Delayed dopamine signaling of energy level builds appetitive long-term memory in Drosophila.

22 Pages·2015·2.47 MB·English

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Delayed Dopamine Signaling of Energy Level Builds
Drosophila
Appetitive Long-Term Memory in
Graphical Abstract Authors
Pierre-YvesMusso,PaulTchenio,
ThomasPreat
Correspondence
[email protected](P.T.),
[email protected](T.P.)
In Brief
Appetitivelong-termmemory(LTM)
formsinDrosophilaafterpresentationof
anodorantwithsugar.Mussoetal.now
showthat,duringLTMformation,apairof
dopaminergicneuronsinformsthe
olfactorymemorycenter,themushroom
body,abouttheenergycontentofthe
ingestedfood.
Highlights
d Delayedenergeticsupplyallowsappetitivelong-term
memoryformation
d MB-MP1dopaminergicneuronscontrolappetitivelong-term
memoryconsolidation
d MB-MP1oscillationsareenhancedafterconditioningwithan
energeticsugar
d TheDAMBdopaminergicreceptorisrequiredforappetitive
long-termmemory
Mussoetal.,2015,CellReports10,1–9
February24,2015ª2015TheAuthors
http://dx.doi.org/10.1016/j.celrep.2015.01.036
Pleasecitethisarticleinpressas:Mussoetal.,DelayedDopamineSignalingofEnergyLevelBuildsAppetitiveLong-TermMemoryinDrosophila,Cell
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Cell Reports
Report
Delayed Dopamine Signaling of Energy Level Builds
Drosophila
Appetitive Long-Term Memory in
Pierre-YvesMusso,1PaulTchenio,1,2,3,*andThomasPreat1,3,*
1GenesandDynamicsofMemorySystems,BrainPlasticityUnit,CNRS,ESPCI-ParisTech,PSLResearchUniversity,75005Paris,France
2LaboratoireAime´ Cotton,CNRS,Universite´ Paris-Sud,ENS-Cachan,91400Orsay,France
3Co-seniorauthor
*Correspondence:[email protected](P.T.),[email protected](T.P.)
http://dx.doi.org/10.1016/j.celrep.2015.01.036
ThisisanopenaccessarticleundertheCCBY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/3.0/).
SUMMARY (Scott, 2011; Domingos et al., 2011, 2013; Uematsu et al.,
2014),thecomplexityoftheseneuralnetworkshashindereda
Sensory cues relevant to a food source, such as global view of the mechanisms underlying these associations,
odors,canbeassociatedwithpost-ingestionsignals especiallyregardingrewardassociations.
related either to food energetic value or toxicity. Recently,BurkeandWaddell(2011)showedthatDrosophila
Despite numerous behavioral studies, a global un- formsshort-termmemories(STM)byassociatinganodorwith
derstanding of the mechanisms underlying these asweetfoodsource,whereaslong-termmemory(LTM)isonly
formed if the food source isnutritious. The authors concluded
long delay associations remains out of reach. Here,
wedemonstrateinDrosophilathatthelong-termas- that nutrient value is assessed within less than 2 min. In this
report, we demonstrate that LTM formation relies not only on
sociationbetweenanodorandanutritioussugarde-
sensory processing during or immediately after feeding but
pendsondelayedpost-ingestionsignalingofenergy
alsoondelayedpost-ingestionsignalingofenergylevel,which
level.Weshowattheneuralcircuitlevelthattheac-
canoccurasmuchas5hraftertraining.Ourtwo-stepcondition-
tivityoftwopairsofdopaminergicneuronsisneces- ingprotocol,combinedwithaprecisetemporalinhibition/activa-
saryandsufficienttosignalenergyleveltotheolfac- tion of neuronal circuits and in vivo brain imaging, has now
torymemorycenter.Accordingly,wehaveidentified enabledacomprehensiveviewofappetitiveLTMmechanisms.
in these dopaminergic neurons a delayed calcium
tracethatcorrelateswithappetitivelong-termmem- RESULTS
ory formation. Altogether, these findings demon-
strate that the Drosophila brain remembers food We investigated the post-ingestion component of appetitive
LTM processing using either D- or L-glucose as a reward.
qualitythroughatwo-stepmechanismthatconsists
L-glucoseisanenantiomerofD-glucosethatcannotbemetab-
oftheintegrationofolfactoryandgustatorysensory
olizedandthereforedoesnotprovideanyenergy.Becausethe
informationandthenpost-ingestionenergeticvalue.
