Dopamine and norepinephrine receptors participate in methylphenidate enhancement of in vivo hippocampal synaptic plasticity

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by  Daniel Jenson

Neuropharmacology90(2015)23e32
ContentslistsavailableatScienceDirect
Neuropharmacology
journal homepage: www.elsevier.com/locate/neuropharm
Dopamine and norepinephrine receptors participate in
methylphenidate enhancement of in vivo hippocampal synaptic
plasticity
Daniel Jenson a, Kechun Yang b, Alexandra Acevedo-Rodriguez a, Amber Levine a,
John I. Broussard a, Jianrong Tang c, John A. Dani b,*
aDepartmentofNeuroscience,CenteronAddiction,Learning,Memory,BaylorCollegeofMedicine,Houston,TX77030,USA
bDepartmentofNeuroscience,MahoneyInstituteforNeurosciences,PerelmanSchoolofMedicine,Philadelphia,PA19104,USA
cJanandDanDuncanNeurologicalResearchInstitute,TexasChildren'sHospital,DepartmentofPediatrics,BaylorCollegeofMedicine,
Houston,TX77030,USA
a r t i c l e i n f o a b s t r a c t
Articlehistory: Attention-deficithyperactivedisorder(ADHD)isthemostcommonlystudiedanddiagnosedpsychiatric
Received20January2014 disorderinchildren.Methylphenidate(MPH,e.g.,Ritalin)hasbeenusedtotreatADHDforover50years.
Receivedinrevisedform ItisthemostcommonlyprescribedtreatmentforADHD,andinthepastdecadeitwasthedrugmost
29October2014
commonlyprescribedtoteenagers.Inaddition,MPHhasbecomeoneofthemostwidelyabuseddrugs
Accepted30October2014
oncollegecampuses.Inthisstudy,weexaminedtheeffectsofMPHonhippocampalsynapticplasticity,
Availableonline11November2014
which serves as a measurable quantification of memory mechanisms. Field potentials were recorded
withpermanentlyimplantedelectrodesinfreely-movingmicetoquantifyMPHmodulationofperforant
Keywords:
pathsynaptictransmissionontogranulecellsofthedentategyrus.OurhypothesiswasthatMPHaffects
Dentategyrus
Perforantpath hippocampal synaptic plasticity underlying learning because MPH boosts catecholamine signaling by
Memory blocking the dopamine and norepinephrine transporters (DAT and NET respectively). In vitro hippo-
Reward campalsliceexperimentsindicatedMPHenhancesperforantpathplasticity,andthisMPHenhancement
Long-termpotentiation arose from action via D1-type dopamine receptors and b-type adrenergic receptors. Similarly, MPH
LTP boostedinvivoinitiationoflong-termpotentiation(LTP).Whiletherewasaneffectviabothdopamine
andadrenergicreceptorsinvivo,LTPinductionwasmoredependentontheMPH-inducedactionviaD1-
type dopamine receptors. Under biologicallyreasonable experimental conditions, MPH enhances hip-
pocampalsynapticplasticityviacatecholaminereceptors.
©2014TheAuthors.PublishedbyElsevierLtd.ThisisanopenaccessarticleundertheCCBY-NC-SA
license(http://creativecommons.org/licenses/by-nc-sa/3.0/).
1. Introduction et al., 2003; Volkow et al., 1995; Volkow and Swanson, 2003).
Human studies using PET imaging show that intravenous MPH
Attentiondeficit/hyperactivitydisorder(ADHD)isabehavioral increases DA neurotransmission in the striatum (Volkow et al.,
disorder characterized by inattention, hyperactivity, and impul- 2004),whichisanearmarkofaddictivedrugs(DiChiara,2002).
sivity.Althoughnewerdrugshavebeendeveloped,methylpheni- MPHincreaseshippocampalNEandDAinvivo(Kuczenskiand
date (MPH) is one of the most highly prescribed drugs for the Segal, 2002; Weikop et al., 2007), both of which are known to
treatmentofADHD(Chaietal.,2012).MPHdose-dependentlyin- affect synaptic plasticity such as long-term depression (LTD) and
creases extracellular dopamine (DA) and norepinephrine (NE) long-termpotentiation(LTP)(HopkinsandJohnston,1984;Hyman
indirectly by blocking the transporters, DATand NET (Biederman etal.,2006;Izumietal.,1992;JonesandBonci,2005;Kauer,2004;
andSpencer,1999;KuczenskiandSegal,1997,2002).Functionally, LismanandGrace,2005;Thomasetal.,1996).Aswellasinfluencing
MPH is remarkably similar to cocaine in both DAT blockade and hippocampal plasticity (Kulla and Manahan-Vaughan, 2000; Saji-
subjective “high” when administered by the same route (Stoops kumarandFrey,2004;TangandDani,2009),DAneurotransmission
alsoinfluenceshippocampal-relatedfunction(Rossatoetal.,2009).
Theseresultsareconsistentwithevidenceindicatingthataddictive
drugsactuponsynapticplasticitymechanismsthatnormallyun-
* Correspondingauthor.Tel.:þ12158988498. derlielearningandmemory(DaniandHarris,2005;Hymanetal.,
E-mailaddress:[email protected](J.A.Dani).
http://dx.doi.org/10.1016/j.neuropharm.2014.10.029
0028-3908/©2014TheAuthors.PublishedbyElsevierLtd.ThisisanopenaccessarticleundertheCCBY-NC-SAlicense(http://creativecommons.org/licenses/by-nc-sa/3.0/).
24 D.Jensonetal./Neuropharmacology90(2015)23e32
2006;Jay,2003;JonesandBonci,2005;Kauer,2004;Kelley,2004; 2.3. Fast-scancyclicvoltammetryinstriatalslices
Unglessetal.,2004;Winderetal.,2002). Wild-typeC57BL/6Jmalemicebetweentheagesof3and5monthswereused
One of the most important pathways for the formation of for these experiments. Animals were anesthetized via injection of a ketamine,
associativememoryistheperforantpath,whichoriginatesinthe xylazineandacepromazinecombinationanesthetic.Whentheanimalwasdeeply
anesthetized,itwasdecapitated,andthebrainquicklydissectedout.Horizontal
entorhinalcortexandtransmitsconvergentinformationfromthe slices(300mm)containingthedorsalstriatumwereincubatedat32±0.5(cid:2)Cfor
neocortextothehippocampus(Deadwyleretal.,1979;Lavenexand 10minandheldatroomtemperaturefor(cid:3)1h.Thesliceswerestudiedat34±1(cid:2)C
Amaral, 2000). The accumulated evidence supports that synaptic inACSF:125mMNaCl,2.5mMKCl,1.3mMMgCl2,2.5mMCaCl2,1.25mMNa2HPO3,
plasticity along the perforant path is a substrate for memory 26mMNaHCO3and10mMglucosesaturatedwith95%O2and5%CO2.
(Lynch, 2004; Martinez and Derrick,1996; McHugh et al., 2008; Fast-Scan cyclic voltammetry was performed using homemade carbon-fiber
microelectrodes.Thepotentialappliedtotheelectrodewasscannedlinearlyfrom
Rumpel et al., 2005), and the medial perforant path inparticular 0to(cid:4)400to1000to(cid:4)400to0mVatarateof300mV/msagainstasilver/silver
carriesplaceandspatialinformation(Hargreavesetal.,2005)that chloridereference.Thetipofthecarbonfiberrecordingelectrodewascentered
isimportantfordrugassociatedmemory.BothDAandNEreleasein approximately 200 mmawayfrom the tips of the bipolarstimulating electrode.
the hippocampus enhance LTP and learning, which establishes a Voltammogramsweresampledat50kHzandthebackgroundcurrentwasdigitally
subtracted. An oxidationpeak between 400 and 600 mV and a reductionpeak
functionallinkbetweenmemorysystemsandthecatecholamine-
between(cid:4)200and(cid:4)250mVwereusedtoidentifythesignalasadopaminesignal.
releasing “reward” centers (Lisman and Grace, 2005; Moore and
ThepeakoxidationcurrentsforDAineachvoltammogramwereconvertedtoaDA
Bloom,1979).Recentworksuggeststhatthesetwocatecholamine concentrationduringapostexperimentalcalibrationofthecarbon-fiberelectrode
systemsmaybemoreinteractiveinthehippocampusthanprevi- againstsolutionsof0.1e10mMDA(Zhangetal.,2012,2009,2010).
ouslyanticipated(Agnatietal.,1995;Borgkvistetal.,2012;Smith Thedorsalstriatumwasusuallystimulated(1-msduration,0.6-mAconstant
and Greene, 2012). In this study, we examined MPH's influence current) with a bipolar tungsten electrode with 120 s between stimulations to
ensurerecoveryoftheDAsignal.AfterrecordingstableDAreleasefor10minin
overperforantpathsynapticplasticityowingtosignalingviaDAor ACSF,MPHwasbathadministeredandallowedtoequilibratefor45minbeforedata
NEreceptors. werecollectedforMPHinducedchangesinDArelease.Forsomeoftheexperiments,
stimulustrainswereapplied.Whenstimulationtrainswereapplied,theycombined
atonicandphasiccomponent.Thetonicstimulationwasgivenat1.8Hz,andthe
2. Materialsandmethods phasicstimulationwasgivenas5pulsesat20Hz.Thephasicstimulationmatches
the average intraburst firing frequency of approximately 20 Hz that has been
2.1. Experimentalprocedures measuredinvivofromrodents(Benoit-Marandetal.,2001;Hylandetal.,2002;
AllanimalexperimentswerebeencarriedoutinaccordancewiththeNIHguide Zhangetal.,2009).Theaveragefrequencyofthetotalstimulationtrainwas4Hz,
forthecareanduseoflaboratoryanimalsandaccordingtoprotocolssubmittedto whichisconsistentwiththeaveragebaselinefiringrateofDAneuronsinvivo.The
theInstitutionalAnimalCareandUseCommitteeatBaylorCollegeofMedicine.All stimulationtrainwasappliedbeforeandafterbathadministrationofMPH(50nM).
effortsweremadetominimizeanimalsuffering,toreducethenumberofanimals This MPH concentration is close to the peak serum concentrations observed
used,andtousealternativestoinvivotechniqueswhenavailable. followingmoderatetherapeuticoraldosages(SwansonandVolkow,2003).
