ContentslistsavailableatScienceDirect
Behavioural
Brain
Research
j o ur na l h o me p a g e :w w w . e l s e v i e r . c o m / l o c a t e / b b r
Short
communication
Associative
learning
in
the
multichamber
tank:
A
new
learning
paradigm
for
zebrafish
Yohaan
M.
Fernandes
a,
Mindy
Rampersad
a,
Ana
C.
Luchiari
b,
Robert
Gerlai
a,∗aDepartmentofPsychology,UniversityofToronto,Mississauga,Canada
bDepartamentodeFisiologia,UniversidadeFederaldoRioGrandedoNorte,Natal,Brazil
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•Asimple,easytomanufacturelearningapparatuswasdesigned.
•Visualdiscriminationbasedassociativelearningwasstudied.
•Zebrafishwerefoundtoexhibitprocedurallearningandmemory.
•ZebrafishwerefoundtoexhibitacquisitionofCS-USassociation.
•Mazeisarguedtobesimpleenoughforhighthroughputapplications.
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Articlehistory:Received17January2016
Receivedinrevisedform24April2016 Accepted20June2016
Availableonline21June2016 Keywords: Associativelearning Behaviouralphenotyping Colourdiscrimination Memory Zebrafish
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Thezebrafishhasbeengainingprominenceinthefieldofbehaviouralbrainresearchasthisspecies offersagoodbalancebetweensystemcomplexityandpracticalsimplicity.Whilethenumberofstudies examiningthebehaviourofzebrafishhasexponentiallyincreasedoverthepastdecade,theneedisstill substantialforparadigmscapableofassessingcognitiveandmnemoniccharacteristicsofthisspecies. Herewedescribeandutilizeanovelvisualdiscriminationtaskwithwhichweevaluatedacquisitionof CS(colour)-US(sightofconspecifics)associationinadultzebrafish.Wereportsignificantacquisitionof CS-USassociationindicatedbytheincreasedtimethefishspentinandtheincreasedfrequencyofvisits ofthetargetchamberduringaprobetrialintheabsenceofreward.Giventhesimplicityoftheapparatus andprocedure,weconcludethatthenewtaskmaybeemployedtoassaylearningandmemoryinadult zebrafishinanefficientmanner.
©2016PublishedbyElsevierB.V.
1. Introduction
Despiteconcertedeffortsbyalargenumberoflaboratories,the mechanismsoflearningandmemoryarestillnotclearly under-stood.Forexample,thenumberofgenesandgeneproductknown tobeinvolvedinneuronalplasticity([1])isasmallfractionofthe numberofgenesfoundexpressedinthevertebratebrainincluding thezebrafishbrain(e.g[2]).Notably,alargeproportionofthese expressedgenesarenotyetfunctionallyannotated,butare sus-pectedtobeinvolved in mnemonic or cognitiveprocesses. For thesereasons,laboratoryspeciesthatwouldaidgeneidentification vialarge-scalemutagenesisscreenshavebeenproposed[3].The
∗ Correspondingauthorat:DepartmentofPsychology,UniversityofToronto Mississauga,3359MississaugaRoad,RmCCT4004,Mississauga,OntarioL5L1C6, Canada.
E-mailaddress:[email protected](R.Gerlai).
zebrafishisonesuchspecies.Overthepastfewdecadessubstantial amountofgeneticinformationhasbeencollectedandnumerous recombinantDNAmethodshavebeendevelopedforthisspecies [4].
The bottleneckin zebrafish behaviourgenetics research has beenthebehaviouralcharacterizationofthisspeciesandthe devel-opmentofappropriatebehaviouraltestingproceduresandapparati [3,5].Althoughthesequestionsarestillrelativelyunderexplored, thenumberofstudiesattemptingtoaddressthemhasbeenrapidly increasingoverthepastfewyears.Forexample,studiesof learn-ingandmemoryinzebrafishnowshowthatzebrafisharecapable ofactiveavoidance[6],simpleCS-USassociativelearningaswell ascomplexlatentspatiallearning[7].[alsoreviewedin8]. Fur-thermorevariousapparatiincludingtheplus-maze(+)[9,10],the T-maze[11],and theshuttle-box [12] have beendeveloped. In addition,differentstimuliincludinganimated imagespresented onacomputerscreenhavebeenexploredforzebrafishlearning paradigms[13,14].Despitetheserecentdevelopments,thenumber http://dx.doi.org/10.1016/j.bbr.2016.06.038
280 Y.M.Fernandesetal./BehaviouralBrainResearch312(2016)279–284 oflearningparadigmsavailableforzebrafishissignificantlysmaller
comparedtothosedevelopedfortraditionalvertebratelaboratory organismsincludingtheratorthemouse[3].Proper characteriza-tionofchangesinmnemonicand/orcognitiveprocessesinduced bymutationsordrugsmayrequiremultiplebehaviouraltasksthat tapintosimilarcentralnervoussystemfunctionswhilehaving dif-ferentperformance demands[15]. Giventhecomplexnatureof learningandmemoryandtherelativepaucityoftestparadigms thatcanmeasuretheseprocessesinthezebrafish,thereisa sub-stantialneedforthedevelopmentofefficientandrobustlearning andmemorytasksaswellasforthecharacterizationofthese pro-cessesinzebrafish.
