Impulsivity and home-cage activity are decreased by lentivirus-mediated silencing of serotonin transporter in the rat hippocampus

Impulsivity

and

home-cage

activity

are

decreased

by

lentivirus-mediated

silencing

of

serotonin

transporter

in

the

rat

hippocampus

Francesca

Zoratto

_,

_Amanda

_L.

_Tringle

_,

_Giancarlo

_Bellenchi

_,

_Luisa

_Speranza

_,

Domenica

Travaglini

_,

_Umberto

_diPorzio

_,

_Carla

_{Perrone-Capano}

_,

_Giovanni

_Laviola

_,

Jean-Luc

Dreyer

_,

_Walter

_Adriani

a_{Dept.CellBiology&Neurosciences,IstitutoSuperiorediSanità,Rome,Italy} b_{InstituteofGeneticsandBiophysics,CNR,Naples,Italy}

c_{Dept.BiologicalSciences,University“FedericoII”,Naples,Italy} d_{Dept.Medicine,UniversityofFribourg,Switzerland} e_{BambinoGesùChildren’sHospitalIRCCS,Rome,Italy}

h

i

g

h

l

i

g

h

t

s

•Lenti-SERTvectorsweredesignedtosuppressSERTgeneexpressioninvivo.

•WeevaluatedaratmodelofADHDthroughhippocampalinoculationofLenti-SERT.

•Lenti-SERTratsexhibitedlesspronouncedpeaksofcircadianactivitythancontrols.

•Lenti-SERTratsdisplayedatransientdecreaseincognitiveimpulsivity.

•Suchphenotypeisconsistentbothwith5-HTmanipulationsandhippocampallesions.

Brainserotonin(5-HT)systemsmodulateemotional,motivationalandcognitiveprocesses.Mutations

intheserotonintransporter(SERT)genehavebeenassociatedwithsusceptibilitytowardsthe

develop-mentofseveralpsychiatricdisorders,bothinhumansandanimalmodels.Presentapproachexploiteda

bilateralintra-hippocampusstereotaxicinoculationoflentiviruses,forenduringinvivosilencingofSERT.

Controlratswerebilaterallyinoculatedwithheat-inactivatedlentiviruses.TheseLenti-SERTvectorswere

intendedtoeventuallymanipulatetheneurotransmitterreuptakeatsynapticlevel,thusenhancingtonic

5-HTtransmission.Weinvestigatedwhethersuchmanipulationcouldinducebehaviouralalterations

relevanttothemodellingofADHD,inparticularsymptomsofhyperactivityandimpulsivity.Wistarrats

weremonitoredforspontaneoushome-cagelocomotoractivityandstudiedforimpulsivity

(Intolerance-to-Delaytask).ResultsshowthatratsinoculatedwithLenti-SERTvectorsexhibitedlesspronounced

circadianpeaksofactivitythancontrols.Moreover,Lenti-SERTcomparedtocontrolratsexhibiteda

transientincreaseinchoiceforadelayed-largerrewardoveranimmediate-smallreward.Thissuggests

thatenhancedhippocampalserotonergictransmissionproducedaprofileofrestfulnessandadecrease

incognitiveimpulsivity.Thisphenotypeisconsistentwithavailabledatabothon5-HTmanipulations

andhippocampallesions.Inconclusion,presentfindingsmaypossiblydisclosenovelavenuestowards

thedevelopmentofinnovativetherapeuticalapproachesforbehaviouralsymptomsrelevanttoADHD.

∗ Correspondingauthorat:vialeReginaElena299,I-00161Rome,Italy. Tel.:+390649903171.

E-mailaddress:walter.adriani@iss.it(W.Adriani).

1. Introduction

Attention-deficit/hyperactivitydisorder(ADHD)isa heteroge-neoussyndrome,affecting2–5%ofinfantsandadolescentsaswell asabout2%ofadultsworldwide[59].Togetherwithinattention andmotorhyperactivity,onekeysymptomisimpulsivity,a mul-tifacetedconceptimplicatedinmanydisorderscharacterizedby inappropriateinhibitorycontrol[16].It hasbeensuggestedthat



Published in "1HXURVFLHQFH/HWWHUV±"

which should be cited to refer to this work.

impulsivity isnot a unitary construct,but describesa range of behavioursandencompassesavarietyofrelatedphenomenathat maydifferintheirbiologicalbasis[12,60].

