Reward signal in a recurrent circuit drives appetitive long-term memory formation

18 Pages·2015·3.57 MB·English

RESEARCHARTICLE
elifesciences.org
Reward signal in a recurrent circuit drives
appetitive long-term memory formation
Toshiharu Ichinose1,2, Yoshinori Aso3, Nobuhiro Yamagata1,2, Ayako Abe1,
Gerald M Rubin3, Hiromu Tanimoto1,2*
1Graduate School of Life Sciences, Tohoku University, Sendai, Japan; 2Max Planck
Institute of Neurobiology, Martinsried, Germany; 3Janelia Research Campus, Howard
Hughes Medical Institute, Ashburn, United States
Abstract Dopaminesignals reward inanimalbrains. A single presentation ofasugar rewardto
Drosophila activatesdistinct subsetsofdopamine neurons thatindependentlyinduceshort- and
long-term olfactory memories(STMand LTM,respectively). In thisstudy,we show thatarecurrent
rewardcircuitunderliestheformationandconsolidationofLTM.Thisfeedbackcircuitiscomposedof
asingleclassofreward-signalingdopamineneurons(PAM-α1)projectingtoarestrictedregionofthe
mushroombody(MB),andaspecificMBoutputcelltype,MBON-α1,whosedendritesarborizethat
sameMBcompartment.BothMBON-α1andPAM-α1neuronsarerequiredduringtheacquisitionand
consolidation of appetitiveLTM. MBON-α1additionally mediates theretrieval ofLTM,which is
dependenton the dopamine receptorsignalinginthe MBα/βneurons. Ourresultssuggest thata
reward signal transformsa nascentmemory trace intoastableLTM usingafeedback circuit at the
cost ofmemory specificity.
DOI:10.7554/eLife.10719.001
Introduction
Environmentally relevant information, such as poison or food, can trigger formation of long-term
*Forcorrespondence: memory(LTM)inasingleassociationevent(Garciaetal.,1955;Alexanderetal.,1984).BecauseLTM
[email protected] formationisenergeticallycostly(MeryandKawecki,2005;PlacaisandPreat,2013),itisadvantageous
foranimalstorefertosurroundingconditionswhenselectingwhatinformationtoretainasstableLTM.
Competinginterests:The
Appetitive olfactory learning in Drosophila melanogaster provides an opportunity to uncover the
authorsdeclarethatno
mechanismsunderlyingsuchintegrationofinformation,asasinglepresentationofnutritioussugarand
competinginterestsexist.
anodordrivesrobustshort-termmemory(STM)andLTM,whilenon-nutritioussugarinducesonlySTM
Funding:Seepage14 (KrashesandWaddell,2008;Colombetal.,2009;BurkeandWaddell,2011).
Received:10August2015 InDrosophila,sugarrewardismediatedbyagroupofdopamineneuronscalledtheprotocerebral
Accepted:05October2015 anterior medial (PAM) cluster (Burke et al., 2012; Liu et al., 2012). Artificial activation of the PAM
Published:17November2015 clusterneuronsinthepresenceofodordrivesrobustappetitiveSTMandLTMformation.Recentstudies
subdividedthePAMclusterneuronsintoSTM-andLTM-inducingcelltypesandidentifiedseveralcell
Reviewingeditor:LeslieC
types responsible for LTM formation, including PAM-α1 (Huetteroth et al., 2015; Yamagata et al.,
Griffith,BrandeisUniversity,
2015). Interestingly, the induced LTM is undetectable immediately after conditioning but gradually
UnitedStates
developswithtime(Dasetal.,2014;Huetterothetal.,2015;Yamagataetal.,2015).
CopyrightIchinoseetal.This
Dopamine reward signals are conveyed by PAM cluster neurons to a brain structure called
articleisdistributedunderthe
mushroombody(MB),wheretheconvergenceoftherewardandtheodorisassumedtotakeplace
termsoftheCreativeCommons
(Schwaerzel et al., 2003; Krashes et al., 2007; Trannoy et al., 2011; Cervantes-Sandoval et al.,
AttributionLicense,which
2013;Huangetal.,2013).TheMBrepresentsodorsusingasparsesubsetof∼2000intrinsicneurons,
permitsunrestricteduseand
redistributionprovidedthatthe Kenyon cells (KCs) (Turner et al., 2008; Aso et al., 2009; Honegger et al., 2011; Campbell et al.,
originalauthorandsourceare 2013). KCs send parallel fibers to form lobe structures, where multiple MB-output neuron (MBON)
credited. typeselaboratespatiallysegregateddendriticarbors(Asoetal.,2014a).TheKC-MBONsynapsesare
Ichinoseetal.eLife2015;4:e10719.DOI:10.7554/eLife.10719 1of18
Researcharticle Neuroscience
eLife digest Ananimalthatfindsparticularlynutritiousandpalatablefoodwilloftendevelopa
long-lasting memory—evenifthey experience thateventonlyonce.