sweetness of L-glucose approximates that of D-glucose, as
measured in particular by the fly proboscis extension reflex
INTRODUCTION andthelabellarnerveresponse,itisavaluabletoolforidentifying
energetic food-dependent processes (Fujita and Tanimura,
Visual,olfactory,andgustatorycuescandrivetheefficientselec- 2011; Stafford et al., 2012). Our initial attempt to investigate
tionoffoodsources.However,thetruenutritivevalue(ortoxicity) whether LTM is formed with energetic D-glucose rather than
of food can only be evaluated after absorption. Accordingly, L-glucose employed a classical protocol (Colomb et al.,2009)
post-ingestionsignalshaveanessentialroleinfoodsourceeval- withthefollowingsequence:21hrstarvationfollowedbytraining
uation (Sclafani and Ackroff, 2004). This well-documented with an odorant and sugar; starvation; and, finally, a memory
feature has been demonstrated by conditioned taste or odor testwiththeodorant24hraftertraining.However,thisstraight-
aversion assays, in both mammals (Scott, 2011) and inverte- forwardstrategywasunsuitablebecausethetotal45-hrfasting
brates (Wright et al., 2010; Simo˜es et al., 2012). Additionally, durationof fliestrainedwithnon-metabolizableL-glucosewas
severalresearchgroupshaveshownthatpost-ingestionsignals likely to affect their performance during the memory test in a
attributearewardvaluetonutritiousorenergeticfoodsources non-specific manner (Figure S1A). Thus, the low performance
(Sclafani and Ackroff, 2004; de Araujo et al., 2008; Dus et al., offliestrainedonL-glucose(FigureS1B)cannotbeinterpreted
2011; Fujita and Tanimura, 2011). Because sensory cues as a bona fide appetitive LTM defect, as it may be a conse-
perceivedduringfeedingmustbeassociatedwithmetabolicin- quenceofthedeleterious45-hrfasting.Bycontrast,controlflies
formationthatisonlyavailableafteralongdelay,aspecificform trainedonnutritiousD-glucosewerere-fedduringtrainingand
of associative memory is required to remember food source so were not starved for more than 24 hr; these flies thus dis-
quality.Despiterecentprogressintheidentificationofmolecular played normal LTM (Figure S1B). The non-specific effect due
mechanisms and neural circuits involved in these processes to prolonged starvation of L-glucose-trained flies could be
CellReports10,1–9,February24,2015ª2015TheAuthors 1
Pleasecitethisarticleinpressas:Mussoetal.,DelayedDopamineSignalingofEnergyLevelBuildsAppetitiveLong-TermMemoryinDrosophila,Cell
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A 24hr memory B 24hr memory
Conditioningfeeding 0.4 0.4
D-glucose medium test
n.s.
n.s.
CondTit0ioningfee3dhirng 24hr ex 0.3 dex 0.3
L-glucose medium test d n
e In Conditioningfeeding ce I
CondTit0ioning 3hr feeding 24hr anc 0.2 L-glucoseD-glucose test man 0.2
D-glTu0cose me6dhiurm 2te4shtr erform 0.1 ** T0 3hr 24hr Perfor 0.1
P
Conditioning feeding Conditioning
L-glucose medium test D-glucose test
0.0 0.0
T0 6hr 24hr T0 24hr
C 24hr memory D 24hr memory
Conditioning feeding 0.4 0.3
D-glucose medium test
n.s.
T0 6hr 24hr 0.3 x
D-gluCcoonsdei t+io npihnlgorizin mfeeeddiiunmg test dex nde 0.2
CDo-nfrduTTitc00iotonsineg mfeee66ddhhiiurrnmg 22te44shhtrr Performance In 00..21 ** CLo-ngdlTuit0cioonsieng Dff-eeg3eeluhddcriinnoggse 2te4shtr Performance I 0.1 *
Conditioning feeding Conditioning D-glucose
D-fructose + phlorizin medium test L-glucose + phlorizin test
0.0 0.0
T0 6hr 24hr T0 3hr 24hr
Figure1. AppetitiveLTMIsFormedwhenEnergyIsDelivered3hrafterTrainingbyaPost-ingestionMechanism
(A)L-glucose-conditionedfliesfedwithfood(for30min)6hrafteracquisitiondidnotformLTM(scoreisnotstatisticallydifferentfrom0,ttest,t26=1.684,p=
0.104andissignificantlylowerthanallothergroupsF(3,92)=6.904,p=0.0003;nR21).L-glucose-conditionedfliesfedwithfoodat3hr(for30min)didformLTM,
andtheirscoreisnotsignificantlydifferentfromD-glucose-conditionedflies(F(3,92)=6.904;p=0.0003).nR21foralldata.
(B)L-glucose-conditionedfliesfedwithD-glucose(for1min)3hrafteracquisitionperformedaswellasD-glucose-conditionedfliesintheLTMtest(ttest;t25=
1.134;p=0.267;nR12).
(C)FliesconditionedwithamixtureofD-glucoseandphlorizindidnotformLTM(scorestatisticallynotdifferentfrom0;ttest;t15=1.708;p=0.1083)and
presentedasignificantlylowerscorethanD-glucose-conditionedflies(ttest;t31=2.283;p=0.008).FliesconditionedwithamixtureofD-fructoseandphlorizin
exhibitedanon-statisticallydifferentscorefromD-fructose-conditionedflies(ttest;t =0.661;p=0.513).nR14foralldata.Allgroupswereplacedonfly
30
medium6hraftertrainingfor30min.
(D)L-glucose-conditionedfliesfedamixtureofD-glucoseandphlorizin(for1min)3hrafteracquisitionexhibitedasignificantlylowermemoryascomparedto
D-glucosere-fedflies(ttest;t =2.442;p=0.023;nR11).
22
SeealsoFigureS1.Means±SEM;statisticaltest:ttestandone-wayANOVA;n.s.:pR0.05;*p<0.05;**p<0.01incomparisonbetweentwogroupsforttestand
inposthoccomparisonswithbothparentalcontrolsforANOVA.
suppressed by re-feeding with fly medium after training. We nutritive value of the sugar reward (cf. Burke and Waddell,
therefore reasoned that these non-specific effects could be 2011). These results suggest that it is the energetic value of
rescuedwiththeproperdelaybetweentrainingandre-feeding. food that triggers appetitive LTM formation. However, this
Indeed, this delay must be long enough to not interfere with does not resolve whether the energetic value is associated
any potential post-feeding signaling that could be associated withtheodorduringtrainingorwhetherthereisadelayedmech-
with the odorant but short enough to maintain fly health. We anismthatallowsthebraintousepost-feedingsignalstointe-
foundthata6-hrdelaybetweentrainingandre-feedingonstan- gratethevalueofthefood,asinmammals(SclafaniandAckroff,
dard fly medium (30-min duration) met these requirements. 2004;deAraujoetal.,2008).
