ContentslistsavailableatScienceDirect
Behavioural
Processes
j ou rn a l h o m e pa ge :w w w . e l s e v i e r . c o m / l o c a t e / b e h a v p r o c
Time
place
learning
and
activity
profile
under
constant
light
and
constant
dark
in
zebrafish
(Danio
rerio)
Clarissa
de
Almeida
Moura,
Jéssica
Polyana
da
Silva
Lima,
Vanessa
Augusta
Magalhães
Silveira,
Mário
André
Leocadio
Miguel,
Ana
Carolina
Luchiari
∗DepartamentodeFisiologia,CentrodeBiociências,UniversidadeFederaldoRioGrandedoNorte,Natal,RN,Brazil
a
r
t
i
c
l
e
i
n
f
o
Articlehistory:
Received19July2016
Receivedinrevisedform
27November2016
Accepted16February2017
Availableonline20February2017
Keywords: Time-placelearning Circadianrythym Chronotype Constantlight Constantdark
a
b
s
t
r
a
c
t
Theabilitytolearnaboutthesignsofvariabilityinspaceandtimeisknownastimeplacelearning
(TPL).Toadjusttheircircadianrhythms,animalsusestimulithatchangeregularly,suchasthelight-dark
cycle,temperature,foodavailabilityorevensocialstimuli.Becauselight-darkcycleisthemostimportant
environmentaltemporalcue,weaskedhowadiurnalanimalwouldperformTPLifthiscuewasremoved.
Zebrafishhasbeenextensivelystudiedinthechronobiologyareaduetoitdiurnalchronotype,thus,we
studiedtheeffectsofconstantlightandconstantdarkonthetime-placelearningandactivityprofilein
zebrafish.OurdatashowthatwhileunderconstantlightanddarkconditionzebrafishwasnotableofTPL,
after30daysundertheconstantconditions,constantlightledtohigheractivitylevelandlesssignificant
(robust)24hrhythm.
©2017ElsevierB.V.Allrightsreserved.
1. Introduction
Theavailabilityoffood,sexualpartners,predatorsandother
bio-logicallyrelevantstimulivarybothintimeandinspace(Carrand
Wilkie,1997).Toprocesstemporalandspatialinformation,animals
useexternalcuesthatvaryregularly,suchaslightand
tempera-ture,toadjusttheinternalclockandestimatetime(Aschoff,1954;
Dunlap,1999;Reebs,2002;Kuhlmanetal.,2015).Theabilityto
learnaboutthevariabilityofsignsinthespaceandtimeisknownas
timeplacelearning(TPL).AccordingtoGallistel(1990),the
occur-renceofabiologicallysignificanteventpromotestheformationof
amemorycodethatincludesthetypeofevent,thetimeandplace
oftheoccurrence.Thisabilityisrelatedtotheconnectionbetween
theinternalcircadiansystemandassociativememory(Anokhin,
1974).
Duetothe24hdurationofthesiderealday,light-darkcycleis
themostremarkablezeitgeber(Kuhlmanetal.,2015);themajority
oftheanimalspresentphotoreceptivecells,andthuscanperceive
lightfluctuationsthroughouttheday(Bell-Pedersenetal.,2005).
∗ Correspondingauthorat:DepartamentodeFisiologia,CentrodeBiociências,
UniversidadeFederaldoRioGrandedoNorte,POBOX1511,59078−970Natal,Rio
GrandedoNorte,Brazil.
E-mailaddress:[email protected](A.C.Luchiari).
However,underconstantlightconditions(24hlightor24hdark),
theorganismsstillmaintainrhythmicity,guidedbyendogenous
regulatorsofthebiologicalcycle(Johnsonetal.,2004;Wegeretal.,
2013).Therefore,eveninfreerunningconditioning,asetof
self-regulatedmolecularmechanismsgeneratesthecircadianrhythm
throughgeneexpression(Amaraletal.,2014),andallows
organ-ismstopredictandanticipateeventsthatoccurwithinaperiodof
24h(KoukkariandSouthern,2006).