Oneexample ofthisisthe abilityofthe fruitfly Drosophila toformalong-term association
between asugar rewardand aspecific odor thatwaspresentwhenthey receivedthe reward. The
consumptionofsugartriggersthereleaseofachemicalcalleddopamineonspecificcompartments
of abrain structurecalled the mushroombody.Dopamine thenacts tomodifythe connection
betweencellscalled“Kenyoncells”,whichencodespecificodors,andtheneuronsthatsendsignals
outfromthemushroombody(calledMBONs).Theresultistheformationofamemorythatlinksthe
odor with thereward.
However, littleisknown about howthisprocess differsfor long-termvs.short-term memories,
and howitcanoccur whenthefly hasexperienced theodor and rewardtogether on only asingle
occasion. Tofind out, Ichinoseet al.combinedbehavioral testingof fruitflieswith genetics. The
resultsconfirmedthatthedopamineneuronsandtheMBONsthatprojecttoasinglecompartment
of themushroom body,called α1,are bothrequired forthe formationof long-termodor-reward
memories, but nottheir short-termequivalents. These neurons are called PAM-α1and MBON-α1,
respectively.
Unexpectedly, anatomical data revealed thatPAM-α1dopamine neurons receiveinput from
MBON-α1;thatis,long-termmemoryformationinvolvesafeedbackcircuit:fromPAM-α1toKenyon
cells,thentoMBON-α1andbacktoPAM-α1.BlockingfeedbackfromtheMBON-α1ontothePAM-
α1neurons shortlyafter odor-reward trainingdisrupted long-term memoryformation. Conversely,
blocking feedbackat alater stagedid not. Thissuggests thatprolongedactivation of PAM-α1by
MBON-α1helpstostrengthennewlyestablishedmemories,convertingthemintomemoriesthatwill
last for along time.
Thediscoveryofaspecificcircuitthatsupportslong-term,butnotshort-term,memoryformation
infruit fliesisconsistent withevidence ofdistinct mechanisms underlyingthese processes in
mammals. Furtherwork isnowrequired todeterminewhether feedback circuits similar tothose in
fruit fliesalso contributetoreward-basedlearning inother animals.
DOI:10.7554/eLife.10719.002
presumed to be modified in the course of memory formation by the reinforcing dopamine neurons
(TomchikandDavis,2009;Gervasietal.,2010;Sejourneetal.,2011;Cervantes-Sandovaletal.,
2013;Placais etal., 2013; Botoet al.,2014).
While there has been progress on the molecular and cellular mechanisms underlying LTM
formation resulting from repetitive conditioning (Pagani et al., 2009; Placais et al., 2012; Philips
etal.,2013),howasinglepresentationofarewardcantriggerLTMremainstobeunderstood.Here,
weidentifyandbehaviorallycharacterizearecurrentrewardcircuitthatisessentialforappetitiveLTM
formationandconsolidation.TherecurrentcircuitconsistsofPAM-α1dopamineneuronsandasingle
MBON type, MBON-α1. This recurrent dopamine reward circuit may provide insights into a general
mechanism forLTMformation.