Accordingly, interrupting the 45-hr starvation session after To address this issue, we trained flies with non-energetic
27hrwitha30minre-feedingrescuedthenon-specificdefect L-glucoseandre-fedthem3hraftertraining,inordertogenerate
previouslyobservedforL-glucosetrainingafter45-hrstarvation potential post-feeding reinforced signaling. Strikingly, flies
(FigureS1C).Whenflieswerestarvedfor21hr,trained,re-fed trainedonL-glucoseperformedaswellat24hrasthosetrained
6hrlater,andfinallytestedafter18hrofadditionalstarvation, onD-glucose,whentheywerere-fed3hraftertrainingfor30min
weobservedthatfliestrainedwithL-glucoseexhibitedanLTM onstandardflymedium(Figure1A).Asimilareffectwasobserved
scorethatwassignificantlylowerthanthescoreoffliestrained whenflieswerere-fedfor1minwithD-glucose3hraftertraining
onD-glucose(Figure1A).Thus,byusingglucoseenantiomers, (Figure1B),indicatingthatthefoodenergeticvalueisthecritical
we confirmed that appetitive LTM formation depends on the parametertogenerateLTM.Weanalyzedthetemporalwindow
2 CellReports10,1–9,February24,2015ª2015TheAuthors
Pleasecitethisarticleinpressas:Mussoetal.,DelayedDopamineSignalingofEnergyLevelBuildsAppetitiveLong-TermMemoryinDrosophila,Cell
Reports(2015),http://dx.doi.org/10.1016/j.celrep.2015.01.036
whereenergeticpost-trainingre-feedingallowsLTMformation when training occurs at 33(cid:1)C, the transition to the permissive
for L-glucose-trained flies. LTM score remained high for re- temperature is generally progressive, and neurotransmission
feedingdelaysaslongas4hr,afterwhichitdecayedandvan- maybeinhibitedduringthefirst20–30minoftheconsolidation
ishedfora6-hrdelay(FigureS1D).Thepossibilitytodelaythe phase.ToseparatethecontributionofPPL1DAneurotransmis-
energeticfoodsupplywithrespecttotrainingsuggestedthata sion during learning from the early consolidation phase, we
post-ingestion mechanism was involved. To demonstrate this, designedprotocolswithasharptemperaturetransitionimmedi-
weutilizedphlorizin,aspecificblockeroftheintestinalglucose atelyaftertraining(seeExperimentalProcedures).Inthesharp
transporter, which does not affect the intestine-to-hemolymph version of the training blockade, flies were maintained at the
transportofothersugarslikefructose(Dusetal.,2013).Recently, restrictivetemperaturefor30minbeforeandduringthetraining
phlorizinwassuccessfullyusedtoblockglucoseentryintohemo- but were then returned to the permissive temperature within
lymph in Drosophila (Dus et al., 2013). First, we checked that 3 min of odorant and sugar presentation. The sharp training
phlorizindidnotinterferewithLTMformationdespiteitsslightly blockadeofTH-GAL4neuronsdidnotimpairLTM(Figure2B).
bittertaste.Forthis,weconditionedflieswithamixtureoftheen- WeverifiedthatthesharptrainingblockadeofthePAMcluster
ergeticfructoseandphlorizin.ThesefliesperformedLTMaswell withthe58E02-GAL4driverdidaffectLTM(FigureS2E),aspre-
asfructose-conditionedflies(Figure1C).Then,flieswerecondi- viouslyreportedusingaclassicalprotocol(Liuetal.,2012).Inthe
tioned with a mixture of D-glucose and phlorizin. Interestingly, sharpversionoftheconsolidationblockade,fliesweretrainedat
whenglucosenolongerenteredintothehemolymph,fliesfailed the permissive temperature and transferred to the restrictive
toformLTM(Figure1C).Thisindicatesthathemolymphsugar temperaturewithin3minoftraining;finally,theyweremaintained
level controlled LTM formation. Accordingly, re-feeding flies at the restrictive temperature for 3 hr. Sharp blockade of TH-
withamixtureofD-glucoseandphlorizin3hraftertrainingwith GAL4neuronsaftertrainingimpairedLTM(Figure2C),demon-
L-glucosefailedtorescueLTM(Figure1D).Together,thesere- strating that only post-training activity of TH-GAL4 neurons
sultsdemonstratethatsugarenergeticvaluewasdetectedbya wasrequiredforappetitiveLTM.
