.
MioNonaka1,RichardFitzpatrick1,JenniferLapira3,DamianWheeler3,PatrickA.
Spooner1,MartaCorcoles-Parada1,MónicaMuñoz-López2,TimTully3,
MarcoPeters3andRichardG.M.Morris1,4
1CentreforCognitiveandNeuralSystems,
EdinburghNeuroscience,TheUniversityofEdinburgh,1GeorgeSquare,Edinburgh,EH89JZ,Scotland(UK)
2HumanAnatomyLaboratory,
FacultyofMedicineandRegionalCentreforBiomedicalResearch,UniversityofCastilla-La-Mancha,
14AvenidaAlmansa,02071Albacete,Spain
3DartNeuroScienceLLC12278ScrippsSummitDrive,SanDiego,CA92131,USA
4InstituteforNeuroscience,
CSIC-ULM,Alicante,03550Spain
CorrespondingAuthor:[email protected]
KeyWords:Learning,event-arena,spacedtraining,
RNAseq,gene-expression
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ABSTRACT
Thetestingofcognitiveenhancerscouldbenefitfromthedevelopmentof
novelbehaviouraltasksthatdisplaybettertranslationalrelevancefordaily
memory,andpermittheexaminationofpotentialtargetsinawithin-subjects
mannerwithlessvariability.Wehereoutlineanoptimizedspatial‘everyday
memory’task.Wecalibrateitsystematicallybyinterrogatingcertainwell-
establisheddeterminantsofmemory,andconsideritspotentialforrevealing
novelfeaturesofencoding-relatedgeneactivation.Ratsweretrainedinanevent
arenainwhichfoodwashiddeninsandwellsinadifferentlocationeveryday.
Theyfoundthefoodduringaninitialmemory-encodingtrialandwerethen
requiredtorememberthelocationin6-alternativechoice-orprobetrialsat
varioustime-pointslater.Trainingcontinueddailyoveraperiodof4months,
realisingastablehighlevelofperformanceandcharacterisedbydelay-
dependentforgettingover24h.Spacedbutnotmassedaccesstomultiple
rewardsenhancedthepersistenceofmemory,asdidpost-encoding
administrationofthePDE4inhibitorRolipram.QuantitativePCRandthen
genomewide-analysisofgene-expressionledtoanewobservation-stronger
gene-activationinhippocampusandretrosplenialcortexfollowingspacedthan
massedtraining.Inasubsidiarystudy,aseparategroupofanimalsreplicated
aspectsofthistrainingprofile,goingontoshowenhancedmemorywhentraining
wassubjecttopost-encodingenvironmentalnovelty.Distinctivefeaturesofthis
protocolincludeitspotentialvalidityasamodelofmemoryencodingused
routinelybyhumansubjectseveryday,andthepossibilityofmultiplewithin-
subjectcomparisonstospeedupassaysofnovelcompounds.
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INTRODUCTION
Transientmemoriesareformedaswegoaboutourdailylifeathomeorwork.
Theygenerallyoccuragainstthebackgroundofrelevantpriorknowledge.Manyof
these‘everyday’memories,beingoflittleconsequence,areforgottenrapidlyas
Ebbinghausfirstreportedwithexperimentsonmemorytrainingfornonsensesyllables
(Ebbinghaus1885).However,asubsetofeverydaymemoriesmayberetained
overnightorforlonger(Wixted2004).Whatisresponsiblefortheselectivityofdaily
memory?Mightbetterunderstandingoftherelevantneuralmechanismsbehelpfulfor
thedevelopmentofeffectivecognitiveenhancers?Thismethodsreport/technical
spotlightarticlehighlightsanoptimizedprotocolformonitoringeverydaymemoryin
animals.Itsdevelopmentwas,inpart,inspiredbythe“RivermeadEverydayMemory
Task”usedinclinicalpracticeandresearch(Wilson,Cockburnetal.1991).
Memoryofsuccessiveitemsovertimereflectsvariationsintheeffectivenessof
encodingand/orretentionofmemory.Synergisticorcompetitiveeffectshavelongbeen
studied,suchastheclassicalphenomenaofretro-orproactiveinterference(Mullerand
Pilzecker1900,McGeoch1932,Underwood1969).Neurobiologicalstudieshave
identifiedchangesinmemoryretentioncausedbyongoingeventsatthetimeof
encoding(suchaschangesinattentionormetaplasticity:(AbrahamandBear1996,
Kentros,Hargreavesetal.1998),byeventsshortlybeforeorafterencodingthattrigger
plasticityprotein-synthesis(synaptictaggingandcapture:(FreyandMorris1997,
RedondoandMorris2011),andbyneocorticalmechanismsengagedforlongtime
periodsafterencoding(suchassystemsconsolidation:(Squire1992,McGaugh2000,
DudaiandMorris2001).Alterationsintheeffectivenessofmemoryretrievalcanalso
occurinassociationwithdifferentialaccesstoappropriateretrievalcues(Tulving1983,
SpearandRiccio1994).Tounderstandtheneuraldynamicsofeverydaymemory
processinginmoredetail,weneedbetter,theoreticallyguidedanimalmodels.Wenow
reportanewandpotentiallyusefulmodel.
Twokeyprincipleshaveguidedourapproach.Thefirstisthat,justlikehumans
facingdailymemorychallenges,theanimalsshouldnotbe‘experimentallynaïve’.A
strikinglyhighproportionofbehaviouralneurosciencestudiesusenaïveanimals,many
forgoodreasonbutmanyoutofhabit.Memoryinexperimentallynaïveanimalsis
frequentlytestedintaskssuchnovelobjectrecognition,conditionedtasteaversion,or
variousparadigmsoffearconditioning.Inthisstudy,wedeliberatelyallowmemory
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processingtooccuragainstabackgroundofexperimentallycontrolledpriorexperience
thatpermitsrepeatednovelencodingandretrievalacrossdays.Thisbackgroundlikely
affectstheefficiencyofencodingorconsolidation(orboth).Asecondprinciplewasto
developaprotocolinwhichwhatisrememberedfromonemomenttothenextis
usuallybutnotalwaysforgotten.Thisisnotonlymore‘realistic’ofeverydaylife,where
weforgetagreatdeal,butalsomotivatedbyaspecifictheoreticalmodelofmemory
formationbasedonthetime-courseofactivity-dependentsynapticplasticity(Morris
2006).Whilevariouswidelyused‘everyday’taskscouldbeincludedinsuchaprotocol,
includingrecognitionmemory,ourstartingpointwasspatialmemory–analogousto
the“wheredidIleavemyglasses?”kindofmemory(asexaminedintheRivermead
Test).Trainingtookplaceeveryday,overweeksandmonthsinarepetitivemanner,
withonlyoccasionaltestsofthememorabilityofaspecificitem.
The‘delayedmatching-to-place’protocolinthewatermaze(SteeleandMorris
1999,daSilva,Bastetal.2014)isanexampleofaneverydaymemorytaskasdefined,
butanappetitivelymotivatedalternativeismorerealisticthanataskinvolvingescape
fromwater.Weusedan‘eventarena’inwhichratsaretrainedtodigforfoodin
sandwellswhoselocationvariedfromdaytoday.Eachday,formemoryencoding,they
explorethearenatofindoneortwosamplesandwells(ofwhichonlyonesandwellwas
rewarded),bothlocatedinavirtualarrayofupto43locationsacrosssessions.For
memoryretrieval,atanintervalof60-90minlater,arewardedchoicetrialwastypically
givenwith6sandwellsofwhichonlythecorrectonefromthesampletrialwas
rewarded.Win-staytrainingcontinuedlikethisovermanydaysandweeksasthe
animalsfirstlearnedthetaskandthenperformeditwell–learninganeweachdayand
rapidlyforgettingwherethefoodhadbeenovertheprecedingdays.Apartfromdaily
choice-trials,memorywasalsotestedoccasionallyusingananalogoustechniquetothe
watermaze-non-rewardedprobetestsatvariousdelaysupto24h.Inthese,thetime
spentdiggingateachof6availablesandwells,noneofwhichcontainedaccessiblefood
duringthetest,wascarefullymeasured.
Suchaprotocolinvolvesnewspatialmemoryencodingeveryday–wherethe
‘event’islearningwheretodigeachdayandthenrapidlyforgettingwherethefoodhad
beenovertheprecedingdays(Bast,daSilvaetal.2005).Withmodestrewardonthe
sampletrial,delay-dependentforgettingresultsinthememoryofthedailylocationof
foodbeingatchancelevelswithin24h.Thepresentprotocolhastheimportant
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refinementthatthesampleprotocolhastwosandwellsratherthanjustone.Pilotwork
revealedthatthisreducedvariabilitybecausetheanimalslearnnottokeepdiggingat
oneSandwell,buttoswitchbetweensandwellsasanoptimumstrategytosecure
rewardduringbothmemoryencodingandretrieval.Aspectsoftheencodingprotocol
werethensystematicallyvariedinwaysthatservedtocalibratethenewtask(singlevs.
multiplerewards,thetemporalspacingofmultiplerewards)toidentifywhetherthese
wouldaffectmemoryretention.Toexplorethefuturepotentialofthenewtechnique,
RT-qPCRandRNA-Seqanalyseswereconductedtoexaminetraininginducedgene-
induction(ShengandGreenberg1990,LanahanandWorley1998).Assessmentofthe
effectofaphosphodiesterase4inhibitor,theputativecognitiveenhancerRolipram
(Rutten,Prickaertsetal.2006)furtherservedtovalidatethebehaviouralprotocol.A
separatecohortofanimalswastrainedinasubsidiarystudytoestablishareplication
andtoexploretheimpactuponretentionofpost-encodingnoveltythatisknownto
driveimmediate-earlyandplasticity-proteinsynthesis(Guzowski,McNaughtonetal.
1999,Guzowski,McNaughtonetal.2001,Wang,Redondoetal.2010,Takeuchi,
Duszkiewiczetal.2016).
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METHODS
Subjects
Themainstudyused24adultmaleListerHoodedratsweighing200g-250gon
arrival.Theywereapproximately2monthsoldandtrainingtookplaceoveraperiodof
4months;allbutoneratcompletedthetesting(dataisreportedonn=23).Theywere
grouphoused(3-4/cage;12-hlight-darkcycle;alltraininginthelightphase).After
habituationtotheanimalfacilitywithfreefeedingandregularhandling,theywerefood
restrictedtomaintainbodyweightat90%ofthefreefeedingweightthroughoutthe
trainingandtestingperiod.Ifananimal’sweightdroppedbelow85%,itwas
individuallyfedtogain>85%bodyweight.Thesubsidiarystudyused11adultmale
ListerHoodedrats(250gonarrival)maintainedidentically.Allanimalexperiments
wereperformedinaccordancewiththeregulationsandguidelinesoftheUKHome
Officeregulations(PPLnumber,60/4566).