Results
MBON-α1 provides a glutamatergic feedback to PAM-α1
PAM-α1 delivers reward signals essential for appetitive LTM formation (Yamagata et al., 2015). To
understand the neuronal circuit that regulates rewards for LTM formation, we first sought to
anatomicallyidentifytheinputneuronstoPAM-α1.ThepresynapticterminalsofPAM-α1arelocalizedto
the basal compartment of the MB α lobe (α1) (Liu et al., 2012), whereas the dendrites stretch
horizontally across the superior medial, intermediate, and lateral protocerebra (SMP, SIP, and SLP,
respectively)(Asoetal.,2014a;Yamagataetal.,2015)(Figure1A,B,H).Bysegmentingthedendrites
ofPAM-α1inconfocalimages(Asoetal.,2014a),wequeriedadatabaseofGAL4expressionpatterns
(Jenettetal.,2012)tocomputationallysearchforcandidateupstreamneurons.Strikingly,oneofthe
inputcandidatesweidentifiedwastheoutputneuronsfromtheα1compartmentoftheMB(MBON-α1)
(Figure1C,D).Transgenicexpressionofapresynapticmarker(Robinsonetal.,2002)byMB310C-GAL4
Ichinoseetal.eLife2015;4:e10719.DOI:10.7554/eLife.10719 2of18
Researcharticle Neuroscience
Figure1.MBON-α1receivesinputsfromMB-α1andprojectstothedendritesofPAM-α1.(A–D)AnatomyofPAM-α1
(A,B)andMBON-α1(C,D).Dottedlineindicatesα/βlobesinthemushroombody(MB)(A,C)orSMP,SIP,andSLP
(superiormedial,intermediate,andlateralprotocerebra,respectively)(B,D).Arrowsindicateα1andarrowheadsthe
SIPandSLP(A,C).PAM-α1andMBON-α1arevisualizedbypJFRC2-10xUAS-mCD8GFPinVK00005andMB299B-
GAL4(A,B)orMB310C-GAL4(C,D),respectively.(E)PresynapticterminalsofMBON-α1arehighlylocalizedinSIP
andSLP.MB310C-GAL4isusedtodriveageneralmembranemarker(green)andapresynapticmarker(magenta)in
MBON-α1.(F)DoublelabelingofthemembraneofMBON-α1(green)andanti-vesicularglutamatetransporter(dVGluT,
magenta)(F ),membranestaining(F ),andanti-dVGluTstaining(F ).Arrowheadshighlighttheoverlap.(G)Two
1 2 3
individualMBON-α1neuronsvisualizedbymulti-colorflip-outwithdifferentcolors(redandcyan).(H,I)Schematicsof
PAM-α1andMBON-α1,respectively.Theα/βlobeoftheMBisoutlinedwithlightorange.Scalebars,10μm.
DOI:10.7554/eLife.10719.003
Figure1.continuedonnextpage
Ichinoseetal.eLife2015;4:e10719.DOI:10.7554/eLife.10719 3of18
Researcharticle Neuroscience
Figure1.Continued
Thefollowingfiguresupplementisavailableforfigure1:
Figuresupplement1.MBON-α1isneitherGABAergicnorcholinergic.
DOI:10.7554/eLife.10719.004
revealedthatthepresynapticterminalsofMBON-α1lieincloseappositiontothedendritesofPAM-α1
(Figure1E,I).TheterminalsofMBON-α1wereimmunoreactiveforthevesicularglutamatetransporter
(Figure1F)(Danielsetal.,2008)butnotformarkersforGABAergicorcholinergicneurons(glutamic
acid decarboxylase [Jackson et al., 1990] or choline acetyltransferase [Takagawa and Salvaterra,
1996], respectively) (Figure1—figuresupplement1),consistentwithMBON-α1beingglutamatergic
(Aso et al., 2014a). Differential labeling of the individual MBON-α1 neurons in the same brain (Nern
etal.,2015)revealedthatMBON-α1comprisestwocellswithverysimilarmorphology(Figure1G).
TopreciselydeterminetherelativearrangementofPAM-α1andMBON-α1neurites,weperformed
a double-labeling experiment using R72D01-LexA, which labels PAM-α1, and the split-GAL4 driver
MB310C-GAL4, which labels MBON-α1 (Aso et al., 2014a). Confocal images revealed that the
processesofMBON-α1andPAM-α1substantiallyintermingledbothintheSIPandSLP,andintheα1
compartment of the MB—the input and output sites of PAM-α1, respectively (Figure 2A–E,
Figure2—figuresupplement1,Video1).ToobservetheseprocessesinSIPandSLPinevengreater
detail, we employed a microscope with a super resolution detection system and found bouton-like
structures of MBON-α1 attached to the dendrites of PAM-α1 (Figure 2B–D). Furthermore, a GFP
reconstitution across synaptic partners (GRASP) (Feinberg et al., 2008; Gordon and Scott, 2009)
experimentusingR72D01-LexAandMB310C-GAL4revealedstrongGRASPsignalsinSIP,SLP,andtheα1
compartment(Figure2F).ThisfurthersupportstheclosejuxtapositionbetweenPAM-α1andMBON-α1.
