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ImpulsivechoicesinmicelackingimprintedNesp55Dent CLa, Humby Tb, Lewis Ka, Plagge Ac, Fischer-Colbrie Rd, Wilkins JFe,
WilkinsonLSa,b&IslesARa*
aBehavioural Genetics Group, MRC Centre for Neuropsychiatric Genetics and
Genomics,NeuroscienceandMentalHealthResearchInstitute,CardiffUniversity,
HadynEllisBuilding,MaindyRoad,Cardiff,UnitedKingdombBehavioural Genetics Group, School of Psychology, Cardiff University, Tower
Building,Cardiff,UnitedKingdomcDepartment of Cellular and Molecular Physiology, Institute of Translational
Medicine,UniversityofLiverpool,Liverpool,UnitedKingdomdDepartmentofPharmacology,InnsbruckMedicalUniversity,Innsbruck,AustriaeRoninInstitute,Montclair,NJ07043,USA
*Authorforcorrespondence:AnthonyR.Isles,[email protected]
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ABSTRACT
Genomic imprinting is theprocesswherebygermlineepigenetic events lead to
parent-of-origin specificmonallelic expression of a number of keymammalian
genes.The imprintedgeneNespisexpressed fromthematernalalleleonlyand
encodes for Nesp55 protein. In the brain Nesp55 is found predominately in
discrete areas of the hypothalamus andmidbrain. Previously, we have shown
thatlossofNesp55givesrisetoalterationsinnovelty-relatedbehavior.Herewe
extendthesefindingsanddemonstrate,usingtheNespm/+mousemodel,thatloss
ofNesp55 leads to impulsive choices asmeasuredby a delayed-reinforcement
task,wherebyNespm/+micewerelesswillingtowaitforadelayed,largerreward,
preferring instead to choosean immediate, smaller reward.Theseeffectswere
highlyspecificasperformanceinanothercomponentofimpulsivebehavior,the
abilitytostoparesponseoncestartedasassayedinthestop-signalreactiontime
task, was equivalent to controls. We also showed changes in the serotonin
system,akeyneurotransmitterpathwaymediatingimpulsivebehavior.First,we
demonstrated that Nesp55 is co-localisedwith serotonin and thenwent on to
showthatinmidbrainregionstherewerereductionsinmRNAexpressionofthe
serotoninspecificgenesTph2andSlc6a4,butnotthedopaminespecificgeneTh
inNespm/+mice;suggestinganalteredserotonergicsystemcouldcontribute, in
part,tothechangesinimpulsivebehavior.Thesedataprovideanovelmodeof
action for genomic imprinting in the brain and may have implications for
pathologicalconditionscharacterizedbymaladaptiveresponsecontrol.
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INTRODUCTION
Genomicimprintingistheprocessbywhichsomegenesaremarkedinaparent
oforiginspecificmannerasaconsequenceofepigeneticeventsthattakeplacein
the mammalian germ line (Ferguson-Smith, 2011). For canonical imprinted
genes, in the somatic cell lineages this epigeneticmarking leads tomonoallelic
expressionfromoneparentalalleleonly.Forsomeimprintedgenes,expression
is solely from thematernal allele, whilst others are solely expressed from the
paternalallele.
There are approximately 150 canonical imprinted genes andnon-codingRNAs
known in the mouse, with similar numbers in humans, although recent next-
generationsequencingexperimentshaveexpanded thisnumber (Wilkinsetal.,
2016). Though relatively small in number, imprinted genes are critical for
normaldevelopmenttotakeplace(Mcgrath&Solter,1984,Suranietal.,1984).
Furthermore, functionally, imprinted genes converge on specific biological
processesthathaveprominentimportanceinmammals,suchasinuterogrowth,
metabolismandbehaviour(Peters,2014,Wilkinsetal.,2016).
