ContentslistsavailableatScienceDirect
Behavioural
Brain
Research
j o ur na l h o me p a g e :w w w . e l s e v i e r . c o m / l o c a t e / b b r
Research
report
Spatial
learning
by
mice
in
three
dimensions
Jonathan
J.
Wilson,
Elizabeth
Harding,
Mathilde
Fortier,
Benjamin
James,
Megan
Donnett,
Alasdair
Kerslake,
Alice
O’Leary,
Ningyu
Zhang,
Kate
Jeffery
∗InstituteofBehaviouralNeuroscience,ResearchDepartmentofExperimentalPsychology,DivisionofPsychologyandLanguageSciences,UniversityCollege
London,UnitedKingdom
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•Mousespatialmemorywastestedonanovel3Dradialarmmaze.
•Miceexhibitedlearningonworkingandreferencememorytasksonthe3Dmaze.
•Workingmemorywasnotimpairedonthe3Dmazewhencomparedwitha2Danalogue.
•Referencememorywasimpairedonthe3Dmazewhencomparedwiththe2Dmaze.
•Thismaybeexplainedbyadifferentialencodingofverticalandhorizontalspace.
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Articlehistory:
Received27January2015
Receivedinrevisedform24March2015 Accepted20April2015
Availableonline27April2015 Keywords: Navigation 3D Mice Memory
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Wetestedwhethermicecanrepresentlocationsdistributedthroughoutthree-dimensionalspace,by
developinganovelthree-dimensionalradialarmmaze.Thethree-dimensionalradialmaze,or
“radio-larian”maze,consistsofacentralsphericalcorefromwhicharmsprojectinalldirections.Micelearn
toretrievefoodfromtheendsofthearmswithoutomittinganyarmsorre-visitingdepletedones.We
showherethatmicecanlearnbothastandardworkingmemorytask,inwhichallarmsareinitially
baited,andalsoareferencememoryversioninwhichonlyasubsetareeverbaited.Comparisonwitha
two-dimensionalanalogueoftheradiolarianmaze,thehexagonmaze,revealedequallygood
working-memoryperformanceinbothmazesifallthearmswereinitiallybaited,butreducedworkingand
referencememoryinthepartiallybaitedradiolarianmaze.Thissuggestsintactthree-dimensionalspatial
representationinmiceovershorttimescalesbutimpairmentoftheformationand/oruseoflong-term
spatialmemoryofthemaze.Wediscusspotentialmechanismsforhowmicesolvethethree-dimensional
task,andreasonsfortheimpairmentrelativetoitstwo-dimensionalcounterpart,concludingwithsome
speculationsabouthowmammalsmayrepresentthree-dimensionalspace.
©2015TheAuthors.PublishedbyElsevierB.V.ThisisanopenaccessarticleundertheCCBYlicense
(http://creativecommons.org/licenses/by/4.0/).
1. Introduction
Theabilitytoaccuratelynavigatethroughtheworldisvitalfor thesurvival ofmobileanimals,andrequirestheperceptionand encodingofspatialcuesassociatedwithimportantlocationssuch asfoodnestinglocation.Studiesovermanyyearshaveconcluded thatmammalscreateaninternalrepresentationofspace (some-timesknownasthecognitivemap;[1]).Thisworkhasprimarily focusedonspatialnavigationinhorizontalplanarenvironments: however, recently, interest hasbeen growing in the meansby
whichlargerand moredimensionallycomplex spacesmightbe
∗Correspondingauthorat:26BedfordWay,London,WC1H0AP,UnitedKingdom. Tel.:+442076795308.
E-mailaddress:[email protected](K.Jeffery).
representedandusedinnavigation(seeJefferyetal.[2]forreview). Fewlaboratorystudiesofthree-dimensionalspatialencodinghave beenconductedtodate,andsotheaimofthepresentexperiment wastodeterminewhethermicecouldperforma3Dversionofa widelyusedlaboratory-basednavigationtask,theradialmazetask
[3].
TheadvantageoftheOltonradialmazeparadigmisthatitcan testbothlong-term(reference)andshort-term(working) mem-oryconcurrently.Inthe3Dversionofthemaze,which because ofitssphericalsymmetrywehavenamedtheradiolarianmaze,1
foodrewards are locatedat theend of armsthat project from
1Radiolariaarezooplanktonhavingradialsymmetry:seehttps://en.wikipedia. org/wiki/Radiolaria.
http://dx.doi.org/10.1016/j.bbr.2015.04.035
areskilledclimbersandhaveanaturally3Decology.
Theanimalsweretrainedontwoversionsoftheradialmaze task,thestandardversioninwhichallarmsbeginbybeingbaited andaredepletedwithoutreplacementasthetrialprogresses,and areferencememorytaskinwhichonlysomeofthearmsareever baited.Wereasonedthatifmicecannotencodethevertical com-ponentofthespacethentheyshouldhavedifficultylearningboth versionsofthetaskduetoconfusionbetweentheupperandlower armsatagivenazimuth(horizontaldirection).Comparisonswitha versionoftheoriginalradialarmmazeandwithatwo-dimensional analogueoftheradiolarianmazerevealedthatmiceareequally abletolearntheworkingmemoryversionofthetaskinthe radio-larianmazeasintwo-dimensionalmazes.Inthereferencememory tasktherateofreductioninreferencememoryerrorswere compa-rablefortheradiolarianmazeanditstwo-dimensionalanalogue; however,overalllearningwasreducedintheradiolarianmaze.We suggestthatmiceareabletosimultaneouslyrepresentboth verti-calandhorizontalcomponentsofaspatialtask,butthathavingto encodebothcomponentscreatesdifficultiesforthem.Weconclude withadiscussionofhowthe3Dstructureofthemazetaskmaybe represented.