Inthecurrentstudy,wedevelopedandtestedanewapparatus thatwe employed toanalyzevisual discrimination,an associa-tivelearningtaskperformedinacheapaquariumretrofittedwith acrylicinserts.Althoughnotnovelinitsconceptualfeatures,the taskhassomeadvantagesand/orcomplementaryfeaturesto cur-rentlyexistingparadigms.Forexample,comparedtotheT-mazeit allowstheinvestigatortostudythesubjects’choicesbetweenmore thantwooptions.Comparedtotheplusorradialmazes,thetest apparatushasasmallerphysicalfootprint,anditisalsoeasierto construct.Last,althoughsimpleintermsofexperimentalsetupand easytoconduct,theshuttleboxparadigmdevelopedforzebrafish wasfoundtoleadtolessthanrobustacquisitionofmemory[12].
Insummary,thepurposeofthecurrentstudyistodetermine whetherthissimplemaze-designinwhichzebrafisharerequired toassociateavisualcue(CS)withareward(alsoavisualstimulus, thesightofconspecifics)maybeemployedefficientlytoinduceand quantifyassociativelearninginzebrafish.
2. Methods
2.1. Animalsandhousing
Sixteenzebrafish(Daniorerio)oftheABstrainwereusedin thisexperiment.Allfishtestedweresexuallymature,6–10months old,youngadults(malesandfemales50–50%).Thefishwerebred andraisedattheUniversityofTorontoMississauga(UTM) vivar-ium.Theywerehousedinhigh-densityracks(AquaneeringInc.,San Diego,CA,USA)equippedwithmultistagefiltrationthatincludeda mechanicalfilter,fluidizedglassbedbiologicalfilter,anactivated carbonfilter,aswellasafluorescentUVlightsterilizingunit.Ten percentofthewaterwasreplaceddailywithdeionizedwater sup-plementedwith60mg/lInstantOceanSeaSalt(BigAl’sPetStore, Mississauga,ON).
During the habituation phase, fish were housed in groups of 8–10, in 2.8l rearing tanks (Aquaneering Inc.) placed in a high-densityracksystem.Afterhabituationfishwereindividually housedforidentificationpurposes,in1ltanksthatwerepartofa high-densityracksystem(AquaneeringInc.).Thewater tempera-turewasmaintainedat26±2◦C.Illuminationwasprovidedby fluorescentlighttubesfromtheceilingwithlightsturnedonat 07:00handoffat19:00h.Fishwerefedamixtureofgroundflake food(4parts,TetraminTropicalFlakes,Tetra,USA)andpowdered spirulina(1part,JehmcoInc.,Lambertville,NJ,USA).
Inthecurrentparadigmweallowedtheexperimentalfishto interactwithagroupofconspecificcalledthestimulusfish.We usedfivezebrafishasstimulus.Innaturezebrafishhavebeenfound toformshoalsranginginsizebetweenonlyafewtohundredsof members.In thelaboratory, varyingthenumber ofmembersof animatedshoalsbetween3–8wasalsofoundnottohavea signif-icanteffectonthestrengthoftheshoalingresponse[16],butin numerouspaststudiesweemployed5stimulusfish[13,14,17,18], andthuswedecidedtousethesamenumberoffishhereaswell. Todistinguish“stimulusfish”fromtheexperimentalsubject,we
injectedtheformerwith0.05lofbluemarkingtissuedye(Sigma Aldrich)nearthecaudalfinasdescribedbefore[19].