Amongthestudiesonlearningrelatedtothecircadianrhythm,
beeswerethepioneerstoshowTPL(Wahl,1932;Finke,1958),
suggestingtheypossessacircadianoscillatorthatallowsfor
mon-itoringtime(Pittendrighetal.,1958).Afterthesestudies,several
othershavefoundsignsofbothtemporalandspatiallearninglinked
totheendogenousclocks(Kramer,1950;BoulosandLogothetis,
1990;Reebs,1999;Gomez-LaplazaandMorgan,2005;Heydarnejad
and Purses, 2008). In additionto abioticzeitgebers thatfavour
rhythmicityandlearning,recurrentvisual,olfactory,auditoryor
tactilesignalsfromoneindividualtoanothercanentrainan
ani-malactivity/restcycle,andthusbeconsideredasocialsynchronizer
(RajaratnamandRedman,1999).Severalsocialspecieshavetheir
rhythmsinfluencedbysocialcues,suchasrodents(Crowleyand
Bovet,1980;Mrosovsky,1988)andprimates(ErkertandSchardt, 1991;Meloetal.,2013).However,evenbeingconsidereda
syn-chronizer,thereisnoevidencethesocialstimulicanactlonelyas
azeitgeber,withoutthemostoutstandingsignalthatisthe
light-http://dx.doi.org/10.1016/j.beproc.2017.02.015
darkcycle.Knowingthatzebrafish(Daniorerio)isahighlysocial
species(Pritchardetal.,2001;Larsonetal.,2006;Gerlai,2014)that
presentsTPLresponsebasedonsocialreward(MouraandLuchiari,
2016),theaimofthisstudywastotesttheTPLabilityofzebrafish
intheabsenceoflightsignals(constantlightandconstantdark).In
thiscase,socialstimuli wouldbetheuniquezeitgeber,andfish
wouldhave touseit toestimatetime and adjust thecircadian
rhythmintheabsenceoflightsignals.
Zebrafishisconsideredapromisinganimalmodel,bothforits
highpracticalityofstorageand maintenanceasthehigh
physi-ologicalandbehaviouralsimilaritytomammals(Ingham,2009),
allowingtranslationalstudies.Inadditiontotheseadvantages,the
zebrafishhasbeenextensivelystudiedinthechronobiology(Vatine
etal.,2011)becauseofit diurnalactivitypattern(Paciorekand
Mcrobert,2012),whichfavoursitstranslationalresearch,opposite
torodentsthatarenocturnal.Inthissense,weusethezebrafish
tostudytheeffectsofconstantlightorconstantdarkforthe
time-placelearning,offeringsocialstimulusaszeitgeber andreward.
Ourhypothesisarethat(1)socialstimuluscanactasa
synchro-nizerelement,allowingrhythm,and(2)fishwillshowTPLdueto
theestimationontimegivenbythesocialstimuliandestimation
ofplacegivenbythelocationofthestimuli.
2. Materialandmethods
2.1. Animalsandprocedures
ZebrafishDaniorerio(Hamilton,1822) wereobtainedfroma
localfishfarm(Natal,RioGrandedoNortestate)andkeptin
stock-ingtanks(2fish/L)withairedandfilteredwater.Four50Ltanks
makeuponestockingunitintheclosedwatercirculationsystem,
withmechanical,biologicalandchemicalfiltration,inadditionto
UVdisinfection.Waterwasmaintainedat28±1◦C,withpH7.2
andlowlevelsofammoniumandnitrite.Thelightcycle
(fluores-centlight,150Lux)wasfixedatlight-dark(12:12LD),withthe
startofthelightphaseat7am.Thefishwerefedcommercial
pel-letstwiceaday(38%protein,4%lipids,NutricomPet)andArtemia
salina.
Eighteenadultzebrafishofbothsexesfromtheaforementioned
stockwereusedtotesttime-placelearning.Alltheprocedureswith
theanimalswereauthorizedbytheAnimalEthicsCommitteeof
UniversidadeFederaldoRioGrandedoNorte(CEUA039/2015).
2.2. Experimentaldesign
Theexperimentalanimalswereindividuallytransferredto
test-ing tanks (100×25×25cm; length x width x height), divided
horizontallyintothreesame-sizecompartments(33cmlong):one
centraland twolateral(sameprocedureofMouraandLuchiari,
2016).Thecompartmentswereseparatedbyopaquedividers,each
withan8cm-diameter circularpassagethatallowedthefishto
swimbetweenthecompartments.Thepassagewaslocatedonthe
rightoftherightsidedividerandontheleftoftheleftsidedivider,
suchthatthefishcouldnotvisualizemorethantwocompartments
atthesametime,therebypreventingthestimulusplacedinone
ofthesidecompartmentsfrombeingseenwhentheanimalwas
intheoppositesidecompartment.Acylindricalopen-front
recep-tacle(10cmindiameterand10cmhigh)wasfixedtotheupper
partofthelateralwalls,andusedtoofferthestimulus(conspecific
group)atspecifictimes.Thesidecompartmentswererandomly
denominatedmorningcompartmentandafternooncompartment.
Eachtankwasconstantlyaeratedthroughanexternalfilter(JEBO
50,250L/h)locatedinthecentralcompartmentandairstonesin
eachsidecompartment.
Animalswere keptfor 30days under the above
experimen-talconditions.Agroupof5zebrafish (samesize andage)were
introducedeverydayintothereceptaclelocatedinthemorning
compartmentat8amandremovedat9am,andintothereceptacle
oftheafternooncompartmentat5pmandremovedat6pm,acting
asastimulusfortheexperimentalfishtooccupythecompartment
wherethegroupwasplaced.Thegroupwasintroducedthrougha
receptacle(500ml)connectedtoahandle(2m)sothatthe
experi-mentercouldnotbeseenbytheanimals,whichwereseparatedby
anopaquecurtain.Food(artemia)wasoffereddaily(onceaday)
atrandomtimesbetween10amand4pm,alwaysinthecentral
compartmentsofoodwouldnotbeassociatedwithanystimulus
ortime.