Appetitive LTM formation requires feedback from MBON-α1
IftheMBfeedbackmodulatesPAM-α1,theoutputofMBON-α1mightberequiredfortheacquisition
ofLTMofthesugarreward.WeexaminedtherequirementofPAM-α1andMBON-α1byexpressinga
temperature-sensitive dominant negative dynamin, Shibirets1 (Kitamoto, 2001) using the split-GAL4
drivers MB299B-GAL4 and MB310C-GAL4 (Figure 3B,F). MB299B-GAL4 has strong expression in
PAM-α1(Asoetal.,2014a;Yamagataetal.,2015)(Figure3B).Consistentwithourpreviousresults
(Yamagataetal.,2015),theblockadeofPAM-α1duringconditioningdidnotsignificantlyaffectSTM
(Figure 3C), but led to a severe impairment of sucrose-rewarded LTM (Figure 3D). The unimpaired
performance in appetitive STM demonstrates that flies of these genotypes have normal sugar and
odor perception at the restrictive temperature. Intriguingly, we found a similar preferential
impairment of LTM upon the blockade of MBON-α1, using MB310C-GAL4 driver (Figure 3H). The
sameblockadedidnotsignificantlyimpairSTM(Figure3G).Weconfirmedthepreferentialrequirement
of MBON-α1 for LTM using another driver, MB323B-GAL4 (Figure 3—figure supplement 1A), and
obtained similar results (Figure 3G,H); neither MB310C/UAS-shi nor MB323B/UAS-shi flies showed a
significantLTMimpairmentwhentrainedandtestedatthepermissivetemperature(Figure3—figure
supplement1B).
WenextexaminedwhetherglutamatergicinputtoPAM-α1isrequiredforLTMformationbydown-
regulating the expression of glutamate receptors using MB299B-GAL4. For efficient and cell-type-
specificmanipulation,weusedUAS-RNAiflystrainsbasedontheshRNAtechnique(Nietal.,2011).
Intriguingly, knocking down either dNR-1 or dNR-2, which together form the functional NMDA
receptor (Xia et al., 2005), impaired LTM (Figure 4C) but not STM (Figure 4B). This preferential
requirement in LTM is similar to the blockade of MBON-α1 and PAM-α1, suggesting the direct
glutamatergic feedback.
Given that thermogenetic stimulation of PAM-α1 with a temperature-sensitive cation channel
dTrpA1 (Hamada et al., 2008) can provide the reward signal for LTM formation (Yamagata et al.,
2015), we asked if activation of MBON-α1 would also be capable of inducing appetitive LTM
(Figure5A).Indeed,wefoundappetitiveLTMuponthermo-activationofMBON-α1inthepresenceof
an odor (Figure 5B). These results, together with the consistent requirements of MBON-α1 and
NMDAR-signalinginPAM-α1,aremosteasilyexplainediftheglutamatergicfeedbackregulationfrom
MBON-α1augments dopamine rewardsignaling byPAM-α1.
Ichinoseetal.eLife2015;4:e10719.DOI:10.7554/eLife.10719 4of18
Researcharticle Neuroscience
Figure2.ProcessesofPAM-α1andMBON-α1intermingleeachother.(A)DoublelabelingofPAM-α1andMBON-
α1.PAM-α1(magenta)andMBON-α1(green)arevisualizedusingR72D01-LexAandMB310C-GAL4,respectively.
(B–D)MagnifiedsubstackimagesinSIPandSLP,obtainedbyasuper-resolutiondetectionsystem.(E)Magnified
substackimageintheα1compartmentintheMB.(F)GFPreconstitutionacrosssynapticpartner(GRASP)signalsin
SIP,SLP,andMBα1supportthecontactsbetweenPAM-α1andMBON-α1.Scalebars,10μm(A,B,E,F),1μm(C,D).
DOI:10.7554/eLife.10719.005
Thefollowingfiguresupplementisavailableforfigure2:
Figuresupplement1.StereotypedprojectionsofPAM-α1andMBON-α1.