2. Methods
2.1. Subjects
Subjectswere40maleC57BL/6Jmiceobtainedat8–10weeksof agefromCharlesRiverLaboratories,individuallyhousedandmildly foodrestricted tomaintainbody weightat 90% offree feeding weight.A12hreversedlight/darkcyclewasusedwith30min sim-ulateddawnat23:30andsimulatedduskat11:30;allmicewere trainedduringtheirdarkcyclebetween12:30and15:00. Experi-ment1usedthreecohortsofmice(eachn=8)whileExperiment 2usedtwo(eachn=8).Allmicewerenaïvetotheexperimental apparatuspriortohabituation.Allprocedurescarriedoutduring theseexperimentswerelicensedbytheUKHomeOffice,subjectto therestrictionsandprovisionscontainedintheAnimals(Scientific Procedures)Actof1986.
2.2. Apparatus
Theapparatuscomprisedthreeversionsoftheclassicradialarm maze:a3Dversionnamedtheradiolarianmaze(Fig.1A),a13-arm versionreferredtothroughoutastheclassicmaze(Fig.1B),and atwo-dimensionalanalogueoftheradiolarianmaze,namedthe hexagonmaze(Fig.1C).Armswerebaitedwithcondensedmilk appliedtodressmakers’pinsinsertedattheendofeacharm.All threemazeswereusedforExperiment1,andjusttheradiolarian andhexagonmazesforExperiment2.Allexperimentswerecarried outinthesamewelllitroom,withconsistentvisualextramazecues availabletomicethroughoutdatacollection.
Theradiolarianmaze(Fig.1A)wasconstructedfromlightweight materialsandcoatedwithpapier-mâchéfollowedbycrèpe ban-dage, to provide grip. The central section comprised a 30cm diameterspherefromwhichradiated14equidistantlyplaced cylin-dricalarms,each14cminlengthand3.5cmindiameter.Themaze wassuspendedbynylonlineinthecentreofanempty19-inchrack, withthelowermostarm30cmabovetheflooroftherack.
wasplacedinthecentreoftheexperimentalroom.
Thehexagonmaze(Fig.1C),atwo-dimensionalanalogueofthe radiolarianmaze,had12arms.Themazeconstitutedahexagonal ring,with30cmsideseach3.5cminwidth,withsix14cmarms, withawidthof 3.5cm, extendedoutwards andsix 14cm arms extendedinwardsfromthecornersofthering.Thus,onreturning fromanarmexcursionmicewouldhavefourchoices—turnleft, turnright,gostraightaheadorturnback.Thismazewastherefore moregeometricallysimilartotheradiolarianmazethanwasthe classicmaze.Themazewascoveredincrèpebandage,soasto main-tainconsistencywiththeradiolarianandclassicmazes,andwas againraised30cmaboveatableinthecentreoftheexperimental room.
2.3. Habituation
Forbothexperiments,subjectswerehabituatedtoeachmaze for5daysbeforetrainingcommenced.Inthefirst2daysnoarms werebaitedandmicewereallowedtofreelytraversethemazefor 15min.Inthefinal3dayseachmousewasintroducedtothemaze fromeachofthearmsinturn.Onceitmadeitswayfromanarmto thecentreofthemazeitwasremovedandplacedonanother ran-domlyselectedarm.Thiswasrepeateduntilallanimalswillingly navigatedfromeachofthearmstothecentrewithin1min.
2.4. Experiment1—workingmemorytask 2.4.1. Subjectsandtraining
Eachcohortofmice(n=8percohort)wastrainedononeofthe threemazesonly.
Oncehabituationwascompleted,theworkingmemoryphaseof trainingbegan.Fortheworkingmemorytask,allofthearmsofeach ofthemazeswerebaitedwithcondensedmilk.Micewererequired toretrievefoodfromallarmsofthemaze.Anarmvisitwasonly recordedwhenananimal’sheadreachedtheendofanarm.The numberofre-entryerrors(repeatedvisitstoanalready-depleted arm)andomissionerrors(numberofunvisitedarms)andorder ofvisitswerescoredmanuallybytwoexperimenters,whosatin opposingcornersoftheexperimentalroom.Micewereremoved fromthemazeaftereither15minoroncetheyhadcollectedthe foodrewardfromallarmsofthemaze,whicheverwassoonest. Miceweretrainedonthistaskforonetrialperdayforatleast7days oruntilthenumberofomissionandre-entryerrorshadreacheda 3-dayplateau,definedasanon-significantdifferencebetweenthe final3daysoftrials(usingrepeatedmeasuresANOVA).
2.4.2. Analysis
Pairedt-testscomparingthefirstthreetrialstothelastthree trialswereusedtoassesslearning.Valuesfortasklatency,thetotal numberofvisits,thenumberofomissionerrorsandtherateof re-entry(workingmemory)errorsasapercentageoftotalvisitswere
comparedbetweenmazesusingrepeatedmeasuresANOVA.