Apparatus–theeventarena
Allexperimentswereconductedusingan‘eventarena’(1.6mx1.6m)whose
floorsurfaceconsistedof49squarePlexiglaspanels(20x20cm).Someofthesepanels
hadholesof6cmdiameterintowhichsandwellscouldbeplaced(Fig.1A,Fig.S1A).The
floorpanelswerecleanedregularlyandreplacedindifferentlocationstorandomizeany
residualolfactorybias.Therewereprominent2Dand3Dextra-arenacues(Fig.S1B),
andtwointramazelandmarks(square-towerinrow4,column2;golf-balltowerinrow
4,column6).Thearenahadtransparentside-walls(30cmhigh),andfourstart-boxesat
thecenteroftheNorth(N),S,EandWwallsmadeofblackPlexiglas(transparentlid,
carpetedwithsawdust,availablewatercup,andautomateddoors,andthusdarker
insidethanthemainarena).Aceilingmountedvideocamerawasusedtomonitor
animalmovementsandchoices,andthisconnectedtoaDVDdriveandaLabView
event-recordingsystem.
InsertFigure1abouthere
Plexiglassandwells(6cmdiameter,4cmdepth),thatcontainedhiddenfood
pellets,wereplacedinthefloorpanelswithholes(Fig.1B,Fig.S1C).Tomaskthesmell
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ofthereward,thesandwellswerefilledwithbirdsandmixedwithGaramMasala(P&B
(Foods)Ltd.,150g/5kgsandinitially,with3g/5kgaddedeverysession(toreplenish
themaskingeffect,andthesandlostduringcleaning).Eachsandwellhadaspherical
plasticbowlatthebottominwhichrewardpellets(5x0.5g)wereplacedtobe
accessible(‘rewardedsandwell’).Thisplasticbowlalsomadeitpossibleforanequal
numberofrewardpelletstobeplacedunderneath,andtherebyinaccessible(‘non-
rewardedsandwell’).Theplasticbowlshadholesandsowereporoustoodors,
ensuringthattherewarededandnon-rewarededsandwells,notonlycontainedthe
samenumberofrewardpelletsatapproximatelythesamedepthinthesandbutwould
alsosmellthesame(Fig.1B,Fig.S1C).Thesandwellwasdesignedwithanextrapanelat
thebottomtoenableeasychangingofthebottompellets,withfreshpelletsusedin
everysession.Extensiverandomizingandcounterbalancingwasconductedtominimize
anyolfactoryartifacts;thisincludedensuringthatthesandwellsusedinthesampletrial
werenotusedforthechoicetrialofthesamesession;wipingthearenafloorwithan
alcoholimpregnatedtowelbetweeneachanimaltrial,andbeforechoiceandprobe
trials.
Behaviourtraining,sampletrialsandchoicetrials
Therewasaseriesofdistinctphasesoftraining:(1)habituation,(2)trainingto
dig,(3)maintrainingsessionswithsampleandchoicetrials,andinterspersedtests
involving(4)within-subjectmanipulationofdiverseparametersthatmightaffectthe
strengthorpersistenceofmemory(e.g.spacingofrewardavailabilityinasampletrial)
withassociatedmemoryprobetests.
Habituation(session-8):Theanimals,at90%offree-feedingbodyweight,were
broughttothearenaandplacedinthestart-boxfor30s,andthenallowedtoexplorein
thearenafor10min,beforebeingplacedbackinthestart-boxfor30s,andthengivena
rewardpelletinthehomecage(Fig.1C).Trainingtodig(sessions-7to-1):Theanimals
weregivenonepelletinthestart-box,thenallowedtoexploretolookforthereward
pellets(BioServ,SupremeMini-Treat0.5gm)atthesinglesandwellplacedinarandom
locationofthearena,andtaughttobringthepellet,oneatatime,backtothestart-box
ofthedaytoconsume3-5pellets(Fig.1C).Pelletswereinitiallyvisibleonthesand,and
thenburiedlowerdaybyday,encouragingtheanimalstodigdeeptofindthepelletsat
thebottomofthebowl.Bytheendofhabituationsessions,alltheratshadlearnedtodig
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withconjointexperienceofthesandwellsinthehomecage.Theyalsostartedtolearn
thewin-staystrategyofreturningtoarewardedsandwellthatwasexplicitlytrained
duringthesubsequentsessions.
Trainingsessionswithsampleandchoicetrials(sessions1-81):Trainingthen
commenced,andrepeatedacrossdays,5-7daysperweek,forseveralmonths.A
trainingsessionconsistedofasampletrial(ST=memoryencodingtrial)and,60-90min
later,achoicetrial(CT=memoryretrievalchoicetrial)(Fig.1D).OnST,theratswere
presentedinthearenawithonerewardedsandwell(sessions1-18)ortwosandwellsof
whichonlyonecontainedaccessiblereward(session18onward).Theratswerefirst
givenafoodpellet(0.5g)ofaspecificflavorinthestart-box.Thedoorwasthenopened
permittingaccesstothearenawheretheratcouldfindanddiguptherewardpelletthat
itthenimmediately,orwithgentleencouragementforthefirstfewtrials,tookbackto
thestart-boxtoeat(Whishaw,Kolbetal.1983).Thereafter,between1and5pellets
wereavailableduringanST,asdescribedinlaterprotocols.OnCTs,theratswere
presentedwith6sandwellsinthearena,withthecorrectsandwellbeinginthesame
locationastherewardedsandwelloftheearlierST(thus,thecorrectsandwelliscalled
the‘cued’sandwellinCTandPTalthoughthisisamemorycue;thereisnoexplicit
sensorycuemarkingit)(MovieS1).
Eachfloorpanelwitha6-cmholeforsandwellplacementcouldbeplacedinany
slotinthearena,theonlyconstraintsbeingthatthelocationsoftheintramazecues
(Row4,Columns2and5)andthosedirectlyinfrontofeachofthe4possiblestart-box
locationswereneverused(i.e.43possibleoutof49).Thesandwelllocations(map)
changedacrosssessionstoexcludeproceduralsearchingstrategiessuchasright/left,
close/far,specificquadrants,centre/corner,ordistancefromtheintramazecues.The
rewardedandnon-rewardedlocationswerecounterbalancedacrossanimalswithina
day.Twelvedifferentflavorsofrewardpelletwerealsoused,tohelpdiscriminability
andsopotentiallytoenhancememory(theseflavorswerecurry,blueberry,bacon,
nutmeg,anise,maple,garlic,ginger,cinnamon,mustard,coffee,andpeach).Flavors
wererepeatedonlyafter12sessions.
Behaviourwascarefullymonitoredthroughoutforerrorsandcorrect-digsfrom
whichaperformanceindex(PI)andLatencywerecalculated.OnCTs,theexperimenter
notedtheidentityandnumberofsandwellsatwhichananimaldug(errors)before
diggingatthecuedsandwell(correctdig),andthetimebeforediggingatthecued
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sandwell.Thechancelevelforerrorsis2.5,butweaimedinthis6-alternativeforced-
trialprocedureforastablelevelofperformanceataround1error/trial.ThePIwas
calculatedas100*(5-errors/5)%,forwhichchanceis50%.Thelatencyto‘first-dig’at
thecorrectsandwellintheCTdroppedgraduallytoastablelevelof<20s(Fig.S1D),
exceptingonthesinglecontrolsession(S60).(Seeresultssectionfordetails.)Thiswasa
sessiontocheckforolfactoryartifactsbydeletingthesampletrialandrunningonlythe
choice-trial.Itwasexpectedthatperformancewouldbeslowandatchance.
Probetrialsessions
Oncestableperformancewasreached,occasionalProbeTrial(PT)sessionswere
interleavedwithfurtherS+CTtraining–thesebeingassociatedwithvariationsin
protocolfortheSTonawithin-subjectbasis(seebelow).Thesesessionsconsistedofan
STandthen,afteramemoryinterval,thePTitself.All6sandwellshadinaccessiblefood
pellets(NonrewardedsandwellsinFig.1D).Theanimalscoulddigatoneormoreof
thesandwells,butrewardwasinaccessibleateverylocation.Effectivememorywas
preferentialdiggingatthepreviouslyrewardedsamplelocation(cuedlocation).The
ratsweregivenonepellet(0.5g)inthestart-box,andthenallowed60secinthearena
(MovieS2).Diggingtimewasdefinedasdirectcontactwiththesandwiththeforepaws;
incontrast,sniffing,anosepoke,oragentlefoot-printassociatedwithonlyavisitwere
notconsideredas‘digging.’Thediggingtimescoring(0.1sresolution)wasperformed
inblindmannerbythesingleexperimenter(MN,mainstudy;MC,subsidiarystudy),and
checkedwithfurtheroff-lineblindscoringbyanindependentobserver(RF,seeFig.1F
foroneexampleofexperimenter/observercross-correlation).After60sec,thedoorof
thestart-boxwasclosedandtheratsreturnedtoitbyhand.Toreduce‘extinction’,the
experimenterthenplaced3accessiblepelletsatthebottomofthesandwellatthe
correctlocationinthearena,openedthestart-boxdoor,andtheratwasagainallowed
tofindandretrieveall3pellets.Allprobetestswereinterleavedwithtwoormore
regulartrainingsessions(STsfollowedbyCTs)(Fig.1D).
Memoryparametersexaminedbehaviourallyandbiochemically
OncetheanimalshadreachedstableasymptoticperformanceondailySTandCT
sessions,aseriesofPTswereconductedprecededbydistinctmanipulationsonorafter
thedailysampletrials(STs).
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First,timedelays:weexaminedmemoryduration–schedulingthePTina
counterbalancedmanner24min,2.4h,and24hafteranSTinwhichtheanimalswere
rewardedwithasingle0.5gfoodpellet.Thisandfuturetestsalsoservedtocheckon
theaccuracyofmemoryscoringduringthePTs.
Second,massedvs.spaced:wecomparedtheclassicparameterofmassedvs.
spacedaccesstorewardonST,with3pelletsavailableoneachST.Afterretrievingthe
firstrewardpellet,andreturningtothestart-boxtoeatit,thestart-boxdoorwasclosed.