post-ingestion mechanism and not through external or diges- Next, we identified which PPL1 DA neurons are involved in
tive-tract sensory receptors. Our two-step protocol therefore LTMformationaftertraining.LTMimpairmentwasreproduced
enabledinvestigatinghowtheDrosophilabrainusespost-inges- by blocking NP0047-GAL4 neurons, which include only three
tionsignalstoremembertheenergeticvalueoffood. types of DA neurons shared with TH-GAL4: MB-V1; MB-MV1;
Appetitive olfactory memory in Drosophila is encoded in the and MB-MP1 (Figures S2F–S2K; Tanaka et al., 2008; Plac¸ais
mushroom bodies (MBs), a symmetrical structure comprising etal.,2012).WeconfirmedthattheLTMdefectcouldbeattrib-
2,000 neurons (Aso et al., 2009). The main inputs to the MBs utedspecificallytoDAneuronswithintheNP0047-GAL4expres-
are cholinergic projection neurons from the antennal lobes, sionpattern,byusingtheTH-GAL80constructthatinhibitsGAL4
whichencodeolfactoryinformation,aswellasvariousdopami- transcriptional activity in DA neurons. Indeed, the LTM defect
nergic (DA) neurons that encode aversive or appetitive signals was abolished when Shits was expressed in non-DA NP0047
(Waddell, 2010). We demonstrate here that post-ingestion neurons(FigureS2I).TherearetwoMB-MP1neuronsperbrain
signalinginvolvesneurotransmissionfromthePPL1DAcluster. hemisphere (labeled by the NP2758-GAL4 and C061-GAL4
Expression of the thermo-sensitive shibire protein (Shits) in a drivers;Tanakaetal.,2008;Krashesetal.,2009),asingleMP-
givensetofneuronsallowstheblockadeoftheirneurotransmis- MV1neuron(labeledbytheMZ604-GAL4driver;Tanakaetal.,
sionattherestrictivetemperature(33(cid:1)C),whichisreleasedatthe 2008), and a single MB-V1 neuron (labeled with the 15E10-
permissive temperature (25(cid:1)C or below). We expressed Shits GAL4and50B03-GAL4drivers;Jenettetal.,2012).Byblocking
withtheTH-GAL4driver(Asoetal.,2010)inalargepopulation individual subtypes of NP0047 DA neurons using these more
ofDAneurons,includingwithinthePPL1clusterandafewneu- spatiallyrefineddrivers,wehaveestablishedthatMB-MP1neu-
rons of the PAM cluster, and subsequently trained flies at the ronsspecificallycontrolappetitiveLTMformation(FiguresS2L–
restrictivetemperature.Consequently,LTMinfliestrainedonsu- S2O; see Figures S2P–S2U for controls). More precisely, the
crose(anutritioussugar)wasimpairedfollowingthisblockade NP2758-GAL4 driver allowed us to determine that LTM is
(Figure 2A), but not STM (Figure S2A). We checked that LTM impaired by sharp blockade of MB-MP1 after training, but not
was normalin flies trained atthe permissive temperature (Fig- duringtraining(Figures2Dand2E).
ure S2B) and that sugar response (Figure S2C) and olfactory These results implicate the activity of specific mechanisms
acuity(FigureS2D)werenormalattherestrictivetemperature. involving DA signaling to the MBs, following training to form
This LTM impairment was unexpected, because it was previ- appetitiveLTM.Tofurtherinvestigatethesemechanisms,wein-
ously established that PAM DA neurons not labeled by TH- hibited D1-type DA receptors in adult MBs neurons with RNAi
GAL4 are responsible for delivering the reward signal to the and the thermo-inducible TARGET system (which uses the
MBs during appetitive training for both STM and LTM (Liu 238Y-GAL4;tub-GAL80tsdriver;McGuireetal.,2003).Inhibition
etal.,2012).OurresultsthuspotentiallysuggestthatPPL1neu- ofthedumbDAreceptorinadultMBsinduceda2-hrSTMdefect
ronsareinvolvedduringtrainingorearlyconsolidationforLTM (Figure3A;seeFiguresS3A–S3Cforcontrols),aspreviouslyre-
formation,althoughPPL1neuronsarenotinvolvedinappetitive ported elsewhere for dumb constitutive mutants (Kim et al.,
STMformation(Schwaerzeletal.,2003).Here,thespatialreso- 2007;Liuetal.,2012).Here,weconfirmedthatthedumb3mutant
lutionofthethermo-sensitiveShitsisprovidedbytheGAL4driver displays an STM defect (Figure S3D). Strikingly, flies with
specificity.However,itisnotpossibletoachievethesametem- reduceddumbexpressioninadultMBsanddumb3mutantsdis-
poral resolution as with other techniques (e.g., optogenetics): played normal LTM (Figure 3B; see Figures S3E and S3F for
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Figure2. SpecificRequirementofMB-MP1DopaminergicNeurons
duringLTMConsolidation
(A) Blocking TH-GAL4 neurons during training and early consolidation
(approximately30min)impairedLTM(F =6.842;p=0.003;nR11).
(2,34)
(B) Blocking TH-GAL4 neurons strictly during training did not affect LTM
(F =0.127;p=0.880;n=13).
(2,36)
(C)StrictblockadeofTH-GAL4neuronsduringthefirst3hrofconsolidation
impairedLTM(F(2,44)=6.450;p=0.003;n=15).
(D)MB-MP1neurons(NP2758-GAL4)werenotrequiredduringLTMacquisi-
tion(F(2,53)=0.1621;p=0.8508;n=18).
(E)ThesameMB-MP1neuronswererequiredduringconsolidation(F(2,52)=
3.686;p=0.0321;nR17).
SeealsoFigureS2.Means±SEM;statisticaltest:one-wayANOVA;n.s.:pR
0.05;*p<0.05;**p<0.01inposthoccomparisonswithbothparentalcontrols.
control), suggesting that another DA receptor is active down-
stream of MB-MP1 neurons. We then expressed an RNAi
construct that targets the DAMB DA receptor (DAMBRNAi1) in
adult MBs (Han et al., 1996) and observed normal appetitive
STM,whereasLTMwasstronglyaffected(Figures3Cand3D).
LTM at the permissive temperature as well as sugar response
andolfactoryacuitycontrolswereallnormalintheDAMBRNAi1
mutant (Figures S3G–S3I). A specific LTM defect was also
observed for flies expressing an alternative non-overlapping
RNAi construct that targets DAMB (Figures S3J–S3O). Thus,
the DAMB receptor is specifically required for appetitive LTM
and the dumb receptor is specifically required for appetitive
STM. Thissuggests a post-training role for DAMBduring MB-
MP1 DA signaling, although the time resolution for RNAi
experiments is too low to independently analyze the role of
DAMBduringthetrainingandpost-trainingphases.
Wenextusedinvivoimagingtofurtherinvestigatethepost-
training implication of MB-MP1 during LTM processing.