2.4. Invivoelectrophysiology
2.2. Slicepreparationandperforatedpatchclamprecording 2.4.1. Animalcareandsurgery
Hippocampalslicescontainingthedentategyruswerepreparedaspreviously AdultmaleC57BL/6Jmice(JacksonLaboratory),3e4monthsold,wereusedin
described(Zhangetal.,2010).Briefly,C57BL/6miceofeithersex(24e30dayold) alloftheseexperiments.Forinvivoelectrophysiology,themicewerehousedindi-
wereanesthetizedviainjectionofamixtureofketamine(42.8mg/ml),xylazine vidually with food and water ad libitum in a temperature-controlled room
(8.6mg/ml),andacepromazine(1.4mg/ml).Whentheanimalwasdeeplyanes- (23±1(cid:2)C)withareversed12hartificiallightedarkcycle(lightonat9p.m.).
thetized,itwasdecapitated.Thenthebrainwasrapidlyremovedandslicedwitha Onday0micewereanesthetizedwithsodiumpentobarbital(80mg/kg,intra-
vibratome(LeicaVT1000S).Fourhorizontalbrainslices(270mmthick)werefirst peritonealinjection,i.p.,withadditional15mg/kgsupplementsasnecessary)and
recoveredinahomemadechamberfilledwithlow-calcium,high-magnesiumarti- securedintothestereotaxicapparatus.Aconcentricbipolarstimulatingelectrode
ficialcerebrospinalfluid(ACSF)containing(inmM):125NaCl,25NaHCO3,2.5KCl, consistingof a tungstenwireplaced inside a 30 gaugestainless steeltubewas
1.25NaH2PO4,7MgSO4,0.5CaCl2,and25D-(þ)-glucosecontinuouslysaturated positionedinthemedialpartoftheperforantpath(0.2mmposteriorandbetween
pweirthatu9r5e%(2O22±an1d(cid:2)5C%)fCoOr2ataltea3s2t(cid:2)1Chfournt3i0ltmheinreacnodrdtihnegnswmearientbaeinguedn.atroomtem- 2el.8ectarnodde3(.T0eflmomn-claotaetreadltoufnglasmtebndwa,ir1e.,0bea1r.e3dmiammebteerlo5w0mtmhe)adtutarcah).edThtoearestcaoirndliensgs
Therecordingsweremadewitha200Bamplifier(AxonInst.)appliedtoahip- steelguidetube(outerdiameter310mm)wastargetedipsilaterallytothehilusofthe
pocampalslicethatwasplacedinahomemaderecordingchambercontinuously dentate gyrus (DG) (1.8e2.0 mm posterior, 1.4e1.6 mm lateral of bregma,
perfusedwithwelloxygenatedACSF(1e2ml/min)containing(inmM):125NaCl,25 2.2e2.3mmbelowthe skull)(FranklinandPaxinos,1997).Thefinaldepthand
NaHCO3,2.5KCl,1.25NaH2PO4,1MgSO4,2CaCl2,25D-(þ)-glucose,maintainedat positionoftheelectrodesweredeterminedbyjudgingthereal-timeelectrophysi-
32e34(cid:2)Cbyanautomatictemperaturecontroller(TC-324B,WarnerInstrument ologicalsignal.Asilverelectrodeplacedcontralaterallyinthecortexservedasthe
Corp,Hamden,CT).Picrotoxin(100mM,SigmaeAldrich),aGABAAreceptorantag- recordingreferenceandground.Dentalcementwasusedtoanchortheelectrodes
onist, was routinely included in the ACSF. Patch-clamp recording electrodes andtheconnectingdeviceforchronicrecordings.Miceweregivenatleast2weeks
(3e4MU)werefilledwiththefollowingintracellularsolution(inmM):140po- torecoverbeforebeginningtheexperimentalrecordings.
tassiumgluconate,10KCl,10HEPES,5MgCl2,(pH7.2withKOH)freshlysupple-
mentedwith200mg/mlamphotericinB(SigmaeAldrich)(Yangetal.,2009).Therest 2.4.2. Handling,habituation,stimulation,andrecordingprocedures
ofthepipettesolutioncontainingamphotericinBwaskeptina4(cid:2)Crefrigeratorand Beginning on day 10 post-surgery and continuing for 5 days, each animal
wasdiscardedafter6h. received5minofhandlingfollowedbyadministrationof0.3mlsaline(i.p.)inthe
After achieving a gigaohm seal (>2 GU), a perforated whole-cell recording homecageintheexperimentalroombothinthemorningandafternoon.Thenthe
conformationwasobtainedwithin5e20minasjudgedbygradualreductionofthe micewereindividuallyhabituatedtotherecordingchamber:aPlexiglascylinder
accessresistanceto<20MU.Undercurrentclamp,excitatorypostsynapticpoten- withbeddingwithinasound-reducingbox(MedAssociates,VT).Atthesametime
tials(EPSPs)wereevokedbystimulationpulsesgeneratedbyastimulusisolator themicewerehabituatedtotherecordingheadstagesystem.Micereceived1hof
(A365,WPI,FL)every30sviaastereotrodetungstenelectrode(WPI,FL)placedat habituationdailyfor4consecutivedaysstartingfromday11.Monophasicsquare
themedialperforantpathway.Astablebaselinewasrequiredfor10minbefore pulses(100ms)weredeliveredtotheperforantpath(A360R,WPI,FL).Recorded
beginning the experiment by applying drugs or beginning the spike timing signalswereamplified((cid:5)100),filtered(bandpass0.1Hze5kHz),digitizedat10kHz,
dependent plasticity (STDP) protocol. In order to monitor the perforated patch andstoredondiskforoff-lineanalysis.Onday15,aninputeoutputcurve(I/O)was
recording conditions, a small hyperpolarizing current (0.01 nA) was injected generated.Singleteststimuliwerethendeliveredat30sintervalsatanintensity
(200ms)throughtherecordingpipettetomeasureinputresistance.Iftheinput (35e100mA)thatevoked40%ofthemaximalamplitudeofthepopulationspike.
resistancechangedby30%ormore,thentheexperimentwasstoppedandthedata Inatypicalexperimentexaminingevokedsynapticresponsesintheperforant
werediscarded.Alldrugswerebathappliedforatleast15min,andtheSTDPpro- path-dentatepathway,thereweretwoorthreecounterbalancedsessionsforeach
tocol(presynapticstimulationfollowedbypostsynapticdepolarizationseparatedby mouse:e.g.,saline,5mg/kgMPH,and20mg/kgMPH.Eachdataacquisitionsession
a10-mstimeinterval)wasdelivered60timesat0.33Hzin3min.Themagnitudeof continuedfor4days.Ondayminusone((cid:4)1d),thebaselineamplitudeofthepop-
the synaptic plasticity change was defined as the mean peak amplitude of 40 ulationspikewasexaminedforstabilityfor30min.On0d,another30minstable
consecutive EPSPs taken 30e60 min after the end of STDP induction protocol baselinerecordingwasrequiredbeforeadministeringsaline,5mg/kg,or20mg/kg
normalizedbytheaverageoftheinitial20consecutiveEPSPs(i.e.,baseline). MPH(i.p.,0.1mlper10gbodyweight).Theresponseswereusuallymonitoredfor
D.Jensonetal./Neuropharmacology90(2015)23e32 25
another3hfollowingtheinjection,butinsomecasestheresponsesweremonitored electricalafterdischargesandtoensurethemousewasina“still-alert”state(Doyere
for5h.Insomecases,thebaselinesalsowererecordedbetweeninjectionofdrugor etal.,2003).