Thedoorwasthenre-openedforaccesstothetwofurtherpelletsaftereither30s
(massed)or10min(spacedaccess)–theseaccessdelaysbeingscheduledwithin-
subjectsinacounterbalancedmanner.Aftercollectingandeatingthethirdpelletinthe
start-box,theanimalswerereturnedtothehomecage.Betweenrewards,the
experimenterquicklycleanedanysanddugupbytheanimal.Thiscleaningprocesswas
introducedtolimittheemergenceofanystrategyinvolvingpreferentialdiggingata
sandwellsurroundedbysand,ratherthanguidancebyspatialmemory(MovieS3).
Third,biochemicalassayfortask-associatedgeneexpression:welookedat
theimpactofmemoryencodingongeneexpression.Asampletrialwasgivenusing
eitherthemassedorspacedtrainingprotocolwithtotalof3rewardpelletsretrieved.
Weconsideredcarefullytheappropriateprotocolsandtime-delaysbeforesacrificefor
conductingthisassay,decidingtokeeptheintervalafterthe2ndrewardpellet
consistentacrossconditions(seeFig.4A).Thismeansthat,asaconfoundingofthe
intervalbetweenrewardpellets,thereislongerinthestart-boxforthemassed
condition,andthismayhave,insomewaycontributedtothedifferentialpatterntobe
reported.Thereareseveralpotentialwaystoaddressthisproblem,butallinvolve
addeddifficulties(e.g.equatingtimeinthestart-boxbytakingtheanimalsinandout;
leavingtheanimalsinthestart-boxforalongperiodafterthesampletrialbefore
returningtothehome-cage,whichwouldhaveaddednovelty.Wechosethesimplest
protocol.Thus,forty-five(45)minafterthe3rdrewardpellet,theanimalswerebriefly
anaesthetizedinanisofluoranechamberanddecapitated.Thebrainswererapidly
takenoutandplacedinice-coldPBSfor30s.Acoronalblock(A/Pfrom-2mmto-7mm
fromBregma)wascutusingachilledbrainmatrix,dorsalhippocampusand
retrosplenialcortexweredissected,andsnapfrozenintheliquidnitrogen.Thesamples
wereshippedtoDartNeuroScience,andthenanalyzedusingRT-qPCRandRNA-Seq
(seebelow).
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Fourth,cognitiveenhancement:priorto(3)above,welookedattheimpactof
i.p.injectionofaphosphodiesterase4inhibitor(PDE4inhibitor)Rolipramattwotime
pointsaftermemoryencoding.Thedrugsolutionwasfreshlypreparedonthedayof
injectioninthesamewayasdescribedbyRuttenetal.(Rutten,Prickaertsetal.2006).
Rolipram(Tocris,#0905)wasfirstdissolvedinethanol,withinjectablegradewater
addedtoduringrigorousvortexing.Thissolutionwasthenmixedwith2%Tylose
(Sigma,MH300,#93800)inwaterthathadbeenpreparedbyovernightgentlemixing
onarotatorat4oC.Afinalconcentrationof68.1mM(0.019mg/mL)in5%Ethanol/1%
Tylosewascreated,with0.03mg/kginjectedintraperitoneallyinthecontrolroomof
thelaboratoryat45minor3haftertheST(2-sandwellprocedure,onepelletretrieval).
Fifth,noveltyexposure:asbraintissuewastakeninstep3abovetomeasure
differentialgeneexpression,theimpactofnoveltyonretentionwastestedinthe
subsidiarystudy.Environmentalnoveltyinvolvedplacingtheanimalsfor5minintoa
largesquarePerspexbox30minafteradailyST(post-encodingnovelty).Thebox
measured1.4msquare,andcontaineddifferentfloorsurfacesonsuccessivevisits.Rats
areverysensitivetothesurfaceonwhichtheywalkandtheuseofverydifferent
surfacestothatofthemaineventarenaevokedactivityandexplorationoneach
occasion,providedthesearenotscheduledtoofrequently.Twoconditionswere
compared–onepairoftestsinwhicheithernoveltyexposureorremainingthehome-
cagefollowed30minafteranSTwith1pelletreward(Lowreward);andanotherwith
3pelletsreward(Highreward).Thetwo-sandwellprotocolwasusedthroughoutfor
which1pelletrewardisinsufficienttoproducemuchmorethanatransientmemory.
RT-qPCRandRNA-Seq
RNAExtraction:FrozentissueswerehomogenizedinQiazol(Qiagen)using
LysingMatrixDceramicspheresintandemwithFastPrep24TM5Ghomogenizer(MP
Biomedicals).RNAextractionwashstepsandelutionwereperformedroboticallywith
Qiacube(Qiagen)withanRNeasyMinikit(Qiagen)andDNasetreatment(Qiagen)as
permanufacturer’sinstructions.
QuantitativePCR(qPCR):TheDNasetreatedRNAswerereversetranscribedwith
randomhexamers(TaqmanReverseTranscriptionKit,ThermoFisherScientific).qPCR
reactionswererunintriplicateusingtheTaqmanFastAdvancedMasterMix
(ThermoFisherScientific)withTaqmanprobesfortheimmediateearlygenes,Arc,c-Fos,
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Npas4andthereferencehousekeepinggenesTbp,Gapdh,andActb(ThermoFisher
Scientific)onaStepOnePlusRealTimePCRSystem(ThermoFisherScientific).We
performed40cyclesofPCR:1X(50°Cfor2min,95°Cfor20s)and40X(95°Cfor1s,
60°Cfor20s),anddeterminedrelativemRNAlevelsbynormalizingcyclethreshold(Ct)
valuesofthetargetandreferencehousekeepinggenesusingthe∆∆Ctmethod.Briefly,
wecalculated∆Ct(thedifferenceinCtbetweentargetgenesandreferencegenes)for
experimentalandcontrolconditions.Thedifferencebetweenthese∆Ctvaluesfor
controlandexperimentalconditionsisthe∆∆CtandchangesinmRNAlevelsaredirectly
proportionalto2-∆∆Ct.
RNA-Seq:RNAconcentrationandqualityweremeasuredwithaQubit
Fluorometer(LifeTechnologies)andaBioanalyzer(Agilent),respectively.RNA-Seq
librarieswerepreparedwithnon-strandedNEBNext®Ultra™RNALibraryPrepKitfor
Illumina®(NEB).Briefly,mRNAwaspurifiedfrom200ngtotalRNAandthen
fragmentedat94°C.cDNAwaspreparedwithrandomhexamers.Illumina-compatible
adaptorsanduniqueindexeswereaddedandDNAfragmentswereamplifiedwith12
roundsofPCR.Ampurebeads(BeckmanCoulter)wereusedforsizeexclusion.Libraries
wererunontheBioanalyzerandhadmeanfragmentsizesbetween384and457bp.3-
20pMofeachlibraryunderwentpairedend(2x50)sequencingonaHighSeq2000
(Illumina)with8librariesmultiplexedperlane.FastQfileswerealignedtotheEnseml
transcriptomeviaBowtieanddifferentialexpressionwasdeterminedusingDE-Seq.To
avoidcomplicationsfromfalsepositives,wesetalowp-valuecut-offofp<0.0001for
significanceandfocusedouranalysisonthosehitsthatwereconcordantbetween
hippocampusandretrosplenialcortex.
Randomising,CounterbalancingandStatisticalAnalysis
Behaviouralexperimentsrequirecarefulattentiontodetailwithrespecttoboth
theirconductandanalysis.Pointstonoteincludethatthecomparisonsofconditions
areallwithin-subjectsuchthatthereisnoissueofrandomisingtheallocationof
animalsacrossgroups–thereisonlyonegroupinboththemainandsubsidiarystudies.
Counterbalancingisalsosimpleinprinciplebutcomplicatedinpractice,theaimbeing
toensurethat,foranyoneofthefourstart-boxpositionsthatareusedacrossdays,half
theanimalsaretrainedoneachdaywiththecuedsandwellinanearlocationandthe
otheranimalsinafarlocation.Similarly,theplacementofthenon-rewardedsample
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maybenearorfar,andtotheleftorrightoftherewarded(correct)samplelocation.
Withrespecttostatisticalanalysis,thedataalwayscompliedwithassumptionsof
homogeneityofvarianceallowingparametricANOVAstobeusedthroughout.Some
analysesfocusedonlyonchoiceperformanceusingthePerformanceIndex(PI),while
othersfocusedon(1)timespentdiggingattherewardedlocation(hereafter‘cued
location’),or(2)therelativediggingtimeatthecuedlocation,thenon-rewardedsample
location(non-cuedtrained)andtheremainingincorrectsandwells(non-cueduntrained
locations).Notetheterm‘cued’doesnotrefertoanystimulusmarkingtherewarded
location;tothecontrary,allcueingiswithinmemory.Asweusedawithin-subjects
design,Greenhouse-Geissercorrectionsofdegreesoffreedomwereroutinelyused
withinSPSStoensureaconservativeapproachtotheanalysis,buttheiruseleadsto
somewhat‘odd’valuesfordegreesoffreedom,asapparentbelow.
14
RESULTS
Theratsrapidlylearnedtorunfromthestart-boxesintothearenaandtodigfor
foodthattheywouldalwayscarrybacktothe(darker)start-boxtoeat.Following
habituationduringwhich,asnotedintheMethods,somelearningwouldhaveoccurred,
webegantrainingwithasinglesamplesandwellastheencodingtrial(ST)followedbya
choicetrial(CT),butswitchedafter18sessionstousingtwosamplesandwellsofwhich
onlyonewasrewarded(Fig.1E).