Toverify if theTPL occursin constant light conditions, two
groupsweretested:constantlightgroup(LL;n=8)andconstant
darkgroup(DD;n=10).Inconstantlightgroup,theanimalswere
exposedtoconstantlight(150lux)duringthe30-dayexperiment
andbehaviouralwasrecord.Theconstantdarkgroupfollowedthe
sameprotocol,butwithanimalsexposedtothetotalabsenceof
lightduringthe30days.
Ondays15and30oftheexperimentalperiod,thebehaviour
oftheanimalswasrecordedonvideofor1hand15min,starting
at7:45amand4:45pm,inordertoobserveanimalsfor15min
beforethearrivalofthegroup(stimulus)andduringtheirentire
presence.Behaviouronday15wasrecordedinthepresenceofthe
grouptoestimatethestrengthofthisstimulus.However,onday
30,theanimalswererecordedwithoutthepresenceofthegroup,
inordertoassessTPLintheexperimentalzebrafish.
Forthevideorecords,weusedahandycamcorder(Sony
DCR-SX45DigitalVideoCameraRecorders)placed1.5mawayandin
frontofthetanks.Thebehaviouralanalyseswereconductedusing
theZebTracksoftware,developedinMatLab.Thefollowing
param-eterswereassessed:residencetimeandfrequencyofentryinthe
morningandafternooncompartments.
2.3. Activityregistry
Fromthe8fishundertheLLconditionand10fishundertheDD
condition,4fishofeachgroupwerealsorecordedduringthelast
6daysoftheTPLexperiment.Another4zebrafishfromthestock
conditionwereusedtocomposetheLDcondition,inordertohavea
controlgroup.These12zebrafishwereusedtoevaluatetheeffects
ofconstantlightconditionsontheactivitypattern.Fishheldunder
light-dark(12:12LD;n=4)werealsosubmittedtotheTPLtest,in
ordertohavethesameconditionsoftheothergroups.Theactivity
ofeachfishwasrecordedusingSonyKitinfraredsecuritycameras
CCD,coupledtotheDVRunit,for144h(thelast6consecutivedays
ofthe30TPLdays).Thebehaviourrecordswereanalysedusingthe
ZebTracksoftware.Weconsideredtheaveragespeed(cm/s)ofeach
fishevery15minofthe144h.Thedatawereplottedondiagrams
ofactogram,cosinorandwaveform.
Actogram is a graphical representation of activity (average
speed,yaxes)along24hlengthofeachplotline(xaxes),and
suc-cessivecyclesareplottedbeloweachother.Thecosinor(Halberg
etal.,1967)isamodeltoanalysethebiologicalrhythmsconsisting
ofcosinecurveswithknownperiods(inourstudy,24h)toestimate
rhythmicparametersandthepatternofthesmoothrhythm.Each
pointofacosinusoidalcurveofacosinorisafunctionofthe
aver-agevalueofthevariableofinterest.Thesevariablesare:MESOR
(M,MidlineEstimatingStatisticofRhythm:therhythm-adjusted
meanthatdiffersfromthearithmeticmeanwhenthedataarenot
equidistantand/ordonotcoveranintegernumberofcycles),the
amplitudeoftheoscillation(A),andtheacrophase(,timeatwhich
thepeakofarhythmoccurs)(Refinettietal.,2007).Thewaveform
istheprototypicalcycle ofa rhythm,definedbytheamplitude
accountforthenon-sinusoidalityofthesignal).Then,thewaveform
canbeconsideredanextendedcosinoranalysisininferential
sta-tisticalchronobiology(Refinettietal.,2007).TheSokolove-Bushell
periodogramanalysiswasalsodevelopedtodeterminecircadian
rhythmicity.
2.4. Statisticalanalysis
Data were analysed for normality(Shapiroand Wilk, 1965;
Doornik and Hansen, 2008) and homoscedasticity (Brown and Forsythe,1974;Anderson,2003)andparametrictestswereused
duetoitsnormalandhomoscedasticdistribution.Behaviouraldata
forresidencetime inthecompartmentsandfrequencyof entry
ineachcompartmentwerecomparedinthemorningand
after-noon,ondays15 and30, usingthepairedstudent’sT-test. We
excludedthedatafromthecentralcompartment,becauseitwas
apassageareaandfeedingsite atrandomtimes,thus, the
ani-malcouldvisitthisareatopassfromthemorningcompartment
totheafternoonortosearchforfood.Theaveragespeeddatafor
activityregistryforthelast6daysofTPLexperimentwere
com-paredbetweenLD,LLandDDgroupsusingtheone-wayANOVA.To
verifytheacrophaseofeachexperimentalgrouptheRayleightest
andWatson-Williamstestwereused(circularstatisticalanalysis).