salineandthetaburststimulation(TBS)toensurestability(e.g.,Fig.4).Intheex-
perimentsforFig.4,afterthebaselinewasrecorded,miceweredisconnectedfrom 2.4.3. Thetaburststimulation
therecordingamplifier,givenaninjectionofeithersalineorMPH,reconnectedto Theta burst stimulation (TBS) was designed to model the firing patterns of
therecordingamplifier,andbaselinerecordingcontinuedforthe15minbeforethe hippocampalneuronsrecordedduringexploratorybehaviorinintactanimals.A
TBSwasadministered.However,duringthemultipleinjectionsnecessaryforthe singleTBStrainconsistedof6individual0.1mspulsesina400Hzburstwith6
experimentsofFigs.5and6,wedidnotrecordduringthemanipulationstoavoid burstsat5Hzpertrain.Thistrainwasrepeatedatanintervalof20stoinduce
electricalnoise.Themicewereleftintherecordingchamber,butunconnectedtothe differentamountsofsynapticpotentiation.Asingletrainproducednochangein
recordingsysteminbetweentheinjectionsandTBS.Themicewerereconnected synaptictransmission,while6trains(astrongTBSprotocol)consistentlyinduced
1e2minpriortoTBSadministration.Baselinerecordingsweretakenon1dand2d LTP of the perforant path connection to dentate granule cells. We titrated the
aftertreatmenttofollowtheoverallstabilityoftherecordings.I/Ocurveswere strengthoftheTBSandfoundthat2trainsonaverageproducedslightlong-term
recordedsystematicallybetweensessionstoverifythestabilityandtherecovery depressionanddidnotinducesignificantLTPonitsown.ThisversionofTBSwas
afterthetreatmentofeachsession.Throughouttheelectrophysiologicalrecording, usedinalloftheTBSexperiments.
themouse'sbehaviorwasvideomonitored,andtheEEGderivedfromthedentate
gyrusrecordingalsowasdisplayedonanoscilloscopetoensuretheabsenceof
2.4.4. Drugadministrationandhistology
AllthedrugswerefromSigma(St.Louis,MO).Fori.p.injection,sodiumpen-
tobarbitol, dl-threo-methylphenidate hydrochloride, R(þ)-SCH-23390 hydrochlo-
ride,andtimololmaleateweredissolvedinnonpyrogenicsalineandadjustedfor
injectionvolumeof0.1mlper10gbodyweight.Afterallexperimentation,themice
werekilledwithanoverdoseofisoflurane.Ananodalcurrent(30mA,10s)was
passedthroughthetungstenwireforidentificationoftheelectrodeplacements.
Frozen30mmcoronalsectionswerecutandstainedwithhematoxylin.
2.4.5. Dataanalysis
Duringtheinvivoexperiments,theamplitudeofthepopulation(pop)spikewas
measuredforeachstimulusevokedpostsynapticresponsebycalculatingthedepth
ofthelocalfieldpotential.Thepopspikeamplitudewasmeasuredfromthepoint
directly above the minimum of the local field potential on an imaginary line
extending between the local maxima before and after the pop spike itself. The
calculatedvaluesfromeverytenconsecutivepopspikeswereaveraged.Theaver-
agedvaluesbeforeanydrugtreatmentwerenormalizedto100%,andthoseaveraged
valuesdefinedthebaseline.Themeanbaselinevalueswereobtainedduringthe
30minofrecordingimmediatelybeforethefirstdrugtreatment.
TheinvivodataofFigs.3e6werecollectedin3continuoussectionsonday
0(i.e.,thetreatmentday).Thebaseline(30min)collectedbeforethetreatments
began,thetreatmentsection,andthepost-treatmentsection(3h).Thebaselinewas
alwaysnormalizedto100%,andthevalueforsynaptictransmission(i.e.,theLTPor
LTDvalue)wascalculatedbyaveragingthedatacollectedinthelasthourofthepost-
treatmentsection.Todeterminewhetheratreatmentdatasethadaneffect,we
ultimatelycomparedthetreatmentgroupanditspairedcontrolgroup.Thedata
groups were analyzed with multi-way repeated measures analysis of variance
(ANOVA).Inthecaseswhere3ormoregroupswerecompared(i.e.,Figs.4e6),the
multi-wayANOVAindicatedwhetherthegroupsweredifferent.Then,aTukeyHSD
post-hoctestwasperformedtodeterminetherelationshipofsignificantdifferences
betweengroups,andthisprocessyieldedthereportedpvalue.Thatis,theprocess
indicatedwhetherthetreatmentwasdifferentfromitspairedcontrol,whichalso
indicatedtheinductionofLTPorLTD.
3. Results
3.1. MPHenhancedSTDPinhippocampalslices
EPSPswereevokedbystimulationpulsesappliedevery30svia
anelectrodeplacedinthemedialperforantpathwayandrecorded
undercurrentclampthroughaperforatedpatchondentategranule
cells(Fig.1A).Fig.1BindicatesthetimingoftheSTDPprotocolthat
wasapplied.Thepresynapticstimulationoccurred10msbeforethe
postsynaptic depolarization. Postsynaptic current was injected to
causethepostsynapticcelltofiretwoactionpotentialsat100Hz.
This pairing was repeated 60 times at 0.33 Hz over 3 min after
Fig.1. MPHenhancedSTDPinhippocampalslices.(A)Aschematicdiagramofapatch-
recording the baseline for 15 min to ensure stability of the
clamprecording(R)fromagranulecellinamousedentategyrus(DG)slice.Astim-
recording.EPSPsevokedbystimulationwererecordedfor60min
ulatingelectrode(S)wasplacedinthemedialperforantpath(mPP)originatinginthe
medialentorhinalcortex(EC).(B)AnillustrationoftheSTDPinductionprotocolwith following the STDP pairing. The STDP stimulation protocol alone
Dt¼10ms,meaningthepresynapticstimulationprecededby10msthepostsynaptic did not produce consistent synaptic plasticity, with the average
actionpotential(AC)firinginducedbycurrentinjectionviatherecordingpipette.STDP being notsignificantly differentfrombaseline (94% ± 19%, n ¼ 7,
(inSCdHuc)tioþn5b0y0thneMproMtoPcHol(iDn),th2e0prmeMsentciemooflo5l00(TInMM) MþPH50(0C),n2M0mMMPHSCH(E-)2,33an9d0 p¼0.77;graycircle,Fig.1G).
SCHþTIMþMPH(F),respectively.Theopenhorizontalbarineachleftpanelindicates The literature indicates MPH doses for hippocampal slice ex-
theperiodofdrugapplication,andthearrowsindicateapplicationofthe10msSTPD perimentsrangingfromabout50nMto300mM(Dommettetal.,
protocol. The right panel of the corresponding figures shows tabulated individual 2008; Ishimatsu et al., 2002). MPH has a higher affinity for the
psulamstmicaitryyovafltuheesmaenadnamveargangietuddveaolufeSsTDoPfitnhdeumceadgbnyituthdee1o0fmsysnparpottioccopllasshtoicwitny.in(GC)eAF DAT(Ki¼34nM)thanfortheNET(Ki¼339nM)(Bymasteretal.,
andforcontrolexperiments.*p<0.05comparedtothebaseline,#p<0.05comparison 2002;Pristupaetal.,1994),however,themedianeffectivedosesfor
betweenthetwogroupsindicated. DATand NET blockade are close (Hannestad et al., 2010; Volkow
26 D.Jensonetal./Neuropharmacology90(2015)23e32
Fig.2. TheMPHdosedependenceforinfluencingDAreleasemeasuredbyfast-scancyclicvoltammetry.(A)ExampleDAtracesbefore(0MPH)andafterbathadministrationofthe
indicatedconcentrationofMPH.TheaverageDAresponsespeakmagnitude(B)anddopaminesignalarea(C)areplottedagainsttheMPHconcentrations.(D)MPH(50nM)in-
creasesDArelease.AnexampleDAtraceisshownbefore(blacktrace)andafterMPHbathadministration(graytrace).ThebargraphsshowthattheaverageDApeakandareaunder
theDAcurveincreaseafterMPHadministration.(E)MPH(50nM)increasesthephasic/tonicratioofDArelease.Thephasic-to-tonicratioincreasesfollowingtreatmentwith
50nMMPH.Theindividualpairedtracesareaveragedandplottedovereachother.Whentreatedwith50nMMPH(largeramplitudegraytrace),thetonicandphasicDArelease
bothincreased.However,thephasicDAreleaseincreasedmore,andthisresultisreflectedintheincreasedphasic-to-tonicratiosofthepeakamplitudesandareasfollowingMPH
application(bargraphs,right).
etal.,1998),andtherapeuticdosesinjectedi.p.inratsshowsimilar antagonistalsoreducedtheSTDPenhancementsothatitwasno
increases in DA and NE concentrations in the prefrontal cortex longer significant (TIM, Fig.1E: 148% ± 46% of baseline, n ¼ 8,
(Bymaster et al., 2002). Based on the literature and our voltam- p¼0.17;blackupwardtriangle,Fig.1G).Withasingleantagonist,
metry experiments below, we used a moderate concentration of thestimulationprotocolforagivenrecordingcouldproduceLTP,
0.5mMMPH,andfoundthatMPH(0.5mM)significantlyenhanced LTD, or no change, with the average being not significantly
LTPinducedbytheSTDPprotocol(154%±21%ofbaseline,n¼7, different from baseline (Fig. 1D and E, right panels). However,
p¼0.02,Fig.1C;blackcircle,Fig.1G). when both antagonists (SCH-23390 and timolol) were applied
SCH-23390, a D1/D5 receptor antagonist, reduced the STDP alongwithMPHbeforetheSTDPprotocol,nochangeinsynaptic
enhancementsothatitwasnolongersignificantlydifferentfrom strengthwasobserved,andindividualrecordingsweremuchless
baseline (SCH, Fig.1D: 128% ± 48% of baseline, n ¼ 8, p ¼ 0.29; likelytovarygreatly(Fig.1F:81%±15%ofbaseline,n¼6,p¼0.13;
black square, Fig. 1G). Timolol, a nonspecific NE b receptor blackdownwardtriangle,Fig.1G).
D.Jensonetal./Neuropharmacology90(2015)23e32 27
Fig.3. HighdoseofMPHinducedLTP.(A)Anatomicaltrackverificationofarecordingelectrodelocationinthedentategyrus(dottedcircle,left).Theblackdotsshowtherangeof
recordinglocationsinthedentategyrus(right).(B)Anatomicallesionofastimulatingelectrodeinthemedialperforantpath(arrow,left).Theblackdotsshowtherangeof
stimulation-sitelocationsinthemedialperforantpath(right).(C)MPH-inducedpotentiationoftheperforantpathrecordedintheDG(graysquares).Thetimecourseofeach
procedureisacross4days,fromthedaybefore((cid:4)1d)until2daysafter(2d)theMPHadministration.A30minbaselinewasobtainedon(cid:4)1dandconfirmedtobestablefor30min
onday0(0d).Saline(black,n¼5)or20mg/kgMPH(gray,n¼5)wasinjected(i.p.)at30minonday0(verticaldashedline).TheamplitudesofthepopulationspikeofthefEPSCare
plottedovertimefor4days((cid:4)1dto2d).SignificantLTPwasmeasuredfor20mg/kgMPHon0d(F(2,389)¼7.8,p<0.0005).Thestabilityoftherecordingsandthereturntobaseline
areshownon1dand2d.
The compiled data (Fig. 1G) show that MPH with the STDP DAneuronsdischargetonicallyatlowfrequenciesthatconsist
protocol(blackcircle)significantlyenhancedSTDP-inducedLTPin mainly of individual action potentials (Grace and Bunney,1984).
hippocampalslicescomparedtothebaseline,comparedtoacon- Periodically,DAneuronsfirephasicburststhataveragenear20Hz
trol in which the STDP protocol was applied without MPH (light inrodents(Hylandetal.,2002;Robinsonetal.,2004;Zhangetal.,
graycircle),andcomparedtoacontrolinwhichMPHwasperfused 2009).AfterbathapplicationofMPH(50nM),theDAreleaseinthe
inthebathbutnoSTDPstimulationwasapplied(lightgraysquare). dorsolateral striatum increased (Fig. 2D). For a single electrical
AddingeitheraDAD1/D5receptorantagonistoraNEbreceptor stimulus (1p), the peak DA concentration increased to
antagonistreducedMPH-enhancementbelowthelevelofsignifi- 1.40 ± 0.15 mM from 0.99 ± 0.11 mM in the control (n ¼ 13,
cance.Combiningthetwoantagonists (blackdownward triangle) p<0.0002),andthetotalDAsignal,measuredasareaunderthe
potentlyblockedtheincreaseinsynapticstrengthinducedbyMPH curve,increasedto0.89±0.13mM*sfrom0.47±0.07mM*sinthe
duringtheSTPDprotocol. control(n¼13,p<0.005).
To examine the tonic and phasic components of DA release, a
3.2. MPHincreaseddopaminereleaseinstriatalslices tonic1.8Hzstimulationwasgivenbeforeaphasicburst(5pulsesat
20 Hz). In the presence of MPH (50 nM), each DA response was
TobetterunderstandMPH'sinfluenceoverDAreleaseandMPH larger(Fig.2E).Alsothephasic-to-tonicratiosofthepeakampli-
dosingforourexperiments,weusedfast-scancyclicvoltammetry tudes and areas were significantly increased following MPH
(FSCV)tomeasuredynamicDAreleasefrominvitrostriatalslices, application. The phasic/tonic ratio of the DA concentrationpeaks
which provide robust, measurable DA release. Our intent was to increased to 3.07 ± 0.36 in MPH from 2.05 ± 0.26 in the control
findtheMPHdosethatincreasesDAsignalingwithoutproducing solutions(n¼11,p¼0.009)andthephasic/tonicratioofthearea
anoverlyseverealterationintheDAsignalingprocess.Inthatway, under the DA curves increased to 5.91 ± 1.02 in MPH from
wehopedtobettermodelthemoreconservativedosingexpected 3.40±0.59inthecontrol(n¼11,p¼0.014).Thus,MPHincreased
intherapeuticsituations.WeusedFSCVtoobtainhighfidelityin- phasic DA release more than it increased tonic DA release under
formationonthetimecourseoftheevokedDAresponse.Firstwe theseconditions.
completed a doseeresponse curve for concentrations of MPH
rangingfrom1nMto300mM(Fig.2AeC),whichcoversthedose 3.3. BehavioralobservationsdefinedinvivoMPHdosing
rangecommonlyusedforinvitrohippocampalstudies(Dommett
et al., 2008; Ishimatsu et al., 2002). The doseeresponse curve We set out to find an appropriate in vivo MPH dose that re-
showedaninvertedU-shapecurve.Upto1mMMPH,theDArelease capitulates some of the behavioral effects of cocaine, but not so
iswellbehaved,butabovethatconcentrationtheDApeakfallswith highastoinducestereotypyorimpairperformanceonabehavioral
increasingMPHconcentrations.Therefore,weusedMPHconcen- memory task. Increased locomotion and stereotypies are side ef-
trations well below 1 mM for the slice experiments, and we fectsofmoderatetohightherapeuticdosesofMPHinhumans.The
considereddosesinthisrangetobebiologicallyreasonable. therapeuticrangeofMPHdosesinhumanshasbeensuggestedto
28 D.Jensonetal./Neuropharmacology90(2015)23e32
Fig. 6. Inhibiting NE b-receptors decreased but did not prevent MPH's effect. (A)
Systemicinjectionofanon-specificNEb-receptorantagonist,timolol,alonedidnot
affectsynaptictransmissionfollowingtheweakTBS(n¼5).(B)Salineadministered
beforeMPHandTBS(black,n¼5)gaverobustLTP,whiletimololadministeredbefore
MPHandTBS(gray,n¼7)decreasedMPH'seffectontheresultingsynaptictrans-
mission,butLTPwasnotblocked.Treatmentswerecounterbalancedandgivenatleast
7daysapartforeachmouse.InjectionoftimololbeforetheMPH/TBSLTPinduction
protocol significantly decreased the resulting synaptic transmission in the dentate
gyruswhencomparedtothesalinecontrol,(F(1,339)¼252.4,p<0.05).
Fig.4. ModeratedoseofMPHenhancedTBSinducedLTP.(A)Exampletracescompare
before(blacktrace)andaftertreatment(gray)traceforMPH(5mg/kg)alonewithout
TBS,forsalinewithTBS,andforMPH(5mg/kg)þTBS.(B)Saline(gray,n¼5)or5mg/
kgMPH(black,n¼5)wasinjected15minbeforeTBSstimulationwasgiven.This 2010; Wright and White, 2003). A boost in T-maze performance
lowerdoseofMPHwillnotinduceLTPalone(opencircles,n¼5).Eachmousereceived shouldmeanwehaveamoderatetherapeuticdose,asindicatedin
2seriesofTBSwithsalineorMPHinacounterbalancedmannerover2weeks.An the literature by behavioral experiments in mice (Heredia et al.,
injectionof5mg/kgMPHfollowedbyTBSresultsinsignificantLTPofthepopulation 2013; Huang and Huang, 2012; Lloyd et al., 2013). A dose of
spikeamplitude(F(1,551)¼176.17,p<0.0001). 1 mg/kg did not significantly increase locomotor behavior, while
5 mg/kg increased locomotor behavior without inducing stereo-
be0.5e2.5mg/kg(KuczenskiandSegal,2001).However,theroute typy.Wefound,however,thatadoseof20mg/kgdidinduceste-
of administration and species differences in drug metabolism reotypy,andwethereforelabeled20mg/kgahighdoseofMPH.For
significantlyaffectplasmaandbrainMPHconcentrations.Although theremainingexperimentsweused5mg/kgasourmediumdose
MPHhashigheraffinityforthehumanDATovertheNET,3mg/kg ofMPH.Afterbeingthoroughlytrainedonthetask,administration
MPH injected i.p. in rats showed similar increases in DA and NE of5mg/kgMPH(i.p.)significantlyincreasedcorrectarmchoicein
concentrations in the prefrontal cortex, striatum, and nucleus theT-mazecomparedtomicethatreceivedsaline(88%±7%after
accumbens as a percentage of baseline (Bymaster et al., 2002). MPHversus69%±11%aftersalineasacontrol,p<0.05,n¼16for
ResultsalsosuggestthatmicearelesssensitivetoMPHthanrats, bothgroups,datanotshown).Thisgaveusourbasisfordosingto
andchronicinjectionsof3mg/kgpreferentiallyincreasedDAand begintheinvivoelectrophysiologicalexperiments.