Performanceonthe6-sandwellCTs,circa1hafterthesampletrial,was
measuredusingtheperformanceindex(PI;seeMethods).Thiswasalreadyjustabove
chanceinsomeanimalsafterhabituation,butthenassumedbyallanimalsand
sustainedatanaveragelevelof77.9%fromsession10onwardswithameanz-score
relativetochanceof4.8(Fig.1E).ThisremarkablestabilityofthePIacross3monthsof
testingisimportantasitconstitutesthebackgroundagainstwhichthesuccessivedaily
1-trialencodingcanbeevaluated.Onsession60,thecontrolconditionwasscheduledin
whichtheSTwasnotgivenbutaCTstillrun;theanimalsshouldthennotknowwhich
sandwellwascorrectandshouldfalltochance.The‘virtual’correctsandwelllocation
wasdeterminedaccordingtothesamemappingruleasinregulartraining–namelythat
anynewrewardedsamplesandwelllocationcouldnotbeinthesamequadrantinthe
arenaasthatoftheprevioussession.Alltheconditionbiases-left/rightfromthe
startbox,corner/center,nearfarfromthestartbox-werecounterbalancedamong4
sub-batcheswithinthesession.Insuchdesign,theperformanceindexofsession60was
calculatedusingthe‘virtual’error.Virtualerroristhenumberof‘virtual’errorsmade
beforediggingatthe‘virtual’correctone(whichwasinpracticerewarded).Ananalysis
ofthePIsfromsession58to62showsthat,aspredicted,performancedroppedto
chanceonsession60(repeatedmeasures(RM)-ANOVA:F=9.52,df2.9/61.2,
p=0.000036;degreesoffreedomadjustedbyuseofGreenhouse-Geissercorrection;
one-samplet-testvs.chance:t22=1.16,p=0.26forsession60andt22>4,ps<0.0001for
allothersessions;Fig.1E).InterspersedPTs(grayshading)werethereafterusedasthe
primarymeasureofmemory.ThescoringduringPTswasdouble-checkedforasub-set
ofanimalsusingindependentblind-scoring.Averyhighcross-correlation(R2=0.992)
wasobservedbetweentheinitialsemi-blindscorederivedfromscoringduringtesting
(byMN)andthatderivedfromoff-lineblindanalysisofDVDrecordeddatabyan
independentobserver(RF;Fig.1F).Thus,theratssuccessfullyencodedthelocationof
15
therewardedsandwellduringanST(withoneortwosamplesandwells);thisencoding
wasnecessarytomakeacorrectchoiceduringthesucceedingCT;andPTscoringwas
objective.
Theuseofasinglesandwell(containing1-5rewardpellets)asthesampletrial
(ST)hadtheunintendedeffectofencouraging,duringCTsandPTs,persistentdiggingat
whicheversandwellwaschosenfirst.Asthisincludedtrialswhenerrorsweremade,
persistentdiggingataninitiallyselectedlocationresultedinhighvariabilityoftest
scores.Theswitchtousing2sandwellsonSTsresultedinastrategychangetofrequent
shiftingbetweensandwellstosecurereward.Thisgreatlyreducederrorvariability
duringlaterPTsatthecostofaslightdecreaseinaveragetimespentdiggingatthe
correct(cued)sandwell.
Delay-dependentforgettingwasobservedaftersampleencodingusingboththe
1-and2-sandwellprocedures,eachinvolvingasinglesmallreward(1pellet).Two
counterbalancedseriesof3within-subjectPTs(nofoodavailable)wereconductedat
delaysof24min,2.4hand24hafterSTs,with2interveningregulartrainingsessions
(consistingofSandCTs,Fig.2A).Memorywasmeasuredintermsofthepercenttime
spentdiggingatthecorrect(cued)vs.theincorrect(non-cued)sandwellsduringthe
PTs(Fig.2B-C).TheoverallpooledrepeatedmeasuresANOVAofthetwosetsofdata
showedsignificantdelay-dependentforgettingover24h(F=3.87,df1.6/35.7,
p=0.038);Greenhouse-Geissercorrectionforrepeatedmeasures),withastrong
downwardlineartrendovertime(F=10.12,df1/22,p=0.0043)).Subsequentt-tests
comparingcorrect(cued)sandwellperformanceateachmemoryintervalindicated
abovechanceperformanceinthePTsafterboth1-sandwelland2-sandwellsample
trialsat24minand2.4h(1sandwell:t22=5.12and4.95,ps=0.00004and0.00006,
respectively)forboth;2sandwell:t22=4.34and3.24,ps=0.00027and0.0039but
chanceperformanceat24h(1SWt22=1.96,p=0.063;2SWst22=1.73,p=N.S.).
InsertFigure2abouthere
Theimpactofmassedvs.spacedaccesstorewardwasthenexamined,using
onlythe2-sandwellprocedureforSTs(Fig.3A).Memoryretentionimprovedwhen
rewardavailabilitywasincreasedfrom1to3pellets,witheithermassedaccessto
reward(inter-reward-intervalof30sec)orspacedaccess(IRI=10min)(MovieS3).
16
However,whilemassedrewardproducedgoodmemoryat2.4h,therewasstill
forgettingover24hr.Incontrast,spacedrewardhadadramaticeffecton24hmemory
whichwasbothabovechanceat24handnotmeasurablydifferentfromthatat2.4h
(Fig.3B).Importantly,theANOVAcomparingmassedvs.spacedrewardandtime-
intervalsrevealedasignificantinteraction(F=7.39,df1/22,p=0.013).Separatet-
testsshowedthatmemorywasabovechanceat24honlyfor3spacedrewards(Spaced:
t22=4.52,p=0.00017;Massed:t22=1.73,p=0.099),whereaslocationmemoryat2.4h
didnotdifferaftermassedvs.spacedreward(Massed=35.3± 3.3%;Spaced=32.1±
2.7%;N.S.p=0.45).Thus,theimpactofspacedrewardseemstobeonthepersistenceof
everydaymemoryat24hratherthanimmediatememorystrengthat2.4h.
Overthe3rewardsoftheST,wealsoexaminedtherateofdeclineoferrorsin
choosingbetweenthe2sandwells(Fig.3C).Tobeexpected,theanimalswereatchance
forthe1strewardpellet,butimprovedforpellets2and3.Ifanything,thetrendwasfor
thisimprovementtobeslightlygreaterformassedthanspacedtrials,butthisdidnot
reachsignificance(F=1.15,df1/22,p=0.295).However,theimprovementacross
successivepellets(1st,2ndand3rd)wassignificant(F=48.51,df2/44,p=7.4E-12).
InsertFigure3abouthere
Becausememoryconsolidationrequiresproteinsynthesisandtranscription
(DavisandSquire1984,AlberiniandKandel2015),wethenexaminedwhetherdaily
encodingofspatialmemoryresultedinchangesingeneexpression,andmore
interestingly,ifthereweredifferencesintheresponsetomassedandspacedtraining.
Attheendofstudy,ratsweregiveneithermassedorspacedaccesstothereward
presentationsinthe2-sandwellprotocolandeuthanized45minafterthesecondof
threetrials(Fig.4A;anintervalchosentominimizeanypossibleimpactofslight
differencesintimingbetweenparadigms).Thethreegroups(Control(noSTgivenand
Naive),Massed,andSpaced)wereassignedsoasnottodifferwithrespecttopriortask
performance(PIsforCtrl,MassedandSpacedwere77.4,77.8,77.5%,respectively
(one-wayANOVA,N.S.);andPTaveragescoreswere30.1,29.4,28.6%,respectively
(alsoN.S.).Numberofsubjects=11,8,8(Note:Thenaïverats(n=4)didnotdifferfrom
thetrainedanimals(n=7)givenno-STpriortoeuthanasiaandtheRT-qPCRanalyses,
sopooledtogetherasacontrolgroup(Ctrln=11)).Ouranalysisfocusedonthedorsal
17
hippocampusandtheretrosplenialcortex-tworegionsthatarecriticalforspatial
navigationandmemory(VannandAggleton2002,FranklandandBontempi2005,Vann,
Aggletonetal.2009)(Fig.4A).
InitiallyweperformedRT-qPCRonthreeimmediate-earlygenes(IEGs),Arc,c-
FosandNpas4aswellasthreehousekeepinggenes(HKP),Gapdh,ActbandTbp.All
threeIEGsweremarkedlyupregulatedbymassedandspacedtraininginbothtissues,
whereasthelevelsofthehousekeepinggeneswereunchanged(Fig.4B-C).TheDDCt
wascalculatedwithnormalizationtoGapdh,butsimilardatawasobservedwheneither
oftheothertwohousekeepinggenes(ActbandTbp)wasusedasareference.Thedata
fromallthreeIEGswaspooled,andthatforthethreeHKPgenes,andanRM-ANOVA
conductedacrossthethreeconditions(Ctrl,Massed,Spaced).Thisrevealedahighly
significantIEGvs.HKPxConditionsinteractioninbothbrainregions(HPC:F=24.08,df
2/24,p=000002;RSC:F=14.13,df2/24,p=0.000088).Thisjustifiedlookingatthe
IEGsalone,revealingsignificanteffectsoftrainingcondition,i.e.betweenControl,
MassedandSpacedconditions(HPC:F=19.64,df2/24,p=0.000009;RSC:F=13.17,df
2/24,p=0.000138).Subsequentorthogonalcomparisonsshowedthat,forHPC,the
Controlconditionwassignificantlylowerthanthecombinedmassedandspaced
conditions(F=34.1,df1/24,p<0.001)andthatactivationwithspacedrewardwas
higherthanwithmassed(F=5.12,df1/24,p<0.05).Basedonthesedata,wedecidedto
performRNA-Seqexperimentsonthesamesamples.