Theserotonergicsystemiswellknownformodulationof emo-tional,cognitiveandmotivationalprocesses[11].Dysfunctionsin thissystemplayacrucialroleinmanypsychiatricdisorders, includ-ingaffectiveandimpulse-controldisorders[39,50].Bymediating theinterplaybetweenlimbicandcognitiveloops,forebrain sero-tonin(5-HT)hasakeyroleinthetop-downinhibitorycontrolover behaviouralinitiationandexecution,whichisimportantfor with-holdingofinstinctivereactionsand foranappropriatefeedback regulationofbehaviour[11,22].Assuch,itiscentralto psychomo-torcontroloversubcorticalprocessingofrewardandreinforcement [47].

Permanentinvivointerferencewithgeneandprotein expres-sionandfunctionisnowadayspossibleusinglentiviralvectors[42]. Wehaverecentlyusedthisapproachexploitingbraininoculation oflentivirusestargetingthedopaminetransporter(DAT):its over-expressioninrats’nucleusaccumbensleadstoanimpulsiveand risk-pronephenotype[1,2].Similarly,wepreparedanovelvector designedtosuppresstheserotonintransporter(SERT)gene expres-sion.Indeed,mutationsintheSERTgeneinfluencetherateof5-HT reuptakeandhavebeenassociatedwithsusceptibilitytowardsthe developmentofseveralpsychiatricdisorders[10,53].Specifically, acommonpolymorphism(5-HTTLPR),localisedinthepromoterof theSERT-encodinggene,givesrisetotwomajorallelicvariants[31] thatsignificantlychangetheprobabilityofbeingaffectedbyADHD [18,40,54].Inparticular,thehippocampuswasselectedasthe inoc-ulationsite,sinceitslesionsareknowntocausehyperactivityand impulsivechoiceinrats(e.g.[9,27,46]).

Experimental studies with different serotonergic manipula-tionshave demonstratedan inverserelationship between5-HT levels and impulsivity, with a reduction in the neurotransmit-tercausinganincreaseinimpulsivityandviceversa(e.g.[8,55]). However, in humans, an increase in impulsivity appears tobe associatedwiththeshort(s)allelicvariantof5-HTTLPR,leading toreduced SERT gene transcription [41,51,57]. Thus, we aimed todetermine whethera partialsilencing of theSERT-encoding gene withinthehippocampuscouldinducealterations relevant to the modelling of ADHD, in particular symptoms of hyper-activityand impulsivity.Specifically,for measuringbehavioural impulsivity,theIntolerance-to-Delay(ID)taskwasused,inwhich impulsive subjectsare detected bytheir intolerancetoperiods of forced waiting before the delivery of a large reward [17]. Indeed, delay-discounting paradigms in generalare among the most successfully utilised tools for measurement of impulsive choice[60].

2. Materialsandmethods 2.1. Lenti-SERTvectors

2.1.1. ConstructionofpTK431-SERT-siRNAs

TosilenceSERTexpressioninvivo,threetargetsweredesigned accordingtotheSERTmRNAsequence.Thefollowingtargetswere selected,basedonHannon’sdesigncriterion:1sttarget:bp64–29, 2ndtarget:bp2667–2629,3rdtarget:bp1805–1829.Toeacholigo, aXhoIrestrictionsitewasaddedat3andaU6-3-specific10mer at5.UsingthepSilencer1.0-U6(Ambion,UK)asatemplateand aU6promoter-specificforwardprimercontainingBamHI restric-tionsite(5-CGCCGCCGCGGATCCCGCTCTAGAACTAGTGC-3), eachsiRNAtargetwasaddedtothemouseU6promoterbyPCR, usingthefollowingprogram:120sat94◦C(initialdenaturation) followedby35cycles(45sat94◦C,45sat64◦Cand45sat72◦C) in4%dimethylsulfoxide(Sigma, Switzerland).ThePCRproduct

wasdigestedwithBamHIandXhoI,clonedintosimilarsitesinto pTK431,andsequencedtoverifytheintegrityofeachconstruct. 2.1.2. Lentivirusproduction