The imprinted gene Nesp, encoding Nesp55, is part of the complex GNAS
imprintingclusterandisexpressedexclusivelyfromthematernalallele(Peters
etal., 1999).Nesp is expressed indiscrete areasof themidbrain, including the
Edinger-Westphalnucleus,dorsalRaphénucleus(DRN),andthelocuscoeruleus
(LC), and also in the hypothalamus (Plagge et al., 2005). Our previous work
demonstrated thatmice null formaternally expressedNesp55 show increased
activitywhenplacedinnovelenvironment,butareducedpropensitytoexplorea
novelenvironmentwhengiven thechoice (Plaggeetal.,2005).Alterednovelty
exploration may involve changes in the balance between self-control and
impulsive responding (Flagel et al., 2010, Stoffel & Cunningham, 2008).
Furthermore,Nespisexpressedinkeybrainstructures,namelytheDRNandLC,
thatareimplicatedinmediatingresponsecontrol(Bari&Robbins,2013)andthe
ability towait for a reward (Fonseca etal., 2015), respectively.Apriori, these
datasuggestpossiblelinksbetweenNesp55andimpulsivebehavior.
The term ‘impulsivity’, defined as actionwith limited forethought, is a natural
part of behavior. However, in certain individuals, impulsive behavior becomes
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pathological and maladaptive and can manifest in psychiatric disease such as
schizophrenia and attention deficit/hyperactivity disorder (ADHD), as well as
addictivedisorderssuchaspathologicalgamblinganddrugaddiction.Agrowing
bodyofevidencesuggeststhatimpulsivityisnotunitary,andinsteadisamulti-
facetedconstructdissociableintermsofbehaviorandunderlyingneurobiology
(Bari & Robbins, 2013). There have been attempts to segregate impulsive
behaviorintotwoseparatecategories,impulsivechoiceandimpulsiveaction
(Broos et al., 2012, Dent & Isles, 2014b). Impulsive choice often manifests as
impulsive decisions resulting from a distorted evaluation of the delayed
consequences of behavior and an increased preference for smaller immediate
rewards over larger delayed rewards; on the other hand, impulsive action
reflectsthemotoricaspectofimpulsivity,andtypicallymanifestsinpoorability
to override a pre-potent response (Humby & Wilkinson, 2011). A number of
behavioral tasks exist to tease apart these discrete aspects of impulsivity
(Humby&Wilkinson,2011),manyofwhicharereadilyavailableinmice(Dent&
Isles,2014b).
Hereweusedadelayed-reinforcementtask(Islesetal.,2003)andastop-signal
reaction time (SSRT) task (Humby etal., 2013), to assay impulsive choice and
impulsive action, respectively, in mice null for maternally expressed Nesp55
(Nespm/+mice).WefoundthatNespm/+miceshowedamarkedincreaseinchoice
impulsivity,manifestindecisionstochooseanimmediate,smallerrewardovera
largerbutdelayedreward,butexhibitednosignificantdifferencesfromcontrol
mice (Nesp+/p) in impulsive action,where subjectshad to suppress anongoing
pre-potentmotorresponse.WealsodemonstratedthatNesp55co-localizeswith
5HT inmidbrain regions, and furtherdemonstrated that thebehavioral effects
observed in the Nespm/+ mice were accompanied by selective effects on the
expression of key serotonergic genes. The data are consistent with a role for
Nesp55 in mediating specific aspects of impulsive behavior, possibly via
interactionswithmidbrainserotonergicpathways.
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MATERIALSANDMETHODS
Animals
AllprocedureswereconductedinaccordancewiththerequirementsoftheUK
Animals(ScientificProcedures)Act1986,undertheremitofHomeOfficelicence
number30/2673withadditionalethicalapprovalatCardiffUniversity.
As described previously (Plagge et al., 2005), expression of Nesp55 was
eliminatedbyadeletionof26bpatthestartcodonoftheORF(bp2362to2387
in AJ251761). Mice carrying a maternally derived null allele (Nespm/+) lacked
Nesp55 expression, whereas those carrying a paternally derived null allele
Nesp+/p have the same level of expression as wild-type mice (Figure S1). As
before (Plagge et al., 2005), in the present study Nespm/+ were compared to
Nesp+/p mice as themost appropriate control (see Supporting Information for
moredetails)andthesearereferredtoascontrolmicethroughout.