DOI:10.7554/eLife.10719.006
DopR1-mediated appetitive LTM trace is formed in α/β KCs
Several lines of evidence suggest that D1-like dopamine receptor (DopR1) is required in KCs for
appetitive memory (Kim et al., 2007; Liu et al., 2012; Boto et al., 2014). However, previous studies
focusedononlySTM.ToidentifythereceivingsiteofdopamineforLTMformation,weknockeddown
DopR1,eitherinα/βKCsorinMBON-α1,thepotentialtargetsofdopaminefromPAM-α1,andmeasured
appetitive LTM. To target the expression specifically to the α/β KCs, we employed two driver lines,
MB008B-GAL4 (Aso et al., 2014a; Vogt et al., 2014) (Figure 6—figure supplement 1A) and c739-
GAL4 (Yang et al., 1995). Interestingly, knocking down DopR1 in the α/β KCs, using c739-GAL4 or
MB008B-GAL4, resulted in a severe impairment of appetitive LTM (Figure 6A) while leaving STM
unaffected(Figure6—figuresupplement1B).Theseresultsareconsistentwiththeobservationthatthe
coincidence detector adenylate cyclase encoded by rutabaga, thought to act downstream of the
dopaminereceptor(Tomchikand Davis, 2009;Gervasi etal., 2010; Botoetal., 2014), functions for
LTMintheα/βKCs(Trannoyetal.,2011).Ontheotherhand,knockingdownDopR1inMBON-α1did
notsignificantlyimpaireitherappetitiveLTMorSTM(Figure6AandFigure6—figuresupplement1B).
TofurtherlocalizetheroleofDopR1inKCs,weperformedarescueexperiment,usingapiggyBac
insertion mutant, dumb2 (Liu et al., 2012; Qin et al., 2012). dumb2 mutant flies showed severely
impairedappetitiveLTM.Targetingwild-typeDopR1geneexpressioninKCsfullyrescuedthedefects
Ichinoseetal.eLife2015;4:e10719.DOI:10.7554/eLife.10719 5of18
Researcharticle Neuroscience
(Figure 6B). Together, although we cannot
exclude the possible role of other dopamine
receptorsinMBON-α1,weproposethatDopR1-
mediatedmemorytraceisformedinα/βKCsnot
inMBON-α1.
Output from the α/β KCs is
required during conditioning
Previousstudieshavehighlightedtheroleofthe
output from α/β KCs for memory retrieval
Video 1. Volume-rendered image of processes of (Krashes et al., 2007; Trannoy et al., 2011;
PAM-α1(magenta)andMBON-α1(green)inSIPandSLP.
Cervantes-Sandovaletal.,2013;Perisseetal.,
DOI:10.7554/eLife.10719.007 2013). Given the requirement of MBON-α1 for
the acquisition of appetitive LTM, the output of
the α/β KCs during conditioning may be impor-
tant for LTM formation. To test this possibility, we blocked the output of the α/β KCs with Shits1.
Strikingly,theblockadeoftheα/βKCsduringconditioningsignificantlyimpairedappetitiveLTM,but
notSTM(Figure7A,B),similartotheblockadeofPAM-α1andMBON-α1(Figure3).Whentheflies
weretrainedandtestedatthepermissivetemperature,nosignificantimpairmentofappetitiveLTM
wasobserved(Figure7—figuresupplement1).Therequirementforα/βKCsynapticsignalingduring
LTM formation supports our model that MBON-α1 provides information about the memory trace,
formed intheα/βKCs, tothe rewardsignaling PAM-α1neurons.
LTM retrieval requires MBON-α1
Ifthenascentmemorytraceinα/βKCsiscommunicatedbyMBON-α1duringconditioning,MBON-α1
mightalsoconveytheread-outofLTM.Intriguingly,whenMBON-α1wasblockedonlyduringthetest
phaseofLTM,wefoundasevereimpairmentofconditionedodorapproach(Figure8A).Incontrast,
the blockade of PAM-α1 during LTM retrieval did not have a significant effect (Figure 8B). These
resultsimplythatMBON-α1playsadualroleinbothformationandretrievalofLTM;differentpost-
synaptictargetsofMBON-α1wouldbeusedinthesetworoles,withtherecurrentPAM-α1-MBON-α1
circuit selectively involved inLTM formation.
Post-training dopamine release consolidates the nascent memory trace
at the cost of memory specificity
The glutamatergic input from MBON-α1 might provide an excitatory feedback on the PAM-α1
neuronsviatheNMDAreceptortherebyprolongingthedopaminereleaseafterthecessationofsugar
presentation.Ifthismodelwerecorrect,thepost-trainingblockadeofthefeedbackcircuitmightbe
expected to impair appetitive LTM. Consistent with this idea, blocking any component of the
feedback circuit for 1 hr immediately following conditioning resulted in a significant impairment of
appetitiveLTM(Figure9A,C,E),whereasthesameblockade22hraftertrainingdidnot(Figure9B,D,
F).Theseresultsstronglysuggestthattherecurrentcircuithasanongoingactivity,whichplaysakey
role inearly phase ofappetitiveLTM consolidation.
