ROTHMAN MICHAEL H BAUMANN CHRISTINA M DERSCH DANA V ROMERO KENNER C RICE F IVY CARROLL AND JOHN S PARTILLA Clinical Psychopharmacology Section Intramural Research Program NIDA NIH Baltimore Maryland Laboratory of Medicinal Chemistry NIDDK NIH Bethes ID: 49219
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amphetamine,3,4-methylenedioxymethamphetamine(MDMA),and()-methamphetamine(()-METH)aswellasstimulantswhichhavereceivedscantstudywithmodernneurochemicalmethods:ephedrine,phen-termine,andaminorex.Usingpublisheddata,wethencorrelatedthepotencyoftheagentsforreleasingNEwiththeoraldosewhichproducesamphetamine-typesubjectiveeffectsinhumans.Drugsthatinteractwiththeintegralmembranepro-teinsthatfunctionastransportersforthebiogenicaminesDA,NE,andserotonin(5-HT)canbedividedintotwogroups.Uptakeinhibitorsbindtothetransporter,yetarenottransported.Theseagentselevatesynapticneu-rotransmittersbyinterferingwiththeirremovalfromthesynapticextracellularspace.Substrates,incontrast,aretransportedbytheseproteinsintothenerveterminal,wheretheypromotethereleaseofneurotransmitterbyatwo-prongedmechanism.Substratesincreasecytoplas-micneurotransmitterbyinterferingwiththeaccumula-tionofneurotransmitterinstoragevesiclesandtheypro-moteaprocessofcarrier-mediatedexchange(RudnickandClark,1993).Asnotedabove,manystimulantsde-velopedinthe1950sand1960shavenotbeenstudiedwithmodernneurochemicalmethodsandcouldacteitherasuptakeinhibitorsorsubstratesofthebiogenicaminetransporters.Wethereforecharacterizedtheactionsoftheseagentsusinganinvitromethodthatdiscriminatesbetweentransporteruptakeinhibitorsandsubstrates.MATERIALSANDMETHODSMETHODS3H]DA,[H]5-HTand[H]NEreuptakeassaysTheeffectoftestagentson[H]DAand[uptakewasevaluatedusingpublishedmethods(Roth-manetal.,1993).Brie¯y,synaptosomeswerepreparedfromratcaudatefor[H]DAreuptakeorfromwholeratbrainminuscaudateandcerebellumfor[H]5-HTreuptake.Freshtissuewashomogenizedinice-cold10%sucroseusingaPotter-Elvehjemhomogenizer.Homog-enateswerecentrifugedat1,000for10minat4ÉCandsupernatantswereretainedonice.Polystyrenetesttubes(1275mm)werepre®lledwith50lofKrebs-phosphatebuffer(®nalpH7.4)consistingof0.5mM,0.5mMKH,126mMNaCl,2.4mMKCl,0.83mMCaCl,0.8mMMgCl,11.1mMglucoseatpH7.4,with1mg/mlascorbicacid,1mg/mlbovineserumalbumin(BSA),and50Mpargylineadded(uptakebuffer),750lof[H]DA(5nM)or[H]5-HT(2nM)dilutedinuptakebufferwithoutBSA,and100loftestagentinuptakebuffer.Nonspeci®cuptakewasde®nedusing10Mtyramine([H]DA)or100tyramine([([3H]NEuptakeproceededwithminormodi®cationsoftheaboveprocedure:thetissuesourcewaswholeratbrainminuscaudateandcerebellum,5nMRTI-229wasusedtoblockuptakeof[H]NEintoDAergicnerves,theincubationsproceededfor10minat25ÉC,theconcentrationof[H]NEwas5nM,andnonspeci®cuptakewasde®nedusing10Mindatralineor10Theuptakeassaywasinitiatedbyadding100lofthesynaptosomalpreparationtothetubes.Inhibitioncurvesweregeneratedbyincubating[H]ligandwithtestagent(1nMto100M®naltubeconcentration)dilutedinuptakebuffer.The[H]5-HTreuptakeexperimentswereconductedinthepresenceof100nMnomi-fensineand100nMGBR12935topreventuptakeintonoradrenerigicorDAnerveterminals.Incubationsof15or30minwerecarriedoutat25ÉCfor[H]DAandand3H]5-HT,respectively.Theincubationswereterminatedbyadding4mlofwashbuffercontaining10mMTrisHCl(pH7.4)in0.9%NaClat25ÉC,followedbyrapid®ltrationoverWhatmanGF/B®ltersandtwoadditionalwashcycles.Thetritiumretainedonthe®lterswascountedinabetacounter(Taurus,Titertek,Huntsville,AL)at40%ef®ciencyafteranovernightextractionintoICNCytoscintcocktail(ICNBiomedi-cals,CostaMesa,CA).CA).3H]DA,[H]NEand[H]5-HTreleaseassaysRatcaudate(for[H]DArelease)orwholebrainminuscerebellumandcaudate(for[H]NEandrelease)washomogenizedinice-cold10%sucrosecon-taining1Mreserpine.Nomifensine(100nM)andGBR12935(100nM)werealsoaddedtothesucrosesolutionfor[H]5HTreleaseexperimentstoblockanypotential[H]5HTreuptakeintoNEandDAnerveterminals.After12strokeswithaPotter-Elvehjemho-mogenizer,homogenateswerecentrifugedat1,00010minat0±4ÉCandthesupernatantswereretainedonice(synaptosomalpreparation).Eachratbrain(ap-proximately1,200mg)producedenoughtissueprepa-rationfor250testtubesforthe[H]DAand[releaseassaysandfor125testtubesforthe[releaseassay.Synaptosomalpreparationswereincubatedtosteady-statewith5nM[H]DA(30min),7nM[(60min),or5nM[H]5HT(60min)inuptakebuffer(withoutBSA)plus1Mreserpineinapolypropylenebeakerwithstirringat25ÉC.Nomifensine(100nM)andGBR12935(100nM)wereaddedtothebufferforfor3H]5HTreleaseexperiments.RTI-229(5nM)wasaddedtothebufferfor[H]NEreleaseexperimentstoprevent[H]NEreuptakeintodopaminenerveterminals.Afterincubationtosteady-state,850lofsynap-tosomespreloadedwith[H]neurotransmitterwereaddedto1275mmpolystyrenetesttubeswhichcontained150ltestdruginuptakebuffer.After5minmin3H]DAand[H]5-HT)or30min([H]NE)thereleasereactionwasterminatedbydilutionwith4mlwashbuffer(10mMTris-HClpH7.4containing0.9%NaClat25ÉC)followedbyrapidvacuum®ltrationoverWhat-manGF/B®ltersusingaBrandelHarvester.The®lterswererinsedtwicewith4mlwashbufferusingtheBrandelHarvesterandtheretainedtritiumwasNOREPINEPHRINEANDSTIMULANTADDICTION