TheperiodogramresultswereBonferronicorrected,andone-way
ANOVAwasusedtocomparethegroups.
3. Results
3.1. Constantlight(LL)
On the day15, during the 15min beforethe groupof
con-specificsarrived,therewasnosignificantdifferenceinresidence
time betweenthemorning andafternoon compartmentsinthe
morning(Student’st-test,t=1.20p=0.27)orafternoon(Student’s
t-test,t=−0.28p=0.79),respectively(Fig.1a).Thefrequencyofentry
intothecompartmentsdidnotdifferinthemorning(Student’s
t-test,t=1.21p=0.26;Fig.1c),neitherintheafternoon(Student’s
t-test,t=−0.61p=0.56;Fig.1c).
Duringpresentationofthegroup,residencetimeinthemorning
compartmentwashigherinthemorning(Student’st-test,t=8.82
p<0.001),andintheafternoonitwashigherintheafternoon
com-partment(Student’st-testt=−4.99p=0.002)(Fig.1b).Itwasfound
higherfrequencyofentryinthemorningcompartmentduringthe
morning(Student’st-test,t=4.32p=0.003),butitdidnotdifferin
theafternoon(Student’st=−0.36p=0.73)(Fig.1d).
Ontheday30,inthe15minbeforethegroupwasintroduced
intothetank,therewasnodifferencebetweenthetimespentin
eachcompartmentinboththemorning(Student’st-test,t=−1.22
p=0.26) and the afternoon (Student’s t-test, t=−0.01 p=0.99;
Fig.2a).Thefrequencyofentrieswashigherinthemorning
com-partmentinthemorning(Student’st-test,t=3.75p=0.007),butit
didnotdifferintheafternoon(Student’st-test,t=−1.22p=0.26;
Fig.2c).
Duringthe60minthatthegroupshouldbepresented(absence
ofthestimulus),thefishremainedforalongertimeinthe
after-nooncompartmentbothinthemorning(Student’st-test,t=−3.41
p=0.01)andafternoon(Student’st-test,t=−2.37p=0.05;Fig.2b)
times.Thefrequencyofentrydidnotdifferinthemorning
(Stu-dent’st-test,t=0.97 p=0.36) orthe afternoon(Student’st-test,
t=−1.87p=0.10;Fig.2d).
3.2. Constantdark(DD)
On the day15, during the 15min beforethe groupof
con-specificsarrived,therewasnosignificantdifferenceinresidence
time betweenthemorning andafternoon compartmentsinthe
morning(Student’st-test,t=0.40p=0.70)orafternoon(Student’s
t-test,t=−0.05p=0.96;Fig.3a. Thefrequencyofentryintothe
rightcompartments didnot differ inthe morning(Student’s
t-test,t=−1.51p=0.15),neitherintheafternoon(Student’st-test,
t=0.52p=0.61;Fig.3c).
Duringpresentationofthegroup,residencetimeinthe
com-partmentsdidnotdifferinthemorning(Student’st-test,t=−1.84
p=0.08),butitwashigherintheafternooncompartmentinthe
afternoon(Student’st-testt=2.85p=0.01;Fig.3b).Withrespect
tothefrequencyofentryintothecompartments,itwashigherin
themorningcompartmentinthemorning(Student’st-test,t=2,54
p=0.02),but intheafternoon it didnot differ(Student’s t-test,
t=−0.80p=0,43;Fig.3d).
Ontheday30,inthe15minbeforethegrouppresenceintothe
tank,therewasnodifferencebetweenthetimespentineach
com-partmentbothinthemorning(Student’st-test,t=−1.03p=0.31)
andintheafternoon(Student’st-test,t=−1.40p=0.18;Fig.4a).
Thefrequencydidnotdifferinthecompartmentsinthemorning
(Student’st-test,t=−0.21p=0.83),orafternoon(Student’st-test,
t=0.02p=0.98)(Fig.4c).
Duringthe60minthatthegroupwasexpected(absenceofthe
stimulus),thefishremainedforalongertimeinthemorning
com-partmentbothinthemorning(Student’st-test,t=−2.33p=0.03)
andafternoon(Student’st-test,t=−2.07p=0.05;Fig.4b).However,
therewerenodifferencesinthefrequencyofentriesinthe
com-partmentsinthemorning(Student’st-test,t=−0.31p=0.76)orthe
afternoon(Student’st-test,t=0.10p=0.93;Fig.4d).