Forthemovementpatternanalysisontheradiolarianmaze,the lowerverticallyprojectingarmwasremovedfromanalysisasthis armwasonlyevervisitedbytwomice,andwasconsistentlythelast tobevisited.Forcomparisonofmovementswithinandbetween layers,sevenarmscomprisedtheupperlayer—theupwards pro-jectingarmatthetopofthemazeandthesixoutwardsprojecting armsonthetopsectionofthemaze.Thesixarmsprojecting out-wardsinthelowerhalfofthemazecomprisedthelowerlayer.The
Fig.1. (A)Radiolarianmaze;(B)classicmaze;(C)hexagonmaze.
percentagesoftotal visitsthatcouldbeassignedtowithin-and between-layermovementswerethencalculatedasaproportionof chanceforeachmovementtype,withachancelevelof50%forboth within-layerandbetween-layermovements,astherewasan over-all6.5/13chanceofmovementbetweenlayers(duetotheseven armspresentontheupperlayer,andsixarmsonthelowerlayer), anda6.5/13chanceofmovementwithinlayers.
Furtheranalysesofmovementpatternstestedfor neighbouring-armbiases.Intheradiolarianmaze,ontheupperlayertheanimals could visit one of seven neighbouring arms (two horizontally adjacent,twodiagonallyadjacent,onedirectlybelowthecurrent arm,onevertically orientedarm,oranimmediaterevisittothe just-visitedarm). Whenonthe lower layeranimalscould visit one of six neighbouring arms (two horizontally adjacent, two diagonallyadjacent, one directly above the current arm, or an immediaterevisit).
Intheclassicmaze,threeoutof13armswereneighbouring:the twoimmediatelyadjacentarms,andthejust-visitedarm.
Inthehexagonmaze,sixoutof12armswereneighbouring:the armoppositethepreviouslyvisitedarm,twodiagonallyadjacent arms,twoadjacentarmswhichwerewithinthesamesubsetof arms(i.e.movingfromanoutwardsprojectingarmtooneofthe twoadjacentoutwardsprojectingarms),andthejust-visitedarm. Armvisitswereexpressedasaproportionofchance.
2.5. Experiment2—referencememorytask 2.5.1. Subjectsandtraining
Eachcohortofmice(n=8percohort)wastrainedononeofthe twomazesonly.Sixbaitedarmswereassignedtoeachmouse.In theradiolarianmaze,onlyarmsonthetwocentrallayersofthe mazewerebaited(threeupperandthreelower).Inthehexagon maze,threeoftheoutersixarmsandthreeoftheinnersixarms werebaited.Thesixgoallocationsineachmazewerespecifiedin allocentriccoordinates,suchthatthebaitedarmsforeachmouse weredefinedbytheirrelationtoextramazecues.Eachmazewas rotatedhorizontallyby180◦eithereverytrialoreveryothertrialto controlforintramazecues,suchastactilecuesandolfactorycues relatedtoscentmarksleftbythemice.Foreachmaze,twotrials werecarriedoutperdayover25days,withthefirstsession com-mencingat1pmandthesecondat3pm.Eachtriallastedfor5min oruntilallsixrewardshadbeenretrieved,whicheverwassooner. Performancewasscoredasworkingmemoryerrors(visitsto already-visitedarms)andreferencememoryerrors(visitsto never-baitedarms).Theorderofarmvisits,numberoftotalvisits,number ofomissionerrors(baitedarmsthatwerenotvisited),tasklatency, andthetotalnumberoferroneousvisits(commissionerrors)were alsocollected.
2.5.2. Probetrials
Two5-minprobetrialswerecarriedoutthedayafter comple-tionofthereferencememorytask,inordertoruleoutthe(remote)
possibilitythattheanimalshadusedolfactiontofindthefood.All armswereleftunbaited,andthemazewasrotated180◦between probetrialstocontrolforintramazecues.Referencememoryerrors, re-entryerrors,commissionerrors,omissionerrors,tasklatency, orderofarmvisitsandthetotalnumberofvisitswerescored.
2.5.3. Analysis
Valuesfortasklatency,thetotalnumberofvisits,numberof omissionerrors,andthepercentagesofreferencememory, work-ingmemoryandcommissionerrorswereanalysed.Pairedt-tests comparingthefirstthreeandlast3daysoftrainingwereusedto assesslearning.Abetween-subjectsanalysiscomparedtherateof learningforeachofthesevariablesbetweenmazes.
3. Results
3.1. Experiment1—workingmemorytask 3.1.1. Radiolarianmaze
Toassesslearning,wecomparedthefirstandlast3daysof train-ing.Thefourvariablesanalysedwerethetotalnumberofvisits,task latency,omissionerrors,andre-entryerrors,measuredasa per-centageofthetotal.Pairedt-testsfoundnodifferenceinthetotal numberofarmvisitsbetweenthefirstthreetrials(16±2visits) andfinalthreetrials(17±1,t(7)=−0.520,p=619),buttasklatency
decreased significantly from 842±40 to 496±68s (t(7)=5.129, p=0.001).