ProlongeddopaminereleaseintheabsenceofodormayalsomodulateKCsthatdonotrespond
totherewardedodor.Ifso,theodorspecificityofappetitiveLTMmightbecompromisedcompared
to STM. Thus, we measured the generalization profile of STM and LTM by testing with the graded
mixturesof‘contaminating’odors(Figure10).Odormixtureswerecreatedbysystematicallyvarying
the relative contents of the contaminant (trained odor: contaminant = 100:0, 80:20, 60:40, 40:60,
20:80,or0:100):ifthememoryisspecifictothetrainedodor,theperformancequicklydecreaseswith
theincreasingcontaminantratio.Strikingly,theperformancedeclineisshallowerinLTMcomparedto
STM (Figure 10C,D). We confirmed these results with another set of odorants, suggesting that
compromised stimulus specificity in LTM is general (Figure 10—figure supplement 1). Considering
thattheonlydifferencebetweenthesegroupsistheretentiontime,theremaybeatrade-offbetween
consolidation and specificity, possibly due to the prolonged dopamine release in the MB following
memory acquisition.
Ichinoseetal.eLife2015;4:e10719.DOI:10.7554/eLife.10719 6of18
Researcharticle Neuroscience
Figure3.FeedbackfromMBON-α1isrequiredforappetitiveLTMformation.(A)Diagramoftheexperiment.(B)ExpressionpatternofMB299B-GAL4.
(C)BlockadeofPAM-α1doesnotimpairSTMsignificantly.5-minappetitivememorywasmeasured(n=10,10,12).c:conditioning,t:test.(D)Blockadeof
PAM-α1duringconditioningimpairsLTM.24-hrappetitiveLTMwasmeasured(n=9,12,12).(E)Diagramofexperiment.(F)Expressionpatternof
MB310C-GAL4.(G)BlockadeofMBON-α1doesnotimpairSTMsignificantly(n=6,7,10,6,10).(H)BlockadeofMBON-α1duringconditioning
impairsLTM.MB323B-GAL4isaseconddriverlinethatexpressesinMBON-α1(seefiguresupplement;n=23,24,13,14,24).Bargraphsare
mean± s.e.m.*:p<0.05,***:p<0.001,n.s.:p>0.05.Scalebars,20μm.
DOI:10.7554/eLife.10719.008
Thefollowingfiguresupplementisavailableforfigure3:
Figuresupplement1.(A)ExpressionpatternofMB323B-GAL4.
DOI:10.7554/eLife.10719.009
Discussion
Function of a recurrent reward circuit
Prior studies on the role of MBONs in learning and memory have focused on memory retrieval
(Sejourneetal.,2011;Paietal.,2013;Placaisetal.,2013;Asoetal.,2014b).Here,weextendthe
known role of MBONs and show that MB output through MBON-α1 is required not only for the
retrieval, but also for the acquisition and early consolidation of long-term appetitive olfactory
memory.ThisunanticipatedaspectoftheMBoutputforLTMformationappearstofunctionthrougha
recurrent loop (Figure 11): MBON-α1 dendrites and the terminals of PAM-α1 occupy the same MB
compartment (Aso et al., 2014a); the DopR1 dopamine receptor is required in the KCs of that
compartment(Figure6);MBON-α1inturnsendsaportionofitsoutputstothedendritesofPAM-α1
(Figures 1, 2) closing the loop; synaptic signaling by all three cell types—α/β KCs, MBON-α1, and
PAM-α1—is required during LTM formation and consolidation (Figures 3, 7, 9). These findings
stronglysupportourmodelthatfeedbacksignalsfromα/βKCs,mediatedbyMBON-α1,playacritical
role in controlling the rewarding dopamine signals from PAM-α1 (Figure 11). Inhibitory recurrent
circuits in the MBs have been implicated in appetitive learning of honeybees (Grunewald, 1999a,
1999b).Ourpresentstudyrevealsanewcircuitmotif,afeedbackcircuitthatisrequiredfordopamine
neurons toprovide reinforcement duringappetitive LTMformation.