ToensurethatourRNA-SeqdataprovidedaccuratemeasurementofmRNA,we
plottedthenormalizedFPKMvaluescalculatedwithRSEMforthegenesalreadytested
withqPCR(Fig.4D-E).WefoundthattheRNA-SeqandRT-qPCRmethodologiesgave
nearlyidenticalresults,andproceededtomeasuretheeffectsoftrainingonallprotein
codinggenesusingDE-Seq2.Inthemassedcondition,theexpressionof45geneswas
increasedinHPCand82inRSC.Ofthesegenes,29weresignificantlyinducedinboth
structures.Inthespacedcondition,wefound113upregulatedgenesinHPCand137in
RSC-with54genesinducedinboth.Becauseanyrandomfalse-positivesinagiven
tissuewouldbeexceedinglyunlikelytoreplicateinindependentRNAsamplesfrom
anothertissue,weexaminedfurtheronlythe29‘Massed’and54‘Spaced’genesthat
wereup-regulatedinboththeHPCandRSC,andfoundthatofthegenesmeetingour
strictcriteriainmassedtraining,27werealsoup-regulatedinspaced(Fig.4F).Because
moregeneswereupregulatedinthespacedconditionthaninthemassed,wewantedto
18
determineiftherewereanygenesonlyregulatedbyspacedfoodreward.Wefocusedon
the56genesthatweresignificantinbothtissueswitheithertrainingparadigmand
correlatedthefold-changeinducedbymassedandspacedtraininginHPC(Fig.4G)and
RSC(Fig.4H).Thisanalysisshowedthatthegenesthatwereup-regulatedsignificantly
inratstrainedwithspacedtrialswerealsoup-regulatedbymassedtraining,butdidnot
alwaysreachourstrictstatisticalsignificancecut-off.Thisindicatesthatthe
constellationofgenesinducedbymassedandspacedtrainingissimilar.However,a
linearfitofthedata(R2=0.94and0.96forHPCandRSC,respectively)revealedthatthe
relationshipbetweentheeffectsofmassedandspacedtrialsfollowedapowerlawof
1.33inbothtissues,indicatingalargerincreaseingeneexpressionfollowingspaced
foodreward(TableS1).Only2ofthe56geneswereincreasedtoagreaterextentinthe
massedconditionand,interestingly,thesetwogenesweresignificantinbothHPCand
RSC.Incontrast,ahighernumberofgeneswereincreasedtoagreaterextentafter
spacedtraining.Orthogonalcomparisonsacrossall56genesrevealedthat7geneswere
significantlymoreinducedinspacedthanmassedinbothHPCandRSC,allofwhichare
CREB-targetgenes;14genesweresignificantlymoreinducedinspacedthanmassedin
eitherHPCorinRSC;someofthesearealsoCREB-targets.Amongstthose,weidentified
severalmembersoftheEgrfamilyofgenes,Dusp1(aregulatoroftheMAPKpathway),
andtheheatshockproteinsHspa1a(Hsp70)andHspb1(Hsp27),chaperonescriticalfor
thefoldingofnewlysynthesizedproteinsandcellulardifferentiation(Fig.4G-H).These
dataindicatethatthemassedandspacedtrainingparadigmsinitiallyrecruitvery
similargeneexpressionresponses,butthatthemagnitudeoftheresponseisgreater
withspacedtraining.
InsertFigure4abouthere–RT-qPCRandRNA-Seq
TheimpactoftheputativecognitiveenhancerRolipram(0.03mg/kg)wasthen
investigatedusingtwopost-encodinginjectiontimes,45minand3h,usingonlysingle
rewardencodingintheSTtomaximizeforgettingover24h(Fig.5A;butstillusing2
sandwellswithonlyonerewardedintheencodingtrial).Noenhancementwas
observedwiththe3hpost-encodinginjections,butastrikingimprovementwas
apparentat45minrelativetovehicleinjections(28.5±2.8%,comparedto19.8±2.4%).
AnoverallANOVAofdiggingintheprobetrialsatjustthecued-locationofthesample
19
trialshowedatrendintheinteractionbetweenDrugandTime-Delay(F=3.87,df1/22,
p=0.061842).However,anRM-ANOVAcomparingdiggingattheCued-trained,Non-
cuedtrained,andtheNon-cueduntrainedsandwellswassignificantforthe45minpost-
encodinginjection(F=10.92,df1.415/31.12,p=0.001),togetherwithaninteraction
reflectingtheimpactofRolipramcomparedtovehicle(F=3.67,df1.615/35.54,p=
0.044)(Fig.5B).Theanalysisofperformancewiththe3hpost-encodinginjections
showednodrugeffect(F<1),althoughtheusualdifferenceacrosssandwells(F=9.36,df
1.42/31.24,p=0.0019).Thus,theretentionof‘everyday’spatialmemorycanbe
enhancedbyinhibitionofPDE4andsuchaugmentationofmemoryispossiblein
behaviourallyexperiencedanimals.
InsertFigure5abouthere
Thesubsidiarystudyinvolvedtrainingaseparatecohortofanimalsinsameway
toinvestigatetheimpactof5minofpost-encodingenvironmentalnoveltyonmemory
retentionafterweak(1pellet)orstrongreward(3-pellet,massed)inthe2-sandwell
sampletrialprocedure(Fig.6A).TheexcellentstabilityofperformanceonTraining
DaysisshownintheFig.S2.Afteraninitiallearningperiod,dailyperformancewasat
oraround75-85%throughoutalongseriesofmemoryprobetests(Chance
performanceis50%).Thisstabilitygivesconfidencethatprobetestsconductedearly
orlateintrainingwereagainstastableperformancebaseline.
AsshowninFig.6B-C,forthestrongprotocol,therewasexcellentmemoryfor
therewardedsamplelocationat24min(58.2± 7.1%;chance=16.7%).Memory
declinedover24htochance(25.6± 6.3%)asexpectedformassedreward(an‘internal
replication’).However,if5minnoveltyexposurewasscheduled30minpost-encoding,
memorywassustainedabovechance(39.2± 4.0%).Thedataplottedisanaverageof
thefirst60secsearching,during2separateprobetestsforthe24htests,withonlyone
testbeingsufficientforthe24mintest.TheRM-ANOVAofdiggingacrossthesandwell
locationsshowedawithin-subjectsconditionseffect(F=6.72,df1.97/19.70,p=
0.0061)thatdeclinedinamonotonicfashionacrossconditions(24min,24h+Novelty,
24h-only:LinearF=11.89,df1/10,p=0.062).Thus,retentionwasgreaterwithpost-
encodingnovelty.Separatetestsestablishedthatperformancewasabovechanceinthe
24minand24h+Noveltyconditions(p=0.00017and0.00022,respectively),butat
20
chanceforthe24hcondition(p=0.19,NS).Noveltyscheduled30minafterencoding
appearedtoimprovetheabilityoftheanimalstodiscriminatetherewardedandnon-
rewardedsamplelocations(Fig.6C;plotting%timespentattheNon-cuedtrained
locationfromthatattheCuedlocation,whileignoringtheotherNon-cueduntrained
locations.Specifically,thisdiscriminationisclear24minafterencodingbutatchance
after24h.However,post-encodingnoveltyliftsthecapacitytodistinguishNon-cued
trainedfromCued-trainedatthe24hrtestinterval(ANOVArevealedadeclineacross
memorydelays(F=4.16,df1.99/19.95,p=0.031),decliningmonotonically(LinearF=
7.98,df1/10,p=0.018).Subsequentplannedorthogonalcomparisonsrevealedthatthe
24minand24h+NoveltyconditionsdidnotdifferfortheCuedlocation(F=2.01,df
1/20,N.S.),butthemeandiscriminationbetweenthetwosamplelocationsinthesetwo
conditionswassignificantlygreaterthanthatinthe24honlycondition(F=6.29,df
1/20,p<0.01).
Separateprobetestswereconductedusingtheweak1-pelletrewardprotocol
(Fig.6D,E).Theserevealedmodestmemoryat24min,nothingat24h,andnoimpact
ofnoveltyoneitherabsolutelevelsforeachsandwellorwithrespecttothedifference
score(ANOVAs:F=1.66,df2/20,p=0.216(N.S.);andF=2.69,df2/20,p=0.092).
Takentogether,thesefindingsimplythatpost-encodingnoveltycanaugmentretention
buttherehastobesomeminimallevelofmemoryforthistobeeffective.
InsertFigure6abouthere
DISCUSSION
Thisreport/technicalspotlightreportsaneverydaymemoryprotocolthat
enables1-trialmemorytobeinvestigatedrepeatedlyovermonthsinexperienced
animalstherebyenablingwithin-subjectcomparisons.Inthisstudy,wehaveconducted
both‘calibration’experimentsinrelationtowell-establishedprinciplesofmemory,and
a‘prospective’studyillustratingitspotentialforfuturework.Bywayofcalibration,we
observe:(1)delay-dependentforgettingofone-trialappetitivelymotivatedspatial
memoryover24h;(2)morepersistentmemoryovertime(i.e.betterretention)with
multiplespacedaccesstorewardbutnotmassedaccesstoreward;(3)enhanced
retentionwhenthePDE4inhibitorRolipramwasadministeredshortly(45min)after
21
encoding.Withrespecttoits‘prospective’useforfuturediscoveryresearch,wealso
report(4)upregulationofspecificIEGsandawiderpatternofgene-inductionin
associationwitheverydaymemorytrainingthatisquantitativelygreaterforspaced
comparedtomassedexperience.Toourknowledge,thisisthefirstreportofsuch
differentialgeneexpressionasafunctionoftrialspacinginmammals.Thesedatawere
securedagainstabackgroundofdemonstrablystablelevelsofchoiceperformance(our
PerformanceIndex(PI)measure),withtheobjectivescoringoftheMemoryProbeTests
(PTs)securedbymeansoftwoindependentbutwell-correlatedobservers.The
reliabilityofthetaskwasreplicatedinthesecondcohortofanimalswhowentonto
display,usingthe2-sandwellprotocol,that(5)post-encodingnoveltyenhances
retention.Weconsidertheseresultsinrelationtothevalueofour‘everydaymemory’
protocol,itslikelygenerality,andthusitspotentialformakingdiscoverieswithrespect
tocognitiveenhancement.
Everydaymemoryischaracterizedbysubstantialdailyforgetting
DavidMarrsuggested,asanassumptionwithinhismathematicalmodelof
‘archicortex’(Marr1971),thathumansmayencodeamaximumof1memorypersec
throughouttheday,withovernightconsolidationintoneocortex,therebyrequiringa
maximaldailystoragecapacityinhippocampusof105-see(Willshaw,Dayanetal.
2015).Thisassumption,whilelikelyinaccurateindetail,servesasareminderthatthe
mammalianepisodic-likememorysystemshouldbecapableofprocessingnumerous
eventsthroughthedaywithminimalinterference.Thisideaiscapturedinseveral
behaviouraltasks,includingspatialmemoryastestedhere((Olton,Beckeretal.1979,
SteeleandMorris1999,Bast,daSilvaetal.2005),andrecognitionmemorytasksas
developedfornon-humanprimates(MishkinandDelacour1975)anditsrodent
equivalent(EnnaceurandDelacour1988).Ourapproachto‘everydaymemory’
incorporatestheideathatdistincteventscanbeencoded,rememberedandforgotten
eachdayincludingsuchinformationas“what,where,when,whatcontext”(Griffiths,
Dickinsonetal.1999,EacottandEaston2007).