Omissionerrors(failurestoenteroneofthearms)decreased sig-nificantlyfromanaverageof3.9±1.2errorsonthefirstthreetrials to0.4±0.4errors onthefinalthreetrials(t(7)=3.816,p=0.007; Fig.2A).Re-entryerrorsdeclinedsignificantlyfromanaverageof 44.7±7.9to24.5±2.9%(t(7)=2.538,p=0.039;Fig.2B).
We finally analysed movement patterns,in light of findings from previousstudiesof a bias towardshorizontal movements inthree-dimensionalenvironments[4,5].Wecalculatedthe
per-centage of the total number of visits that were horizontal
within-layer movements, and expressed these as a
propor-tion ofchance (chance=50%).Within-layer movementsdidnot exceed chance (proportion of chance: 1.04±0.04, t(7)=0.756, p=0.474), but there was a significant preference for visiting neighbouringarmsregardlessoflayer(mean1.4±0.05,t(7)=8.20, p<0.001).
In conclusion, mice showed good working memory
perfor-manceontheradiolarianmaze,showingadecreaseintasklatency, omission errors and re-entry errors. Thus, they were able to representthespatial aspectsof thetaskandtrack theseacross trials, eventhough thearmsweredistributed in3D space,and eachhorizontalarmpositionoccurredattwo verticallocations; theydidnotappeartousestereotypedchoicestrategiesanddid notshowdifferencesbetweenhorizontalandverticalmovement patterns.
0 1 2 3 4 5 6 7 8 9 10 R e-e Trial 0 1 2 3 4 5 6 7 8 9 10 Om Trial
Fig.2. Comparisonofworkingmemorytasklearningbetweentheradiolarianmaze(black),classicmaze(blackdotted)andhexagonmaze(grey).(A)Numberofomission errorsand(B)rateofarm-re-entryerrorsasapercentageofthetotalnumberofvisits.Arrowsrepresentintroductionofbarrierstotheclassicmaze.
3.1.2. Classicmaze
Becauseoftheunexpectedlygoodperformanceoftheanimals onthe3Dmaze,weconductedaclassic-mazeexperiment,using a13-armversionofastandardradialmaze,inordertodevelop
abenchmarkof standard two-dimensionalperformance against
whichtocomparetheradiolarianmazefindings.Micereceived10 daysoftrials.
Observationoftheanimalsduringlearningrevealedamarked propensityforthemicetoadoptstereotypedmovementpatterns, showingastrongpreferenceforvisitingneighbouringarms,unlike thepatternstypicallyshownbyratsinasimilarapparatus[6].Over thefirstfourtrialstheproportionofvisitstoneighbouringarms increasedfrom0.9±0.1onthefirsttrialto3.0±0.3onthefourth trial.Onesamplet-testsforall10trialscombinedrevealeda sig-nificantlyabove-chancepreferenceforvisitingneighbouringarms (mean2.3±0.18,t(7)=7.28,p<0.001).
Tothwartuseofanadjacent-armrule,weintroduced barri-erstotheentranceofeacharmonthefifthdaytotryandreduce stereotypedbehaviour.Whilethisinitiallyreducedtheproportion ofneighbouringarmvisitsto1.4±0.3onthefifthtrial,animals quicklyreturned tothis stereotypedbehaviour,reachinga peak proportionof3.4±0.3neighbouringarmvisitsbytrial10.
Therewasnodifferenceinthenumberoftotalvisitsbetweenthe firstthreetrials(27±2)andthefinalthreetrials(19±2,t(7)=2.254, p=0.059). Therewas,however,a decrease intasklatencyfrom thefirstthreetrials(412±49s)tothefinalthreetrials(210±49s,
t(7)=3.505,p=0.010).
Owingtothestereotypydiscussedabove,micewereableto completetheworkingmemorytaskwithzeroomissionerrorsfrom thefirsttrial,withanaverageof0.04±0.04omissionerrorsfirst threetrialsandanaverageof0±0omissionerrorsonthefinal threetrials (Fig.2A).Thesevalues werenotsignificantly differ-ent(t(7)=1.000,p=0.351).Re-entryerrorsbetweenthefirstthree
trials(45.9±4.9%)andthefinalthree trials(23.1±6.0%)almost approachedsignificance(t(7)=2.350,p=0.051;Fig.2B).
Insummary,micecompletedtheworkingmemorytaskonthe classicmazewithoutomissionerrorsandwithnodecreasein re-entryerrors, whilemovement patternanalysisindicated ahigh tendencytochooseneighbouringarmsinastereotypedmanner, which accountedfor theerror pattern.To overcomethis prob-lemandassesstruespatiallearning,weadaptedthemazesoas toreducetheaffordanceofthemazeforstereotypyandmakeit structurallymoresimilartotheradiolarianmaze,whileretaining thetwo-dimensionallayout.Resultsfromthis“hexagonmaze”are describedinthefollowingsection.
3.1.3. Hexagonmaze
Thehexagonmazeismoreakintotheradiolarianmazeinwhich theanimalshadmultipleneighbouringarmstochoosefromandwe hopeditwouldprevent,oratleastreduce,theapplicationofsimple rules.Micereceived10 daysoftrialsonthismaze,andanalysis proceededasbefore.