countedbyaTaurusliquidscintillationcounterat40%ef®ciencyafteranovernightextractionin3mlCyto-scint(ICN).Typicalsignal-to-noiseratiosarereportedinTableI.DataanalysisandstatisticsAspreviouslydescribed(Rothmanetal.,1993),valuesweredeterminedusingthenonlinearleast-squarescurve-®ttingprogramMLAB-PC(CivilizedSoftware,Bethesda,MD).Inuptakeexperiments,valueswerecalculatedaccordingtotheformula(ChengandPrusoff,1973):)wheretheconcentrationoftheradiolabeleddrug([([3H]NEor[H]5-HT).Inthe[H]NEuptakeassays,surfaceswere®ttoone-andtwo-componentmodels(Akunneetal.,1994).TheF-testwasusedtodeterminethebetter-®ttingmodel.Inreleaseexperiments,theofantagonistswascalculatedaccordingtothefollowingformula:[Antagonist]/(1)whereistheintheabsenceofantagonistandistheinthepresenceofDrugsandreagents)-Fen¯uramineHCl,()-fen¯uramine,chlorphen-termineHCl(220.2),()-amphetaminesul-fate,phentermineHCl,()-MDMA,()-METH,and)-METHwereobtainedfromtheAddictionResearchCenterPharmacy(NIDA,NIH,Baltimore,MD).Ami-norex,()-norepinephrine,dopamine,desipramine,¯uoxetine,()-ephedrine,tyramine,andindatralinewerepurchasedfromResearchBiochemicals(Natick,MA).RTI-55andRTI-229wereprovidedbyDr.F.IvyCarroll.[H]DA(Ci/mmol),[H]NE(55Ci/mmol)and[H]5-HT(Ci/mmol)werepurchasedfromDupontNewEnglandNuclear(Boston,MA).Thesourcesofotherreagentsarepublished(Rothmanetal.,1993,1994).1994).3H]NEuptakeexperimentsInitialexperimentsindicatedthat[H]NEuptakewascomposedoftwocomponents.Tocharacterizethesecomponents,uptakeªsurfacesºweregeneratedaccordingtotheexperimentaldesignoutlinedinTableII.Twoconcentrationsof[H]NE(5nMand105nM)wereeachinhibitedbyNE,theNE-selectiveuptakeinhibitordesipramine,andtheDA-selectiveuptakein-hibitorRTI-229intheabsenceandpresenceoftheindicatedconcentrationsofªblockingºagents.AsreportedinTableIII,[H]NEuptakeinhibition®tatwo-componentmodel.NEhadthehigheraf®nityforthelowercapacitycomponent(63.9nM)andloweraf®nityforthehighercapacitycomponent706nM).Thehighaf®nityandselectivityofdesipramineforthelowercapacitycomponentidenti-®esthisastheNEtransporter.Thehighaf®nityandselectivityofRTI-229forthehighercapacitycompo-nentidenti®esthisastheDAtransporter.InitialreleaseexperimentsTime-courseexperimentswith[H]NEwereconductedintheabsenceandpresenceof1Mreserpinetodeterminethetimerequiredtoreachsteady-state.AsreportedinFigure1A,[H]NEfailedtoachieveTABLEI.Representativesignal-to-noiseratiosobservedinthereleaseassays 3H]NE3,0001,5001,5002.02.03H]DA3,7001,9001,8001.91.93H]5-HT13,0006,5006,5002.0Representativecountsperminute(cpm)obtainedwhenthereleaseassaysareconductedasdescribedinMethods.TABLEII.Experimentaldesignusedtocharacterize[H]NEuptake Surface/[NE]nMPrimaryinhibitorBlocker1NENone5nM[3H]NE(10±10,000nM)2NENone5nM[3H]NE100nMNE(10±10,000nM)3NE25nM5nM[3H]NE(10±10,000nM)Desipramine4NE25nM5nM[3H]NE100nMNE(10±10,000nM)Desipramine5NE20nMRTI2295nM[3H]NE(10±10,000nM)6NE20nMRTI2295nM[3H]NE100nMNE(10±10,000nM)7DesipramineNone5nM[3H]NE(0.5±50,000nM)8DesipramineNone5nM[3H]NE100nMNE(0.5±50,000nM)9Desipramine20nMRTI2295nM[3H]NE(0.5±50,000nM)10Desipramine20nMRTI2295nM[3H]NE100nMNE(0.5±50,000nM)11RTI229None5nM[3H]NE(0.1±1,000nM)12RTI229None5nM[3H]NE100nMNE(0.1±1,000nM)13RTI22925nM5nM[3H]NE(0.1±1,000nM)desipramine14RTI22925nM5nM[3H]NE100nMNE(0.1±1,000nM)desipramineUptakeªsurfacesºweregeneratedaccordingtotheexperimentaldesignde-scribedabove.Twoconcentrationsof[H]NE(5nMand105nM)wereeachinhibitedbyeightconcentrationsofNE,theNE-selectiveuptakeinhibitordesi-pramine,andtheDA-selectiveuptakeinhibitorRTI-229intheabsenceandpresenceoftheindicatedconcentrationsofªblockingºagents.TABLEIII.Best-fitparameterestimatesfor[uptakeinhibition ParameterNEtransporterDAtransportermax(fmol/mgprotein)2,6005010,200NE(,nM)63.91.6706Desipramine(,nM)0.057,220RTI-229(,nM)0.60.35ThedataofthethreeexperimentsdescribedinTableIIwerepooledtoyield504points.Theentiresetofdatawas®ttoaone-componentmodel(sum-of-8,220)andatwo-componentmodel(sum-of-squares536).Thetwo-componentmodel®tsigni®cantlybetterthantheone-componentmodelEachvalueisR.B.ROTHMANETAL. steady-stateby60minintheabsenceofreserpine,butdidsoby60mininthepresenceof1Mreserpine(Fig.1B).Reserpinereducedretained[H]NEby65%at60minandincreasedtheabilityof()-METHtoreleaserelease3H]NE(Fig.2).Wethereforeconductedreuptaketime-courseexperimentswith[H]5-HTand[H]DAinthepresenceofreserpine.Thesedata(notshown)demon-stratedthatsteady-statewasachievedby30minwith5nM[H]DAandby60minwith5nM[H]5HT.Otherexperimentsshowedthataccumulationof[transmitteratsteady-statewasdirectlyproportionaltoprotein(datanotshown).Typicalproteinconcentra-tionsusedwere:0.75mg/tubefor[H]NE,0.35mg/tubefor[H]5-HT,and0.07mg/tubefor[Effectofincubationtimeondrug-induceddrug-induced3H]DAreleaseTodeterminetheeffectofincubationtimeondrug-induced[H]DArelease,dose±responsecurvesweregeneratedwithGBR12909,aDAuptakeinhibitor,and)-METH,aDAreleaser,withtheincubationsbeingterminatedafter5,10,15,or30min.AsreportedinFigure3A,longerincubationtimeswithGBR12909increasedthepotencyofGBR12909forstimulatingstimulating3H]DArelease.Thereleasecurvewasbiphasicwiththeleveloftheplateauincreasingwithshortertimeincubations.Witha5-minincubation,evenhighcon-centrationsofGBR12909releasednomorethan20%ofaccumulated[H]DA.Incontrast,asreportedinFigure3B,()-METH-induced[H]DAreleasewasalmostidenticalatallincubationtimesandcompletereleasewasachieved.Basedontheseresults,[H]DAandand3H]5-HTreleaseassayswereterminatedaftera5-minincubationwithtestdrugs.Similarexperimentsdem-onstratedthata30-minincubationwithtestdrugswasoptimumforthe[H]NEreleaseassay(datanotPharmacologicalblockadeofreleaseUptakeinhibitorsblockthereleasingeffectofsub-stratesbypreventingtheentryoftransportersub-stratesintothenerveterminal(HurdandUngerstedt,1989a).Toseewhetherthisblockadealsooccurredwiththeinvitroreleaseassays,substratedose±re-sponsecurvesweregeneratedintheabsenceandpres-enceoflowconcentrationsofselectiveuptakeinhibi-tors.AsreportedinTableIVA,concentrationsoftheNE-selectiveuptakeinhibitordesipramine(DMI)whichdidnotalter[H]NErelease,increasedthevaluesfor()-METH-induced[H]NErelease.Similarly,concentrationsoftheDA-selectiveuptakeinhib-itorGBR12909whichdidnotalter[H]DArelease,increasedthevaluesfor((3H]DArelease(TableIVB).Finally,asshowninTableIVC,concentrationsofthe5-HT-selectiveuptakeinhib-itor¯uoxetinewhichdidnotalter[H]5-HTreleaseincreasedthevaluesfor((3H]5-HTrelease.Basedontheshifttotherightinthe Fig.1.Time-courseof[H]NEaccumulationbyratbrainsynaptoIntheabsenceofreserpine.Inthepresenceof1reserpine.EachpointisthemeanSD( Fig.2.Effectof()-METHon[H]NEreleaseasafunctionoftimeintheabsenceandpresenceof1Mreserpine.EachpointistheSD(3).*whencomparedtothenoreserpinecontrol(two-tailedNOREPINEPHRINEANDSTIMULANTADDICTION dose±responsecurves,itwaspossibletocalculateanapparentfunctionalvalueforeachuptakeinhibitor.TheapparentvaluesofDMI(about3nM),GBR12909(about0.3nM)and¯uoxetine(about3nM)weresimilartotheirvaluesforinhibitingtheuptakeof[H]NE,[H]DAand[H]5-HT,respectively(TableV).PharmacologicalprofileofCNSstimulantsandrelateddrugsWedeterminedtheactivityofavarietyoftrans-porterligands,bothuptakeinhibitorsandsubstrates,at[H]NEreleaseanduptake,[H]DAreleaseanduptake,and[H]5-HTreleaseanduptake(TableV).DA-selectiveuptakeinhibitorssuchasGBR12909,GBR12935,andRTI-229wereallinactiveinthereleaseassays.5-HT-selectiveuptakeinhibitorssuchas¯uox-etineandcitalopramwerealsoinactiveinthereleaseassays.Similarly,theNE-selectiveuptakeinhibitorsdesipramineandmazindolwereinactiveinthereleaseassays.NonselectiveuptakeinhibitorssuchascocaineandRTI-55werealsoinactiveinthereleaseassays.Theendogenoussubstratesofthetransporterswerealsotested.5-HTpotentlyinhibited[H]5-HTuptake16.7nM)andwasabout160-foldlesspotentatinhibiting[H]DAand[H]NEuptake.Consistentwiththesedata,5-HTpotentlyreleased[H]5-HT(44.4nM)andwasessentiallyinactiveasareleaserofof3H]NEand[H]DA.BothNEandDAweaklyinhibitedinhibited3H]5-HTuptakeandwereinactiveasreleasersofof3H]5-HT.DAmorepotentlyreleased[H]NE(66.2nM)than[H]DA(nM).Incontrast,NEmorepotentlyreleased[H]NE(164nM)than[H]DA(869nM).Ofnote(seeDiscussion),DAwaspotentlyaccumulatedbynoradrenergicnerves.Incontrasttouptakeinhibitors,whichwereessen-tiallyinactiveinthereleaseassays,compoundsknowntobesubstrateswereactiveinbothuptakeandreleaseassays,althoughtheyweregenerally5±10-foldmorepotentinthereleaseassays.()-METHwasmostpo-tentatNErelease(nM),followedbyDArelease(nM),and5-HTrelease(736nM).()-Amphetaminewasmorepotentthan(METHandhadasimilarpro®le:NErelease(7.1nM),DArelease(nM),and5-HTrelease(nM).EphedrinewasapotentandselectivereleaserofNE:NErelease(DArelease(nM),and5-HTrelease Fig.3.EffectoftimeonGBR12909-induced()and(induced((3H]DArelease.EachpointisthemeanSD(TABLEIV.Effectofuptakeinhibitorsonon3H]neurotransmitterrelease A.EffectofDMIon()-METH-induced[H]NEreleaserelease(1)-METH(IC506SD)N6SDDMIapparentKi(nM)010.60.60.891nM15.71.10.860.052.110nM36.85.80.700.0814.9100nM249350.780.094.4 B.EffectofGBR12909on()-METH-induced[H]DAreleaseGBR12909(nM)018.11.40.925288301.080.110.33nM201,4501280.910.070.25nM