NEintheprefrontalcortexwithoutaffectinglevelsinthestriatum
andwithoutcausingsensitizationover21daysofdailyinjections
(Kodaetal.,2010).Guidedbythesedataweperformedopenfield 3.4. MPHenhancedLTPinvivo
experimentsonseveraldosesofMPHlookingfor increasedloco-
motorbehaviorwithoutstereotypies,definedasrepetitiveturning Fieldpotentialsevokedbystimulationofthemedialperforant
pathwererecordedinthehilarregionofthehippocampaldentate
intightcirclesorexcessiveself-grooming.
gyrusfromfreelymovingC57BL/6mice(Fig.3A,B).Anexampleof
As a second test of a moderate dose of MPH, we ran mice
thelesionfromarecordingsiteisshowninFig.3A(left),andallthe
throughahippocampaldependent,delayednon-matchtosample
recordingsitesinthedentategyrusareindicatedinFig.3A(right).
T-maze task designed to test spatial working memory, a type of
memorythatMPHisspecificallyknowntoenhance(Repantisetal., AnexampleofthelesionfromastimulationsiteisshowninFig.3B
(arrow,left), andall thestimulationsites inthemedialperforant
path are indicated in Fig. 3B (right). We focused on the medial
perforantpathbecauseitrelaysconvergentinformationfromthe
neocortex that is rich in contextual, place, and spatial content
(Hargreaves et al., 2005), and evidence indicates that such infor-
mationisassociatedwiththedrugexperience(Bialaetal., 2005;
Kilts et al., 2001; Tang and Dani, 2009). Field recordings were
madefromthehilusofthedentategyrustofollowthesynaptically
generated field excitatory postsynaptic potential (fEPSP) and the
population (pop) spike, which corresponds to the synchronous
discharge of a large number of granule cells (Buzsaki and Czeh,
1981). The magnitudes of these components are directly related
tothenumberofneuronsactivatedbythestimulus(Andersenetal.,
1971; Lomo, 1971). Synaptic transmission was quantified by
Fig.5. InhibitingDAD1-typereceptorsblockedMPH'seffect.(A)Systemicinjectionof measuringthepopspikeamplitudebecausetheslopeofthefEPSP
aD1/D5receptorantagonist,SCH-23390,alonedidnotaffectsynaptictransmission isoftenobscuredinvivobyanincreaseinthepopspikethatoccurs
followingtheweakTBS(n¼5).(B)SalineadministeredbeforeMPHandTBS(black,
aftersynapticpotentiationinductioninawakeanimals.
n¼5)gaverobustLTP,whereasSCH-23390administeredbeforeMPHandTBS(gray,
n¼5)didnotshowanysynaptictransmissionchange(F(1,319)¼248.19,p<0.0001). Two weeks after surgical implantation of the electrodes and
Treatmentswerecounterbalancedandgivenatleast7daysapartforeachmouse. habituation to the recording situation, mice were treated with
D.Jensonetal./Neuropharmacology90(2015)23e32 29
three 4-daycounterbalanced sessions of systemic i.p. injection of Therefore, the influence of MPH over plasticity of the perforant
saline,5,or20mg/kgMPH.WetestedwhetherMPHalonewould pathtothedentategyruswasexamined.Wefocusedonthemedial
induceanydirectionalplasticitythatcouldbemeasuredbythepop perforantpathbecauseit specificallycarriesplaceandspatial in-
spikeofthefEPSPs.Neithersalinenor5mg/kgMPHaffectedsyn- formation(Hargreavesetal.,2005)thatisimportantforassociative
aptictransmission:forsaline,104%±1.1%,n¼3,p>0.05(Fig.3C, memory. The accumulated evidence also supports that synaptic
saline,blacksquares);for5mg/kgMPH(seeFig.4B,opencircles,to plasticityalongthispathwayisacontributingsubstrateofmemory
simplify Fig. 3C). However, 20 mg/kg MPH induced long-term (Lynch, 2004; Martinez and Derrick,1996; McHugh et al., 2008;
synaptic potentiation (Fig. 3C, gray squares) as measured in the Rumpeletal.,2005).UsinginvitrosliceswefoundthatMPHen-
popspikeamplitude2hafteradministration:109%±1.1%,n¼5, hancestheinductionofSTDP.ASTDPprotocolthatinducednonet
p<0.001for20mg/kg. synaptic change by itself would induce LTP when the stimulus
To simulate a signal along the perforant path to the dentate protocolwasappliedinthepresenceofMPH(0.5mM).Wheneither
gyrus,weappliedrelativelyweakthetaburststimulation(TBS)and theD1-typeDAreceptorswereinhibitedwithSCH-23390ortheNE
combinedthatTBSwithMPHadministration.Basedontheinvitro b receptors wereinhibited bytimolol, the averageLTPamplitude
experiments(Fig.1),wehypothesizedthatMPHshouldboostthis induced by the MPH protocol was reduced (Fig.1). In slices, it is
sub-threshold TBS paradigm to produce long-term potentiation noteworthy that with either D1-type receptors or NE b receptors
(LTP) owing to blockade of the NET and/or DAT (Kulla and inhibited there is still a trend toward potentiation, and there is
Manahan-Vaughan,2000;MooreandBloom,1979;Rossatoetal., significant scatter among the individual plasticity recordings
2009;SajikumarandFrey,2004).Asalineinjection15minbefore (Fig.1D, E, right bar plots). When both sets of catecholamine re-
the standard theta burst stimulation resulted in slight long-term ceptors were inhibited, however, LTP induction by the STDP pro-
depression (Fig. 4B, light gray circles, 93.1% ± 2.0% of baseline, tocol was eliminated on average and for each individual
n¼5,p¼0.008).AmoderatedoseofMPH(5mg/kg)alonedidnot experiment(Fig.1F,rightbarplot).Chronicrecordingsfromfreely
produce significant synaptic change (Fig. 4B, open circles): movingmiceshowedthatsimilarcatecholaminemechanismsare
104%±1.1%,n¼3,p>0.05.However,whenthatdosewasgiven atplayinvivo.Fieldpotentialsevokedbystimulationofthemedial
15minbeforethestandardTBS,itproducedlonglastingpotenti- perforant path were recorded in the hilar region of the dentate
ationoftheperforantpathtodentategyruspathway(Fig.4B,black gyrus (Tang and Dani, 2009). Weak theta burst stimulation (TBS)
circles):131%±2.3%,n¼5,p<0.0001. thatinducednosynapticchangeorslightLTDonitsown,induced
To determine whether primarily DA or NE receptors were LTP when applied in the presence of MPH (5 mg/kg). The LTP
responsible for the MPH induced synaptic potentiation, we sys- induced by TBS þ MPH was reduced when NE b receptors were
temicallyinhibitedDAD1/D5receptors(0.2mg/kg,i.p.,SCH-23390) inhibitedbytimolol(Fig.6),buttheLTPinductionwasabsentwhen
orNEbreceptors(10or20mg/kg,timolol).InjectionsofSCH-23390 D1-typeDAreceptorswereinhibitedwithSCH-23390(Fig.5).
(0.2 mg/kg, i.p) alone did not significantly alter synaptic trans- Whileboththeinvitroand invivoexperiments suggest influ-
mission(Fig.5A):102.5%±2.8%ofbaseline,n¼5,p>0.05.Inthe ence by both catecholamines, the results are quantitatively
pairedcontrol experiments wheresaline (i.p.) preceded the MPH different. The in vitro experiments show influence by inhibiting
administration, MPH induced a significant potentiation over the eitherthe D1-type receptorsor NE b receptors, butthe strongest
baseline(Fig.5B,blacksquares):164.2%±2.9%ofbaseline,n¼5, inhibition of STDP occurred when both receptor types were
p<0.0001.SystemicinjectionofSCH-23390precedingMPH(5mg/ inhibited.Theinvivoexperimentsshowgreaterandcompletein-
kg) administration completely prevented synaptic potentiation hibition of the MPH-influenced plasticity when the D1-type re-
induced by MPH combined with TBS (Fig. 5B, gray squares): ceptors were inhibited. These differences could arise from the
100.1%±18.6%ofbaseline,n¼5,p>0.05.Injectionsoftimolol(10 differenceinthepreparations.Theinvitrosliceswerefrom24to30
or 20 mg/kg, i.p.) alone did not significantlyalter synaptic trans- day old mice while the in vivo experiments were conducted on
mission(Fig.6A):103.2%±5.9%ofbaseline,n¼5,p>0.05.Inthe youngadultmice(3e4monthsold).Possiblymoreimportantly,the
paired control experiments where saline (i.p.) preceded MPH scatteramongtheindividualexperimentsinvitro(Fig.1D,E,right)
administration,MPHfollowedbyTBSinducedasignificantpoten- suggest that the individual placements of stimulation and
tiation over baseline (Fig. 6B, black circles): 174% ± 13%, n ¼ 7, recordingelectrodes, coupled with the reduced slicepreparation,
p<0.0001.Systemicinjectionoftimololdecreasedthechangein couldproducemoreextremesinthelocalcatecholaminesignaling.
synaptictransmissioninducedbyMPHprecedingTBS,butsignifi- This variability appears less likely in the intact, in vivo situation
cant LTP was still induced: 151% ± 3.4% of baseline, n ¼ 5, wheremature,moreconsistentcatecholaminesignalingispresent.
p¼0.0037.The3-wayANOVAandTukeyHSDpost-hoctestsreport Inthatcase,theactionsofthereceptorsandthereceptorinhibitors
that all 3 groups are statistically different, indicating that when may reflect the final more refined and consistent catecholamine
timololinhibitsNEbreceptorsthereisadifferenceinMPH'seffect. signalingintothedentategyrusseenintheadultmice.