3.3. Activityregistry
Duringthelast6daysoftheexperimentalperiodforTPL,the
animalsunderconstantlight(LL),constantdark(DD)and
light-darkcycle(LD)showedcircadianrhythmandtheiractivityprofile
is represented by theactogram in Fig. 5. The activity(average
speed)meanvaluesstatisticallydifferedbetweenthethreetested
conditions(OnewayANOVAF=4.35p=0.04;Fig.6a–c;Table1):
animalsunderLLshowedhigheractivitythantheanimalsunder
DD,butnoneofthemdifferedfromLD.While,animalsunderLD
(Rayleighr=0.997p=0.007)andDD(Rayleighr=0.938p=0.017)
groupsshowedsignificantdirectionalityintheacrophase
distribu-tion(withina24hcircle),thesamedidnothappenforLLgroup
(Rayleighr=0.477p=0.427;Fig.6d–f),howevertheacrophase
dis-tributiondidnotdifferbetweenthegroups(Watson-Williamstest
F=0.575p=0.585)(Table2).Thecentre ofgravitywasalso
sig-nificantdifferentbetweenthegroups (OnewayANOVAF=4.83
p=0.04;Table1).Therewasnosignificantdifferencebetweenthe
groupsregardingthetotalareaunderthecurve(OnewayANOVA
F=3.92p=0.06).Theperiodogramanalysisshowedthatanimals
under LD had stronger circadian rhythmicity (Sokolove-Bushell
periodogramwithBonferronicorrection,showinghigher
percent-ageofthetotalvariance)thantheanimalsunderLLandDD,but
therewerenodifferencebetweenLL andDD(OnewayANOVA
F=8.17p<0.05).
Regardingthe subjectivelight phase (7am to7pm), both the
meaninterval(I-m(w))(OnewayANOVAF=3.96p=0.06)andthe
areaunderthecurve(I-a(w))(OnewayANOVAF=3.94p=0.06)did
notdifferbetweenthegroups(Table1).Thepercentageof
activ-ity,asmeasuredbypercentageofthetotalarea(I-a(w)%),showed
significantdifferencebetweentheconditions(Oneway ANOVA
F=10,60p=0.004;Table1).
Onthelastdayoftheexperiment(probeday),inwhichthe
stim-uluswasnotpresentedtotheexperimentalanimals,theactivityof
thegroupswassignificantlydifferent(OnewayANOVAF=29.98
p<0.001).TheanimalsunderLLandLDshowedhigheractivitythan
0 500 1000 1500 2000 2500 3000
Morning Afternoon
Re si dence ti me ( s)
a) 15' before shoal arrival Morning compartiment
Afternoon compartiment
Morning Afternoon b) 60' with shoal 0 10 20 30 40 50 60 70 80 90 100 Morning Afternoon Frequenc y of entry Time of the day
c) 15' before shoal arrival
Morning Afternoon
Time of the day
d) 60' with shoal
Fig.1.Zebrafishresidencetime(aandb)andfrequencyofentry(candd)inthemorningandafternooncompartmentsonday15oftheTPLtest(n=8)underconstant
light.Observationsweremadebetween7:45and9:00am,and4:45and6:00pmThefirst15minofobservationindicatetheirabilitytoanticipatethearrivalofthesocial
stimulus(aandc).Duringthefollowing60min,thesocialstimulus(groupwith5conspecifics)wasmaintainedinsidetheexperimentaltank(bandd).*indicatesstatistical
significance(Student’st-test,p<0.05)betweenthecompartmentscorrespondingtoeachexperimentalperiod.
Table1
Activityvariablesmeasuredinzebrafishsubmittedtolight-darkcycle(LD),constantlight(LL)andconstantdark(DD).
Meanactivity Centerofgravity Totalareaunderthecurve Meaninterval Areaunderthecurve Percentageoftotalarea
LD 3.94±0,11ab 740.90ab 364.63 5.00 245.35 64.29a
LL 4.17±0,39a 724.45b 401.07 4.35 213.45 52.66c
DD 3.20±0,12b 761.82a 307.41 3.76 184.62 59.45b
Differentlettersindicatestatisticaldifferencesbetweenthegroupsinthesamevariableevaluated(OnewayANOVA,p<0.05).
Table2
Cosinorsummaryofthezebrafishsubmittedtolight-darkcycle(LD),constantlight(LL)andconstantdark(DD).
Animals Mesor Amplitude Acrophase %Ve(total)
LD 1 4.11:4.05–4.17 1.83:1.72–1.93 797.23:783.94–810.53 97.20 2 4.04:3.98–4.10 1.52:1.41–1.63 807.38:791.02–823.74 96.88 3 4.027:3.96–4.090 1.46:1.35–1.57 823.06:805.42–840.70 96.65 4 3.60:3.51–3.70 1.36:1.19–1.54 783.08:754.07–812.09 90.81 LL 1 3.75:3.68–3.82 0.65:0.53–0.77 908.43:865.19–951.67 95.25 2 3.96:3.90–4.020 0.309:0.20–0.41 784.82:706.22–863.43 96.83 3 5.32:5.25–5.40 0.60:0.47–0.73 970.31:921.28–1019.34 97.37 4 3.67:3.58–3.75 0.30:0.15–0.464 241.75:118.32–365.18 91.98 DD 1 3.52:3.35–3.68 1.19:0.90–1.48 920.57:864.73–976.4 76.94 2 3.01:2.88–3.15 0.89:0.64–1.14 884.12:818.77–949.47 76.24 3 3.27:3.12–3.42 1.23:0.96–1.50 832.051:781.49–882.61 76.83 4 2.99:2.88–3.12 0.35:0.14–0.57 1051.76:902.03–1201.5 80.28 Periodanalysed:1440min.