Therewasa significantdecreaseinthetotalnumberof arm-visits(t(7)=5.667,p=0.001),from26±2visitsonthefirstthree
trialstoanaverageof17±1onthefinalthreetrials.Therewasalso adecreaseintasklatencyfrom776±31to269±91s(t(7)=6.396, p<0.001).Omissionerrorsdecreasedfromanaverageof1.5±0.4to 0.2±0.2(t(7)=4.651,p=0.002;Fig.2A).Analysisofre-entryerrors
revealedthat,unlikeintheclassicmaze,therewasevidenceof spatiallearning,withasignificantdecreaseinthepercentageof re-entryerrorsfromanaverageof55.5±2.0to27.8±3.9%(t(7)=6.905, p<0.001;Fig.2B).Nevertheless,movementpatternanalysisfound thatdespitethechangeinmazedesign,thereremaineda signif-icantpreferenceforvisitingneighbouringarms(mean1.5±0.03,
t(7)=17.13,p<0.001).
3.1.4. Comparisonbetweenmazes
WenextcarriedoutrepeatedmeasuresANOVAstocomparethe learningbetweenthethreeradialarmmazes.Wefocusedonthe twoerrortypes(omissionerrorsandre-entryerrors),followedby comparisonofneighbouring-armvisits.
Comparison of omission errors betweenmazes for the first three versuslast threetrials revealeda significantreduction in thenumber of omissionerrors (F(1,21)=25.389, p<0.001).
Anal-ysis of between-maze effects revealed a significant difference betweenmazes (F(2,21)=5.342,p=0.013;Fig.2A),inwhich
Bon-ferroniadjustedposthoccomparisonsrevealedsignificantlyfewer omissionerrors ontheclassic maze than theradiolarianmaze (p=0.012),withnodifferencesbetweenthehexagonandclassic maze(p=0.638)orhexagonandradiolarianmazes(p=0.190).For re-entryerrors,repeatedmeasuresANOVAalsoshoweda signifi-cantoverallreduction(F(1,21)=28.801,p<0.001)withnodifference
betweenmazes(F(2,21)=1.517,p=0.242;Fig.2B).
Finally,comparisonsoftheneighbouringarmbiasrevealeda significantdifferencebetween mazes(F(2,21)=21.026,p<0.001),
with Bonferroni-corrected independent-groups t-tests (only
p<0.017wereconsideredtobesignificant)revealingastronger preference for visiting neighbouring arms in the classic maze thantheradiolarian(t(7.97)=4.951,p=0.001)andhexagonmazes
(t(7.39)=4.347,p=0.003)andnodifferencebetweentheradiolarian
andhexagonmazes(t(14)=2.281,p=0.039;Fig.3AandB). 3.1.5. Summary
Together, these data show that mice can hold a
short-termworking-memoryrepresentationofrewardlocationsonthe hexagonand radiolarianmazes, and that their ability todo so increaseswithexperienceofthetask.Boththehexagonmazeand radiolarianmazeyieldedsimilarresultsforreductionsinworking memoryerrors,andforneighbouringarmbiases,suggestingthat thegreatergeometriccomplexityofthemazescomparedtothe classicmazecanreducethebehaviouralbiasesobservedonthe latter.
Wenextlookedatwhethermicecanformlong-termspatial memoryrepresentationsinthreeaswellastwodimensions.
0 1 2 3 4
Radiolarian Classic Hexagon
Visits (p ro poron chance) 0 1 2 3 4 1 2 3 4 5 6 7 8 9 10 Visits (p ro poron chance) Day
B
A
BarrierFig.3.Movementpatternsduringtheworkingmemorytask.(A)Neighbouring-armvisitsontheradiolarian(black),classic(blackdotted)andhexagonmazes(grey)foreach dayoftrials,representedasaproportionofchance.Arrowsrepresentthetimepointsatwhichbarrierswereaddedtotheclassicmaze.(B)Taskaverageofneighbouringarm visitsrepresentedasaproportionofchanceforeachofthethreemazes.Chancelevelsarerepresentedbyahorizontaldashedline.
3.2. Experiment2—referencememoryexperiment
Thisexperimentcomparedlong-termspatialmemory,in addi-tiontoworkingmemory,betweenthe3Dradiolarianmazeandits 2Dcounterpart,thehexagonmaze.Sixteennaïvemice,splitinto twocohortsofn=8each,weretrainedoneithertheradiolarian mazeorhexagonmazeover25consecutivedays,withtwotrials perday.Bothcohortsofmicewereableandmotivatedtomove aroundtheirdesignatedmazebytheendofhabituation.The clas-sicmazewasnotusedforthereferencememorytask,duetothe strongneighbouring-armbiasobservedinExperiment1.
3.2.1. Radiolarianmaze
In the radiolarian maze, total arm visits did not decrease between the first 3 days (9±1) and the last 3 days (10±1,
t(7)=−0.768,p=0.468)buttherewasareductionintasklatency
from295±3to168±22s(t(7)=5.841,p=0.001).
Omission errors decreased significantly from an average of 2.3±0.3errorsonthefirst3daysoftrialsto0.3±0.2onthefinal 3daysoftrials(t(7)=8.064,p<0.001;Fig.4A).Commissionerrors (percentageofallvisitsthatwereerroneous)declinedsignificantly from57.1±2.8%onthefirst3daysoftrialsto37.7±4.0%onthe final3daysoftrials(t(7)=4.393,p=0.003;Fig.4B).Thesetypesof
errorswerefurthersplitintotwocomponentparts—re-entryerrors andreferencememoryerrors(visitstoarmsthathadneverbeen baited).Thepercentageofre-entryerrorsdecreasedsignificantly from15.3±2.5 to 7.0±2.0% (t(7)=2.7, p=0.032; Fig.4C), while
referencememoryerrorsdecreasedfrom56.0±2.6to31.5±2.9% (t(7)=7.141,p<0.001;Fig.4D).Therewasthusclearevidenceof3D
spatiallearning:workingmemoryperformanceimprovedslightly andreferencememoryimprovedconsiderablyacrossthecourseof training.