All behavioural experiments were video-recorded from an overheadcamera(JVCEverioGZ-MG500,Yokohama,Japan),and laterreplayedforobservation-basedquantificationusingObserver ColourProXT(NoldusInfoTech.,Wageningen,TheNetherlands). 2.2. Experimentalmaze
The new maze (Fig. 1) was made of Plexiglas, rect-angularly shaped, measuring 60cm×47cm×25cm (length×width×height). While designing the maze, we kept simplicityinmind.Forexample,theinteriorconstructionofthe mazeutilizesacrylicinsertsthatarerectangularanddonothave tobegluedincomplicated shapeor angles.Furthermore,these insertsmaybeloweredintoastandard40lpetstorevarietytank madeofglassandthusunlikeinYorT-mazesdonotrequire preci-sionwater-tightmanufacturing.Alongthelengthofthemaze,on eithersidewerefourchambers(15cm×10.5cm×25cm),which wereseparatedbyanopencompartment(60cm×25cm×25cm). Onesidecontainedthefourstartchambers,labeledonethrough four. Directly opposite the four start chambers were the four targetchambers.Eachtargetchamberhadonecoloured(yellow, blue,red,orgreen)removablecuecard.Thechoiceofcuecolour wasbasedonpreviousresearch[20].Thecolourcuemarkingthe chamberwherethestimulusfishwereplacedwedesignatedas CS+,whiletheothercolourcuesthatmarktargetchamberswithout theUSarereferredtoasCS0.Thetargetchamberswereaccessible fromtheopenareathrougha9cmtubethatconnectedeachtarget chamber to the open compartment. The openingbetween the tubeandthetargetchamber wasclosedbya transparentdoor, whichallowedtheexperimentalsubjecttoseethestimulusfish (the unconditionedstimulus,US) and thecuewhile preventing thestimulusfish fromleaving thetarget chamber. Atthetube entrance,whichconnectedtotheopencompartmentwasaslot foraseconddoor.Theseconddoorwasplacedattheentrance, oncetheexperimentalsubjectenteredthetube.
2.3. Procedure
The experimental procedure had three phases: habituation, training,andtheprobe.Duringthehabituationphasethe experi-mentalfishwereplacedinthemazeingroups,andthesizeofthese groups graduallydecreasedfrom 16(firstday,one 20minlong explorationsession),to8(secondday,twoseparate10min explo-rationsessions),to4(thirdday,twoseparate10minexploration sessions),to2(fourthday,fourexplorationsessionof5mineach) to1(fifthday,four5minlongexplorationsessions).Foreach habit-uationtrial,thefishwere/wasplacedinadifferentstartchamber. Duringthehabituation/explorationsessions,onlythestart cham-berfromwhichthefishwerereleasedremainedopen,whilethe otherthreewereclosed.Allrewardchamberswereopen.
Following the habituation, experimental fish entered the training phase. Experimental subjects were housed in the behaviouraltestingroom.Ablackmetaldivider(120cm×180cm, width×height) blockedtheviewof themazefromtheholding tanks.Fishwerenettedfromtheirholdingtank,thenetwiththe fishwasplacedintoa500mlbeaker,andthefishweretransported whileimmersedintothebeakerwiththenettotheexperimental tank.Theexperimentalfishwasplacedsinglyintothestart cham-berofthetestapparatusandwasreleasedbyraisingtheguillotine door.Alltargetchamberswereopenandaccessibletothe experi-mentalfish.Oneofthetargetchamberscontainedastimulusshoal, i.e.thereward(US).Atransparentdoorpreventedthesefishfrom leavingthetargetchamber.Theparticularcolourcue(CS+) mark-ingthelocationofthetargetchambercontainingthestimulusfish
Fig.1. Theblue-printoftheexperimentaltankshowingthedifferentcompartments(startchambersandcolourcuemarkedtargetchambers)fromabove(A)andfromthe side(B).Thedimensionsofthemazeareindicatedincm.Thecolourdesignationsrepresentanexample.Thecolourcuesmaybemovedatwill.Thetunnelsareshorttubes throughwhichtheexperimentalsubjectscouldswimintothecolourcuemarkedtargetchambers.
remainedconstantforeachexperimentalfishacrossall20trials butwhichcolourcuewasusedasCS+ variedacrossthe experi-mentalfish.Theotherthree(non-rewarded)chamberswerealso markedbyacolourcue(CS0)butthecoloursdifferedfromCS+. Therewerefourstartinglocations.Duringthecourseofatraining dayeachexperimentalfishreceivedfourtrainingsessions,starting oncefromeverystartinglocation.Thetrialended5minafterthe startchamberwasopened.Eachexperimentalfishwasgiven20 trainingtrialsintotal,i.e.four,5mintrialsperdayoverthecourse of5consecutivedays.