Previously, we demonstrated that MB-MP1 neurons display
spontaneouscalciumoscillations(Plac¸aisetal.,2012)thatare
sensitive to satiety state (Plac¸ais and Preat, 2013). Here, MB-
MP1 activity was monitored for 90 min post-training with a
reinforcing stimulus that was either energetic (D-glucose) or
non-energetic (L-glucose; Figures 4B and S4C). We observed
30 min after conditioning that the frequency and quality factor
ofMB-MP1neuronoscillationsweresignificantlyhigher(Figures
4CandS4D)innutritiousD-glucose-trainedflieswithrespectto
those in L-glucose-trained flies, whereas the oscillation ampli-
tude did not differ (Figure S4E). At longer time points (60 and
90min),fliesfrombothgroupsdisplayedanequivalentoscilla-
tionfrequency(Figure4C),amplitude,andqualityfactor(Figures
S4DandS4E).Theseresultsdemonstrateanearlypost-training
activity of MB-MP1 neurons that correlated with the energetic
valueofthesugarreward.Accordingly,L-glucose-conditioned
flies re-fed 3 hr after training with D-glucose (Figures 4E and
S4F), which exhibited normal LTM scores (cf. Figure 1B), dis-
played a higher oscillation frequency within MB-MP1 neurons
30minafterthere-feedingthanfliesre-fedwithL-glucose(Fig-
ure4F).Qualityfactorandamplitudeoscillationswereequivalent
inbothgroups(FiguresS4GandS4H).Together,theseresults
confirmMB-MP1implicationintheprocessingofenergeticlevel
informationinappetitiveLTM.
Here,weshowedthatMB-MP1activityisrequiredforener-
getic level signaling during appetitive LTM consolidation. We
4 CellReports10,1–9,February24,2015ª2015TheAuthors
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A B LTM (Figure 5), unlike the genotypic control groups (Figure 5)
or flies kept at the permissive (20(cid:1)C) temperature (Figure S5).
Thus,MB-MP1activationmimickedenergeticpost-trainingre-
feedingandwassufficientforLTMformationintheabsenceof
anyenergysupplyduringthe6hrfollowingtraining.
DISCUSSION
Our behavioral results with enantiomeric sugars confirm that
appetitive LTM can be formed only if the sugar reward is
nutritious(BurkeandWaddell,2011).Moreover,ourresultsun-
coverfundamentalaspectsofappetitiveLTMformation,aswe
demonstrate that the processing of nutritive value depends
on post-ingestion signaling of energy level. Thus, we estab-
lished that hemolymph energetic sugar level controls appeti-
tive LTM consolidation by taking advantage of the specific
blocking of glucose intestinal transport to the hemolymph via
phlorizin.
Remarkably,wewereabletocreateartificialolfactoryLTMby
utilizinganon-energeticsugarduringtrainingandbyfeedingflies
C D
with energetic food in a critical period following training that
lastedabout5hr.Thesefindingssuggestthat,afterodor/sugar
association,thereisanopentimewindowof5hrinwhichanen-
ergeticsupplyallowstheconsolidationoftheinitialassociation
into LTM. This two-step protocol allowed us to differentiate
trainingsignalingrelatedtoinitialassociationfromenergy-level
signaling. We established that MB-MP1 DA neurons play a
critical role in signaling energy level to the MBs during LTM-
consolidation phase. It is worth noting that the same neurons
werepreviouslyidentifiedascontrollingmotivationduringappe-
titive STM recall (Krashes et al., 2009). The functions of these
neurons during LTM formation and recall are compatible.
Indeed, if MB-MP1 neurons are active when energy level is
high,therecallofappetitiveSTM(whichnormallyrequiresthat
fliesarestarvedtobemotivated)occurswhenMB-MP1activity
is low, because there is no available energy. Because energy
levelappearslowtoMBswhentheyare‘‘misled’’byMB-MP1
blockade (whatever the real energy level of the fly), STM can
be recalled in fed flies. Conversely, activation of MB-MP1 im-
Figure 3. DAMB Is Required Specifically for Appetitive LTM For- pairsSTMrecallinstarvedflies(Krashesetal.,2009).
mation
AppetitiveLTMisconsolidatedwhenenergylevelishigh,and
(A)STMwasimpairedbyRNAiinhibitionofdumbinMBsofadultflies(F(2,48)= weobservedMB-MP1tobemoreactiveduringLTMconsolida-
4.909;p=0.011;nR16).
tioninaspecifictimewindowof30minaftertraining.Interest-
(B)LTMwasnotimpairedbyRNAiinhibitionofdumbinMBsofadultflies
(F(2,40)=1.463;p=0.244;nR13). ingly,LTMisformedinfliestrainedwithanon-energeticsugar
(C)STMwasnotimpairedbyRNAiinhibitionofDAMBinMBsofadultflies but re-fed 3 hr later with energetic food; accordingly, we
(F(2,34)=0.299;p=0.743;nR11). observedanincreaseinMB-MP1activitywithina30-mintime
(D)LTMwasimpairedbyRNAiinhibitionofDAMBinMBsofadultflies(F(2,48)= window delayed by 3 hr with respect to training. The fact that
6.87;p=0.002;nR15).