Thevectorplasmids(pTK431-U6-siSERT1,pTK431-U6-siSERT2, pTK431-U6-siSERT3andpTK433-GFP),togetherwiththe packag-ingconstructplasmidp891and theenvelopeplasmidPMD2-G, wereco-transfectedintoHEK293Tcellstoproducetheviral par-ticles[6,7].Onceharvestedandconcentrated,theseviralparticles weretestedinvitro.Theexperimentofinfectionandtransfection wasrunwith3,10and25␮lofeachlentivirusstock(LV-siSERT1, LV-siSERT2, LV-siSERT3) and 20␮l of a mix of thethree siLVs. HEK293TcellswereplatedandinfectedwithLV-siSERTsandthen transfected witha plasmidthat expressesSERT.ThesiLVs were addedwithPolybrene(Sigma,Switzerland,at10␮g/mlfinal con-centration) to the cells. After 7h, cells were transfected with pcDNA3-rat-SERT(2␮g/well).Thenextday,cellsweresubjected tototalRNAisolation,reversetranscriptionandreal-timePCR,to quantifySERTexpression/silencing(seeSupplementaryData). 2.2. Subjects

SeventeenadultmaleWistarrats(400g;forhousingconditions, seeSupplementaryData)wererandomlyassignedto experimen-talgroups:onegroupreceivedbilateralinoculationofLenti-SERT vectors(1␮lofamixofthethreeLV-siSERTs)intendedtoabolish the genetic expression of SERT. Inoculations were made bilat-erally at coordinates AP−3.3, ML±2.2, DV−4.0 from bregma [45].Theothergroup(controls)receivedabilateralinoculationof heat-inactivatedlentiviruses(1␮l)atthesamecoordinates(see SupplementaryData).Aftersurgery,ratsweresingle-housedand leftundisturbedforatleastonemonthpriortobehavioural exper-iments.

2.3. Circadiancycle

Ratswerecontinuouslymonitoredforspontaneoushome-cage locomotoractivity[3,4]bymeansofanautomaticdeviceequipped with small passive infrared sensors placed on a standard rack over thetop ofeach home-cage (ActiviScope system; TechnoS-mart,Rome,Italy).Thesesensors(20Hz)detectedanymovement ofrats:scoreswereautomaticallydividedinto60-minintervals. Thespontaneoushome-cageactivitywascontinuouslymeasured inLenti-SERTandcontrolsubjects,startingfromday50after inoc-ulationandover15days.Fromthisperiod,weextractedthecentral 5-daysinterval,fromwhichameandaywascalculated.

2.4. IDtaskforimpulsivity

Twomonthslater,ratsweredailytested(between10:30and 15:30)exploitingclassicalSkinner-boxes(forapparatusand food-restrictionschedule,seeSupplementaryData).Nose-pokinginone hole(termed“Small&Soon”,SS)resultedintheimmediatedelivery ofonepelletinonemagazine,whereasnose-pokingintheother hole(termed “Large&Late”,LL)resultedin thedeliveryoffive pelletsintheothermagazine.Afternose-pokingandbeforefood delivery,thechamberlightcorrespondingtothenose-pokedhole wasswitchedonfor1s.Followingfooddelivery,thecorresponding magazinelightwasturnedonfor30s,tosignalthelengthof time-out(TO)duringwhichadditionalnose-pokingwasrecordedbut hadnoscheduledconsequences(i.e.inadequate,see Supplemen-taryData)[49,50].Thetestingphasewasprecededbythreetraining sessionsatdelay0s,whichallowedsubjectstoreachasignificant preferenceforLL.Duringthetestingphase(eightdailysessions, 40mineach),adelaywasinsertedbetweennose-pokingintheLL holeanddeliveryofthe5-pelletreward.Thechamberlightoverthe



LLholewaskeptontosignaltheentirelengthofthisdelay,which wasfixedforeachdailysessionandwasprogressivelyincreased acrosssubsequentdays,from0sto90s.Thesmall-rewarddelivery wasunchanged.