TheNesp55nulllinewasmaintainedonaC57Bl/6J(CharlesRiverLaboratories,
U.K.) background with separate cohorts of Nesp55 and control mice being
utilizedinthetwobehavioraltasks.Subjectsweremalemiceagedbetween4-10
monthsduringtesting;thelargeagerangebeingduetothetimetakentolearn
and perform the operant tasks. Standard laboratory chow was available ad
libitum,but justpriortoandduringtheexperiment,waterwasrestrictedto2h
access per day. This regime maintained the subjects at ≈90% of free-feeding
bodyweight.Allbehaviouraltestingwasperformedinthelightphase(lightson
07:00,for12-hours).
Behavioraltasks
Apparatus
Thedelayed-reinforcementtaskandstop-signalreactiontime(SSRT)taskwere
performedin9-holeoperantchambers(CambridgeCognitionLtd,UK)modified
foruse inmice,aspreviouslydescribed(Humbyetal.,2013, Islesetal.,2003).
For the delayed-reinforcement task holes 3, 5 and 7were open,whereas only
holes4and6wereopenfortheSSRTtask.Themicewerepresentedwithvisual
stimuli (lights) recessed into the holes and were trained to respond to this
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stimulus with a nose-poke recorded by infra-red beams spanning the hole.
Rewardwaspresentedinarecessedcompartmentconcealedbyapanel,onthe
wall opposite to thenose-poke/stimulus array; animalswere required topush
the panel open in order to consume the reward. The control of stimuli and
recording of the responses weremanaged by an Acorn Archimedes computer
withadditionalinterfacingbyARACHNID(CambridgeCognitionLtd,UK).
Delayedreinforcementtask
Detailsoftheshapingproceduresandbasicaspectsofthedelayed-reinforcement
taskitselfcanbefoundelsewhere(Islesetal.,2003).Briefly,thetaskcomprised
ofthreesequentialblocksof12trials,witheachtrialconsistingofaninitialnose-
poketothecentrallylocatedstimulus,followedbyasecondnose-poketoeither
theleftorrightapertures.Trials1-4inanyblockwere‘forced’informationtrials,
wheretheinitialnose-pokeresultedinpresentationofonlyoneofthetwochoice
options.Thismeasurewasdesignedtoprovidethesubjectswithpriornoticeof
the extent of any delay associated with choosing the large reward. In the
remaining 8 trials of each block, designated as ‘choice’ trials, the initial centre
nose-pokeledtotheoptionofasecondnose-pokeresponsetoeithertheleftor
rightapertures. Oneresponseresulted inthedeliveryofa largereward(50μL
10%solutionofcondensedmilk;NestleLtd,UK),andtheotherinthedeliveryof
small reward (25μL 10% solution of condensed milk). The response
contingencies were kept constant for each mouse, but were counterbalanced
betweensubjects.Inblock1bothresponsesledtothedeliveryofrewardaftera
1s delay. In blocks 2 and 3 increasing delays were introduced between the
responseandthedeliveryofthelargereward(8s,16s,respectively)whereasthe
delaybetweenresponseanddeliveryofthesmallrewardwasfixedat1s. Asa
probe to test the effect of the delays on behaviour, sessions were conducted
wherethedelayassociatedwiththelargerewardwasfixedat1s,equivalentto
thatassociatedwiththesmallreward,throughoutallthreeblocksofthesession.
Thebiasinchoiceofthelargerrewardateachblock(wherebyalwayschoosing
thelargereward=1;neverchoosingthelargereward=0)wasthemainmeasure
usedtodetermineimpulsiveresponding.Additionalmeasurementsthatrelated
to general motoric competence and motivation within the task were also
7
monitored,includingthe‘Start’and‘Choice’latencies,thetimetakentoinitiatea
trialandthetimetakentomakeachoiceonceatrialwasinitiated,respectively.
Alsomeasuredwerethenumberof ‘Non-started’(no initial,centralnose-poke)
and ‘Omitted’ (no secondary, choice nose-poke, following central nose-poke
initiatingtrial)trials.
For the delayed reinforcement task experimental groups were Nespm/+=11,
controls=7.Animalsweretested6-daysperweek.