4. Discussion
Accordingtoourresults,after30daysunderconstantlightor
darkconditions, zebrafishlosttheability toshowTPLbasedon
socialstimulus(Figs.2and4),howeveritmaintained24hrhythm
inbothconditions(Table1).Althoughzebrafishweretrainedfor
30daystofindaconspecificshoalatdifferenttimeandplacehave
remainedlongerinonlyoneoftheplacesduringthemorningand
theafternoon,fishfromthedarkconditionsearchedforthegroup
Further-0 500 1000 1500 2000 2500 3000 Morning Afternoon Re si dence ti me ( s)
a) 15' before shoal arrival Morning compartiment
Afternoon compartiment
Morning Afternoon
b) 60' without shoal -TPL 0 10 20 30 40 50 60 70 80 90 100 Morning Afternoon Frequenc y of entry Time of the day
c) 15' before shoal arrival
Morning Afternoon
Time of the day
d) 60' without shoal -TPL
Fig.2.Zebrafishresidencetime(aandb)andfrequencyofentry(candd)inthemorningandafternooncompartmentsonday30oftheTPLexperiment(n=10)under
constantlight.Observationsweremadefrom7:45to9:00am,andfrom4:45to6:00pmThefirst15minofobservationindicatestheabilitytoanticipatethearrivalofthe
socialstimulus(aandc),whilethenext60minindicatestheabilitytolearntimeandplaceofthestimuluspresentation(bandd).*indicatesstatisticalsignificance(Student’s
t-test,p<0.05)betweenthecompartmentsineachexperimentalperiod.
0 500 1000 1500 2000 2500 3000 Morning Afternoon Re si dence ti me ( s)
a) 15' before shoal arrival Morning compartiment
Afternoon compartiment
Morning Afternoon
b) 60' with shoal 0 10 20 30 40 50 60 70 80 90 100 Morning Afternoon Frequenc y of entry Time of the day
c) 15' before shoal arrival
Morning Afternoon
Time of the day
d) 60' with shoal
Fig.3. Zebrafishresidencetime(aandb)andfrequencyofentry(candd)inthemorningandafternooncompartmentsonday15oftheexperiment(n=8)underconstant
dark.Observationsweremadebetween7:45and9:00am,and4:45and6:00pmaandcrepresentthe15minbeforeshoalarrival,canddrepresentthe60mininwhichthe
0 500 1000 1500 2000 2500 3000 Morning Afternoon Re si dence ti me ( s)
a) 15' before shoal arrival Morning compartiment
Afternoon compartiment
Morning Afternoon
b) 60' without shoal -TPL 0 10 20 30 40 50 60
Morning Afternoon
Frequenc
y
of
entry
Time of the day
c) 15' before shoal arrival
Morning Afternoon
Time of the day
d) 60' without shoal -TPL
Fig.4.Zebrafishresidencetime(aandb)andfrequencyofentry(candd)inthemorningandafternooncompartmentsonday30oftheexperiment(n=10)underconstant
dark.Observationsweremadefrom7:45to9:00am,andfrom4:45to6:00pmThefirst15minofobservationindicatestheabilitytoanticipatethearrivalofthesocial
stimulus(aandc)andthefollowing60minindicatestimeandplaceassociationwiththereward(bandd).*indicatesstatisticalsignificance(Student’st-test,p<0.05)
betweenthecompartmentscorrespondstoeachexperimentalperiod.
Fig.5.Representativeactogram(averagespeed)ofzebrafishsubmittedtoLD:lightdarkcycle,LL:constantlight,DD:constantdarkduringthelasts6daysofthe30-dayTPL
experiment.
more,fishunderconstantdarkdecreasedoverallactivity,whilethe
fishunderconstantlightdidnotchangeactivitylevelbutitwas
morehomogeneouslydistributedthroughoutthe24h-dayperiod,
despitetheobservedsignificant24-hrhythms.