Experimentalfishwererewardedbybeingabletostayinclose proximityoftheshoal.Theamountoftimetheexperimental sub-jectspentshoalingwasdependentuponhowfasttheylocatedthe shoal.Iftheexperimentalsubjectdidnotfindtheshoalduringthe trialtheydidnotreceiveanyreward.Toentertherewardchamber experimentalsubjectshadtoswimfromtheopencompartment throughthe9cmtubeintotherewardchamber.Oncethe experi-mentalsubjectenteredthe9cmtube,adoorwasfirstplacedatthe tube’sentrance,andsubsequentlythedoortotherewardchamber wasopened.
Duringtheprobetrial,alltargetchamberswiththeircolourcue cardswereopen.Noreward fish(stimulusshoal)werepresent. Theprobetrialbeganwithraisingthedoorofthestartchamber, andended 5minlater.Eachexperimentalfishreceivedasingle probe trial.Anoverhead digital camera(JVC EverioGZ-MG500, Yokohama,Japan)recordedthetrialsforbehaviouralanalysis.To minimizethepossibilityofstraight-linedirectswimmingintothe previouslyrewardedtargetchamber,duringtheprobethestart chamberandthepreviouslyrewardedtargetchamberwerealways twochambersawayfromeachotherforallexperimentalfish.
2.4. Quantificationofbehaviourandstatisticalanalysis
Thedigitallyrecordedvideo fileswereanalyzedbyatrained individual using an event recording software application, The ObserverXT9.0(Noldus,InfoTech.,Wageningen,TheNetherlands). During training we measured thelatency tolocate the reward
chamber,and duringtheprobewemeasuredthepercentageof timespentinthereward chamberaswellasintheotherthree chambersoftheexperimentaltank.Thetimespentintheselater chamberswereaveragedandcomparedtothetimespentinthe targetchamber.
Data were analyzed usingSPSS (version21) for the PC. We employed a repeatedmeasure analysisof variance (ANOVA) to examinetheeffectofTrainingDay(repeatedmeasurewith5levels) onthelatencytoenterthetargetchamber,ameasureweargued maybeusedtoconcludeaboutprocedurallearningduringtraining aswellasaboutmotivationandmotorabilityoftheexperimental subjects.Inaddition,wealsoinvestigatedwhetherthelatencywas reducedastraining progressedcomparingperformanceoneach possiblepairsofdaysusingonetailedrepeatedmeasuresANOVA (twolevels)withBonferronicorrectionformultiplecomparisons.
Todeterminewhetherfishformedanassociationbetweenthe colour cue and the stimulus (reward) shoal we measured the performance ofthefish intheprobe trialduringwhich theUS (conspecificstimulusfish)wasabsentandcomparedthepercent oftimeexperimentalfishspentinthetarget(previouslyrewarded) chamberandtheaverageofthepercentoftimespentinthethree other(non-rewarded)chambersusingapairedtwo-tailedt-test withsignificantdifferenceacceptedwhenp<0.05.Weperformed asimilaranalysisforthefrequencyofvisitstothetarget (previ-ouslyrewarded)versusvisitstotheotherthree(non-rewarded) chambers.Inaddition,wealsocomparedhoweffectiveaCSthe differentcolourswere,i.e.wecomparedthetargetchamber pref-erence(boththepercentoftimeinandthefrequencyofentrytothe targetchamber)acrossfishtrainedwiththefourdifferentcolour cuesusingunivariateANOVAfollowedbyTukeyHSDPosthoctests whenwarranted.