Stop-signalreactiontime(SSRT)task
Detailsof the shapingproceduresandbasic aspectsof themainSSRT task can
alsobefoundelsewhere(Humbyetal.,2013).TheSSRTtask itselfconsistedof
sessions of 100 trials, which involved both ‘go’ and ‘stop’ trials. Go trials
consisted of rapid double nose-pokes (a ‘go’ response) between two separate
stimulilocations,whichwererewardedwithreinforcement(22ul,10%solution
of condensed milk, Nestle Ltd, UK). 20% of trials were stop trials, pseudo-
randomlydistributedthroughouteachsession,whereastop-signal(65dbwhite
noisefor0.3s)waspresentedbetweenthefirstandsecondnose-pokeresponses.
The aim of the stop-signal was to inhibit (‘Stop’) themouse frommaking the
second (‘Go’) nose-poke, and thenwait for the reward. Failure to refrain from
makingthispre-potentresponsewaspunishedbytheabsenceofrewardand5
secondtimeout(chamberlighton).Atbaseline,thestop-signalswerepresented
concurrently with the initial nose-poke response. To maintain high levels of
performanceofbothgoandstopresponding,thegostimulusdurationandwait
periodtorewarddeliveryinastop-signaltrialweredeterminedindividuallyfor
each subject. To assess the ability to stop once an action had been initiated,
sessionswereimplementedinwhichtheonsetofthestop-signalwaspresented
atdifferentpositionswithintheindividualisedgoresponseofeachmouse.Thus,
thestop-signalwaspseudo-randomlypresented10,40,50,60and90%fromthe
onset of the go response of each subject, with the assumption that stopping
would be more difficult the closer the stop-signal presentation was to the
terminationofthegoresponse.
Theamountofcorrectstoppinginstop-signaltrialsandtheSSRTwerethemain
measures of impulsive responding in this task. The SSRT was calculated by
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determiningthe50%stoppingabilityforeachsubjectfromtherangeofsessions
in which the stop-signal onset was varied from baseline (full details of this
calculationcanbefoundintheSupportingInformationand(Humbyetal.,2013).
The proportion of correct go-responses, and latency to respond, were also
assessed.Additionalmeasurementsthatrelatedtogeneralmotoriccompetence
andmotivationwithin the taskwerealsomonitored, including the “intitiation”
and“magazine”latencies,thetimetakentoinitiateatrialandthetimetakento
collect the reward.Alsomeasuredwas thenumberof trials completed for any
givensession.
For theSSRTtaskexperimentalgroupswereNespm/+=17,controls=12.Animals
wererun6-daysperweek.
Immunohistochemistry
Mice were terminally anaesthetised using an intraperitoneal injection of
pentobarbitone and then trans-cardially perfused using 4%PFA in PBS. Brains
weredissectedwholeandplacedin4%PFAovernight,thentransferredtoa30%
sucrose solution (in PBS) at 4˚c for 24 hours in order to dehydrate and
cryoprotectthem.Brainswerethenmountedontoamicrotomeplatformusing
anembeddingmatrixandallowedto freeze fully.Serial sectionsof40µmwere
slicedandputinto25-wellcontainerscontainingcryoprotectant(6sectionsper
well). The free-floating sections in cryoprotectant were stored at -20°C until
required.
Dual-labellingimmunofluorescenceofNespwith5HTwascarriedoutinNesp+/+
(i.e.wild-type) free-floating brain sections in order to localize the endogenous
protein. The Nesp55 primary antibody is a well-characterised anti-body
(generatedandobtainedfromtheReinerFischer-ColbrieLab).Specifically,itisa
rabbit anti-NESP55 polycolonal anti-body, recognizing the free terminal end
(GAIPIRRH) ofNESP55 (Ischia etal., 1997). Sectionswerewashed three times
for10mineachin0.1%PBSbeforebeingincubatedfor15minin0.3Mglycine
in 0.1% PBS at room temperature, to neutralise endogenous aldehyde groups.