Onthe15thdayof behaviourregistry, neithergroups (LLor
DD)showeddifferencesintimespentorfrequencyofentryinthe
compartments15minbeforethestimuluspresentation(Figs.1a,
cand3a,c).Theseresultssuggestthatfishcouldnotanticipate
thesocialstimulusarrival.Under12:12LDcycle,zebrafishshowsa
weakbehaviourofanticipationonthe15thtrainingday(Mouraand
Luchiari,2016),andthusonewouldexpectthatafteronly15daysof
constantlightconditionsfishwouldpresentmuchdifficultyto
fore-castthestimulusevent.Duringthe60minoftheconspecificshoal
presence,zebrafishunderLLremainedsignificantlylongernearthe
groupbothinthemorningand afternoon(Fig.1b),butanimals
Fig.6. Waveformoftheactivity(a–c)foreachgroup,andrepresentativecosinorshowingtheacrophase(d–f)oftheanimalsunderLD(light-darkcycle),LL(constantlight)
andDD(constantdark)conditionsonthelast6daysoftheTPLtest.LDgroupshowedsignificantdifferenceinacrophasebetween12amand2pm(Rayleigh,p<0.05).DD
groupshowedsignificantdifferenceinacrophasebetween2amand4am(Rayleigh,p<0.05).LLgrouphadnosignificantdifferenceinacrophase(Rayleigh,p>0.05).
Fig.7. Activity(averagespeed)oftheanimalsunderLD(light-darkcycle),LL
(con-stantlight)andDD(constantdarkness)ontheprobeday(30thday)ofthetestfor
TPLtest.LLandLDgroupsshowedsignificanthigheractivitythanDDgroup(One
wayANOVA,p<0.05).
(Fig.3b).Duetothesocialnatureofzebrafish(Pritchardetal.,2001;
Larsonetal.,2006;Gerlai,2014;Luchiarietal.,2015),the
pres-enceofashoalisastrongstimulustodriveasinglefishtowardsit.
Thus,weexpectedthefishtojointhegroupuponitspresence,what
didnothappenintheDDconditionprobablybecausetheanimals
hadnovisualcues,butchemicalandmechanicalcuestolocatethe
groupintothetank.Zebrafishishighlyresponsivetolight(Tamai
etal.,2007;MooreandWhitmore,2014)anditsvisualsystemisa
veryaccuratesense,presumablythemostefficientintermsof
stim-ulidetection(FleischandNeuhauss,2006).Whilechemicalcues
quicklydisperseintothewaterandmechanicalcuesmaynothave
passedthroughthecompartments,webelievetheexperimental
zebrafishstrugglefindingthestimulusinthedark.
Many other studies have already demonstrated TPL in fish
(Reebs, 1993, 1996, 1999; Gómez-Laplaza and Morgan, 2005; Delicioetal.,2006;Barretoetal.,2006;DelicioandBarreto,2008; Heydarnejad and Purser, 2008; Ebrahimi et al., 2013; Brannas,
2014),allofthemusingfoodasthereward.TheTPLprotocolused
hereinwasalearningtestbasedonsocialreward.Inaprevious
study,we(MouraandLuchiari,2016)haveshownthatlive
con-specificswereeffectivetoinducerobustTPLbehaviourinzebrafish.
However,recurrentlackofluminositysignalstoindicatedayand
nightmayhaveTPLimplications:zebrafishunderLLandDDdid
notseekforthecorrectplaceinthemorningandintheafternoon
inordertogetthesocialreward.
Ontheprobeday(day30;Figs.2and4),bothtimespentand
fre-quencyofentryinthecorrectcompartmentsinthe15minbefore
thestimulusdidnotdifferintheLLandDDgroups,showingthe
animalscouldnotanticipatetheeventevenafter30daysof
train-ing.Duringthe60minthatthegroupwasexpectedtobepresent,
testingtimes(Fig.2b),whilefishunderDDsettleinthemorning
compartmentatbothtestingtimes(Fig.4b).
ItispossiblethatintheabsenceoftheLDcycle,which
func-tionsascuetopredicttime,theabilityof orientationhadbeen
impaired,since light-dark cycleis one ofmost relevant
zeitge-bersfortheguidanceofindividuals(Hastings,1991).However,itis
worthtonoticethatfishseemtoshowsometemporalassociation
becauseitspentmosttimeinaspecificcompartmentatbothtested
times,butdidnotdiscernthecorrectsideinthecorrecttime,in
otherwords,therewasnotime-placeassociation.Tasksinvolving
appetitive/aversiveevents,inwhichtheindividualneedstemporal
perception,implicateonintervaltimingandcircadianrhythmas
wellasassociativelearningofpredictivecues(Ralphetal.,2013).
AccordingtoCainetal.(2004),timememorycanbeexplainedby
thecircadianoscillatoraction,whichismodulatedbysignificant
experiences.Thus,intheabsenceoflightzeitgebers(strongcue),
theorganismsaredependentonweakercues(suchas
tempera-tureandsocialcue),andtheendogenousclock.Whileourzebrafish
seemnottodisplayfree-running(t=1440;Table1and2),interval
timingtopredicttimeandplacewasnotobserved.Indeed,learning
toassociatetimewithspatiallocationisnotaneasytask(Biebach,
1989),anddependingonthespeciesitmayrequireasignificantly
strongzeitgebertoshowTPL,forinstancetheLDcycle.
