SUPPLEMENTARYMATERIAL

Fig.S1.Theeffectofspecificdownregulatedeptinsignalingonthecocaine-CPPandpalatablefood-inducedCPP

(A)Schematicoftheexperimentalschedule.WT mice received an infusion of ACSF or SMLA (500 ng, i.c.v.) immediatelyafterpre-testandeachconditioningsessions.(B)Quantificationofthe placepreferencescoresinthepre-testandtestsessions.(C-D)Leprflox/floxandWTmicewereinjectedwithAAV-CAG-EGFP-T2A-CreintotheVTA.(C)Schematicofthe palatablefood-inducedCPPexperimentalschedule.(D)QuantificationoftheplacepreferencescoresofLeprflox/floxandWTmiceinthepre-testandtestsessions.The dataarepresentedasthe mean±s.e.m*P0.05.

Fig.S2.Levelofcocaine-inducedmonoamineneurotransmittersafterdownregulation of leptinsignaling

(A)Concentrationofcocaine-inducedNE,5-HTand5-HIAAintheNAcofmicereceivingan infusion ofSMLAorACSFi.c.v..(B-C)Concentrationofthe cocaine-inducedNE,5-HTand5-HIAAintheNAc(B)andAMG(C)ofLeprflox/floxandWTmiceinfectedwithAAV-CreinVTA.The dataarepresentedasthe mean±s.e.m.

Fig.S3.TheexpressionofLepRsinbrainregionsnearbytheAAV-CreinjectedVTA,NAcCoreandCeA

RepresentativeimagesoftheAAV-CAG-EGFP-T2A-CreinfectedVTA,NAcandCeAfromthe Leprflox/floxmice.High-magnificationimagesshowedtheexpressionofLepRs nearby theregionsinfectedbyvirus.EGFP:green;LepR:red;ArrowsindicatetheLepR+cells.Scalebar,100μm(low-magnificationimages)and50μm(high-magnificationimages).

Fig.S4.TheeffectofspecificdownregulatedLepRinVTA,NAcorCeAontheanxietylevelandlocomotoractivityofmice

The openfieldtest inLeprflox/floxandWTmiceinfectedwithAAV-CAG-EGFP-T2A-CreintotheVTA(A,B),NAc(C,D)orCeA (E,F).Entriesintocenter(A,C,E),andthe totaldistancetravelled(B,D,F)were measuredfor30min.The dataarepresentedasthe mean±s.e.m.*P0.05.

SUPPLEMENTARY METHODS

Openfieldtests

Anactivitymonitorsystem(43.2cmlength×43.2cmwidth×30.5cmheight,Med-Associates,St.Albans,VT,USA)wasusedtodetecthorizontalmovement.Inbrief,thissystemusespairedsetsofphotobeamstodetectmovement of miceintheopenfield,andmovementsrecordedasbeambreaks.Eachmousewasplacedinthecenteroftheopenfieldandallowedtoexplorefreelyforpredeterminedtime.Thetotaldistancetraveled, theentriesandthe timespent in the centerzonewererecorded.

Elevatedplusmazetest

Theelevatedplusmazeconsistedofacenterplatformandfourarms(34.5cmlength×6.3cmwidth×19.5cmheight)placed75cmabovethefloor.Twoofthearmshad20cmhighdarkwalls(closedarms),andtwohad0.8cmhighledges(openarms).Thearmswereangledat90°toeachother.Theapparatuswasplacedinaquietanddimmedroom.Micewereplacedinthecenter,andtheirbehaviorswererecordedfor5minwithacameralocatedabovethemaze.EthoVisionXT8.5videotrackingprogram(Leesburg,VA,USA)wasusedtotrack the location,velocityandmovementofhead,bodyandtail.Timespentandentriesinthedifferentcompartments(closedandopenarms)wereassessed.Thearmswerecleanedbetweeneachtesttoensuretheabsenceofolfactorycues.

RNAextractionandreal-timePCRanalysis

Miceweredecapitated,andthebrainswereremovedimmediately.TheNAc,VTA,andCeAweredissectedwithin5mininicedPBS.The intended stereotaxic coordinates were as follows: NAc:fromBregma1.54mmto0.86mm;CeA:fromBregma-1.06mmto-1.82mm;VTA:fromBregma-2.92mmto-3.40mm,andfrozeninliquidnitrogenandstoredat–70°Cuntilthe extraction.TotalRNAwasextractedfromtissuesusingtheTRIzol®Reagent(ThermoFisherScientificInc,Waltham,MA,USA)accordingtothemanufacturer’sinstructions.The RT-PCR wereperformed with theSuperscriptFirst-StrandSynthesis system and the Power SYBR Green PCR Master Mix (TAKARA, Shiga, Japan) usingthe EppendorfMastercyclerepgradientSPCRSystem(Eppendorf,Germany).TheprimersforRT-PCRwere as follows:5'-TGGTCCCAGCAGCTATGGT-3'and5'-ACCCAGAGAAGTTAGCACTGT-3'forLepR,5'-GTGGAGTCATACTGGAACATGTAG-3'and5'-AATGGTGAAGGTCGGTGTG-3'forGAPDH.LepRmRNAexpressionwasnormalizedtotheinternalcontrolGAPDH.

Westernblotting

BraintissueswerelysedinRIPAbuffer(50mMTris,pH7.4，150mMNaCl，1%NP-40，0.5%sodiumdeoxycholate，0.1%SDS，andproteininhibitors).Antibodiesofrabbitanti-phospho-STAT3(Tyr705)andmouseanti-STAT3werefromCellSignalingTechnology(Danvers,MA,USA), IRDye800CW-conjugatedor700CW-conjugatedantibody were from RocklandBiosciences (Gilbertsville,PA,USA). The infraredfluorescenceimageswereobtainedwiththeOdysseyinfraredimagingsystem(Li-CorBioscience,Lincoln,NE,USA).

Leptininjection

Recombinantmurineleptin(PeproTech,RockyHill,NJ,USA)wasdissolvedinto0.1mg/μlinsaline,andinjected(1mg/kg,i.p.)30minbeforecocaineconditioning.Controlmicewereadministeredwith equalvolumeofsaline.

Immunohistochemistry

Micewereanesthetizedwithchoralhydrateandperfusedwithsalinefollowedby4%paraformaldehydein0.1Mphosphate-bufferedsaline(PBS).Thebrainswereremoved,fixedin4%paraformaldehydeovernightandsubjectedtodehydrationinincreasingsucrosesolutions(20%–30%)at4ºCfor72hoursbeforeslicingNAc,VTA,andamygdala.The sliceswereincubatedin PBS solution with3%goatserumand0.2%Triton-Xfor1h.Thentheywereincubatedwithmouseanti-LepR(1:100SantaCruzBiotechnology,Dallas,Texas,USA)antibodyovernightat4ºC.SliceswererinsedinPBSthenincubatedingoatanti-mouseCy3(JacksonImmunoresearch,WestGrove,PA,USA)for1hourand mounted.Imageswereacquiredonamicroscopeusing10×or40 × air objectives(DP-80;Olympus,Japan).