3.2.2. Hexagonmaze
Miceonthehexagonmazeshowedasignificantreductionin thetotalnumberofvisitsbetweenthefirst3days(13.0±0.9)and thelast3days(7.0±0.3;t(7)=5.341,p=0.001)anda significant
reductionintasklatencyfrom271±14to104±12s(t(7)=14.878, p<0.001).
Omissionerrors decreased from1.5±0.3 onthefirst 3days to 0±0 on the final 3 days (t(7)=5.029, p=0.002; Fig. 4A).
Commission errors decreased from 60.2±3.0 to 15.52±2.9%
(t(7)=11.546,p<0.001; Fig.4B). Ofthese, thepercentage of
re-entryerrors decreasedfrom 29.4±2.2 to3.6±1.7%(t(7)=8.943, p<0.001; Fig. 4C), while the percentage of reference memory errorsdecreasedfrom41.9±2.3to12.1±1.7%(t(7)=5.44,p=0.001; Fig.4D).
3.2.3. Probetrials
Probetrialswerethenconductedtoruleouttheuseofolfactory cuesinsolvingthetask;itwasexpectedthatprobetrial perfor-manceshoulddiffer fromperformanceonthefirst daybutnot fromperformanceonthelastday,sothesewereexaminedwith repeated-measuresANOVAcomparingtrialtype(training/probe)
Fig.4.Comparisonoflearningratesbetweenradiolarianmaze(black)andhexagonmaze(grey).(A)Omissionerrors;(B)commissionerrors;(C)workingmemoryerrors; (D)referencememoryerrors.
0
Re
Radiolarian Hexagon
Fig.5.Probetrials torule outrewarddetection(**p<0.01;n.s.denotes non-significant).
againstday(first/last).Fortheradiolarianmaze,therewasamain effectoftrialtypeonthepercentageofreferencememoryerrors (F(2,14)=11.736,p=0.001).Bonferroni-correctedpairedt-tests(in
whichonly valueswitha p<0.017wereconsideredsignificant) foundasignificantly lower rateofreference memoryerrorson thefinaldaywhencomparedtothefirstday(t(7)=3.31,p=0.013)
andasignificantlylowerrateofreferencememoryerrorsonthe probedaywhencomparedtothefirstday(t(7)=4.03,p=0.005).As
expected,therewasnodifferencebetweenthefinaldayoftrials andtheprobetrials(t(7)=0.99,p=0.354).
Forthehexagonmaze,therewasamaineffectoftrialtypeofthe percentageofreferencememoryerrors(F(2,14)=26.868,p<0.001).
Bonferroni-correctpairedt-tests(p<0.017consideredsignificant) showedasignificantlylowerrateofreferencememoryerrorsinthe finaldaycomparedtothefirstday(t(7)=5.44,p=0.001)andinthe
probetrialswhencomparedtothefirstday(t(7)=5.34,p=0.001),
withagain,nodifferenceinprobetrialscomparedtothefinalday oftrials(t(7)=1.44,p=0.194;Fig.5).
Overall,then,probe trialperformance wassimilarto perfor-mancebytheendoftrainingforbothmazetypes.
3.2.4. Comparisonbetweenmazes
Learning between the radiolarian and hexagon mazes was
assessed by examining the interaction in a repeated-measures ANOVAcomparingthefirstandlast3daysoftrials.Fortotalvisits, therewasnooverallbetween-mazeeffect(F(1,14)=1.646,p=0.220)
butthere wasa significantinteraction(F(1,14)=20.03,p=0.001),
reflectingadecreaseinthetotalofnumberofvisitsinthehexagon mazebutnotintheradiolarianmaze.Fortasklatency,therewas asignificantbetween-mazeeffect(F(1,14)=1.409,p=0.017)butno
interaction(F(1,14)=2.72,p=0.122).
Omission errors showed significant between-maze effects
(F(1,14)=4.659,p=0.049),inwhichmiceexhibitedbothfewer
omis-sion errors and shorter trial times in the hexagon maze than theradiolarianmaze,withnointeraction(F(1,14)=2.02,p=0.178; Fig.4A).
Analysisof commission errors found a significant
between-maze effect in which mice made fewer errors in the hexagon
mazethantheradiolarianmazeoverall(F(1,14)=7.621,p=0.015).