Dopamine has been thought to simply relay rewarding or punitive stimuli (Gerber et al., 2004;
Waddell,2013).However,ourresultscallintoquestionthisprevailingcircuitmodel.Morespecifically,
PAM-α1mightintegratesimultaneousinputsfromsugarperceptionandthenascentmemorytraceto
augment and sustain the dopamine release to gate LTM formation (Figure 11). The requirement of
Ichinoseetal.eLife2015;4:e10719.DOI:10.7554/eLife.10719 7of18
Researcharticle Neuroscience
Figure 4. NMDA receptors in PAM-α1 are required for appetitive LTM. (A) The subunits of NMDA receptor are
down-regulatedinPAM-α1.(B,C)KnockingdownNMDAreceptorsubunitsdoesnotimpair5-minmemory(B:n=16,8,
13,12,13)butimpairs24-hrmemorysignificantly(C:n=18,8,22,20,29).Bargraphsaremean±s.e.m.*:p<0.05,**:
p<0.01,n.s.:p>0.05.Scalebars,20μm.
DOI:10.7554/eLife.10719.010
the NMDA receptor in PAM-α1 (Figure 4) further supports this model. Such integration by PAM-α1
might enhance the reward gain selectively in relevant sensory environments. In other words, the
reinforcingsignalforLTMformationmaydependonthepresenceofothersensorystimuli(e.g.,odor).
As LTM formation is energetically costly (Mery
and Kawecki, 2005; Placais and Preat, 2013;
Musso et al., 2015), this gain control of the
reinforcing signal might serve to restrict LTM
formation to ecologically relevant situations.
Although the requirement of NMDA receptor in
PAM-α1 implies that MBON-α1 provides excit-
atory input, the synaptic connectivity between
MBON-α1 and PAM-α1 awaits physiological
demonstration, given functional inhibitory gluta-
matereceptorsignaling (Liuand Wilson,2013).
In addition to the role in memory acquisition,
thePAM-α1outputisrequiredduringconsolida-
tion(Figure9A,B).Thedopaminereleaseislikely
to be sustained by the ongoing activity of the
recurrent circuit (Figure 9C–F). A recent study
reported a consistent finding that the output
from α/β KCs during consolidation has an
essential role for appetitive and aversive LTM
(Huang et al., 2012) in addition to their role in
Figure5.MBON-α1signalsrewardforappetitiveLTM.
retrieval (Krashes et al., 2007; Trannoy et al.,
(A)ActivationofMBON-α1waspairedwithodor
2011; Cervantes-Sandoval et al., 2013; Xie
presentationsand24-hrmemorywasmeasured.
(B)ActivationofMBON-α1inducesappetitiveLTM et al., 2013). Recent studies revealed a gradual
formation(n=16,24,24).Bargraphsaremean±s.e.m. development of appetitive LTM after condition-
*:p<0.05,n.s.:p>0.05.Scalebars,20μm. ing (Das et al., 2014; Huetteroth et al., 2015;
DOI:10.7554/eLife.10719.011 Yamagata et al., 2015). Ongoing activity in the
Ichinoseetal.eLife2015;4:e10719.DOI:10.7554/eLife.10719 8of18
Researcharticle Neuroscience
Figure6.α/βKCsreceivedopaminesignalsthroughDopR1forLTMformation.(A)KnockingdownDopR1inα/βKenyoncells(KCs)butnotinMBON-α1
impairsappetitiveLTM(n=17,10,11,11,18,12,7,12,27).(B)KCexpressionofDopR1inthedumb2mutantbackgroundfullyrescuestheLTMimpairment
(n=12,10,7,13).Bargraphsaremean±s.e.m.*:p<0.05,***:p<0.001.
DOI:10.7554/eLife.10719.012
Thefollowingfiguresupplementisavailableforfigure6:
Figuresupplement1.(A)ExpressionpatternofMB008B-GAL4.
DOI:10.7554/eLife.10719.013
recurrentloopweproposeheremayprovideacircuitmechanismforthegradualLTMdevelopment.
In addition, previous studies reported that LTM consolidation in Drosophila (Placais et al., 2012;
Musso etal., 2015)orinrats (Rossato et al., 2009)requires post-training dopamineinputs.
Our generalization experiments show that appetitive LTM is less specific to the trained odor,
compared to STM (Figure 10). This result fits well into previous studies in mice and bees,
demonstrating that long-lasting memories are more generalized (Stach and Giurfa, 2005; Wiltgen
and Silva, 2007). More generalizable LTM makes sense from an ecological viewpoint: it is worth
taking achancefor survivalevenifthesmell isnot exactly thesameas learned.
Figure7.Outputofα/βKCshasanessentialroleintheacquisitionofappetitiveLTM.(A)Blockadeofα/βKCsdoesnotimpairSTMsignificantly(n=8,7,
7,8,9).(B)Blockadeofα/βKCsduringconditioningimpairsLTM(n=7,7,18,19,20).Bargraphsaremean±s.e.m.*:p<0.05,n.s.:p>0.05.