3. Results
Fig.2showsthelatencytoreachtherewardchamberacross thefivetrainingdays.Trainingdaywasfoundtohaveasignificant effectonthelatencytoreachtherewardchamber(F(4,60)=4.234,
282 Y.M.Fernandesetal./BehaviouralBrainResearch312(2016)279–284 1 2 3 4 5
Training day
60 80 100 120 140 160 180La
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Fig.2. Thelatencytoenterthechambercontainingthestimulusshoal(rewarded targetchamber)significantlydiminishesbythefifthdayoftraining.Mean±S.E.M areshown.n=16. 0 5 10 15 20 25 P e rc en t of tim e
A
Non-rewarded chamber average Rewarded chamber 0 2 4 6 8 10B
Non-rewarded chamber average Rewarded chamberFig.3. Thepercentoftimeexperimentalfishspentinthechambermarkedbythe correctcolourcue(CS+)(PanelA)andthenumberofentries(frequency)tothis
chamber(PanelB)aresignificantlyhighercomparedtotheaveragepercentoftime inandtheaveragenumberofentriestothecambersmarkedbytheothercolour cues(CS0)duringtheprobetrial.Mean±S.E.M.areshown.Theblackbarsrepresent
thevaluesobtainedintherewardedtargetchamber,whereastheaverageofthe performancemeasuredintheotherthreechambersareshownbythegreybars. n=16.Notethatduringtheprobetrial,theUS(thestimulusshoal)wasnotpresent intheexperimentalapparatus.
p=0.004).Posthoctwo-levelrepeatedmeasureonetailedANOVA withBonferronicorrectionrevealedthatthelatencytoreachthe targetchamberwassignificantlysmalleronday5 comparedto allprevioustrainingdays (F(1,15)=8.955,Bonferroni corrected p<0.05)exceptcomparedtoday4.
Fig.3,panelA,showsthepercentageoftimetheexperimental fishspentinthechambermarkedbythecorrectassociativecue (CS+)versustheaverageofthepercentoftimetheyspentinthe otherthreechambersmarkedbytheothercolourcues(CS0) dur-ingtheprobetrial.Notethatduringthistrial,theUS(theconspecific stimulusfish)wasabsent.Thepercentoftimefishspentinthe tar-getchamberinthevicinityoftheCS+,versustheaveragepercentof timetheyspentintheotherthree(non-rewarded)chambersinthe vicinityoftheincorrectcues(CS0)wasfoundsignificantlydifferent
(t=2.610,df=15,p=0.02).Fig.3,panelBshowsthenumber (fre-quency)ofvisitstothetargetchambermarkedbyCS+versusthe averageoftheentriestothethreeother,previouslynon-rewarded, chambersmarkedbyCS0.Thedifferencebetweenthenumberof entriestothesetwo partsofthemazewasfoundsignificantby apairedtwo-tailedt-test(t=2.249,df=15,p=0.04).Alsonotably, comparisonofperformanceoffishtrainedwiththefourdifferent colourcuesasCS+foundnosignificantcolourcueeffectforthe per-centoftimefishspentinthepreviouslyrewardedtargetchamber (F(3,12)=2.587,p>0.10)orforthefrequencyofvisitstothistarget chamber(F(3,12)=2.697,p>0.05)duringtheprobetrial.
4. Discussion
Thesmallnumberofbehaviouralassaysavailableforzebrafish is considereda bottleneckfor using this species in thefield of behaviouralneuroscience[5,8],aproblemespeciallynoticeablein thelightofthevastnumberofgenetictoolsavailableforthisspecies [4].Furthermore,analysisoflearningandmemoryoftenrequires multipletestparadigms.Arguably,asinglelearningtestisnever sufficientforproperinterpretationofpotentialmutationordrug effectsoncognitiveand/ormnemonicprocessesbecause numer-ousperformancefeaturesidiosyncratictotheemployedtaskmay beaffectedbythegeneticorthepharmacologicalmanipulation. Giventhattheseeffectsarenotknowntotheexperimenter,the useofmultiplelearningtaskswithdifferentperformancefeatures havebeenrecommended[3,15].Here,wepresentedandemployed asimpleapparatusthatcanbeusedtomeasurelearningand mem-oryinadultzebrafish.Theparadigmisnotnovelconceptuallyas itisbasedupontheprinciplesofcolourdiscriminationlearning,a typeofassociativelearningthatrequirestheanimaltodistinguish betweenreinforced(CS+)andunreinforcedvisualcues(CS0). How-ever,wearguethattherelativeeasewithwhichtheapparatuscan bemanufacturedandusedmaymakeitausefuladditiontoalready existinglearningtasks.