2.5. Dataanalysis

Datawereanalyzedusingrepeated-measuresANOVA.The gen-eral model was: 24-leveltime (hour; circadian cycle study) or 8-levelsession(delay;IDtask)×2-levelgroup(treatment). Statis-ticalanalysiswasperformedusingStatviewII(AbacusConcepts, USA).Dataareexpressedasmean±SEM.Levelofsignificancewas setatP<0.05.Multipleposthoccomparisonswereperformedwith theTukey’sHSDtest.

3. Results

3.1. QuantificationofSERTsilencing

Compared to SERT expression level in transfected HEK293T cells(100%),infectionwiththethreesiLVsgeneratedthe follow-ingaveragedmRNAexpressionlevels:78%forLV-siSERT1,64%for LV-siSERT2,91%forLV-siSERT3and78%fortheirmix.

3.2. Circadiancycle

DatacollectedfromtheautomatedActiviScopesystemshowed thatcircadianactivitypatternsdiffereddependingontreatment (Fig.1A).Lenti-SERTanimals weresignificantlyless activethan controlsatspecifictimeintervalsthroughouttheday.Specifically, theANOVAyieldedsignificancefor group,time andtheir inter-action(treatment:F(1,15)=9.11,P=0.0087;time:F(23,345)=58.81,

P<0.0001;time×treatment:F(23,345)=2.46,P=0.0003).Lenti-SERT

animalsshowednodifferencesfromcontrolsintheexpected diur-nal rest pattern, as well as in the well-known peak observed at light switch off. As for nocturnal activity pattern, the pro-gressive increase towards the major level of activity (between 10:00 and 15:00) was significantly less marked in the Lenti-SERT when compared to the control group. A second peak was observed in both groups between 17:00 and 20:00, and again the Lenti-SERT rats were significantly less active than controls.

3.3. IDtaskforimpulsivity:preference(%)forLLreward

As expected,all animals showed a clear-cut shiftin prefer-encetowardstheSSrewardasthedelaylengthincreased(group: F(1,14)=1.52, P=0.2372; session: F(7,98)=52.43, P<0.0001;

ses-sion×group:F(7,98)=0.57,P=0.7745).However,animalsbelonging

totheLenti-SERTgroupseemedtodisplaysuchashiftlater (at delay of 45s) than did control animals (atdelay of 30s). Post hoc comparisons, drawn at this latter value of delay, revealed that LLhole wasstillsignificantly preferredbyLenti-SERT rats, whereasadelay-inducedshiftwasalreadypronouncedinthe cor-respondingcontrols(Fig.1B). Therefore, we mayconcludethat Lenti-SERTsubjectsshowedatransientlylessimpulsivechoicethan controls.

4. Discussion

Presently, a new approach exploited a lentivirus-mediated silencing ofSERT within thehippocampus(HPC),withthe aim ofgeneratingananimalmodelforimpulsivityandhyperactivity, whichinturnaresymptomsrelevanttoADHD.Suchmodelisto someextentsimilartoalocalknockoutoftheSERTgene[23],with

Fig.1.(A)Circadianactivitycycle.Circadianactivity(mean±SEM)inLenti-SERT (n=11)andcontrol(n=6)rats,leftundisturbedinsidetheirhome-cages(counts perminute,summedupto1-hpoints).The24-hprofilewasobtainedbyaveraging fivedaysofcontinuousregistration.Markeddifferenceswerefoundinthenocturnal activitypattern:activitylevelsweresignificantlylowerinLenti-SERTratscompared tocontrols.**P<0.01;#_{0.05<P<0.1inposthoctest.(B)ImpulsivityintheIDtask.}