Sectionswerewashedin0.1%PBSandthenincubatedatroomtemperaturefor
1hourin10%blockingsolution;0.5%BSA(BBInternational,Cardiff,UK),0.5%
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TritonX-100(v/v,SigmaAldrich)in0.1%PBS.Sectionswerethentransferredto
a solution containing 1:1000 anti-NESP55 and 1:500 anti-serotonin (Abcam)
dilutedina1%blockingsolution;thiswasallowedtoincubatedovernightat4°C
whilst gently shaking. The next day sections were washed three times for 10
minutes in 0.1% PBS. The relevant fluorescent secondary antibodies (Alexa
Fluor;Lifetechnologies)werediluted1/1000in1%blockingsolution,sections
were incubated in this solution in the dark at room temperature for 2 hours,
whilstgentlyshaking.Sectionswerethenwashedin0.1%PBSasbefore(inthe
dark)andtransferredtopolysinecoatedslidesandallowedtodryover-nightin
a dark dust-free environment. The mounted slides were then dehydrated
throughaprocessofincubationinarisingconcentrationofalcohol,followedby
xylene, then cover-slipped and sealed using DPX (Raymond Lamb DPX), and
allowed todryover-night.Tocontrol fornon-specificbindingof thesecondary
antibodies, secondary-only negative controls were carried out alongside all
experiments.
Immunofluorescenceslideswereviewedand imagescapturedusinganupright
fluorescencemicroscope (LeicaDM5000B).Dual-labelled immunofluorescence
images were acquired through separate channels then subsequently merged
usingImageJ(Image>colour>mergechannels).
QuantitativePCR
RNA from macro-dissected brain regions was isolated using standard Trizol
methods. Equal amounts ofRNAwere reverse transcribedusingRNA to cDNA
EcoDry (double primed) premix strips (Takara Bio Europe, France). Gene
expressionwasassessedusingaRotorgene6000withaCAS1200automatedset
up (Corbett Research, U.K.). PCR reactions were carried out using custom
designed primers and Quantace SensiMix NoRef (Bioline). The genes assayed
were Th (For 5ʹ-AGGAGAGGGATGGAAATGCT-3ʹ; Rev
5ʹ-GCGCACAAAGTACTCCAGGT-3ʹ), Tph2 (For 5ʹ-CTGCTGTGCCAGAAGATCATCA-
3ʹ; Rev 5ʹ-TGCTGCTCTCTGTGGTGTCG-3ʹ) and Slc6a4 (For 5ʹ-
TTGTGCTCATCGTGGTCATC-3ʹ; Rev 5ʹ-GTGGCGTACTCCTCCAGCAG-3ʹ).
Additionally, three housekeeping genes were assessed in order to provide a
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robust measurement of general gene activity. These Gapdh (For 5ʹ-
GAACATCATCCCTGCATCCA-3ʹ; Rev 5ʹ-CCAGTGAGCTTCCCGTTCA-3ʹ), Hprt (For
5ʹ-TTGCTCGAGATGTCATGAAGGA-3ʹ; Rev 5ʹ-AATGTAATCCAGCAGGTCAGCAA-3ʹ)
and β-actin (For 5ʹ-TCTGTGTGGATTGGTGGCTCTA-3ʹ; Rev
5ʹ-CTGCTTGCTGATCCACATCTG-3ʹ).ΔCtvaluesweregeneratedbynormalisingto
thegeometricmeanofthesethreehousekeepinggenes.Allindividualreactions
werecarriedoutintriplicate.RealtimeqPCRdatawasvisualisedusingtheΔΔCt
method(Livak&Schmittgen,2001).
Dataanalysisandstatistics
AllbehavioraldatawereanalysedusingSPSS20(SPSS,USA).Datawereassessed
fornormalityandthenanalysedbyStudent’st-testormixedANOVA,with
between-subjectsfactorsofGENOTYPE(Nespm/+vs.Control),andwithin-subject
factorsDELAY(1s,8s,16s,or1s,1s,1s),CHOICE(choiceoflargeorsmallreward
duringforcedtrialsofthedelayedreinforcementtask),andSTOP-SIGNAL
POSITION(positionofstop-signalrelativetoindividualizedGo-response).For
repeated-measuresanalyses,Mauchly’stestofsphericityofthecovariance
matrixwasapplied;significantviolationsfromtheassumptionofsphericitywere
subjecttotheHuynh–Feldtcorrectiontoallowmoreconservativecomparisons
throughadjustdegreesoffreedom.Duetothenon-parametricnatureofthe
qPCRdata,ΔCtvalueswereanalysedbyMann-WhitneyUtest.Allsignificance
testswereperformedatalphalevelof0.05.