MB-MP1activitycanbedelayedbyseveralhourswithrespect
SeealsoFigureS3.Means±SEM;statisticaltest:one-wayANOVA;n.s.:pR
toLTMformationtrainingraisesthequestionofhowthepost-
0.05;*p<0.05;**p<0.01inposthoccomparisonswithbothparentalcontrols.
ingestion signal can associate specifically with the olfactory
cuepairedwithsugarduringtraining.Thisisaparticularlycritical
nextaskedwhetherMB-MP1activitywassufficienttodriveLTM question,giventhattwoodorantsarepresentedtofliesduringthe
consolidationintheabsenceofenergysupplyduringtheappro- learningphase,onlyoneofwhichisassociatedwithsugar.We
priatetimewindow.Toaddressthisissue,weusedthethermo- proposeherethatassociativeLTMformationinDrosophilaisa
sensitive tool UAS-TrpA1, which allows transient neuronal two-stepprocessthatinvolvestwopropertiesofthesugarun-
activationathigh(30(cid:1)C)temperature(Rosenzweigetal.,2008). conditionedstimulus.First,PAMDAneuronsarenecessarydur-
WeconditionedflieswithL-glucoseat20(cid:1)CandactivatedMB- ingtrainingtoassociatethesweetnessstimuluswiththeodorant
MP1for30min,3hraftertraining.Strikingly,thesefliesformed in the MB (Liu et al., 2012; Figure S2E). The initial step that
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A PAM Figure 4. MB-MP1 Oscillatory Activity Is
EnhancedduringAppetitiveLTMFormation
PPL1 calyx (A) Schematic drawing of mushroom-body-con-
nected neurons. c061-GAL4;MB-GAL80 expres-
sioncorrespondstoasingleDAinthePPL1clus-
ter,MB-MP1(red).
MP1 (B)Trainingprotocolsusedbeforeimagingexper-
lobes iments:flieswereconditionedoneitherL-glucose
orD-glucoseandimagedatthreetimepoints(30,
60,and90minafterconditioning).
PPL2ab TH-GAL4 (C)Ahigheroscillatoryfrequencyisobservedfor
c061-GAL4;MB-GAL80
D-glucose-conditionedflies(red)ascomparedto
L-glucose-conditioned flies (black) during early
B C Frequency consolidation (30 min after training; Mann-Whit-
0.06 ney:p=0.0229),althoughoscillationfrequencies
becomeequivalentforbothgroupsatlatertime
* points(60and90minaftertraining;Mann-Whitney:
Starved Cond Imaging p=0.423;p=0.45,respectively).nR7foralldata.
L-glucose MP1 (D)TwoexamplesofMB-MP1recording,30min
z) 0.04 n.s. n.s. afterL-glucoseconditioning(black)orD-glucose
H
-21h T0 0,5h 1h 1,5h cy ( cirorengduitlaiornninogn-o(rsecdil)l.atBinlgacakctitvraitcyewsithconrorepsepaokn.d to
n
e (E)Trainingprotocolsusedbeforeimagingexper-
u
Starved Cond Imaging q iments:flieswereconditionedonL-glucoseand
e
D-glucose MP1 Fr 0.02 re-fed 3 hr after training on either L-glucose or
D-glucose for 1 min and imaged 30 min after
re-feeding.
-21h T0 0,5h 1h 1,5h (F)Ahigheroscillatoryfrequencyisobservedfor
D-glucose re-fed flies (red) as compared to
0.00 L-glucosere-fedflies(black)(Mann-Whitney:p=
30 60 90 Time (min)
0.011;n=12).
SeealsoFigureS4.Means±SEM;statisticaltest:
Mann-Whitney;**p<0.01incomparisonbetween
twogroups.
D
L-glucose conditioned flies D-glucose conditioned flies
10% 10%
10s 10s
E F Frequency
0.06
*
Starved Cond Feeding Imaging
L-glucose L-glucose MP1
z) 0.04
H
-21h 0h 3h 3,5h y (
c
n
e
u
q
Starved Cond Feeding Imaging Fre 0.02
L-glucose D-glucose MP1
-21h 0h 3h 3,5h
0.00
6 CellReports10,1–9,February24,2015ª2015TheAuthors
Pleasecitethisarticleinpressas:Mussoetal.,DelayedDopamineSignalingofEnergyLevelBuildsAppetitiveLong-TermMemoryinDrosophila,Cell
Reports(2015),http://dx.doi.org/10.1016/j.celrep.2015.01.036
EXPERIMENTALPROCEDURES
FlyStrains
Flystockswereraisedonstandardfoodat18(cid:1)Cand60%relativehumidityun-
dera12hr:12hrlight:darkcycle.TheCanton-Special(CS)strainwasselected
asthewild-typeDrosophilamelanogasterstrain.Alltransgeneswereusedina
CSbackground.ForDAMBexperiments,weusedlinesexpressingtwonon-
overlappingRNAiconstructstargetedagainstDAMB:theJF02043linefrom
theTransgenicRNAiProject(TRiP)andtheKK110947linefromtheVienna
DrosophilaRNAiCenter(VDRC)(Dietzletal.,2007).ForDumbexperiments,
weusedtheKK102341RNAilinefromtheVDRCandthehypomorphicmutant
allele dumb3 PL00420 (Seugnet et al., 2008). We used 50B03-GAL4 and
15E10-GAL4 drivers (Jenett et al., 2012) to manipulate MV1 DA neurons.
TH-GAL4, NP0047-GAL4, NP2758-GAL4, MZ604-GAL4, UAS-TrpA1 and
UAS-shits (Plac¸ais et al., 2012), c061-GAL4;MB-GAL80, and TH-GAL80;
UAS-shits were described in a previous study(Aso et al., 2010). We used
Figure5. MB-MP1ActivationAllowsLTMFormationinAbsenceof tub-GAL80tsforadulttransgeneexpression(McGuireetal.,2003).Forimaging
Energy experiments,flieswereraisedonstandardfoodat25(cid:1)Cand60%relative
WetrainedstarvedflieswithL-glucose,activatedMB-MP1neuronsfor30min
humidityundera12hr:12hrlight:darkcycle.Forcalciumreporting,weused
withTrpA13hrafterconditioning,andfinallyfedfliesfor30min6hrafter
the20XUAS-IVS-GCaMP3line(intheVK00005insertionsite)fromtheJanelia
training.ThisprotocolledtoLTMformation(scoresignificantlydifferentfrom0,
FarmResearchCenter(Akerboometal.,2012).