Usingthenewly developedapparatus, wefoundthatbythe fourthtrainingday,experimentalsubjectsstartedtoreducetheir latencytoenterthetargetchamber,andbythefifthtrainingday thislatencyreductionbecamesignificant.Notably,during train-ingtheconspecificstimulusfish(the rewardingstimulus)were alwayspresent.Theirsightandpotentiallytheirsmellaswellas auditorycueswereallaccessibletotheexperimentalfish.Thus, thereductionoflatencytoenterthetargetchambercontaining thestimulusfishduringtrainingcannotbeinterpretedasasignof acquisitionoftheassociationbetweenthecolourcue(CS+)andthe presenceofthestimulusfish(US).Althoughacquisitionofthe CS-USassociationmighthavecontributedtothedecreaseoflatency toentertherewardedchamber,thelatencyreductionislikely bet-terexplainedbyprocedurallearning,i.e.acquisitionofinformation requiredforthefishtoperformwellinthemazeingeneral.This informationmayincludegettinghabituatedtohumanhandling, thegenerallayoutofthemaze,themovementsthesubjecthadto performwhenswimmingoutfromthestartchamberthrougha shorttunneltothestimulusfish,etc.Inaddition,thedecreasing entrancelatencyalsosignifiesthattheexperimentalfish contin-uedtobemotivatedtoactivelymoveinthemazeandseekoutthe rewardedtargetchamber.Thisisanimportantpointasitsuggests themotivationtofindandtomovetoclosephysicalproximityto shoalmates(thestimulusfish)didnotdiminishthroughoutthe training.Thismotivationalfeatureofthetaskissomewhatnovel comparedtoY,T,+orradialarmmazesinwhichfoodisutilized asrewardforzebrafish[21].Wedecidedtouselivestimulusfish, insteadofmoretraditionalreinforcers(e.g.food)forseveral rea-sons.Previously,Al-Imari&Gerlai[22]showedthatthesightof conspecificsisrewarding.Subsequentstudiesalsorevealedthat
behindthisrewardliesthefunctioningofthedopaminergic sys-tem[17,23,24].Thestrongmotivationofasingleexperimentalfish toseekoutandjoinashoalinanovelenvironmentisnotsurprising giventhatzebrafishformshoalsinnatureandinthelaboratory[25]. Thus,hereweemphasizethattheuseofastimulusshoalhasbeen foundtobeanexcellentmotivatorinzebrafishlearningstudiesas thisstimulusrepresentsanon-satiatingreinforcerthatrequiresno priordeprivation(e.g.restrictedfeeding)thatmayhave deleteri-oushealthconsequences[12].Theproblemwithlivestimulusfish, however,isthatsuchastimulusisinherentlyvariable.Weused stimulusfishthathadbeenwellhabituatedtotheexperimental procedure,andforthisreasonweexpectedthesestimulusfishto behaveinfairlystableandconsistentmanner.Nevertheless,we couldnotpreciselycontrolthebehaviourofthestimulusfish,and thuslikelycouldnotfullyeliminatetrialtotrialvariationintheir behaviour.Suchcontrolwouldonly bepossiblewiththeuseof artificialstimuli,e.g.computeranimatedimagesofzebrafish. Com-puteranimatedimageshavebeensuccessfullyutilizedforinducing strongshoalingresponses[23,26,27]aswellasforacquisitionof shortandlongtermmemoryinzebrafish[13,14].Whetherthis artificialstimulusisappropriateforthecurrentparadigmwillbe empiricallyansweredinthefuture.
MemoryoftheacquisitionofCS-USassociationmaybe eval-uated from the probe trial results. During the probe trial,the rewarding stimulus(US) wasabsent, yetthe experimentalfish spentdisproportionatelyhighpercentageoftimeinthechamber thatcontainedtheCS+.Zebrafishisafastswimmingfish;innature andinthelaboratorytheydonothavestableterritories.Thus,we expectedzebrafishtoactivelyexplorethemaze.Theincreasedtime theyspentinthetargetchambermarkedbyCS+isnotbecausethey remainedinthischamberoncetheyfoundit,butratheritisdue tothesignificantlyincreasednumberofvisitstothischamberas comparedtothevisitstotheotherchambers.Thislatterfinding demonstratesthatthroughouttheprobetrial,zebrafishshoweda consistentpreferenceforthechamberassociatedwiththecorrect colourcue.Thus,weconcludethatexperimentalfishsuccessfully acquiredtheassociationbetweentheCS+andUS.