C.Effectof¯uoxetineon()-fen¯uramine-induced[[(1)-Fen¯uramine(IC506SD)N6SDFluoxetineapparentKi(nM)045.911.80.8910nM135101.000.075.1100nM2,3051940.750.052.0Dose±responsecurvesweregeneratedusing10concentrationsofsubstrate.Thedatafromthreeexperimentswerepooled(30points)and®ttothetwoparameterlogisticequationforthebest-®testimatesoftheandslopefactor().DMIhadnoeffecton[H]NErelease.GBR12909at5nMand20nMinhibitedretained[H]DAby15%.Fluoxetineat10nMand100nMinhibitedretainedretained3H]5-HTby3%and7%respectively.EachvalueisthemeanSDofthreevaluesweregreaterthan0.93.R.B.ROTHMANETAL. nM).Tyraminehadapro®lesimilartothatof()-METH,butwaslesspotent:NEreleasenM),DArelease(119nM)and5-HTrelease(nM).Theneurotoxicamphetamineanalog,MDMA,wasessentiallyequipotentat5-HTandNErelease(56.6and77.4nM,respectively)andconsiderablyweakeratDAre-lease(376nM).Thereleaseassayswerealsousedtopro®lestimu-lantmedicationsdevelopedintheearly1960s.Phen-terminehadanamphetamine-typepro®le,beingmostpotentatNErelease(39.4nM)followedbyDArelease(262nM)andthen5-HTreleasenM).AminorexwasalmostequipotentatNE(26.4nM)andDA(49.4nM)andmorepotentat5-HTrelease(193nM)thanphentermine,()-METH,or(Chlorphenterminewasthemostpotentandselective5-HTreleaser:30.9nMfor5-HTrelease,2,650nMforDArelease,and10,000nMforNErelease.AlthoughineffectiveatNErelease,chlorphen-terminewasamoderatelypotentNEuptakeinhibitor338nM),indicatingthatasingleagentmaybeasubstrateatonetransporterandanuptakeinhib-itoratanother.()-Fen¯uraminepotentlyreleased5-HT(51.7nM)andlesspotentlyreleasedNE302nM).()-Fen¯uraminewasessentiallyinactiveatDAuptakeorrelease.MechanismofactionofstimulantsUsingthereleaseassay,itwaspossibletocharacter-izethemechanismofactionofstimulantswhichhavereceivedscantcontemporaryattention:ephedrine,aminorex,phentermine,andchlorphentermine.Inapreviousstudyweusedadifferentmethod,describedindetailinthatpublication,toclassifyadrugasasubstrateoruptakeinhibitor(Rothmanetal.,1999).Aminorexwasclassi®edasanuptakeinhibitorattheDAtransporteranda5-HTtransportersub-strate.Phenterminewasclassi®edasDAtransportersubstrateandasaweaksubstrateoranuptakeinhib-itoratthe5-HTtransporter.Thismethodclassi®edchlorphentermineasa5-HTtransportersubstrateandDAuptakeinhibitor.Themorere®nedmethodsusedhere,whichdirectlydeterminesubstrate-typeactivity,yieldeddifferentresults:aminorexandchlorphenter-mineareDAtransportersubstratesandphentermineisaweak5-HTtransportersubstrate.Thecore®ndingofourpreviousarticle(Rothmanetal.,1999)thatami-norex,()-fen¯uramine,andchlorphentermineare5-HTtransportersubstrateshasbeencon®rmed.Thelowpotencyof()-METHand(at5-HTreleaseanduptakeinhibitionissimilartothatreportedbyothers(Eshlemanetal.,1999),whochar-acterizedtheseagentsinuptakeinhibitionassaysus-ingclonedDA,5-HT,andNEtransporters.Theresultsforothercompoundsstudiedhereare,ingeneral,sim-ilartothoseobservedusingtheclonedtransporters(Eshlemanetal.,1999).DAistakenupbybothdopa-minergicandnoradrenergicnerveswithaboutequalpotency.Consistentwiththesedata,selectivenorad-renergicuptakeinhibitorsincreaseextracellularDAinthen.accumbens,ventraltegmentalarea,andfrontalcortexofrats(Reithetal.,1997;Carbonietal.,1990;YamamotoandNovotney,1998).TABLEV.PharmacologicalprofileofselectedagentsintheDA,NEand5-HTreleaseanduptakeinhibitionassays NEreleaseNEuptake5-HTrelease5-HTuptakeDAreleaseDAuptakeAminorex26.42.854.54.8193231,24410649.47.521610,0004516630.95.433862,6502733,940Phentermine39.46.6244153,51125313,900510262211,580)-Amphetamine7.070.9538.91.81,765943,83017024.83.534)-Methamphetamine28.52.5234144,64024314,000644416204,840)-Methamphetamine12.30.748.05.1736452,1379824.52.1114)-Fen¯uramine302201,29015251.76.115010,00022,000)-Fen¯uramine739571,98720579.311.526910,00023,700)-Ephedrine72.410.222550,0001,3501244,398Tyramine40.63.572.55.02,7752341,5569511911106)-MDMA77.43.44621856.62.123813376161,572Norepinephrine1641363.950,00086951357Dopamine66.25.440.310,0006,48920086.99.738.310,0003,01326644.45.316.710,0002,70310,00027710,00028910,0003.7010,00079.210,00073.210,0004.310,00077910,00030410,00047810,0002.8810,00027210,00025.910,0008.3210,00035010,0005,94610,00068810,0009.5810,0004,33210,0002.4010,00020,48510,0005.8910,0001.0010,0000.8310,00019.510,00036210,0002.1510,00012.610,0003.100.092,8107771.90EachvalueisthemeanSDofthreeexperiments.Datafrom(Rothmanetal.,1993).DatafromMateckaetal.(1996).NOREPINEPHRINEANDSTIMULANTADDICTION ObservationsthatblockadeofNEtransportersin-creasesextracellularDAcanexplainwhydopaminetransporterknockoutmiceself-administercocaine(Rochaetal.,1998)anddemonstratecocaine-condi-tionedplacepreference(Soraetal.,1998).AlthoughthesedatasuggestthattheDAtransporterisnotcrit-icalformediatingcocainereward,thedatadonotruleoutaroleformesolimbicDAasamediatorofcocainereward.TheinabilityofcocainetoelevateextracellularDAinthestriatumofDATknockoutmice(Rochaetal.,1998)isconsistentwiththelackofNEtransportersinthisbrainregion.Thus,intheabsenceoftheDAtrans-porteritisreasonabletoassumethatNEnerveswouldaccumulatesomeoftheextracellularDAinthen.ac-cumbens.SincecocaineisapotentinhibitoroftheNEtransporter(Eshlemanetal.,1999),administrationofcocainewouldinhibitDAtransportintoNEnerveter-minals,therebyincreasingextracellularDAandtrig-geringcocainereward.Drugself-administrationinanimalsismediatedbyincreasesinmesolimbicsynapticDA(Wise,1996).Thus,onemightexpect,asnotedforDAuptakeinhib-itors(Ritzetal.,1987),thattherankorderofpotencyofthestimulantsstudiedhereattheDAtransportershouldbethesameastheirpotencyasreinforcers.AsreportedbyGrif®thsandassociatesforababoonself-administrationmodel(Grif®thsetal.,1979;Sannerudetal.,1989),therank-orderofpotencyforself-admin-istrationwasamphetamine(24.8nM)(262nM)MDMA(376nM)ephedrine(1,350nM)chlorphentermine(2,650nM)fen¯uraminewasnotself-administered(10,000nM).Thevaluesinparen-thesesarethevaluesforstimulating[H]DArelease.Thus,forthesestimulantstherankorderofpotencyforDArelease(TableV)issimilartotheirrankorderofpotencyinself-administration.Phentermine,whichis10-foldlesspotentthanamphetamineinre-inforcement,is10-foldlesspotentthanamphetamineinreleasing[Relevanceoffindingstostimulant-inducedsubjectiveeffectsinhumansTheroleofDAinmediatingreward/reinforcementbehaviorinanimalmodelsiswellacceptedandsup-portedbythedata.Althoughitistemptingtoassumethattheneurochemicalmechanismsmediatingrein-forcementbehaviorinanimalmodelsofdrug-seekingbehaviorandtheneurochemicalmechanismsmediat-ingthesubjectiveeffectsofstimulantsinhumansarethesame,theneurochemicalmediatorofamphet-amine-likepositivesubjectiveeffectsinhumansre-mainstobeestablished.Indeed,asnotedintheIntro-duction,considerabledatasuggestthatDAmaybenecessary,butnotsuf®cient,toproducestimulant-in-ducedsubjectiveeffectsinhumans(RothmanandGlowa,1995;Rothman,1994;Braueretal.,1997;Vil-lemagneetal.,1999;Ohuohaetal.,1997;Malisonetal.,1997;Priceetal.,1997;BraueranddeWit,1996,1997).TheauthorswishtoemphasizethatwearenotquestioningtheroleofDAasamediatorofreinforce-mentbehaviorinanimalmodelsofdrug-seekingbe-havior,butratherask:Whatneurochemicalscontrib-utetostimulant-inducedsubjectiveeffects(theªhighº)inhumans?Amongthesubstrate-typestimulantstestedhere,phentermine,MDMA,amphetamine,()-METH,andephedrineproduceamphetamine-likesubjectiveeffectsinhumans(Grif®thsetal.,1979;Vollenweideretal.,1998;Braueretal.,1996;Chait,1994).Aminorexactsasalocomotorstimulantinanimalsandgeneralizestothediscriminativecueofamphetamine,andwouldbeexpectedtoproduceamphetamine-likesubjectiveef-fectsinhumans(Woolvertonetal.,1994).Fen¯ura-mine(Grif®thetal.,1975)andchlorphentermine(Grif-®thetal.,1976)donotproduceamphetamine-likesubjectiveeffectsinhumans.Ourresultsshowthatthemostpotentactionofthestimulantswhichproduceamphetamine-likesubjectiveeffectsistoreleaseNE.Importantly,()-amphetamine,()-ephedrine,phen-termine,andMDMAare3.5-fold,19-fold,6.6-fold,and4.8-foldmorepotentatNEreleasethanDArelease.ThesedataraisethepossibilitythatthereleaseofNEcontributestothepositivesubjectiveeffectsproducedbythesesubstrate-typestimulants.Wetermthistheªnoradrenergichypothesis.ºAdirectpredictionofthereleasedataandthenor-adrenergichypothesisisthatoraldosesofthesemed-icationswillproducesympathomimeticeffects,whicharemediatedviareleaseofNE,andamphetamine-likesubjectiveeffects,atlowerdosesthaneffectswhicharemediatedbyDArelease.Inhumans,thenoradrenergiceffectsofthesecompoundscanbeassessedviamea-surementofphysiologicalparameterssuchassystolicbloodpressure.Thedopaminergiceffectsofthesecom-poundscanbeassessedbytheireffectonplasmapro-lactinlevels,whicharedecreasedbydopaminergicago-nists(AscoliandSegaloff,1996),andapparentlynotaffectedbyagentswhichincreaseNE,suchastheselectiveNEuptakeinhibitormaprotiline(Steigeretal.,1993)ortheselectiveNEreleaserandadrenergicagonistephedrine(Angristetal.,1977).Thenoradrenergichypothesisreceivesstrongsup-portfromstudiesconductedinhumans.Oraldosesof-amphetamineintherangeof30±40mgproducesympathomimeticeffectsandsubjectiveeffectswiththesametimecourse(HeishmanandHenning®eld,1991;Martinetal.,1971).Importantly,amineatthisdoserangedoesnotdecreaseplasmaprolactin(Gradyetal.,1996;Masetal.,1999),whichshouldoccurif-amphetaminewerereleasingDA.Similar®ndingsarereportedforMETH(Martinetal.,1971;Gouzoulis-Mayfranketal.,1999).Moreover,MDMA-inducedsubjectiveeffectsoccuratthesamedosesandwiththesametimecourseasitssympatho-R.B.ROTHMANETAL. mimeticeffects(Vollenweideretal.,1998;Masetal.,1999).SinceMDMAreleasesNEand5-HTwiththesamepotency,ourdatapredictthatMDMAshouldreleaseprolactin,aserotonergiceffect(Coccaroetal.,1996),atthesamedoserangeandtimecourseasitssympathomimeticeffects.Indeed,MDMAincreasedplasmaprolactinwiththesametimecourseasitssym-pathomimeticeffects(Masetal.,1999).Asnotedabove,ephedrine-inducedsubjectiveeffectsoccuratthesamedosesandwiththesametimecourseasitssympatho-mimeticeffects(Grif®thsetal.,1979)andephedrinedoesnotdecreaseprolactin(Angristetal.,1977).Com-parableoraldosesofphentermineandamphetamineproducesimilarsubjectiveeffects,yet,asnotedabove,phentermineis10-foldweakeratDAreleasethanam-phetamine,suggestingthatreleaseofDAdoesnotme-diatephentermine-inducedsubjectiveeffects.Consis-tentwiththenoradrenergichypothesis,oraldosesofstimulantswhichproducesubjectiveeffectsarecorre-latedwiththeirpotencyinreleasingNE,notDA(Fig.4).Thesedata,summarizedinTableVI,suggestthatthesemedicationsproduceamphetamine-likesubjec-tiveeffectsatdoseswhichactivatethenoradrenergic,notthedopaminergicsystem.Orallyadministereddextroamphetamine(5±10mgtwoorthreetimesperday)iswidelyusedtotreatattentionde®citdisorder(Eliaetal.,1999).Asnotedabove,amphetamineprobablydoesnotreleaseDAatthisdoserange.Thus,ourresultssuggestthattheprimaryneurochemicalmechanismfortheef®cacyofamphetamineintreatingattentionde®citdisorderre-sultsfromreleaseofNE,perhapsleadingtoactivationofcentralalphaadrenergicreceptors.Consistentwiththisnotion,directalphaagonists,suchasclonidineandguanafacine,effectivelytreatattentionde®citdis-order(Huntetal.,1986,1995).MedicationswhoseprimarymechanismofactionisinhibitionofNEreuptake(antidepressants)donotpro-duceamphetamine-likesubjectiveeffects.Uptakein-hibitorsactivatenegativefeedbackloopswhichde-creasecell®ringratesviasomatodendriticautoreceptors.Thisisobservedforthedopaminergic,serotonergic,andnoradrenergicsystems(Dianaetal.,1991;MoretandBriley,1997;Mongeauetal.,1998;CunninghamandLakoski,1990).Transportersub-strates,however,releaseneurotransmitterinanerve-impulseindependentmannerandarenotsubjecttothesenegativefeedbackmechanisms.Sincetheabilityofuptakeinhibitorstoincreaseextracellularneuro-transmitterisnerveimpulse-dependent(HurdandUn-gerstedt,1989b),reuptakeinhibitorsmaynotelevateextracellularneurotransmittertothelevelachievedbytransportersubstrates.ThismayexplainwhyNEup-takeinhibitorsdonotproduceamphetamine-likesub-jectiveeffects.CocaineblocksthereuptakeofNE,DA,and5-HTwithsimilarpotency(TableV).AsreviewedintheIntroduction,agrowingbodyofdatasuggeststhatDAisnotthesolemediatorofcocainesubjectiveeffectsinhumans.Unlikeantidepressants,cocaineproducesin-tenseamphetamine-likesubjectiveeffects.Assumingforthemomentthatthisisduetoincreasesinextra-cellularNE,thefundamentaldifferencebetweenco-caineandantidepressantsmaybetherateatwhichcocaineentersthebrain(BalsterandSchuster,1973).Alternatively,cocainebindingsitesnotassociatedwiththebiogenicaminetransportersmightcontributetococaine-inducedsubjectiveeffects(Rothmanetal.,Althoughitistemptingtospeculatethatstimulant-inducedpositivesubjectiveeffectsinhumansareme-diatedbyasingleneurotransmitter,themorelikelyscenarioisthatmultipleneurochemicalsandbrain Fig.4.Correlationoforaldosesofstimulantswhichproduceam-phetamine-likesubjectiveeffectswiththeirpotencyinreleasingreleasing3H]NE()and[H]DA(NOREPINEPHRINEANDSTIMULANTADDICTION regionscontributetothesubjectiveexperiencede-scribedastheªhigh.ºThe®ndingthatlowdosesofselectiveNE,DA,and5-HTuptakeinhibitorsenhancethecocainediscriminativecueinratsmaysupportthisnotion(CunninghamandCallahan,1991).Clearly,ad-ditionalexperimentsareindicatedtotesttheªnorad-renergichypothesis.ºInparticular,itwouldbeofin-teresttodevelopahighlyselectiveNEreleaseranddetermineitssubjectiveeffectsinhumans.These®nd-ingsunderscoretheimportance,indevelopingpoten-tialtreatmentagentsfor()-METHabuse,ofneutral-izingtheeffectsof()-METHontheNEsystem(Rothmanetal.,2000).These®ndingsemphasize,moreover,thatstimulant-inducedreinforcementbe-haviorinanimalsandstimulant-inducedpositivesub-jectiveeffectsinhumansmayutilizedifferentneuro-chemicalsystems.ThepossibilitythatNEcontributestostimulant-inducedpositivesubjectiveeffectsinhu-mansdoesnotruleoutaroleforDAineithersubjectiveeffectsorstimulantaddiction.Forexample,itispossi-blethatevenifaªhighºismediatedmostlybyNE,theintenserepetitivedrug-takingbehaviorseeninse-verelyaddictedindividualsismediatedbymesolimbicDA.Perhapsmostimportantly,ourdatahighlighttheimportanceofconsideringtheactionsofcocaineandstimulantsonneuronalsystemsotherthanDA,bothtounderstandtheirmechanismofactionandtodevelopeffectivepharmacotherapeuticmedications(Rothmanetal.,1998a;BaumannandRothman,1998).AkunneHC,DerschCM,CadetJL,BaumannMH,CharGU,PartillaJS,deCostaBR,RiceKC,CarrollFI,RothmanRB.1994.Studiesofthebiogenicaminetransporters.III.Demonstrationoftwobindingsitesfor[H]GBR12935and[H]BTCPinratcaudatemembranes.JPharmacolExpTher268:1462±1475.AngristB,RotrosenJ,KleinbergD,MerriamV,GershonS.1977.DopaminergicagonistpropertiesofephedrineÐtheoreticalimplica-tions.Psychopharmacology(Berl)55:115±120.AscoliM,SegaloffDL.Adenohypophysealhormonesandtheirhypo-thalamicreleasingfactors.In:HardingJG,LimbirdLE,MolinoffPB,RuddonRW,GilmanAG,editors.Goodman&Gilman'sthepharmacologicalbasisoftherapeutics.NewYork:McGraw-Hill;BalsterRL,SchusterCR.1973.Fixed-intervalscheduleofcocainereinforcement:effectofdoseandinfusionduration.JExpAnalBehav20:119±129.BaumannMH,RothmanRB.1998.Alterationsinserotonergicre-sponsivenessduringcocainewithdrawalinrats:similaritiestoma-jordepressioninhumans.BiolPsychiatry44:578±591.BrauerLH,deWitH.1996.Subjectiveresponsestod-amphetaminealoneandafterpimozidepretreatmentinnormal,healthyvolun-teers.BiolPsychiatry39:26±32.BrauerLH,deWitH.1997.Highdosepimozidedoesnotblockamphetamine-inducedeuphoriainnormalvolunteers.PharmacolBiochemBehav56:265