Againstthebackgroundofstabilityacrosstesting,ourfirstfinding–delay-
dependentforgettingof1-trialmemoryover24h-capturesadefiningfeatureofthis
modelofeverydaymemory.Ouruseofthe2-samplesandwellprocedureextendsthe
earlierobservationofdelay-dependentforgettingwithasinglesandwell(Bast,daSilva
22
etal.2005),butnowusingawithin-subjectsprotocolruneverydayfor4+months.We
canthinkoftheprotocolasonethatincludesdailyeventsthatmightbeautomatically
remembered–beingplacedintheapparatus,waitingforthestart-boxdoortoopen,
leavingthestart-box,searching,digging,findingfood,returningtothestart-box,and
eatingthefood.Wecannottestforeachofthese‘everyday’memories,butspatial
memoryinthearenaservesasanindex.Duringtrainingsessions,theanimalsaregiven
theopportunitytochoosebetween6sandwellsapproximately1hrafterencoding.
Behaviourally,theydisplaya‘win-stay’choicecorrectlyfromveryearlyintraining
(therewere7daysof‘habituation’precedingthathelpedteachthisstrategy),eventually
reachingperformanceofaround1errorpertrialthroughouttheremainderoftraining
(circa80%).Thestabilityofperformanceisreflectedinaremarkablez-score,relativeto
chance,of>4.5,thatwasshown,usingasuitablecontrolcondition,wasnotduetoany
‘olfactory’artefact.Whenthechoice-trialwasoccasionallysubstitutedbyaprobetestin
whichreinforcementwasnotavailablefor60sec,theyshowstrongpreferentialdigging
atthecorrectsandwellat24minandat2.4h,butmemorydeclinedtochanceover24h.
The2-sandwellsampleprocedureresultsinnotablylowvariabilityinprobetrial
performanceacrossanimalsastheyacquiredthesandwell-shiftingstrategyinthedaily
STsasdiscussedabove.Thestabilityofperformance(Fig.1E)andthesustained
motivationoftheanimalsshouldenablereliablewithin-subjectcomparisonofdrugs
regardlessofthesequenceofdrugtestingovertime.Performancestabilitywas
replicatedinthesubsidiarystudy(Fig.S2).
Asfurthercalibrationofthetask,weobservedthatmemoryimprovedwith
accessto3spacedrewardsonanencodingtrial(i.e.theanimalcollectsthe3reward
pelletsonebyone).Theimprovementof24hmemoryoccurredwithaninter-
reinforcementinterval(IRI)of10minbutnotwhenonly30sec.Thisfindingconfirmsa
largebodyoffindingsindicatingthatspacedtrainingproducesbettermemorythan
massedtraininginhumansandanimals(Ebbinghaus1885,Greene1989,Commins,
Cunninghametal.2003),aneffectalsoobservedinstudiesoflong-termpotentiation
(Kramar,Babayanetal.2012)thatlikelyreflectstheneedtoengagelong-termmemory
aswellasworkingmemoryduringacquisition.Apotentialcomplicationisthatthe
quantitativeparameterofinter-rewardinterval(30secvs.10min)isconfoundedby
totaltimespentinthestart-boxthatmayhaveeffectsonshort-termsatiation,arousal
orfrustration.However,withsuchasmallproportionoftheanimal’sdailydiet
23
consumedduringthetask,itwasnosurprisethattheanimalsatethepelletsreadilyand
within30secandshort-termsatiationseemsunlikely.Insofarasarousalmayhave
beenaffected,arguablytheshorterintervalwouldhavesustainedarousalbetterthan
the10minwaitinthestart-box,unlessthelongerintervalof10mininducedfrustration.
However,wesuspectthatthemaincontributoristhatreturningtothecorrectsandwell
forthesecondandthirdrewardpelletrequiresretrievalfromlong-termmemoryin
onlythespacedcondition.Retrievalduringanencodingsessionisknowntoenhance
latermemory–theso-called‘testingeffect’(KarpickeandRoediger2008)–andalso
causesdifferentialmemory-relatedgeneexpression.
Enhancedgenetranscriptionassociatedwithtrial-spacingandenhancedmemory
Theprospectivevalueofthisnewbehaviouraltechniqueisshowninthe
observationofgradedupregulationofIEGsgenessuchasArc,c-FosandNPAS-4,and
heatshockproteins,Per1,andDusp1,withnochangeincontrol‘housekeeping’genesin
thehippocampusandretrosplenialcortex.Notonlywasgene-expressionhigherin
animalstrainedinthetaskshortlybeforeeuthanasia,butwasalsosignificantlyhigher
withspacedthanmassedtraining-afindingobservedwithbothRT-qPCRandRNA-Seq.
Toourknowledge,thisisthefirststudyinrodentstofindmoregeneexpressionin
spacedovermassedtraining.Apotentialqualificationisthatwetestedgene-expression
45minafterthemiddletime-pointofthesampletrial,whichconstitutesa9.5min
differencebetweenthefirstandthelasttrialsofmassedorspacedtrainingrespectively.
Whilethisminortimingdifferencecouldpossiblyaccountforthedifferentialexpression
ofhighlytransientIEGs,itisunlikelythatthemanygenesfoundinourRNA-Seqresults
shouldallshowsuchtimesensitivity.Interestingly,inthefruitflydrosophila
melanogaster,memoryencodedwithasinglesessionofolfactoryconditioningis
augmentedbyover-expressionofthetranscriptionfactorCREB(cAMP-response
elementbindingprotein)(Yin,DelVecchioetal.1995).Thisfindingwascorroborated
morerecentlybystudiesdemonstratingthatspaced,butnotmassed,trainingleadsto
CREBdependentactivationofthecalcium/calmodulin-dependentproteinkinaseII
(CaMKII)andperiodgenesintwodorsal-anterior-lateral(DAL)neuronsindrosophila,
andthatproteinsynthesisinDALneuronsisrequiredforLTMconsolidation(Chen,Wu
etal.2012).Inorthogonalexperimentsinmice,eitherspacedtrainingormassed
trainingcombinedwithover-expressionofCREByieldsstrongerfearmemorythan
24
massedtrainingalone(Josselyn,Shietal.2001).Together,theseresultsindicatethat
spacedtrainingisassociatedwithtranscriptionalactivationininvertebratesand
vertebratesalike.
Massedandspacedtrainingtrialsbothincreasedgene-expression,butthe
responsewasmorepronouncedafterspacedencoding.Thedifferentialeffectsof
massedandspacedtrainingonsignal-transductioncascadesforgene-inductionare
poorlyunderstood.However,individualtrialsmayresultincalciumsignalsthatare
alreadyabovethresholdfortriggeringCaMKII,PKAorothersignalingcascades.Insuch
scenario,thespacingofrewardwouldenablesuchcascadestobeactivatedthreetimes
whereas,withmassedtrials,thismaynotoccurduetoarefractoryperiod.Asspaced
trainingcansometimescompensateforthereductionofCREB(e.g.inCREBa/d
knockoutmice(Kogan,Franklandetal.1997,Bourtchouladze,Lidgeetal.2003)),there
isthepossibilitythatotherpathwaysmaybeinvolvedinmemorypersistence.Non-
transcriptionalmechanismsshouldalsobeconsideredsuchasactin-polymerizationand
recruitmentofspinesforencodingduringspacedtraining(Kramar,Babayanetal.
2012),aswellasproteinphosphatases.SpacedlearningtrialsinhibitPP1activityvia
PKAphosphorylationofinhibitor1inmiceleadingtostrongeractivationofCREBand
PKAphosphorylationofGluR1(Genoux,Haditschetal.2002)andindrosophilaspaced
traininginducesrepetitivewavesofMAPKactivationinaSHP2dependentmanner
(Pagani,Oishietal.2009).Dopaminemayalsoplayacriticalroletheregulationof
memorypersistenceafterspacedtraining,somethingsuggestedbyworkinfruitflies
(Placais,Trannoyetal.2012),buttoourknowledgenotyetexploredinmammals.
Clearly,additionalworkisrequiredtofosterourunderstandingofthespacingeffecton
memorypersistence–aphenomenonfirstdescribedmorethan130yearsago.
Cognitiveenhancement:impactofRolipram
WealsoobservedthatthePDE4inhibitorRolipramenhancedeverydaymemory
at24hwhengiven45minafterencodingtrials,butnotwhengiven3haftertraining.It
isalsoworthnotingthat,inmuchoftheliteratureexaminingtheimpactofRolipramon
memory,severalpreviousstudieshavereportedpositiveeffectswhenthedrugis
appliedpriortothelearningtrial,forexample(Barad,Bourtchouladzeetal.1998,
ZhangandO'Donnell2000).Thismayeventuallyprovetobea‘valid’wayforcognitive
enhancerstobeusedinhumans,but,fromtheperspectiveofscientificanalysis,it
25
carriestheambiguityofbeingaprocedurethatconfoundsapotentialimpacton
encoding(attention,memoryacquisition)andaneffectonconsolidation(post-
acquisitionmechanisms).Ourprimaryinterestisthelatterand,forthatreason,weonly
testedpost-trialinjectionsofRolipram.Theeffectivenessofthe45mintime-pointis
alsoofinterestasvariouslinesofresearchonlong-termpotentiationinvitro(at32oC)
suggestthatsynaptictags,setatthetimeofmemoryencoding,havealife-timeof
around1.5h(FreyandMorris1998a).Synaptictagssequesterplasticity-related
proteins(PRPs).IfRolipramupregulatestheavailabilityofPRPsfromthecAMP-PKA-
CREBpathway,thenanintervalof45miniswithinthetimewindowforeffective
capturebyasynaptictag.TheeffectivenessofRolipramasacognitiveenhancerhas
beendemonstratedinvarioustestsofmemoryinnaïveratsandmice,including
contextualconditioning(Barad,Bourtchouladzeetal.1998),objectrecognition
(Bourtchouladze,Lidgeetal.2003,Rutten,Prickaertsetal.2006)andobjectlocation
memory(Rutten,VanDonkelaaretal.2009).Ourstudynowextendsthisefficacy
spectrumtoeverydayspatialmemoryinratswithextensivepriormemorization
experience.WhileRolipramisnotsuitableforclinicaldevelopmentbecauseofits
emesis-inducingpotential(ED50(p.o.)=0.03mg/kg(humannausea),0.48mg/kg(rat
pica);PercieduSert,Holmesetal.2012),nextgenerationPDE4inhibitorswithalesser
emeticpotentialhavebeendevelopedforCNSindications(Tully,Bourtchouladzeetal.
2003,Burgin,Magnussonetal.2010,Peters,Bletschetal.2014).