RESULTS
Nespm/+micearemoreimpulsiveinthedelayedreinforcementtask
In thedelayed-reinforcement task, subjectsmadea choicebetweenreceivinga
smallfoodrewardafterashort(1s)delay,oralargerewardafteralongerdelay
(1,8or16s). Inthisway,theextenttowhichsubjectsmadeimpulsivechoices,
indexedbythepreference forchoosingthe immediatebutsmallrewardovera
delayedlargereward,wasdetermined.Increasingthedelaytothelargereward
within a session decreased the likelihood of choosing that reward for both
Nespm/+andcontrolmice(Figure1a,maineffectofDELAY,F2,32=3.84,P=0.032).
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However,mutantandcontrolmiceshowedsignificantdifferences in theextent
to which they switched their preference to the small, less delayed reward in
responsetoincreaseddelaytothelargereward.Specifically,Nespm/+micechose
the small reward more readily than control mice (Figure 1a, main effect of
GENOTYPE, F1,16=6.74,P=0.019), indicative of increased impulsive responding.
To confirm the specificity of the contingency between reward value and the
delay in reward delivery, performance was also assessed where the cost
associatedwith the large rewardwas removed, thus thedelay associatedwith
the largeand small rewardswasequal (1s) for all trials. In these sessions, all
subjectsdemonstratedanequalhighpreferenceforthelargerewardthroughout
thesession(Figure1b,nomaineffectofDELAY,F2,32=0.478,P=0.624)andthere
were no differences between Nespm/+ and control mice (no main effect of
GENOTYPE,F1,16=0.010,P=0.927).
WealsoconfirmedthatthecontrastingpatternofchoicebehaviorintheNespm/+
mice were not due to any differences between the groups in terms of basic
learning andmotivation to carry out the task, asNespm/+animals acquired the
taskatthesamerateascontrolmice(numberofsessionstolastdayofbaseline,
grandmean=40.3±1.3SEM,t16=1.00,P=0.32).Additionally,variabilitybetween
Nespm/+ and control mice was not related to differences in experiencing the
information trial contingencies. Thus, in the ‘forced’ trials (inwhich no choice
wasavailable)bothNespm/+andcontrolmicemadeequalresponsestothelarge
andsmallreward-relatedstimuliatalldelays(interactionbetweenGENOTYPEx
DELAY x CHOICE, F2,32=1.85, P=0.43; data not shown). Finally, there was no
difference between Nespm/+ and control mice on general measures of task
performance (see Figure S2). As expected, Nespm/+ and control mice
demonstratedageneralmaineffectofDELAYonmanyofthesemeasures(Figure
S2a-d)as follows: increasedstart (F1.7,27.5=22.06,P
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Nespm/+mice show no differences in impulsive responding in the stop-
signalreactiontimetask
TheSSRTtaskmeasurestheabilitytostopanactiononceinitiatedbypresenting
a stop-signal (in this case an auditory tone)during a rapid transitionbetween
two stimuli locations (the ‘go’ response). Correctly inhibiting the go response
earnedrewardina‘stop’trial,andthedifficultyofstoppingwasmanipulatedby
presenting the stop-signal at different times within the go response. Thus
stoppingwas easiestwhen the stop-signal onsetwas near the start andmore
difficultwhenat theendof thego response.Throughout the training stagesof
the SSRT task, all subjects showed equivalent behavior in learning the task.
Nespm/+ mice acquired the task at the same rate as control animals, as
demonstrated by the similar number of sessions taken to complete the task
(Nespm/+:44.3±6.6,control:46.6±6.8).Theeffectsofalteringthepositionofthe
auditory stop-signal during stop trials on stopping efficiency are illustrated in
Figure2a.InlinewithpreviousSSRTtaskexperimentsinhumansandavariety
of other mammalian species, including mice (Bari et al., 2009, Humby et al.,
2013),stoppingbecameincreasinglydifficultasthestop-signalpresentationwas
movedprogressivelyclosertotheexecutionoftheresponse(10,40,50,60and
90%intotheindividualisedgoresponse)leadingtosystematicreductionsinthe
abilitytostopforbothNespm/+andcontrolmice(Figure2a,maineffectofSTOP-
SIGNALPOSITION,F4,108=43.28,P
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stop-signal position was moved from baseline (main effect of STOP-SIGNAL
POSITION,F4,108=0.49,P=0.74andF4,108=1.67,P=0.16,fortheamountandspeed
ofcorrectrespondingingotrials,respectively).