±272.BrauerLH,JohansonCE,SchusterCR,RothmanRB,deWitH.1996.Evaluationofphentermineandfen¯uramine,aloneandincombi-nation,innormal,healthyvolunteers.NeuropsychopharmacologyBrauerLH,GoudieAJ,deWitH.1997.Dopamineligandsandthestimuluseffectsofamphetamine:animalmodelsversushumanlaboratorydata.Psychopharmacology130:2±13.CarboniE,TandaGL,FrauR,DiChiaraG.1990.Blockadeofthenoradrenalinecarrierincreasesextracellulardopamineconcentra-tionsintheprefrontalcortex:evidencethatdopamineistakenupinvivobynoradrenergicterminals.JNeurochem55:1067±1070.ChaitLD.1994.Factorsin¯uencingthereinforcingandsubjectiveeffectsofephedrineinhumans.Psychopharmacology(Berl)113:ChengY,PrusoffWH.1973.Relationshipbetweentheinhibitionconstant(K1)andtheconcentrationofinhibitorwhichcauses50percentinhibition(I50)ofanenzymaticreaction.BiochemPhar-macol22:3099±3108.CoccaroEF,KavoussiRJ,CooperTB,HaugerRL.1996.Hormonalresponsestod-andd,l-fen¯uramineinhealthyhumansubjects.Neuropsychopharmacology15:595±607.CunninghamKA,CallahanPM.1991.Monoaminereuptakeinhibi-torsenhancethediscriminativestateinducedbycocaineintherat.Psychopharmacology104:177±180.CunninghamKA,LakoskiJM.1990.Theinteractionofcocainewithserotonindorsalrapheneurons.Single-unitextracellularrecordingstudies.Neuropsychopharmacology3:41±50.DianaM,PaniL,RossettiZ,PassinoN,GessaGL.1991.Flunarizineattenuatescocaine-inducedinhibitionofA9dopaminergicneurons.PharmacolRes24:197±203.DommisseCS,SchulzSC,NarasimhachariN,BlackardWG,HamerRM.1984.Theneuroendocrineandbehavioralresponsetodextro-amphetamineinnormalindividuals.BiolPsychiatry19:1305±1315.EliaJ,AmbrosiniPJ,RapoportJL.1999.Treatmentofattention-de®cit-hyperactivitydisorder.NEnglJMed340:780±788.EshlemanAJ,CarmolliM,CumbayM,MartensCR,NeveKA,JanowskyA.1999.Characteristicsofdruginteractionswithrecom-binantbiogenicaminetransportersexpressedinthesamecelltype.JPharmacolExpTher289:877±885.Gouzoulis-MayfrankE,ThelenB,HabermeyerE,KunertHJ,KovarKA,LindenblattH,HermleL,SpitzerM,SassH.1999.Psycho-pathological,neuroendocrineandautonomiceffectsof3,4-methyl-TABLEVI.Summaryofthenoradrenergichypothesis OraldoseproducingsubjectiveeffectsOraldoseproducingeffects(NE-mediated)forNEforDADecreaseplasmaprolactin?(DA-Reference134mg34mgNoReference240mg40mg725NoReference330mg30mgNoReference430mg30mg39262NotreportedReference575mg75mg721,350NotreportedReference635±50mg35±50mgNoReference712012077376NoReference815mg15mgNotreportedReference914mg14mg28.5416NoDatasupportingthehypothesisthatoraldosesofstimulantsproduceamphetamine-typesubjectiveeffectswithoutreleasingDAaresummarizedabove.References:1:Gradyetal.(1996);2:Masetal.(1999);3:Dommisseetal.(1984);4:Braueretal.(1996);5:Martinetal.(1971);6:Angristetal.(1977);7:Masetal.(1999);8:Martinetal.(1971);9:Gouzoulis-Mayfranketal.(1999).R.B.ROTHMANETAL. enedioxyethylamphetamine(MDE),psilocybinandd-methamphet-amineinhealthyvolunteers.Resultsofanexperimentaldouble-blindplacebo-controlledstudy.Psychopharmacology(Berl)142:41±GradyTA,BroocksA,CanterSK,PigottTA,DubbertB,HillJL,MurphyDL.1996.BiologicalandbehavioralresponsestoD-am-phetamine,aloneandincombinationwiththeserotonin3receptorantagonistondansetron,inhealthyvolunteers.PsychiatryRes64:Grif®thJD,NuttJG,JasinskiDR.1975.Acomparisionoffen¯ura-mineandamphetamineinman.ClinPharmacolTher18:563±570.Grif®thJD,JasinskiDR,PevnickJS.1976.Chlorphentermine:ab-senceofamphetamine-likepro®leinman[Abstract].ClinPharma-colTher19:107.Grif®thsRR,BradyJV,BradfordLD.1979.Predictingtheabuseliabilityofdrugswithanimalself-administrationprocedures:psy-comotorstimulantsandhallucinogens.AdvBehavPharmacolHeishmanSJ,Henning®eldJE.1991.Discriminativestimuluseffectsofd-amphetamine,methylphenidate,anddiazepaminhumans.Psychopharmacology103:436±442.HuntRD,MinderaaRB,CohenDJ.1986.Thetherapeuticeffectofclonidineinattentionde®citdisorderwithhyperactivity:acompar-isonwithplaceboandmethylphenidate.PsychopharmacolBull22:229±236.HuntRD,ArnstenAF,AsbellMD.1995.Anopentrialofguanfacineinthetreatmentofattention-de®cithyperactivitydisorder[seeComments].JAmAcadChildAdolescPsychiatry34:50±54.HurdYL,UngerstedtU.1989a.Cadependenceoftheamphetamine,nomifensine,andLu19-005effectoninvivodopaminetransmission.EurJPharmacol166:261±269.HurdYL,UngerstedtU.1989b.Cadependenceoftheamphetamine,nomifensine,andLu19-005effectoninvivodopaminetransmission.EurJPharmacol166:261±269.JohansonCE,FischmanMW.1989.Thepharmacologyofcocainerelatedtoitsabuse.PharmacolRev41:3±52.KuharMJ,RitzMC,BojaJW.1991.Thedopaminehypothesisofthereinforcingpropertiesofcocaine.TrendsNeurosci14:299±302.MalisonRT,WrightS,SanacoraG,PotenzaM,MechanicK,HaroonE,CarpenterL,PeltonG,SelfD,KostenT,KrystalJ,NestlerEJ.1997.Reductionsincocaine-inducedcravingfollowingtheselectiveD1dopaminereceptoragonistABT-431inhumancocaineabusers.AmCollNeuropsychopharmacol208-200(Abstract).MartinWR,SloanJW,SapiraJD,JasinskiDR.1971.Physiologic,subjective,andbehavioraleffectsofamphetamine,methamphet-amine,ephedrine,phenmetrazine,andmethylphenidateinman.ClinPharmacolTher12:245±258.MasM,FarreM,delaTorreR,RosetPN,OrtunoJ,SeguraJ,CamiJ.1999.Cardiovascularandneuroendocrineeffectsandpharmaco-kineticsof3,4-methylenedioxymethamphetamineinhumans.JPharmacolExpTher290:136±145.MongeauR,WeissM,deMontignyC,BlierP.1998.Effectofacute,short-andlong-termmilnacipranadministrationonratlocuscoer-uleusnoradrenergicanddorsalrapheserotonergicneurons.Neuro-pharmacology37:905±918.MoretC,BrileyM.1997.Effectsofmilnacipranandpindololonextracellularnoradrenalineandserotoninlevelsinguineapighy-pothalamus.JNeurochem69:815±822.OhuohaDC,MaxwellJA,ThomsonLE,CadetJL,RothmanRB.1997.Effectofdopaminereceptorantagonistsoncocainesubjectiveef-fects:anaturalisticcasestudy.JSubstAbuseTreat14:249±258.PriceLH,PeltonGH,McDougleCJ,MalisonRT,JatlowP,CarpenterL,KirwinPD,O'BrienDW,NadimH,HeningerGR.1997.Effectsofacutepretreatmentwithrisperidoneonresponsestococaineincocaineaddicts.AmCollNeuropsychopharmacol208-200(Ab-ReithMEA,LiMY,YanQS.1997.Extracellulardopamine,norepi-nephrine,andserotoninintheventraltegmentalareaandnucleus-accumbensoffreelymovingratsduringintracerebraldialysisfol-lowingsystemicadministrationofcocaineandotheruptakeblockers.Psychopharmacology134:309±317.RitzMC,LambRJ,GoldbergSR,KuharMJ.1987.Cocainereceptorsondopaminetransportersarerelatedtoself-administrationofco-caine.Science237:1219±1223.RochaBA,FumagalliF,GainetdinovRR,JonesSR,AtorR,GirosB,MillerGW,CaronMG.1998.Cocaineself-administrationindopam-ine-transporterknockoutmice.NatNeurosci1:132±137.RothmanRB.1994.Areviewoftheeffectsofdopamimeticagentsinhumans:implicationsformedicationdevelopment.NIDAResMonogr145:67±85.RothmanRB,GlowaJR.1995.Areviewoftheeffectsofdopaminergicagentsonhumans,animalsanddrugseekingbehavior,anditsimplicationsformedicationdevelopment:focusonGBR12909.MolNeurobiol11:1±19.RothmanRB,LewisB,DerschCM,XuH,RadescaL,deCostaBR,RiceKC,KilburnRB,AkunneHC,PertA.1993.Identi®cationofaGBR12935homolog,LR1111,whichisover4000-foldselectiveforthedopaminetransporter,relativetoserotoninandnorepinephrinetransporters.Synapse14:34±39.RothmanRB,CadetJL,AkunneHC,SilverthornML,BaumannMH,CarrollFI,RiceKC,deCostaBR,PartillaJS,WangJB,UhlG,GlowaJR,DerschCM.1994.Studiesofthebiogenicaminetrans-porters.IV.Demonstrationofamultiplicityofbindingsitesinratcaudatemembranesforthecocaineanalog[I]RTI-55.JPharmacolExpTher270:296±309.RothmanRB,ElmerGI,ShippenbergTS,ReaW,BaumannMH.1998a.Phentermineandfen¯uramine:preclinicalstudiesinanimalmodelsofcocaineaddiction.AnnNYAcadSci844:59±74.RothmanRB,SilverthornML,GlowaJR,MateckaD,RiceKC,CarrollFI,PartillaJS,UhlGR,VandenberghDJ,DerschCM.1998b.Studiesofthebiogenicaminetransporters.VII.Characterizationofanovelcocainebindingsiteidenti®edwith[I]RTI-55inmembranespreparedfromhuman,monkeyandguineapigcaudate.Synapse28:322±338.RothmanRB,AyestasMA,DerschCM,BaumannMH.1999.Amin-orex,fen¯uramine,andchlorphentermineareserotonintransportersubstrates:implicationsforprimarypulmonaryhypertension.Cir-culation100:869±875.RothmanRB,PartillaJS,BaumannMH,DerschCM,CarrollFI,RiceKC.2000.Neurochemicalneutralizationofmethamphetaminewithhighaf®nitynonselectiveinhibitorsofbiogenicaminetransporters:apharmacologicalstrategyfortreatingstimulantabuse.SynapseRudnickG,ClarkJ.1993.Fromsynapsetovesicle:thereuptakeandstorageofbiogenicamineneurotransmitters[Review].BiochimBio-physActa1144:249±263.SannerudCA,BradyJV,Grif®thsRR.1989.Self-injectioninbaboonsofamphetaminesandrelateddesignerdrugs.NIDAResMonogr94:30±42.SegalDS,KuczenskiR.1997.Anescalatingdoseªbingeºmodelofamphetaminepsychosis:behavioralandneurochemicalcharacter-istics.JNeurosci17:2551±2566.SoraI,WichemsC,TakahashiN,LiXF,ZengZ,RevayR,LeschKP,MurphyDL,UhlGR.1998.Cocainerewardmodels:conditionedplacepreferencecanbeestablishedindopamine-andinserotonin-transporterknockoutmice.ProcNatlAcadSciUSA95:7699±7704.SteigerA,GerkenA,BenkertO,HolsboerF.1993.DifferentialeffectsoftheenantiomersR()andS()oxaprotilineonmajorendogenousdepression,thesleepEEGandneuroendocrinesecretion:studiesondepressedpatientsandnormalcontrols.EurNeuropsychopharma-col3:117±126.VillemagneV,RothmanRB,YokoiF,RiceKC,MateckaD,CloughDJ,DannalsRF,WongDF.1999.DosesofGBR12909whichsuppresscocaineself-administrationinnon-humanprimatessubstantiallyoccupyDAtransportersasmeasuredby[C]WIN35,428PETscans.Synapse32:44±50.VollenweiderFX,GammaA,LiechtiM,HuberT.1998.Psychologicalandcardiovasculareffectsandshort-termsequelaeofMDMA(ªec-stasyº)inMDMA-naivehealthyvolunteers.Neuropsychopharma-cology19:241±251.WiseRA.1996.Neurobiologyofaddiction.CurrOpinNeurobiolWoolvertonWL,MasseyBW,WingerG,PatrickGA,HarrisLS.1994.Evaluationoftheabuseliabilityofaminorex.DrugAlcoholDependYamamotoBK,NovotneyS.1998.Regulationofextracellulardopa-minebythenorepinephrinetransporter.JNeurochem71:274±280.NOREPINEPHRINEANDSTIMULANTADDICTION