Theoreticalimportanceoftheimpactofpost-encodingenvironmentalnovelty
Noveltyisofteninvestigatedinrelationtoreward,asinnowclassical
experimentsinwhichananimal’sexpectationsofrewardareviolatedand,forexample,
greaterrewardismadeunexpectedlyavailable(SchultzandDickinson2000).Thisis
nowknowntoactivatedopaminergicsignallingintheventraltegmentalarea(VTA)that
promoteseffectivelearning(MontagueandSejnowski1994).Adifferentkindofnovelty
isunrelatedtothetaskinhand,suchasexposuretoanovelstimulusorenvironmentat
somepointbeforeoraftermemoryencodingofaseparatetask(MoncadaandViola
2007,Wang,Redondoetal.2010).Thisnoveltyaugmentstheretentionoftheunrelated
buttemporallyproximatetaskinadopamine-dependentmannerwithdifferentialparts
oftheCaMKinasepathwayinvolvedintaggingandplasticity-proteinsynthesis
(RedondoandMorris2011).Theneuromodulatorymechanismsarestillunclear,butan
26
intriguingnewpossibilityisthatnoveltyismediatedbyactivationofneuronsinthe
locuscoeruleus(LC)(Takeuchi,Duszkiewiczetal.2016)thatcanco-releasedopamine
(SmithandGreene2012,Kempadoo,Mosharovetal.2016).Thisparadoxical
dopaminergicmodulationcouldtriggerthesynthesisofplasticity-relatedproteins
linkedtothestabilisationandthusretentionofmemory(FreyandMorris1997,
RedondoandMorris2011).Todate,severaltestsdemonstratingenhancedmemory
withpost-encodingnoveltyhaveusedmemoryofasinglestimulusorevent(inhibitory
avoidance,digginginasandwell,object-placememory),butwesoughttotestwhether
noveltycouldalsoenhancethediscriminabilityoftworecentevents,andtheselective
retentionoftherewardedeventrelativetoanon-rewardedevent.Thesubsidiarystudy
usedthe2-sandwellencodingprocedurewith3massedrewardswithonlyone
sandwellrewarded.Whilememoryofthecuedsandwelllocationwasatchanceafter24
h,post-encodingnoveltyenhancedretentionofthecuedsandwellwithoutenhancing
memoryofthenon-cuedtrainedlocation.
Evaluatingcognitiveenhancement
Muchhasbeenwrittenaboutmeritsandpossibledrawbacksofcognitive
enhancement,andaboutthestrategiesforachievingitrangingfrombehavioral
approachessuchasthe‘method-of-loci’andretrievalpractice(RoedigerandButler
2011),throughtodrugsthatactatthetimeofencodingsuchasAMPAkines(Lynch,
Palmeretal.2011)orduringconsolidation(Barco,Pittengeretal.2003,Tully,
Bourtchouladzeetal.2003).Afinalpointrelatestothesuitabilityofanimalmodelsfor
testingputativeenhancers.Acriticalfactorisourpresentinabilityto‘back-translate’
fromhumanclinicaldatatoanimalmodels–largelybecausedrugsthatreliablyenhance
memoryinhumanshaveyettobefound.Minordifferencesinbehaviouralprotocolsin
animalmodelsmayseemtobeunimportantcomparedtotheexcitementofnovel
targetsidentifiedusingthelatestgenetictechniques.However,wenowassertthatthey
haveanimportantplaceinsuccessfullyidentifying‘hits’andavoiding‘falsepositives’.
Thepresentprocedurecanbecontrastedwithseveralothertasksofwhichone,context
fearconditioning,iswidelyusedbyvirtueofbeingsucharapidassayforthescreening
ofgenesanddrugcandidates.However,notonlyisitaslightlyoddtaskcomparedto
anythingthatnormallyhappenstoapersonduringtheday,andthusofdoubtful
‘constructvalidity’,itisgenerallytestedinexperimentallynaiveanimals.
27
The‘lost-in-translation’problemthathasledtotoofewdrugsbeingdeveloped
forhumanusedespitepromisingpre-clinicalresearchmaybeexacerbatedbyafailure
torecognizethatadifferencebetweenanimalandhumantrialswithrespectto
cognitionisnotonlythespeciesbutalsothemannerinwhichthetestingisdone.Prior
knowledgeisakeyparameter.Recentworkonschemalearninginanimalsandhumans
(Tse,Langstonetal.2007,vanKesteren,Ruiteretal.2012)hasbroughtintosharpfocus
theissueof‘priorknowledge’ineverydaymemoryfunction.Thepresenttask,whilenot
schematicinnature,nonethelessreliesonlookingatsingleepisodememoryencoding
inanimalsthathavehadextensivepriortraining.Wenowhaveanoptimizedversionof
theeverydaymemorytaskfeaturing:1)sustainedmotivationandperformanceover
months,2)statisticaladvantages(within-animaldesignandlessvariabilityinprobetest
scoresamonganimals),3)objectivescoringwithgoodinter-observeragreement.
Hopefully,thistaskcouldbridgethegapbetweenrodenttestsandhumantrialsbetter
thanmanyincurrentuseinpreclinicaltrialsThegeneexpressionanalysesnotonly
revealedforthefirsttimeinmammalsthatspacedencodingeventsinducedmoregene
expressionbutthegeneticresourcesobtainedfromRNA-seqwouldoffercluesto
potentialtargetsformemoryenhancement.Ourapproach,possiblysupplementedby
addingnon-spatialrecognitionmemorytotheprotocol,offerstheprospectofrevealing
theeffectivenessofnewdrugsthatcouldtranslateeffectivelyintosuccessfulPhaseII
trialsforhumans.
28
ACKNOWLEDGEMENTSANDROLES
ThisworkwasfundedbyDartNeuroScienceLLCandanERCAdvanced
InvestigatorGrantheldbyRGMMandGuillenFernandez(ERC-2010-AdG-268800-
NEUROSCHEMA).JL,DW,TTandMPareemployeesofDartNeuroScienceLLC.MNwas
supportedbyJSPSPostdoctoralFellowshipsforResearchAbroad.MN,MPandRGMM
designedtheexperiments;PSconstructedtheapparatus;MNandRFranthestudy,and
MCthesubsidiarystudy;JLandDWconductedtheanalysesofgene-expression;MN,MP,
DW,MM-LandRGMMwrotethemanuscript.
CONFLICTSOFINTEREST
JL,DW,MPandTTareemployeesofDartNeuroscienceLLCwhichisinvolvedin
researchanddevelopmentassociatedwithcognitiveenhancement
FIGURELEGENDS
FIGURE1.The‘eventarena’apparatusandperformancedata.
(A)Thearenawas1.6mx1.6mandmadeofclearplexiglass.Itwasopentoextramaze
cuesinthelaboratory.Ithad4start-boxes,withentrydoors,anda7x7gridwhere
floorpanelscanbeplaced.Somefloorpanelshavecircularholesintowhichsandwells
couldbeplaced.Aceilingmountedvideocameraenabledobservationoftheanimals.
(B)Thecircularsandwellswereconstructedwithsemi-circularbowlswithholes(left).
Therewardfood(showninbrown)couldeitherbeinthebowl(accessible)orunderthe
bowl(inaccessible)andwerecoveredwithsandmixedwithmaskingflavor(middleand
right).(C)Habituationprotocol.Onthefirstday[session(-8)],thearenalocationslots
werefilledwithclosedpanels,andtheratshabituatedtothestart-box(1min)andto
thearena(10min).Duringthefollowinghabituationsessions[sessions(-7)to(-1)],one
sandwellwasopeninthearena(invariablelocations).Rewardpelletswereplacedon
topofthesandwells,butgraduallyburieddeepinthesandtoencouragedigging.The
ratswereallowedtoshuttlebetweenthestart-boxandsandwellstoretrievemorethan
onepellet.(D)Trainingschedules.TrainingsessionconsistsofST(greenshade)andCT
thatfollowed60-90minlater.Thelocationoftherewardedsamplesandwell(filled
circle)stayedintheCT.The2-sandwellsampleprotocolisalsoshownasexample.
Whenstableperformancewasachieved,PTsessionswereinsertedonceineverythree
29
sessions.Aprobetrial(PT)isoneinwhichthefiverewardpelletsinallsixsandwells
areinaccessible(opencircles).(E)Performanceoftheanimals(n=23)thatcompleted
allphasesoftraining.Noteseparatephases(colors),thesessionsonwhichprobetests
(PTs)werescheduled(gray),andthe5-sessioncontrolphase(S58-S62)inwhichno
sampletrial(ST)wasgivenonthethirdsession(S60,pinkshade).(F)Inter-observer
correlationofprobetestperformanceinasubsetof12ratstrainedinthetask.X-axis
showsscoressecuredbyMNinasemi-blindmannerduringthetestitself,whileY-axis
showslaterblindscoringbyanindependentobserver(RF).R=0.996,p<0.001).Means
±1S.E.M.SeealsoFigureS1andMovieS1.
FIGURE2.Delay-dependentforgettingandinter-observercorrelation.
(A)WiththeSTgivenasasinglesandwellsample(greenshadingovereventarena,left
panels),wemeasuredintheprobetest,aftervariousmemorydelays,thepercenttime
diggingatthecorrectsandwell(blackarrow)andthatattheother5sandwells.Forthe
two-sandwll(2SW)procedure(rightpanels),diggingwasmeasuredatall6sandwells,
butnowdistinguishedbetweenthecorrectsandwell,thenon-rewardedsandwellinthe
ST(grayarrow),andtheuntrained4sandwells.(B)Forthe1SWprocedure,therats
displayeddelay-dependentforgettingover24h.(C)WiththeSTgivenasa2SWsample
(oneofwhichwasrewarded),delay-dependentforgettingover24hwasalsoobserved,
buttheplotnowseparatesthecued(rewarded)andnon-cued(non-rewarded)
sandwellsoftheSTfromtheother4sandwells.Theaveragevariabilityincorrect
digginginBwas6.9%whereaswasonly3.6%inC.***:p<0.001;****:p<0.0001.
Means±1S.E.M.Chancelevel=16.7%.SeealsoMovieS2.
FIGURE3.Impactofmassedversusspacedaccesstorewardduringthesampletrial.
(A)Experimentaldesignonprobetestswith30sor10minintervalsbetweenthe3
rewardpelletsofthesinglesampletrial.(B)Probetestperformanceat2.4hand24h
asafunctionofmassedvs.spacedtraining.Notesustained24hmemoryforspaced
access.(C)PlotofPerformanceIndex(PI)acrossthe3separateentriestothearena
duringthesampletrial(ST).Theanimalswereatchanceforthe1stpellet(indicating
thatthesandwelldesignwassuccessfulinpreventingodorartifacts),butdeclinedfor
pellets2and3.Thedeclineshowedatrendtowardsafasterdeclineinerrorswithina
30
trialformassedaccess,despitelongtermretentionover24hbeingmoreeffectivefor
spacedaccess.***:p<0.001;****:p<0.0001.Means±1S.E.M.SeealsoMovieS3.