Additionally,theinteractionwithtrialblockwashighly signifi-cant(F(1,14)=18.509,p=0.001)duetoagreaterreductioninthe
hexagonmazethantheradiolarianmaze(Fig.4B).Wefurthersplit thecommissionerrordataintoitscomponentparts,re-entryand referencememoryerrors.Forre-entryerrors,therewere signifi-cantbetween-mazeeffectsoverall(F(1,14)=6.525,p=0.023)anda
significantinteraction(F(1,14)=16.738,p=0.001),wherebyre-entry
errorsdecreasedmoreonthehexagonmaze(Fig.4C).Forreference memoryerrors,micecommittedfewererrorsinthehexagonmaze thanintheradiolarianmazeoverall(F(1,14)=36.036,p<0.001),but
therewasnointeraction(F(1,14)=1.724,p=0.210;Fig.4D).
orytaskonbotha3Dmaze(theradiolarianmaze)anda2Dmaze (thehexagonmaze).Therewere,however,differencesin learn-ingbetweenmazes, withmiceexhibiting morere-entry errors, andmorereferencememoryerrorsonthethree-dimensionalmaze thanthetwo-dimensionalmaze.Theseresultsarefurtherdiscussed below.
4. Discussion
Motivated by previous behavioural and electrophysiological findings,togetherwiththosefromdecadesofbehaviouralresearch intwo-dimensionalenvironmentsusingtheradialarmmaze,we developeda three-dimensionalradialarmmaze,theradiolarian maze,totest whethermice canrepresent locationsdistributed within3Dspaceandwhethertheserepresentationscanbe
main-tained over time. Mice on the radiolarian maze learned both
workingandreferencememoryversionsofthetask,indicatingthat theanimalscouldlearnandremember,bothwithintrialsandacross days,spatiallocationsdistributedwithin3Dspace.However,they didthislesseasilythanacomparabletaskonatwo-dimensional radialmazevariant,thehexagonmaze,whichhadbeenmatchedfor spatialcomplexity.Weconcludethatmicecanrepresent3Dspace butwithmoredifficultythan2Dspace.Below,weexaminethe find-ingsofthestudyandthendiscusshowtheradiolarianmazemay havebeenrepresentedbythemice,togetherwithpossiblereasons fortheapparentdifficultyinforming/usingthisrepresentation.
4.1. Spatialperformanceintwoversusthreedimensions
Experiment1comprisedaworkingmemorytasktoseewhether micecouldrepresentthespatiallayoutofthemazesandholdthisin short-term(working)memory.Becauseoftheevidentapplication ofaprocedural(motoric)ruleintheclassicmaze,theresultsare notinformativewithregardtospatialencodingandsodiscussion isrestrictedtotheothertwomazes.Re-entryerrorsinto already-visitedarmsdeclinedsignificantlyacrossthecourseoflearning, indicating that micewereequally asable toholda short-term representation ofpreviously visited reward locationsin the3D radiolarianmazeastheywereonthe2D mazes.InExperiment 2,thereferencememorytask,micealsoshowedsignificant learn-inginbothmazesandtherateofreductionofreferencememory errorswasthesame.However,althoughtherateoflearningwas thesame,thetotalnumberofbothworkingmemoryandreference memoryerrorsweregreaterintheradiolarianmaze,withthe refer-encememoryerrors—thebestindexoflong-termspatialmemory inthesetasks—asymptotingataround40%ontheradiolarianmaze. Thissuggestsincreaseddifficultyinforming,retainingorusinga long-termrepresentationofrewardlocationsdistributedthrough three-dimensionalspace.
Thatmice wereable tolearn the radiolarianmazesuggests atleastapartialabilitytorepresenttheverticalcomponentofa spatialtask,andsothequestionbecomeshowtheydothis,and whyperformanceislessgoodthanintwodimensions.Thereare severalpotentialreasonsfortheloweredperformance.Oneis sim-plythat thephysicaldemands of movingona difficultsurface, whichrequiredconsiderableeffortnottofalloff,andalsotomove around,mighthavedivertedcognitiveresources(including atten-tion)awayfromthespatialcomponentsofthetaskandtowards itsmoreproceduralaspects.Thesecondistodowithlimitations
inhowthemicecognitivelyrepresentedthemaze.The representa-tionalpossibilities,andlimitations,arediscussedbelow,together with suggestions for future experiments to disentangle these hypotheses.
4.2. Howtheradiolarianmazemightberepresented
Informedbythefindingsofthisstudy,togetherwiththoseof recent behaviouraland electrophysiologicalstudies,we suggest threepotentialmechanismsbywhichthemicemighthavebeen abletorepresenttheradiolarianmazetask.
First, themicemight have createdan integrated volumetric map forrepresenting the3Dmazelayout. Insucha map, each goallocationwouldhaveunique3-componentspatialcoordinates (x,yandz),and/oreacharmofthemazewouldberepresented bya unique vector;animals couldthussolvethetaskby visit-ing each of therelevant locationsin the sameway as theydo onthestandard radialmaze.Weinitiallyconsideredit unlikely thattheywoulddothis:basedontheneuralfindingsofHayman etal.[7]andofthebehaviouralfindingsofGrobétyandSchenk
[5] and Jovalekic et al. [4] we thought it more likely that the animalswould formanessentiallyplanarrepresentation ofthe mazelayout,andthus(a)confusearmshavingthesame horizon-talcoordinates,and/or(b)solvethetaskinaplanarway,visiting all thearms of one layerfirst followed byall the armsof the secondlayer,ashasbeenseeninthepreviousbehavioural stud-ies.Ourpresent findingssuggest thatarmconfusionis unlikely given theequally fastlearning of theworking memorytaskin both3Dand2Dmazes.Aplanarrepresentationmightalsoseem unlikely because analysis of the pattern of responding on the radiolarianmazeshowedthatarmchoiceswerenotdistributed layer-wise;infact,horizontalwithin-layermovementsand verti-calbetween-layermovementsoccurredequallyoften.However,if themicehadformedanintegratedvolumetricmapthenitseems surprisingthattheywouldhavesomuchmoredifficultywiththe referencememorytaskonthe3Dmazethanwithits2D counter-part,sincethemazeshadthesamenumberofarms,andatruly integrated map should make noparticular distinction between dimensions.