ttest,t9=3.311,p=0.009,andsignificantlyhigherthanthatofbothparental
groups,F(2,27)=7.201,p=0.003).n=10foralldata.SeealsoFigureS5. TrainingProtocol
Means±SEM;statisticaltest:ttestandone-wayANOVA;n.s.:pR0.05;*p<
Aftereclosion,adultflieswerekeptovernightinfreshbottlescontainingstan-
0.05incomparisonbetweentwogroupsforttestandinposthoccomparisons dardmediumandthentransferredat25(cid:1)Cintostarvationbottlescontaininga
withbothparentalcontrolsforANOVA.
cottonwooldisksoakedwith6.8mlofmineralwaterfor16–21hr.Flieswere
starvedfor24hrat20(cid:1)Cspecificallyfortheactivationexperiment,toavoid
activatingTrpA1-expressingneuronsduringthestarvationperiod.Thecondi-
associatesgustatoryinformationwithodorcanbeconsideredas tioning apparatus and protocol were previously described (Colomb et al.,
aprimingprocessforLTM.Itdoesnotdependonsugarenergetic 2009).Briefly,groupsof30–40fliesofagivengenotypewereconditioned
valueanddoesnotinvolveMB-MP1neurons.Inasecondstep, inabarrelbyexposuretooneodorpairedwithasugarreward,withsubse-
theassociationformedduringtrainingisconsolidatedintoLTM quentexposuretoasecondodorintheabsenceofsucrose.Thesequenceof
asingletrainingsessionconsistedofaninitial90-speriodofnon-odorized
onlyifapost-ingestionsignalthatreflectsthepresenceofenergy
airflow,60sofoneodor,52sofnon-odorizedairflow,60softhesecond
is sent to the MBs by MB-MP1 DA neurons. We showed that
odor,and52sofnon-odorizedairflow.Odorswereproducedusing3-octanol
MB-MP1activitywasnecessaryandsufficient toengageLTM (>95%purity;Fluka74878,Sigma-Aldrich)at3.60310(cid:3)4Mand4-methylcy-
consolidation, even in the absence of ingestion of energetic clohexanol(99%purity;Fluka66360)at3.25310(cid:3)4Mdilutedinparaffinoil.
food. MB-MP1 activity is thus a ‘‘checkpoint’’ for appetitive For D-glucose, L-glucose, and D-fructose experiments, sucrose was re-
LTMprocessing.The5-hrtemporalwindowinwhichtheenergy placed by L-glucose, D-glucose, or D-fructose as the sugar reward, with
levelsignalingcanoccurprobablyreflectsthelifetimeoftheprim- respect to the protocol described in Colomb et al. (2009). All sugars and
phlorizin were obtained from Sigma-Aldrich. For phlorizin experiments,
ingprocess.Suchatwo-stepmechanismforappetitiveLTMfor-
sucrosewasreplacedbyamixtureofD-glucoseandphlorizinorD-fructose
mation provides additional security to associate true nutritive
andphlorizin.
valuewithodor,ratherthananassociationbasedontastealone. For re-feeding experiments, flies were either re-fed on standard food
Indeed, Drosophila innate food preference strongly correlates medium, nutritious D-glucose, or a mixture of D-glucose and phlorizin.
with the activity of the ‘‘sweetness’’ taste receptor response When re-fed on standard medium, flies were transferred into normal food
(Sloneetal.,2007;Gordesky-Goldetal.,2008),whichisplausible bottlesfor30min,either3or6hrafterconditioning,andthenreturnedto
starvation bottles. For sugar re-feeding experiments, flies were placed for
becausegenerallysweetfoodisenergetic.Weidentifiedpost-
1mininatesttube.ThetubewallswerecoveredwitheitherD-glucoseor
feedingsignalsthatcanmodulatethisinnatebehaviorthrough
a mixture of D-glucose and phlorizin (3 hr after conditioning). Flies were
associativelearning,totakeintoaccounttherealnutritiousvalue. thenreturnedtostarvationbottles.Forimaging,flieswereconditionedwith
Altogether,thesefindingsexplainhowtheDrosophilabraineval- either L- or D-glucose and imaged 30 min after training (see Figures 4B–
uatesandremembersfoodenergeticvalue. 4D).Fortheimagingexperimentwithre-feeding,flieswereconditionedon
Interestingly,agatingmechanismalsoexistsfortheconsoli- L-glucoseandthenre-fedwitheitherL-orD-glucosefor1min,3hrafter
training(seeFigures4Eand4F).
dation of aversive LTM thatrelies on the activity of a group of
Allsugarswereusedat2Mconcentrationinmineralwater.Phlorizinwas
threeDAneuronsthatincludes MB-MP1(Plac¸ais etal.,2012).
pre-dissolvedinmineralwaterandusedata200mMfinalconcentration.
ThisgatingofaversiveLTMallowspreventingaversiveLTMfor-
mationwhenfliesarestarved(Plac¸aisandPreat,2013).Thisis TestofMemoryPerformance
crucial because aversive LTM formation presents a non- During the memory performance test, flies were exposed to both odors
negligiblecostforflies(MeryandKawecki,2005),andfliesthat simultaneouslyinaT-mazefor1min.Theperformanceindex(PI)wascalcu-
form aversive LTM while they are food-deprived exhibit latedasthenumberoffliesattractedtotheconditionedodorminusthenum-
beroffliesattractedtotheunconditionedodor,dividedbythetotalnumber
decreasedsurvival(Plac¸aisandPreat,2013).Similarly,whenin-
of flies in the experiment. AsinglePI valuerepresents the averageof the
gestedfoodisnon-energetic,thetwo-stepmechanismprevents
scores from two groups of flies of identical genotype trained with either
appetitive LTM formation and thus avoids a possible waste of octanolormethylcyclohexanolastheCS+(i.e.,anodorpairedwiththesugar
energy. presentation).