Mean(±SEM)choice(%)ofthelargereward(LL),asafunctionofabsolutedelay duration(s),shownbyLenti-SERT(n=10)andcontrol(n=6)animals.Lenti-SERT ratsexperiencedaclear-cutintoleranceatalongerdelaythandidthecorresponding controls.*P<0.05inposthoctest.

theadvantagesthatsilencingisperformedintheHPCofalready adultanimals;therefore,thisapproachmayimplyless compen-satorymechanismscomparedtobreedingknockoutmodels.We emphasizethatouraimwastoinduceapartial(notatotal) silenc-ing oftheSERT-encodinggene, thusmimicking tosomeextent the short(s)allelicvariant of5-HTTLPR. Our resultsshowthat ratsinoculatedwithLenti-SERTexhibiteddecreasedspontaneous locomotoractivityanddecreasedcognitiveimpulsivity.

SERT,whichselectivelyremoves5-HToutofthesynapticcleft, isamajordeterminantofserotonergicsignallingefficiency,related totheneurotransmitterconcentration(e.g.[52]).Studiesinvitro showedthatthes-variantofthe5-HTTLPRwasassociatedwith reduced SERTgenetranscriptionefficiency,resultinginreduced SERTlevelsandreduced5-HTuptake.Consequently,the extracel-lular levelsof5-HTwerehighercompared tothel-variant (e.g. [20,31]).In thepresent study,thesilencingofSERTgene inthe



Lenti-SERTratswasintendedtoproduceanimprovementof sero-tonergictone,whichwehypothesizedtomimic,toacertainextent, theconditionofhumansubjectscarryingthes-variant.Similarly, homozygous SERT−/− rodents, which are widely considered to resembletheeffectsofthes/sgenotypeinhumans[25],show alter-ationsinmultipleneurobehavioraldomains,includingbehavioural inhibitionanddecisionmaking[28].

4.1. Circadianrhythmofactivity

Byanalyzingthecircadianpattern, thehypolocomotoreffect inLenti-SERTratsappearstobe“intrinsic”toanimals’physiology, sinceitwasobservedinhome-cageconditionsanditwasstable acrossdays.Itwasspecificallylocalizedaroundtwooutofthethree dailypeaksofactivity(inthemiddleofthedarkphaseandbefore thestartofthelitperiod).

5-HTisknowntohavecomplexactionswithrespectto con-trolofactivitylevels[36],althoughnotallformsoflocomotionare equallydependentoncentral5-HTtransmission[14].Forthe pur-posesofthepresentpaper,itisworthwhiletoreportthatSERT−/− miceshowdecreasedhome-cageactivity[21].Increasesin home-cageactivityhavebeenreportedfollowingdepletionofbrain5-HT. Forexample,bothPCPA(parachlorophenylalanine,aninhibitorof the5-HT-synthesizingenzyme)administrationandlesionsofthe median raphe inducean enhancement of locomotoractivity in familiarenvironments[15,19,26].Conversely,chronictreatment withselectiveserotoninreuptakeinhibitors(SSRIs;that,by inhib-itingthereuptake,increase5-HTextracellularlevel)causedrats torunsignificantlylessonarunning-wheelintheirhome-cages [5].Weconcludethatdecreasedspontaneousactivityexhibitedby Lenti-SERTratsisconsistentwithavailabledataon5-HT manipu-lationsinrodents.Moreover,HPClesionsareknowntoincrease (nocturnal)home-cageactivity(e.g.[27]).Thus, thedecreasein home-cageactivityobservedinpresentLenti-SERTratsisalso con-sistentwithapossiblyimprovedHPCfunction,dueinturntohigher amountofextracellular5-HTinthisbrainarea.