Asalways,thereportedLTPvalueabovewascalculatedforthelast EvidenceindicatesthatNETandDATblockadeinfluencesindi-
hourofthe3htimecourseafterTBSadministration(Fig.6B).The vidualareasofthehippocampusdifferently(HopkinsandJohnston,
greatestdifferencecausedbytimololoccursatearliertimesduring 1988; Huang and Kandel,1996; Ishihara et al.,1991; Lacaille and
the first hourafterTBS when inhibition of the NE b receptors by Harley,1985;Munroetal.,2001).InthehippocampalCA1region
timolol resulted in a stronger suppression of the early synaptic ofratslices,MPHboostedtetanicLTPbasedmainlyonitsabilityto
potentiation:136%±3.1%,n¼5,p¼0.0004. block NETs because the NE b receptor inhibitor, timolol, blocked
MPH enhancement of tetanic LTP (Dommett et al., 2008). In our
4. Discussion experimentsappliedtothedentategyrus,however,theDAD1-type
receptors and NE b receptors both seemed important during our
Methylphenidate (MPH) is widely used for the treatment of invitro(relativelyweak)STDPprotocolappliedtoslicesfromyoung
ADHD to improve attention, learning, and memory. The main mice(24e30d).Furthermore,theD1-typereceptorsseemedeven
mechanistic action of MPH is to inhibit DA and NE reuptake moreimportantthantheNEreceptorsduringourinvivoTBSpro-
transporters. Based on its action and on previous findings tocolinadultmice.
(Dommett et al., 2008; Urban et al., 2013), we hypothesized that PreviousreportsshowedmixedresultsfortheabilityofMPHto
MPH directly alters hippocampal long-term synaptic plasticity. increase DA and NE in slice preparations (Heal et al., 2008). We
30 D.Jensonetal./Neuropharmacology90(2015)23e32
found effects of both DA and NE in our hippocampal slice LTP expected to enhance the size and length of adrenergic signaling.
paradigm,suggestingthattheSTDPstimulationprotocolcancause Dopamine and adrenergic receptors are expressed in the hippo-
variable DA and NE release from terminals in the dentate gyrus campus (Huang et al., 1992; Jurgens et al., 2005), and the cate-
slice. Furthermore, MPH significantly increased these catechol- cholamineshavebeenshowntoenhancesynapticplasticityviaD1-
amine signals to enhance synaptic plasticity. In vivo, TBS type DA receptors and b-type NE receptors (Lisman and Grace,
enhancementbyMPHinthedentategyruswasmoredependenton 2005;TangandDani,2009;Thomasetal.,1996).Ourresultssup-
theactivityofDAreceptors.WhethertheDAdrivenenhancement port the importance of catecholamines in synaptic plasticity, and
wasduetoendogenousDAreleaseorpreferentialreleaseofDAby theresultsindicatethatMPHinfluencesthesesynapticprocesses.
theTBSprotocolitselfisunknown.Thesedifferencesintheresults Becausetheinvivoexperimentswereconductedonfreely-moving
that arise from different plasticity induction protocols applied to mice under biologically reasonable conditions, those results pro-
differenthippocampallocationsareconsistentwithregion-specific vide strong support for the importance of MPH acting via cate-
forms of neuromodulation (Hopkins and Johnston, 1988; Huang cholaminesinthehippocampus.
and Kandel,1996; Ishihara et al.,1991; Lacaille and Harley,1985;
Munroetal.,2001).Therefore,thecumulativeresultssuggestthat
MPHinfluenceshippocampalplasticityowingtoeffectsonboththe Acknowledgments
DAandNEsystems.
Throughout these experiments we attempted to use stimulus TheworkwassupportedbygrantsfromtheNationalInstitutes
protocolsandMPHdosesthatarereasonable.ATBSprotocolwas ofHealth,NIDADA09411andNINDSNS21229.
usedinvivotomimicthethetamodeofhippocampalfiringthatis
observed during states of active, alert behavior in freely-moving
References
rodents (Hyman et al., 2003; Orr et al., 2001). That TBS protocol
onlyproducedLTPafteradministrationofMPH.Similarly,theSTDP
Agnati,L.F.,Zoli,M.,Stromberg,I.,Fuxe,K.,1995.Intercellularcommunicationinthe
protocolusedfortheinvitroexperimentswasmild.Inaddition,the brain:wiringversusvolumetransmission.Neuroscience69,711e726.
perforated-patchmethodology,ratherthanthewhole-cellconfig- Andersen,P.,Bliss,T.V.,Skrede,K.K.,1971.Unitanalysisofhippocampalpopulation
uration,wasusedfortheinvitrorecordingstopreservetheintra- spikes.Exp.BrainRes.13,208e221.
Benoit-Marand,M.,Borrelli,E.,Gonon,F.,2001.Inhibitionofdopaminereleasevia
cellular milieu. Unlike tetanic stimulation to induce synaptic presynapticD2receptors:timecourseandfunctionalcharacteristicsinvivo.
plasticity, STDP pairs a presynaptic stimulation followed by a J.Neurosci.21,9134e9141.
postsynapticdepolarizationtomimicthebiologicalcaseinwhich Biala,G.,Betancur,C.,Mansuy,I.M.,Giros,B.,2005.Thereinforcingeffectsofchronic
D-amphetamineandmorphineareimpairedinalineofmemory-deficientmice
the afferent activity drives a postsynaptic response in the target overexpressingcalcineurin.Eur.J.Neurosci.21,3089e3096.
area. The mild STDP protocol used for the in vitro experiments Biederman,J.,Spencer,T.,1999.Attention-deficit/hyperactivitydisorder(ADHD)as
induced synaptic plasticity (LTP) only in the presence of MPH anoradrenergicdisorder.Biol.Psychiatry46,1234e1242.
(0.5mM).ThedosesofMPHarosefromtheliterature,ourvoltam- Borgkvist,A.,Malmlof,T.,Feltmann,K.,Lindskog,M.,Schilstrom,B.,2012.Dopamine
inthehippocampusisclearedbythenorepinephrinetransporter.Int.J.Neu-
metry data, and our behavioral data. Based on our voltammetry ropsychopharmacol.15,531e540.
experiments, the DA release was well behaved up to 1 mM MPH, Buzsaki,G.,Czeh,G.,1981.Commissuralandperforantpathinteractionsintherat
hippocampus.Fieldpotentialsandunitaryactivity.Exp.BrainRes.43,429e438.
whichresultedinthelargestpeakevokedDArelease.Therefore,we
Bymaster, F.P., Katner, J.S., Nelson, D.L., Hemrick-Luecke, S.K., Threlkeld, P.G.,
used 0.5 mM MPH for the hippocampal slice experiments. This Heiligenstein,J.H.,Morin,S.M.,Gehlert,D.R.,Perry,K.W.,2002.Atomoxetine
concentrationishigherthanbloodplasmalevelsinpatientstreated increases extracellular levels of norepinephrine and dopamine in prefrontal
forADHD,whichisusuallyintherangeof50e200nM,butMPH cortexofrat:apotentialmechanismforefficacyinattentiondeficit/hyperac-
tivitydisorder.Neuropsychopharmacology27,699e711.
reaches higher concentrations in the brain than in the blood Chai,G.,Governale,L.,McMahon,A.W.,Trinidad,J.P.,Staffa,J.,Murphy,D.,2012.
(HoffmanandLefkowitz,1996).TheMPHconcentration(5mg/kg) TrendsofoutpatientprescriptiondrugutilizationinUSchildren,2002e2010.
usedfortheinvivoexperimentswithfreely-movingmicehadno Pediatrics130,23e31.
Dani,J.A.,Harris,R.A.,2005.Nicotineaddictionandcomorbiditywithalcoholabuse
effectoverinvivosynaptictransmissionbeforetheTBSwasapplied andmentalillness.Nat.Neurosci.8,1465e1470.
(Fig. 4). This MPH dose (5 mg/kg) increased locomotor behavior Deadwyler,S.A.,West,M.,Lynch,G.,1979.Activityofdentategranulecellsduring
withoutinducingstereotypy,andthisdoseincreasedcorrectarm learning:differentiationofperforantpathinput.BrainRes.169,29e43.
DiChiara,G.,2002.Nucleusaccumbensshellandcoredopamine:differentialrolein
choicesinthedelayednon-matchtosampleT-mazetask.Thattask behaviorandaddiction.Behav.BrainRes.137,75e114.
is designed to test spatial working memory, which is a type of Dommett,E.J.,Henderson,E.L.,Westwell,M.S.,Greenfield,S.A.,2008.Methylphe-
memorythatMPHisspecificallyknowntoenhance(Repantisetal., nidate amplifies long-term plasticity in the hippocampus via noradrenergic
mechanisms.Learn.Mem.15,580e586.