Analternativepossibilityisthattherepresentation isplanar, butitcomprisestwoplanes—onemapforeachlayer—andthemice wereforced, bytheunusualaffordances of themazestructure, tointersperse visitstoone planewiththose oftheother. That is,sometimesthemouse wouldfinditself positionedsuchthat itwaseasiertoswaplayersthantoworkitswayaroundanarm tothenextarminthesamelayer.Ifthisisthecase,theninthe radiolarianmaze,micewouldhaveneededtoaccessandintegrate bothofthesemapsatonce,whileinthehexagonmazemicewere onlyrequiredtoaccessone horizontalmap,withless cognitive loadandhencefasterlearning.Thattheywereunimpairedonthe short-term(working)memoryversionofthetask(Experiment1) suggeststhatmaybetheproblemiswithstoringandretrievingthe informationacrosstime.Alternatively,perhapswiththeworking memory task it is possible to create a more
path-integration-based motoric representation of visited and not-yet-visited
arms.
Apossibleintermediate betweenone volumetricversus two
planar maps is a surface-coding one: that the mice form one
large, planar map in which the surface of the spherical maze is“unwrapped”and representedin 2Dcoordinates. Inthis sce-nario,thepositioninabsoluteverticalspaceisdisregarded,and the positionof the armsis specified relative to the surface of thesphereandreliesonmetricinformationbeingappliedtothe curvedplaneoflocomotionofthemouse.Wecannotruleoutthis possibilityusing behaviouraldata alone;recordingsof spatially sensitiveneurons—especiallygridcells[8]andheaddirectioncells
[9]—wouldbeneededtotestthesurface-codinghypothesis.Ifsuch werethecase, though,it seemssurprisingthatthere would be suchadifferenceinlearningbetweentheradiolarianandhexagon mazes,unlessthesurface-codedmapissomehowhardertoconsult duetothelessorderlyarrayofarms.
5. Conclusionandfuturework
Forafullunderstandingofspatialcognitionitisimportantto understandhowanimalsrepresentandusespaceinallthreeof itsdimensions;accordingly,thepresentexperimentinvestigated
whethermicecouldlearna3Danalogueofacommon2D
labo-ratoryspatialtask,theradialmaze.Wefoundthattheycanlearn thistaskbutdosowithgreaterapparentdifficulty.Futureworkis neededtofindouthowtheysolvethistask,andwhylearningisless effectivethananapparentlyno-less-complex2Dmaze.Itmaybe thatlearningwasaffectedbytheincreasedlocomotorchallenges imposedbythe3Dtask,apossibilitythatcouldbeaddressedby creatinga2Danalogueoftheradiolarianmazeinwhichthemice needtodevoteanequal amountofattentionandeffortto sim-plymovingaround;forexamplebyintroducingbarriersorgaps or otherimpedimentsthat forcetheanimalsto concentrateon howtheymoveaswellaswheretheyaregoing.The representa-tionalexplanationswillrequiremoresophisticatedstudydesign. Inourview,themostlikelyrepresentationalexplanationisthat miceformtwoplanarmapsofthetwolayersofarms,andneedto switchbetweentheseastheymovebetweenthemwithconsequent interference,butanalternativepossibilityisthattheyusea volu-metricmapwhich—duetoitsincreasedcapacityrequirements—is
simplyharder toformand use. Experiments with2D mazesof
varying complexityand capacity requirementswillbe informa-tivehere,buttherecordingofgridandheaddirectioncells will alsobeneeded,inordertodeterminehowthespatialmapforthe radiolarianmazeisconfiguredin3Dspace.Eitherway,thefinding thatmicecansolvea3Dradialmazetaskindicatesthatthey pos-sessthecognitivemachinerytooperateeffectivelyinacomplex3D world.
Competinginterests
Theleadauthorisanon-shareholdingdirectorofAxonaLtd. Authors’contributions
JWandKJconceivedand designedtheexperiments;JW car-riedoutmostoftheexperimentalwork;EH,MF,BJ,MD,AK,AO andNZcontributedtoexperimentaldesignandcarriedoutsome ofexperimentalwork;JWanalysedthedata;JWandKJwrotethe manuscript.Allauthorsgavefinalapprovalforpublication. Funding
The work was supported by BBSRC (BB/J009792/1), MRC
(G1100669)andWellcome(#083540)grantstoKJ,andaBBSRC
CASE studentship (BB/F015968/1) to JW in collaboration with
AxonaLtd. References
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[3]OltonDS,SamuelsonRJ.Remembranceofplacespassed:spatialmemoryinrats. JExpPsycholAnimBehavProcess1976;2(2):97–116.
[4]Jovalekic A, Hayman R, Becares N, Reid H, Thomas G, Wilson J, et al. Horizontalbiasesinrats’useofthree-dimensionalspace.BehavBrain Res 2011;222:279–88.