Despiteconstantlightconditions(LLandDD),theactivity
reg-istryonthelast6daysofthe30-daystestshowedthatzebrafish
maintainedcircadianrhythm(asdetectedbytheCosinormethod−
t=1440).Itmayhaveoccurredduetothepresenceofdailyandfixed
timesofstimuluspresentation,reinforcingthestrengthofthesocial
cuetocircadianrhythmspecies(Mrosovsky,1988).TheLLgroup
showedhigheractivity(averagespeed)thantheDDgroup,but
sim-ilartotheLDgroup(Fig.6and7,Table1).Webelievethispattern
wasrelatedtothediurnalchronotypeofthezebrafish(Hurdetal.,
1998)thatmayhaveinducedtheLLgrouptomaintainlight
respon-siveness.Additionally,althoughitwasnotpossibletoobservea
lengtheningeffectoflightontheactivityphaseofanimalsexposed
toLL,theincreaseinoveralllocomotoractivitylevelisin
accor-dancewiththecircadianrule,whichstandsthattheintensityofthe
lightstimulusispositivelycorrelatedwithlocomotoractivitylevel
indiurnalanimals(Enright, 1980).WhiletheAschoff’srulewas
designedmainlyformammals,itpresentsstatementstodescribe
andpredictananimalcircadianbehaviourwhenhousedunder
con-stantlightconditions.Forinstance,thisrulepredictsthatnocturnal
animalsunderconstantdarkwouldhave periodsinfree-course
smallerthanunderconstantlightandperiodsinfree-coursethat
increasewiththeincreasinglightintensity,andvice-versafor
diur-nalanimals.InastudybyElbazetal.(2013),zebrafishkeptunderLL
cyclebecamemoreactiveandlostcircadianrhythm,althoughour
fishunderLLshowedhigherspeed,activitywasmoredistributed
overthedaytime(notconcentratedintheintervalofthesubjective
day)andtheyhavemaintainedthecircadianrhythmprobablydue
tothesocialcuepresented.Ourresults,therefore,seemtoindicate
thatthezebrafishcircadianrhythmneedsstrongercue,suchasthe
light-darkcycle,butotherenvironmentalcuespreciselyrepeated
overtimemightbeusedtomaintaintheirrhythmicity.However,
weakerzeitgeberssuchasthesocialstimuliusedhereinmaynot
beeffectivetopredicttime,whatmighthaveaffectedthezebrafish
abilityofTPL.
AccordingtoYokogawaetal.(2007),underprolongedconstant
conditions,adultzebrafishsleepovernightinbothLDandDDcycle,
butsleep-wakerhythmisdeletedunderLLandonlyreturnsafter
aboutsevendaysinthiscondition.Underconstantdark,zebrafish
displayrhythmicactivityandincreaseitduringthesubjectiveday
(Cahilletal.,1998;Hurdetal.,1998).Followingthesamepath,
weshowedthat onlyanimalsunder LDand DDhadsignificant
acrophasewithhigheractivityoccurringbetween12amand4pm
(Fig.6).
Althoughzebrafishhasbeenrecentlyusedasaneffectivemodel
in cognitivestudies,nodata associatingLD cyclesinfluence on
learninghasbeenprovidedtodate.Inthispaperweapplieda
previ-ouslyvalidatedprotocoltotestTPLunderconstantlightconditions,
reachingnegativeresultsbothforconstantlightorconstantdark,
thusrefutingourhypothesis.TodemonstrateTPL,ananimalmust
learntoassociatedifferenttimesofthedayatdifferentlocationsof
anevent(Reebs,1996).Wealsoobservedthatconstantdarkleads
todecreasedbutmoreconcentratedactivityoftheanimalsthan
constantlightcondition.
Behaviouralstudiesrepresentanimportantmethodtoidentify
neurofunctionalchanges.Thefindingthatconstantlightconditions
impairTPLimpliesthatlightismorethanonlyan
environmen-talcuetoadjustliferhythm.Moreover,thezebrafishrepresents
ausefulvertebratemodeltofulfilmanyscientificgapsregarding
thelearningprocesses,leadinganopportunitytoresearchabout
themolecularmechanismsinvolvedinthemaintenanceofthe
cir-cadianrhythm.However,ourstudypresentssomefaults,suchas
theneedformoreobservationdaysbeyond15thand30thdays,a
longerperiodof24hactivityregistryinordertofindoutchanges
inbehaviourduetotheimpositionofanalteredlightregime,and
otherLDcyclestotestTPL(e.g.16:08and18:06).Eventhoughother
studiesinthisareaarestillneededtothebetterunderstandingof
light-darkcycleroleonlearning,wepresentedhererobustresults
inrespecttothenegativeeffectsofconstantlightconditionstoTPL.
Furthermore,thispaperrecommendszebrafishasanappropriate
modelforchronobiology,aswellassuggestsfurtherinvestments
ontherelationbetweenlightcycles,clockgenesexpressionand
learning.
Acknowledgements
WethankMsTavares,C.P.M.,MsCoutinho,J.R.S.andMrCanejo,
F.W.G.forhelpincollectingdataforthisarticle.
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