2010; Wright and White, 2003). Therefore, the 5 mg/kg dose of
Doyere,V.,Schafe,G.E.,Sigurdsson,T.,LeDoux,J.E.,2003.Long-termpotentiationin
MPH seems like a biologically appropriate concentration for the freelymovingratsrevealsasymmetriesinthalamicandcorticalinputstothe
behavioraltasksandinvivomeasurementsofsynapticplasticityin lateralamygdala.Eur.J.Neurosci.17,2703e2715.
Floresco,S.B.,West,A.R.,Ash,B.,Moore,H.,Grace,A.A.,2003.Afferentmodulation
mice.Underthesebiologicallyreasonableexperimentalconditions, ofdopamineneuronfiringdifferentiallyregulatestonicandphasicdopamine
MPH enhanced synaptic plasticity involving catecholamine transmission.Nat.Neurosci.6,968e973.
receptors. Franklin,K.B.,Paxinos,G.,1997.TheMouseBraininStereotaxicCoordinates.Aca-
Unpredictedrewardsareknowntoproducebriefburstfiringby demicPress,London.
Garris,P.A.,Ciolkowski,E.L.,Pastore,P.,Wightman,R.M.,1994.Effluxofdopamine
DAneurons(Schultzetal.,1997),andphasicburstsinducegreater fromthesynapticcleftinthenucleusaccumbensoftheratbrain.J.Neurosci.14,
extracellular DA release compared with tonic, single-spike firing 6084e6093.
activity (Floresco et al., 2003; Garris et al., 1994; Gonon, 1988; Gonon, F.G.,1988. Nonlinear relationship between impulse flow and dopamine
releasedbyratmidbraindopaminergicneuronsasstudiedbyinvivoelectro-
Grace,1991;RiceandCragg,2004;Zhangetal.,2009).Dopamine chemistry.Neuroscience24,19e28.
neurons operate in these distinct tonic and phasic timescales to Grace, A.A.,1991. Phasic versus tonic dopamine release and the modulation of
differentiatebehaviorallyrelevantinformation(Schultz,2007).By dopaminesystemresponsivity:ahypothesisfortheetiologyofschizophrenia.
Neuroscience41,1e24.
inhibitingDATsandNETs,MPHincreasesandlengthensthecate- Grace, A.A., Bunney, B.S.,1984.The controlof firingpatternin nigraldopamine
cholaminesignalafterrelease,whichmaysharesimilaritiestothe neurons:singlespikefiring.J.Neurosci.4,2866e2876.
DA/NE signal after a long phasic burst. Our voltammetry results Hannestad, J., Gallezot, J.D., Planeta-Wilson, B., Lin, S.F., Williams, W.A., van
Dyck,C.H.,Malison,R.T.,Carson,R.E.,Ding,Y.S.,2010.Clinicallyrelevantdosesof
showed that MPH enhanced the DA signaling from phasic bursts methylphenidatesignificantlyoccupynorepinephrinetransportersinhumans
even more than it enhanced the tonic signals. Likewise, MPH is invivo.Biol.Psychiatry68,854e860.
D.Jensonetal./Neuropharmacology90(2015)23e32 31
Hargreaves, E.L., Rao, G., Lee, I., Knierim, J.J., 2005. Major dissociation between Lloyd,S.A.,Oltean,C.,Pass,H.,Phillips,B.,Staton,K.,Robertson,C.L.,Shanks,R.A.,
medial and lateral entorhinal input to dorsal hippocampus. Science 308, 2013. Prenatal exposure to psychostimulants increases impulsivity, compul-
1792e1794. sivity,andmotivationforrewardsinadultmice.Physiol.Behav.119C,43e51.
Heal, D.J., Smith, S.L., Kulkarni, R.S., Rowley, H.L., 2008. New perspectives from Lomo, T.,1971. Patterns of activation in a monosynaptic cortical pathway: the
microdialysisstudiesinfreely-moving,spontaneouslyhypertensiveratsonthe perforantpathinputtothedentateareaofthehippocampalformation.Exp.
pharmacologyofdrugsforthetreatmentofADHD.Pharmacol.Biochem.Behav. BrainRes.12,18e45.
90,184e197. Lynch,M.A.,2004.Long-termpotentiationandmemory.Physiol.Rev.84,87e136.
Heredia,L.,Torrente,M.,Colomina,M.T.,Domingo,J.L.,2013.Assessinganxietyin Martinez,J.L.,Derrick,B.E.,1996.Long-termpotentiationandlearning.Annu.Rev.
C57BL/6J mice: a pharmacological characterization of the zero maze test. Psychol.47,173e203.
J.Pharmacol.Toxicol.Methods68,275e283. McHugh,S.B.,Niewoehner,B.,Rawlins,J.N.,Bannerman,D.M.,2008.Dorsalhip-
Hoffman,B.B., Lefkowitz,R.J.,1996.GoodmanandGilman'sthe Pharmacological pocampal N-methyl-D-aspartate receptors underlie spatial working memory
Basis of Therapeutics, Catecholamines, Sympathomimetic Drugs, and Adren- performanceduringnon-matchingtoplacetestingontheT-maze.Behav.Brain
ergicReceptorAntagonists.McGraw-Hill,NewYork. Res.186,41e47.
Hopkins,W.F.,Johnston,D.,1984.Frequency-dependentnoradrenergicmodulation Moore,R.Y.,Bloom,F.E.,1979.Centralcatecholamineneuronsystems:anatomyand
oflong-termpotentiationinthehippocampus.Science226,350e352. physiologyofthenorepinephrineandepinephrinesystems.Annu.Rev.Neu-
Hopkins, W.F., Johnston, D., 1988. Noradrenergic enhancement of long-term rosci.2,113e168.
potentiationatmossyfibersynapsesinthehippocampus.J.Neurophysiol.59, Munro,C.A.,Walling,S.G.,Evans,J.H.,Harley,C.W.,2001.Beta-adrenergicblockade
667e687. in the dentate gyrus in vivo prevents high frequency-induced long-term
Huang,F.L.,Huang,K.P.,2012.Methylphenidateimprovesthebehavioralandcogni- potentiationofEPSPslope,butnotlong-termpotentiationofpopulationspike
tivedeficitsofneurograninknockoutmice.GenesBrainBehav.11,794e805. amplitude.Hippocampus11,322e328.
Huang,Q.,Zhou,D.,Chase,K.,Gusella,J.F.,Aronin,N.,DiFiglia,M.,1992.Immuno- Orr,G.,Rao,G.,Houston,F.P.,McNaughton,B.L.,Barnes,C.A.,2001.Hippocampal
histochemicallocalizationoftheD1dopaminereceptorinratbrainrevealsits synapticplasticityismodulatedbythetarhythminthefasciadentataofadult
axonaltransport,pre-andpostsynapticlocalization,andprevalenceinthebasal andagedfreelybehavingrats.Hippocampus11,647e654.
ganglia,limbicsystem,andthalamicreticularnucleus.Proc.Natl.Acad.Sci.U.S. Pristupa,Z.B.,Wilson,J.M.,Hoffman,B.J.,Kish,S.J.,Niznik,H.B.,1994.Pharmaco-
A.89,11988e11992. logicalheterogeneityoftheclonedandnativehumandopaminetransporter:
Huang,Y.Y.,Kandel,E.R.,1996.Modulationofboththeearlyandthelatephaseof disassociationof[3H]WIN35,428and[3H]GBR12,935binding.Mol.Pharmacol.
mossy fiber LTP by the activation of beta-adrenergic receptors. Neuron 16, 45,125e135.
611e617. Repantis,D.,Schlattmann,P.,Laisney,O.,Heuser,I.,2010.Modafinilandmethyl-
Hyland, B.I., Reynolds, J.N., Hay, J., Perk, C.G., Miller, R., 2002. Firing modes of phenidateforneuroenhancementinhealthyindividuals:asystematicreview.
midbraindopaminecellsinthefreelymovingrat.Neuroscience114,475e492. Pharmacol.Res.62,187e206.
Hyman,J.M.,Wyble,B.P.,Goyal,V.,Rossi,C.A.,Hasselmo,M.E.,2003.Stimulationin Rice,M.E.,Cragg,S.J.,2004.Nicotineamplifiesreward-relateddopaminesignalsin
hippocampalregionCA1inbehavingratsyieldslong-termpotentiationwhen striatum.Nat.Neurosci.7,583e584.
deliveredtothepeakofthetaandlong-termdepressionwhendeliveredtothe Robinson,S.,Smith,D.M.,Mizumori,S.J.,Palmiter,R.D.,2004.Firingpropertiesof
trough.J.Neurosci.23,11725e11731. dopamine neurons in freely moving dopamine-deficient mice: effects of
Hyman,S.E.,Malenka,R.C.,Nestler,E.J.,2006.Neuralmechanismsofaddiction:the dopaminereceptoractivationandanesthesia.Proc.Natl.Acad.Sci.U.S.A.101,
roleofreward-relatedlearningandmemory.Annu.Rev.Neurosci.29,565e598. 13329e13334.