Whatthebasisofthisassociationis,wedonotknow.Itisnotable thatintrinsic(prior-trainingorinnate)colourbiasisunlikelyto explainthepost-traningCS+preference,becausethecolours des-ignatedastheCS+variedacrossthedifferentsubjects.Ouranalysis alsoindicatedalackofsignificantdifferenceintheperformance offishastowhichcolourservedasCS+.Nevertheless,becausethe colourcuesremainedinafixedpositionthroughouttrainingand theprobe,theexperimentalsubjectcouldlocatethereward (tar-get)chamberinthreeprincipallydifferentways:one,colourcue −rewardassociation;two,place−rewardassociation;andthree, ego-centriccue−rewardassociation.Previously,Karnik&Gerlai [28]showedthatzebrafishcanlearntwodistincttypesof associ-ationsduringtrainingdemonstratingthat,similarlytomammals withintacthippocampalfunction,thisfishspeciesiscapableof simultaneousacquisitionofnon-conflictingspatialcuesaswellas aseparatesingleassociativecue.Thecurrentparadigmwillallow theinvestigatortoexplorethesequestionsanddissociatetheeffect ofsuchcues.Forexample,onecouldtrainthefishtofinda partic-ularlocationwithorwithouttheseparatesingleassociativecue markingthelocation,and/oronecouldconflictspatialandsingle associativecuesduringprobe trial,andonecouldperformsuch studiesforego-centriccuesvs.othercues.
Althoughothermazesandassociativelearningtaskshavebeen successfullyemployed,wearguethatthecurrenttestapparatus andparadigmwillhaveaplaceinthetoolboxofzebrafishlearning andmemorymethods.Therearetwoprincipallydifferentreasons forthisargument.Oneispractical,andtheotherismore funda-mental.First, thepracticalreason whywehopethis newmaze willbeusefulisthatitsdesignissimpleandthusthemazecanbe
manufacturedeasilyandcheaply.Thechambers,tubes,anddoors requireminimalengineeringandexpertisetomake,andallthese piecesmaybeloweredintoastandardglassaquarium.Importantly, unlikecomplexY,T,ormultiplearmmazes(+orradialarmmazes), themanufacturingofourapparatusdoesnotinvolvehavingtocut multi-angledcomplexshapes,and alsodoesnotrequire water-tight construction (thestructure is inside a regular glasstank). Thephysical barriersforcetheexperimental fishtomake clear choicesthatareeasytoquantifyusingobservation-basedor video-trackingmethodsbothfroma sideandfromanoverheadview. Theabilitytoclearlyseethefishfromthesideisanaddedbonus becausethisviewenablestheinvestigatortoobserveand quan-tifynumeroussubtlebehaviouralresponsesincludingfinerection displays(aggression),erratic movement(fear),and othermotor patternsdescribedforthezebrafish[29],whichwouldaid inter-pretationofresults.Last,theapparatusissmall,andconducting behaviouraltestsusingmultipletanksissimple.Parallelrunningof multiplelearningapparatifacilitateshigherthroughput,andthus theparadigmmaybeusefulfordrugormutationscreens.The sec-ond,morefundamental,reasonwhywehopethissimpletaskmay beusedintheanalysisoflearningandmemoryinzebrafishisthatit differsfrommostotherlearningtasksinitsproceduraldemands.It isanappetitivetask,butitusesconspecificsastherewardinsteadof themoretraditionalfoodreward.Performanceinthetaskrequires activeswimmingsimilarlytomostothermazesutilizingappetitive conditioning,butunlikeinT,Y,+ormulti-armmazes,thedistances zebrafishneedtotraverseinthecurrentapparatusaresmall,and thefisharerequiredtonavigatethroughtightspaces,making ego-centriccuebasednavigationsomewhatdifficult.
Weacknowledgethatmanyoftheaboveargumentsare specu-lativeatthispoint,andthattheirvaliditywillhavetobeascertained by empiricalstudiescomparingdifferent learningparadigms in asystematicmanner.Nevertheless,giventhesimplicityand ver-satilityof thecurrent paradigm,wehopethatitsutilitywillbe recognized,anditwillbesuccessfullyusedinmutationanddrug screensleadingtobetterunderstandingofthemechanisms under-lyingthecomplexprocessesoflearningandmemory,theultimate goalofthecurrentstudy.
Acknowledgment
SupportedbyNSERCDiscoverygrant(311637)toR.G.
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