DOI:10.7554/eLife.10719.014
Thefollowingfiguresupplementisavailableforfigure7:
Figuresupplement1.Trainingandtestingc739/UAS-shiandMB008B/UAS-shifliesatthepermissivetemperaturedonotimpairappetitiveLTM(n=7,6,8,9,6).
DOI:10.7554/eLife.10719.015
Ichinoseetal.eLife2015;4:e10719.DOI:10.7554/eLife.10719 9of18
Researcharticle Neuroscience
Figure 8. Appetitive LTM is read out through MBON-α1. (A) Blocking MBON-α1 during test impairs appetitive LTM retrieval (n = 20, 21, 11, 13, 20).
(B)BlockingPAM-α1duringtestdoesnotsignificantlyimpairLTMretrieval(n=7,7,10).Bargraphsaremean±s.e.m.*:p<0.05,**:p<0.01,n.s.:p>0.05.
DOI:10.7554/eLife.10719.016
In the fly brain, distinct types of dopamine neurons signal reward for STM and LTM, and these
memoriesareindependentlyformedindifferentMBlobes(Trannoyetal.,2011;Huetterothetal.,
2015; Yamagata et al., 2015). Octopamine has been widely accepted as a positive reinforcement
signalininsects(Hammer,1993;HammerandMenzel,1998;Schwaerzeletal.,2003;Schrolletal.,
2006;PerryandBarron,2013).InDrosophila,itisconsideredtoprovideinputstodopamineneurons
that induce STM formation (Burke et al., 2012; Das et al., 2014; Huetteroth et al., 2015). Recent
studiesinmammalsreachedasimilarconclusionthatmemorieswithdifferenttemporaldynamicsare
mutually conflicting and formed in distinct areas, to which different classes of dopamine neurons
project(Liljenstrom,2003;AbrahamandRobins,2005;KimandHikosaka,2013;Kimetal.,2014).
Independent processingofSTM andLTM mightbeaconserved strategy among animals.
Circuit mechanism of memory-guided behavior
In addition to the function in the recurrent reward circuit, MBON-α1 mediates retrieval of the LTM
traceintheMB(Figure8).MBON-α1samplesfromtheMB-α1andterminatesintheSIPandtheSLP.
Another type of MBONs also mediates appetitive LTM retrieval: MBON-α3 (also known as MB-V3)
(Placais et al., 2013). In contrast to MBON-α1, blocking MBON-α3 during conditioning does not
impairappetitiveLTMacquisition(Placaisetal.,2013).MBON-α3arborizesdendritesinthetipofthe
α lobe and projects to the SMP and the SIP where the terminals of MBON-α1 and MBON-α3 are
intermingled(Asoetal.,2014a).Therefore,appetitiveLTMformedintheα/βKCsisreadoutthrough
atleasttwodifferentclassesofMBONs.TheseMBONsconvergeintheSIP(Asoetal., 2014a)and
might activate shareddownstreamtargetsto guide conditioned odor approach.
Interplay between reinforcing signals and learned information
The recent comprehensive connectivity map of the afferent and efferent neurons of the Drosophila
MBrevealedmanypotentialfeedbackconnections outsidetheMB(Asoetal.,2014a).Considering
the central role of the MB in sensory integration and memory-guided behavior (Aso et al., 2014b),
recurrent computation between learned information and reinforcement signals might be a
fundamental principle ofmemoryprocessing.
In both vertebrates and invertebrates, the activity of reinforcement neurons is modified upon
associative learning (Hammer, 1993; Mirenowicz and Schultz, 1994; Riemensperger et al., 2005;
Cohen et al., 2012; Schultz, 2013). Our results delineated such a feedback circuit, which was
predictedbyaphysiologicalstudyinDrosophila(Riemenspergeretal.,2005).Inthiscircuit,MBON-
α1mediatesmonosynapticfeedbacktothedopamineneurons.Incontrast,thereisnomonosynaptic
feedback to the dopamine neurons found in mammals, rather anatomical studies suggest such a
feedback to be polysynaptic (Lisman and Grace, 2005; Watabe-Uchida et al., 2012). Further
comparativestudiesshouldassesshowtherecurrentcircuitmotifofreinforcementisimplementedin
different animalbrains.
Ichinoseetal.eLife2015;4:e10719.DOI:10.7554/eLife.10719 10of18