Ishihara,K.,Katsuki,H.,Kawabata,A.,Sasa,M.,Satoh,M.,Takaori,S.,1991.Effectsof Rossato, J.I., Bevilaqua, L.R., Izquierdo, I., Medina, J.H., Cammarota, M., 2009.
thyrotropin-releasinghormoneandarelatedanalog,CNK-602A,onlong-term Dopamine controls persistence of long-term memory storage. Science 325,
potentiationinthemossyfiber-CA3pathwayofguineapighippocampalsli- 1017e1020.
ces.BrainRes.554,203e208. Rumpel,S.,LeDoux,J.,Zador,A.,Malinow,R.,2005.Postsynapticreceptortrafficking
Ishimatsu,M.,Kidani,Y.,Tsuda,A.,Akasu,T.,2002.Effectsofmethylphenidateon underlyingaformofassociativelearning.Science308,83e88.
the membrane potential and current in neurons of the rat locus coeruleus. Sajikumar,S.,Frey,J.U.,2004.Late-associativity,synaptictagging,andtheroleof
J.Neurophysiol.87,1206e1212. dopamineduringLTPandLTD.Neurobiol.Learn.Mem.82,12e25.
Izumi,Y.,Clifford,D.B.,Zorumski,C.F.,1992.NorepinephrinereversesN-methyl-D- Schultz,W.,2007.Multipledopaminefunctionsatdifferenttimecourses.Annu.Rev.
aspartate-mediated inhibition of long-term potentiation in rat hippocampal Neurosci.30,259e288.
slices.Neurosci.Lett.142,163e166. Schultz,W.,Dayan,P.,Montague,P.R.,1997.Aneuralsubstrateofpredictionand
Jay,T.M.,2003.Dopamine:apotentialsubstrateforsynapticplasticityandmemory reward.Science275,1593e1599.
mechanisms.Prog.Neurobiol.69,375e390. Smith,C.C.,Greene,R.W.,2012.CNSdopaminetransmissionmediatedbynorad-
Jones,S.,Bonci,A.,2005.Synapticplasticityanddrugaddiction.Curr.Opin.Phar- renergicinnervation.J.Neurosci.32,6072e6080.
macol.5,20e25. Stoops,W.W.,Glaser,P.E.,Rush,C.R.,2003.Reinforcing,subject-rated,andphysio-
Jurgens,C.W.,Rau,K.E.,Knudson,C.A.,King,J.D.,Carr,P.A.,Porter,J.E.,Doze,V.A., logical effects of intranasal methylphenidate in humans: a dose-response
2005.Beta1adrenergicreceptor-mediatedenhancementofhippocampalCA3 analysis.DrugAlcoholDepend.71,179e186.
networkactivity.J.Pharmacol.Exp.Ther.314,552e560. Swanson, J.M., Volkow, N.D., 2003. Serum and brain concentrations of methyl-
Kauer, J.A., 2004. Learning mechanisms in addiction: synaptic plasticity in the phenidate: implications for use and abuse. Neurosci. Biobehav. Rev. 27,
ventraltegmentalareaasaresultofexposuretodrugsofabuse.Annu.Rev. 615e621.
Physiol.66,447e475. Tang,J.,Dani,J.A.,2009.Dopamineenablesinvivosynapticplasticityassociated
Kelley,A.E.,2004.Ventralstriatalcontrolofappetitivemotivation:roleiningestive withtheaddictivedrugnicotine.Neuron63,673e682.
behaviorandreward-relatedlearning.Neurosci.Biobehav.Rev.27,765e776. Thomas, M.J., Moody, T.D., Makhinson, M., O'Dell, T.J.,1996. Activity-dependent
Kilts, C.D., Schweitzer, J.B., Quinn, C.K., Gross, R.E., Faber, T.L., Muhammad, F., beta-adrenergicmodulationoflowfrequencystimulationinducedLTPinthe
Ely, T.D., Hoffman, J.M., Drexler, K.P., 2001. Neural activity related to drug hippocampalCA1region.Neuron17,475e482.
cravingincocaineaddiction.Arch.Gen.Psychiatry58,334e341. Ungless,M.A.,Magill,P.J.,Bolam,J.P.,2004.Uniforminhibitionofdopamineneurons
Koda,K.,Ago,Y.,Cong,Y.,Kita,Y.,Takuma,K.,Matsuda,T.,2010.Effectsofacuteand intheventraltegmentalareabyaversivestimuli.Science303,2040e2042.
chronicadministrationofatomoxetineandmethylphenidateonextracellular Urban,K.R.,Li,Y.C.,Gao,W.J.,2013.Treatmentwithaclinically-relevantdoseof
levelsofnoradrenaline,dopamineandserotoninintheprefrontalcortexand methylphenidatealtersNMDAreceptorcompositionandsynapticplasticityin
striatumofmice.J.Neurochem.114,259e270. thejuvenileratprefrontalcortex.Neurobiol.Learn.Mem.101,65e74.
Kuczenski,R.,Segal,D.S.,1997.Effectsofmethylphenidateonextracellulardopa- Volkow, N.D., Ding, Y.S., Fowler, J.S., Wang, G.J., Logan, J., Gatley, J.S., Dewey, S.,
mine, serotonin, and norepinephrine: comparison with amphetamine. Ashby,C.,Liebermann,J.,Hitzemann,R.,1995.Ismethylphenidatelikecocaine?
J.Neurochem.68,2032e2037. Studiesontheirpharmacokineticsanddistributioninthehumanbrain.Arch.
Kuczenski,R.,Segal,D.S.,2001.Locomotoreffectsofacuteandrepeatedthreshold Gen.Psychiatry52,456e463.
dosesofamphetamineandmethylphenidate:relativerolesofdopamineand Volkow,N.D.,Fowler,J.S.,Wang,G.-J.,Swanson,J.M.,2004.Dopamineindrugabuse
norepinephrine.J.Pharmacol.Exp.Ther.296,876e883. andaddiction:resultsfromimagingstudiesandtreatmentimplications.Mol.
Kuczenski,R.,Segal,D.S.,2002.Exposureofadolescentratstooralmethylpheni- Psychiatry9,557e569.
date: preferential effects on extracellular norepinephrine and absence of Volkow,N.D.,Swanson,J.M.,2003.Variablesthataffecttheclinicaluseandabuseof
sensitization and cross-sensitization to methamphetamine. J. Neurosci. 22, methylphenidateinthetreatmentofADHD.Am.J.Psychiatry160,1909e1918.
7264e7271. Volkow,N.D.,Wang,G.J.,Fowler,J.S.,Gatley,S.J.,Logan,J.,Ding,Y.S.,Hitzemann,R.,
Kulla,A.,Manahan-Vaughan,D.,2000.Depotentiationinthedentategyrusoffreely Pappas, N., 1998. Dopamine transporter occupancies in the human brain
moving rats is modulated by D1/D5 dopamine receptors. Cereb. Cortex 10, inducedbytherapeuticdosesoforalmethylphenidate.Am.J.Psychiatry155,
614e620. 1325e1331.
Lacaille,J.C.,Harley,C.W.,1985.Theactionofnorepinephrineinthedentategyrus: Weikop,P.,Yoshitake,T.,Kehr,J.,2007.Differentialeffectsofadjunctivemethyl-
beta-mediated facilitation of evoked potentials in vitro. Brain Res. 358, phenidateandcitalopramonextracellularlevelsofserotonin,noradrenaline
210e220. anddopamineintheratbrain.Eur.Neuropsychopharmacol.17,658e671.
Lavenex,P.,Amaral,D.G.,2000.Hippocampaleneocorticalinteraction:ahierarchy Winder,D.G.,Egli,R.E.,Schramm,N.L.,Matthews,R.T.,2002.Synapticplasticityin
ofassociativity.Hippocampus10,420e430. drugrewardcircuitry.Curr.Mol.Med.2,667e676.
Lisman,J.E.,Grace,A.A.,2005.Thehippocampal-VTAloop:controllingtheentryof Wright,F.K.,White,K.G.,2003.Effectsofmethylphenidateonworkingmemoryin
informationintolong-termmemory.Neuron46,703e713. pigeons.Cogn.AffectBehav.Neurosci.3,300e308.
32 D.Jensonetal./Neuropharmacology90(2015)23e32
Yang,K.,Hu,J.,Lucero,L.,Liu,Q.,Zheng,C.,Zhen,X.,Jin,G.,Lukas,R.J.,Wu,J.,2009. Zhang,T.,Zhang,L.,Liang,Y.,Siapas,A.G.,Zhou,F.M.,Dani,J.A.,2009.Dopamine
Distinctivenicotinicacetylcholinereceptorfunctionalphenotypesofratventral signalingdifferencesinthenucleusaccumbensanddorsalstriatumexploited
tegmentalareadopaminergicneurons.J.Physiol.587,345e361. bynicotine.J.Neurosci.29,4035e4043.
Zhang,L.,Dong,Y.,Doyon,W.M.,Dani,J.A.,2012.Withdrawalfromchronicnicotine Zhang,T.A.,Tang,J.,Pidoplichko,V.I.,Dani,J.A.,2010.Addictivenicotinealterslocal
exposurealtersdopaminesignalingdynamicsinthenucleusaccumbens.Biol. circuitinhibitionduringtheinductionofinvivohippocampalsynapticpoten-
Psychiatry71,184e191. tiation.J.Neurosci.30,6443e6453.