CellReports10,1–9,February24,2015ª2015TheAuthors 7
Pleasecitethisarticleinpressas:Mussoetal.,DelayedDopamineSignalingofEnergyLevelBuildsAppetitiveLong-TermMemoryinDrosophila,Cell
Reports(2015),http://dx.doi.org/10.1016/j.celrep.2015.01.036
Temperature-ShiftProtocols denotethesmallestsignificantdifferencebetweentherelevantgroupandits
Toblocksynaptictransmissionduringtrainingandearlyconsolidation,flies genotypiccontrols,usingposthocpairwisecomparisons(*p<0.05,**p<
expressing Shits were placed at the restrictive temperature (33(cid:1)C) 30 min 0.01,and***p<0.001;NS,notsignificant).Forimaging,datafrequencyand
before training, transferred into bottles at the restrictive temperature, and qualityfactorwereconsideredasnull(0)innon-oscillatingflies;foramplitude
putbackintoanincubatoratthepermissivetemperature(18(cid:1)CforLTMand calculation,weretainedthecompletesignal,includingnoise.Leftandright
25(cid:1)Cfor2-hrmemoryexperiments)approximately10–15minafteracquisition, MB-MP1recordingsineachflywererecordedindependently,whereasasingle
where they were progressively returned to the incubator temperature. For valueforamplitude,qualityfactor,andfrequencywasderivedforeachfly
sharpblockadeduringtraining,flieswereplacedattherestrictivetemperature byaveragingthevaluesfromthetwohemispheres.Frequencyandquality
30minbeforetrainingandthenconditionedonthesecondodor;immediately factorcomparisonsbetweengroupswereperformedusingtheMann-Whitney
aftertraining,anairflowatthepermissivetemperature(25(cid:1)C)wasinjected non-parametric test, whereas a two-tailed unpaired t test was used for
throughthebarrels.Forsharpblockadeaftertraining,flieswereconditioned amplitude.
on the second odor at the permissive temperature, and immediately after
training, an airflow at the restrictive temperature was passed through the SUPPLEMENTALINFORMATION
barrelsfor3hr.Permissive-temperaturecontrolexperimentswereperformed
at25(cid:1)C.Timecoursesofthetemperatureshiftsemployedineachexperiment SupplementalInformationincludesfivefigures andcan befound withthis
have been provided alongside the graph of memory performance in each articleonlineathttp://dx.doi.org/10.1016/j.celrep.2015.01.036.
relevantfigure.ForRNAiexpressioninadultMBs,fliesweremaintainedat
30.5(cid:1)Cfor2dayspriortotraining,afterwhichexperimentswereperformed
AUTHORCONTRIBUTIONS
at 25(cid:1)C. For non-induced experiments, flies were placed at 18(cid:1)C for
2dayspriortotrainingandtheexperimentswerethenperformedat25(cid:1)C.
P.-Y.M.performedexperiments.P.T.andT.P.supervisedthework.P.-Y.M.,
For LTM experiments, flies were stored at 18(cid:1)C after acquisition, prior to
P.T.,andT.P.designedexperiments,discussedtheresults,andwrotethe
testing.
manuscript.
Toactivatesynaptictransmissionduringconsolidation,fliesexpressingTrpA1
weretrainedatapermissivetemperature(20(cid:1)C),storedfor3hr,andplacedina
roomat30(cid:1)Cfor30minandthenplacedbackat20(cid:1)C.Memorytestswereper- ACKNOWLEDGMENTS
formedat20(cid:1)C.Inthecontrolexperiment,starvation,conditioning,resting,and
testingwereallperformedatthepermissivetemperature(20(cid:1)C). HiromuTanimotoandLaurentSeugnetkindlyprovidedusefulflystrains.We
thank the TRiP consortium at Harvard Medical School (NIH/NIGMS R01-
GM084947)forprovidingtransgenicRNAiandGAL4flystocks.Wearegrateful
SugarResponseTests
to Aure´lie Lampin-Saint-Amaux for help with behavioral experiments and
TestswereperformedonstarvedfliesinaT-mazeapparatusaspreviously
Pierre-Yves Plac¸ais for advice on calcium-imaging experiments. We thank
described(Colombetal.,2009)butwithoutairflow.Fliesweretrappedineither
membersofourteamforcriticalcommentsonthemanuscript.P-Y.M.was
mazearmafter1min.Thearmwithsugarwasplacedalternatelyontherightor
supportedbyafellowshipfromLabexMemolife.T.P.wassupportedbygrants
left.Sugarresponsewascalculatedasforthememorytestandthenusedasa
fromtheFrenchAgenceNationaledelaRechercheandtheFondationPierre-
score.Thesugarresponsetestswereperformedattherestrictivetemperature
forfliescarryingtheUAS-shitstransgene(33(cid:1)C)andat25(cid:1)C(following2days GillesdeGennespourlaRechercheandP.T.byagrantfromtheIle-de-France
ofinductionat30.5(cid:1)C)forfliescarryingthetub-GAL80tstransgene. region.
Received:September11,2014
OlfactoryAcuity
Revised:December18,2014
Testswereperformedaspreviouslydescribed(Colombetal.,2009)atthe
restrictivetemperatureforfliescarryingtheUAS-shitstransgene(33(cid:1)C)and Accepted:January14,2015
at25(cid:1)C(following2daysofinductionat30.5(cid:1)C)forfliescarryingthetub- Published:February19,2015
GAL80ts transgene. Flies were starved for 21 hr before the olfactory test.
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