FIGURE4.Geneupregulationassociatedwithtaskperformance.
(A)Timelineofmassedorspacedsampletrainingandtissuecollection(left).Coronal
ratbraintoillustratelocationofRSCandHPC(right).Atissueblockof4.0mmAPwas
dissectedout,butonlybregma-3.80mmisshownforsimplicity.(B)(HPC)and(C)
(RSC):RT-qPCRanalysisfromHPCtissueshowedupregulationofIEGs(Arc,c-Fos,and
Npas4)aftereithermassedorspacedsampletrialswhencomparedtountrained
controls,whereastwohousekeeping(HPK)genes(ActbandTbp)remainedunchanged.
Gapdhwasusedastheloadingcontrolandthedatashownnormalizedtothecontrol
valueofeachgene.ForthethreeIEGs,therewassignificantlygreaterupregulationin
thespacedthanmassedrewardcondition.(D)(HPC)and(E)(RSC):NormalizedFPKM
valuesfromRNA-SeqanalysisofthesamesetsofIEGsandHPKs(includingGapdh).
NotethesimilartrendsasinBandC,respectively,forthismethodofgene-quantification.
(F)RNA-Seqanalysisofgeneinduction.Venndiagramshowingthenumberofgenes
significantlyinduced(p<0.0001cut-off)aftermassed(left)andspaced(middle)
training.ForgenesinducedinbothHPCandRSCinthemassed(29‘massed’genes)and
thespacedconditions(54‘spaced’genes),2geneswereinducedpreferentiallyby
massedtrainingonly,27genesbyspacedtrainingonly,and27genescalledforboth
massedandspaced(right).(G)Correlationofgene-inductionlevelsformassedvs
spacedconditions(HPC,56genes).Thetwogeneswithmoreexpressioninmassedthan
spacedarehighlightedingray,andcertaincommonIEGsofnoteamongstthe54‘spaced’
genesarenamedandhighlightedinred.Dottedlineindicatesy=xline.Greenlineisthe
linearregressionofall56datapoints.Thedeviationfromunityto1.33reflectsoverall
strongerinductionofgenesafterspacedtraining.(H)SameasGbutforRSCtissue.
FIGURE5.Thephosphodiesterase4inhibitorRolipramenhancesmemoryretention
(A)Experimentaldesignonprobetests24hlaterwithi.p.injectionofRolipram(blue)
at45minor3hafterthesinglesampletrial(ST)ofsinglepelletretrieval.(B)Probetest
performanceat24hmemoryforRolipram/vehicleinjectedgroups.Rolipraminjected
45minaftertheSTsuccessfullyenhancedthememoryat24h.**:p<0.01;Means±1
S.E.M.(Seethemaintextforstatistics.)
31
FIGURE6.Post-encodingenvironmentalnoveltyenhancesmemoryretention.
(A)Experimentaldesignonprobetests(PT)24minor24haftersampletrial(ST)of
eitherHighrewardorLowrewardwithorwithout5minofnoveltyexploration.(B)
Probetestperformance24minor24hafterSTwithorwithoutpost-encodingnovelty,
using3-pelletreward(Highreward).Blackfilledbarsarecued-location,light-greyare
non-cuedtrained;agreysurroundtothebarsdenotesnovelty.(C)Differencescoresof
relativesearchingattheCuedlocationvs.Non-cuedtrainedlocationateachmemory
delay(Highreward).Blackandgreystripingtodenotesubtraction.(D)Probetest
performance24minor24hafterSTwithorwithoutpost-encodingnovelty,using1-
pelletreward(Lowreward).Noteonlytransientmemoryat24min.(E)Difference
scoresofrelativesearchingattheCuedlocationminusthattotheNon-cuedtrained
locationateachmemorydelay(Lowreward).**:p<0.01;Means±1S.E.M.Seealso
FigureS2.
32
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Per
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1SW 2SW 2SW, Massed / Spaced 2SW, Pharmacology
Nonaka et al. - Figure 1
Control
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E
Event arena
Training data
F Inter-observer correlation
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Training schedule
Habituation S(-7) - S(-1)
Stable performance stagepre-training
Habituation S(-8)C
D
20 cmFloor panels
BSandwell design Rewarded sandwellNon-rewarded
sandwell
Sandwells
Locationin event arena:
1 cm
10 cm
A Probe test sessions after 1 or 2SW-sample encoding
Memory Delay
% Cued % Non-cued % Cued % Non-cued % Cued % Non-cued
%di
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C 2SW-sample
B 1SW-sample
Nonaka et al. - Figure 2
24 min
**** N.S.
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Memory Delay
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chance
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%Cued %Non-cuedtrained
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Nonaka et al. - Figure 3
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2.4 hPT
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% Non-cued (untrained)
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*******
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Arc c-Fos Npas40
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y = 1.33x - 0.028R = 0.95642
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16 5329
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ControlMassedSpaced
ControlMassedSpaced
Hippocampus, qPCR (DDCt)
y = 1.33x - 0.024R = 0.94912
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Actb Tbp Arc c-Fos Npas4 Actb Tbp
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HKPIEGArc c-Fos Npas4 Actb Tbp GapdhArc c-Fos Npas4 Actb Tbp
RSC
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Nr4a1Hspb1
Fosb
Arc
Nonaka et al. - Figure 4
Massed
Spaced
45 min
Tissue collection
10 min 10 min
30s 30sProtocol
B
A
45 min
3 h
Rolipram / Vehicle
Vehicle Rolipram Vehicle Rolipram
i.p. 45 min i.p. 3 h
% Cued
% Non-cued (trained)
Rolipram / Vehicle
% d
ig ti
me
Nonaka et al. - Figure 5
ST PT
24 h
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PT
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% Cued% Non-cued (trained)% Non-cued (untrained)
Low reward at encoding (1 pellet)D E Difference score (Low reward)
24 min
Low reward
PT
24 h
24 min PT
24 h PT
High reward
PT
Novelty
Novelty
1 pellet
24 h PT
24 h PT
3 pellets
24 h 24 h +Novelty
24 min
24 h 24 h+Novelty
24 min
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30 min
A Nonaka et al. - Figure 6
ST
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Nonaka et al. - Supplemental Figures, Table, and Movie Legends
Figure S1 (related to Figure 1)
Training apparatus. (A) Photo of the event arena with a random 6 locations open onto
which 6 sandwells were placed. (B) Photo of the distal spatial cues (the patterns on the
walls and hanging cues) in the event arena room. (C) Photo of the sandwells. Rewarded
sandwell (left) has accessible pellet hidden under the sand (not shown in the picture),
and the non-rewarded sandwell (right) has inaccessible pellets below the porous plastic
bowls in the sandwell. (D) Latency to �irst dig at the correct sandwell in the choice trials
throughout the training sessions. Means ± 1 S.E.M.
Figure S2 (related to Figure 6)
Plot of performance index (PI) across 52 sessions of training with the second cohort of
animals used in the novelty study. After 11 sessions of triaining with a single sandwell
during the sample trial, they were switched to a 2 sandwell sample trial, and this used
during the later novelty phase. Note stability of above chance performance throughout
the probe test sessions shown with grey shading. See also Figure 1E. Means ± 1 S.E.M.
Table S1
Statistical information for RNAseq data.
Movie S1 (related to Figure 1)
Training sessions from ceiling camera view.
Two representative cases of the normal training session with 2-sandwell sample, 1-
pellet retrieval paradigm for sample trials (ST). In STs, the rats choose the two
sandwells randomly; in half the cases they �irst dig at the correct sandwell and never dig
the other sandwell, while in other cases they visit and dig the correct sandwell as the
2nd choice. Both cases are shown in this video.
Movie S2 (related to Figure 2)
Probe trial sessions of 2-sandwell protocol from ceiling camera view.
2
(same as above) and the PT that followed 24 h after, indicating the memory decay over
24 h. In the PTs, the six sandwells are identical, but the cued location is indicated with
red arrow and text.
Movie S3 (related to Figure 3)
Probe trial sessions of Massed vs Spaced from ceiling camera view.
Two representative cases of Probe trial (PT) sessions for comparing massed and spaced
access to reward. The �irst case shows the massed ST training (2-sandwell, 3 pellet
retrieval, 30 s inter-reward interval (IRI)) and the PT that followed 24 h later, and the
second case shows the spaced ST training (2-sandwell, 3 pellet retrieval, 10 min-IRI)
and the PT that followed 24 h later.
A
B
Nonaka et al. - Figure S1
Rewarded sandwell Non-rewarded sandwell
(accessible pellets) (inaccessible pellets)
C Sandwell
N E S W
Spatial cues
Event arena
Intramaze cues
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Nonaka et al. - Figure S2
1 5 10 15 30 35 40 45 5020 25
100
0
50
Per
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Probe trial session
1SW 2SW 2SW, Novelty
Chance
Session
4
‘Massed’ vs ‘Spaced’ comparison in HPC: Best-fit values ± SE
Slope 1.333 ± 0.0397 Y-intercept -0.02377 ± 0.02279 X-intercept 0.01783 1/slope 0.7501 95% Confidence Intervals
Slope 1.254 to 1.413 Y-intercept -0.06947 to 0.02192 X-intercept -0.01729 to 0.04973 Goodness of Fit
R square 0.9543 Sy.x 0.09867 Is slope significantly non-zero?
F 1127 DFn, DFd 1, 54 P value <0.0001 Deviation from zero? Significant Equation Y = 1.333*X - 0.02377 ‘Massed’ vs ‘Spaced’ comparison in RSC: Best-fit values ± SE
Slope 1.333 ± 0.03644 Y-intercept -0.02788 ± 0.02664 X-intercept 0.02091 1/slope 0.7501 95% Confidence Intervals
Slope 1.26 to 1.406 Y-intercept -0.0813 to 0.02554 X-intercept -0.02002 to 0.05853 Goodness of Fit
R square 0.9612 Sy.x 0.1243 Is slope significantly non-zero?
F 1338 DFn, DFd 1, 54 P value <0.0001 Deviation from zero? Significant Equation Y = 1.333*X - 0.02788
Nonaka et al. - Table S1
5