4.2. Cognitiveimpulsivity

Lenti-SERT rats preferred a delayed-larger reward over an immediate-smallrewardwhenthewaitingintervalwas30s, sug-gestingthatanenhancedHPCserotonergictransmissionrendered theseanimalsslightlylessimpulsive.Central5-HTmanipulations arereportedtoaffecttheabilitytowaitforreinforcementinstudies performedwithparadigmsforeitherimpulsive-choice(e.g.[8,38]) or impulsive-action (e.g.[13,15]).For example, lesions of 5-HT pathways[8,15,38]aswellasPCPAadministration[8,13]resulted inincreasedimpulsivity.Moreover,SERT−/−ratsshowedincreased correctresponselatencyanddecreasedprematurerespondingin the5-choiceserialreactiontime task(5-CSRTT;[24]).Based on available data, it hasbeen speculated that SERT−/− rats would alsoshowimproved“waiting”whenrewardisdelayed[28]. Con-sistently,acuteincreasein5-HTlevels,throughSSRIstreatment, resultedindecreasedimpulsivityinareward-delaytask[8].

Impulsive behaviour has been associated not only with orbitofrontalcortex(OFC)lesions(e.g.[29,37])butalsowithHPC lesions(e.g.[9,46]).McHughandcolleagues[35]haveshown,using aspatialtaskinvolvingachoicebetweenadelayed-largervs.an immediate-small rewards,that both OFC- or HPC-lesionedrats showedanincrease inimpulsivechoice.Interestingly,thesame group[34]recentlyreportedthat,usinganon-spatialversionof thesametask,HPC-lesionedratsexhibitedimpulsivechoicewhilst OFC-lesionedratsdidnot. Thus,therole of OFCinchoice with delayedrewardscouldbemorelimited[61],andthatoftheHPC moreextensive,thanclassicallythought;thisisprobablyrelated toHPCroleintemporalinformationprocessing[34,61].Wemay

concludethatthetransientincreaseinchoiceforadelayedreward, exhibitedbyLenti-SERT,supportsthehypothesisofaninverse rela-tionshipbetween5-HTandimpulsivity[55],beingconsistentwith available data both on5-HT manipulations(including pathway lesions,SSRIadministrationandSERTknockout)andHPClesions. 4.3. Clinicalimplications

Studiesontheassociationbetweenimpulsivityandthe poly-morphicvariantsof5HTTLPRinhumansfoundrathercontroversial results [30]: many studies describe a significant association betweenimpulsivityandthes-alleleof5-HTTLPR[41,51,57]but aboutasmanystudiesdonotfindsucharelationship[44,48,58]. Asa whole, it maybe notsurprising that thes-allele couldbe associatedwithmoreimpulsiveperformanceinclinicalresearch, giventheanatomicalcomplexityoftheserotonergicprojections, thevarietyofreceptorfamiliesandsubtypes,andtheconsequential breadthofitsfunctions.

Moreover,although5-HTTLPRstudiesinhumanlymphoblasts and platelets have shown that thes-variant is associated with reducedSERTexpressionandfunction(e.g.[20,31]),nodefinitive dataexistshowingthatthes/sgenotypeisassociatedinvivowith decreasedSERTexpressioninhumanCNS.Indeed,post-mortem studiesonbrainSERTmRNAlevels(e.g.[32,33])andPETstudieson centralSERTbinding(e.g.[43,56])havenotalwaysconfirmedthat the5-HTTLPRvariantshavedifferenteffectsonSERTtranscription. 5. Conclusions

Insummary,byallowingaselectiveSERTsilencinginrats’ hip-pocampus, thepresent approach will hopefully contribute toa furtherunderstandingof thebiological mechanisms underlying psychiatricdisorders.Thesameapproachisworthtobeexplored for modelling otherpathologies, above allmood disorders (e.g. depression,anxiety),whicharealsorelatedtoanalterationofSERT function. Noteworthy,ratstransfected with aSERT silencerare morerestfulinhome-cageconditionsanddisplaymore“patience” when facinga reward delay. This may possibly disclose novel avenues towards the development of innovative therapeutical approachesforbehaviouralsymptomsrelevanttoADHD.

Acknowledgements

Funding:Under-40“ADHD-sythe”andERA-net“NeuroGenMRI” (toWA);FIRB-RBIN062YH4andPRIN-2009TBCZJB003(toUdP). AppendixA. Supplementarydata

Supplementary data associated with this article can be found,intheonlineversion,

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