Again, Nespm/+ animals acquired the task at the same rate as control mice
(numberofsessions,grandmean=45.3±1.2SEM,t27=0.90,P=0.38).Additionally,
therewasnodifferencebetweenNespm/+andcontrolmiceongeneralmeasures
of task performance during individualized SSRT session). Thus, the number of
trialscompleted(t27=0.10,P=0.92), latencyto initiatea trial(t27=-0.36,P=0.73)
and magazine latency (t27=-1.12, P=0.27) measures were not significantly
different between Nespm/+ and control mice, but indicated a high degree of
stimuluscontrolforbothgroupsinthetask(seeFigureS3).
Nesp55co-localiseswith5-HT;keyserotoningenesshowreducedexpression
inmid-brainofNespm/+mice
Previouswork(Baueretal.,1999,Plaggeetal.,2005),which isreplicatedhere
(Figure S1 d-f), shows thatNesp55 is strongly expressed in the hypothalamus
andareasofthemidbrain,includingtheEdinger-Westphalnucleus,DRN,andthe
LC. Using immunohistochemistry, we examined whether Nesp55 co-localises
with 5-hydroxytryptamine (5HT) in these regions. Qualitative observation
suggeststhatNesp55isindeedco-localisedwith5HTinthemidbrain(Figure3
a-c).Thereappearstobeverylittle,ornoco-localisationofNesp55and5HTin
the hypothalamus (Figure 3 d-f). Additionally, it is clear that Nesp55 is also
expressednon-5HTexpressingcell-types.
To investigatetherelationshipbetweenNesp55and5HT, theexpression levels
ofsome5HT-relatedgeneswereassessedbyqPCRinsamplesofthemidbrainof
Nespm/+andcontrolmice(Figure4).Thisbrainregionwaschosenasitwasthe
areashowingthegreatestco-localisationbetweenNesp55and5HT.Therewere
10-foldreductionsintheexpressionofgenesencodingtryptophanhydroxylase,
Tph2(U=0.01,P=0.021)andtheserotonintransporter,Slc6a4(U=0.01,P=0.021)
inNespm/+micerelativetocontrolsubjects,butnodifferenceintheexpressionof
Th(U=7.0,P=0.886),thegeneencodingtyrosinehydroxylase.
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DISCUSSION
The imprinted gene Nesp, expressed from the maternal allele only, encodes
Nesp55,andislocatedindiscreteareasofthemidbrainandhypothalamus.Here
wedemonstratethatlossofNesp55expressionfromthematernalallele,butnot
paternalallele, leadstohigher levelsof impulsivechoicebehaviorasmeasured
byadelayedreinforcementtask.TheNespm/+micewerelesswillingtowaitfora
larger but delayed reward, choosing instead the smaller but immediately
availablereward.TheseeffectswerehighlyspecificinthatbehaviorintheSSRT
task, assaying a different form of behavioral control, the ability to stop the
execution of a pre-potent action, was equivalent betweenNespm/+ and control
mice. Inaddition,weshowthatNesp55isco-localisedwith5HTandthatthere
are concomitant reductions in midbrain Tph2 and Slc6a4, but not ThmRNA
expression in Nespm/+ mice, suggesting an altered serotonergic system could
contribute,inpart,tothechangesinimpulsivechoicebehavior.
We have previously shown that Nespm/+ mice have an increased reactivity to
novelty,butareducedpropensitytoexploreanovelenvironmentwhengivena
choice(Plaggeetal.,2005).Alterednoveltyexplorationmayinvolvechangesin
thebalancebetween self-control and impulsive responding (Flageletal., 2010,
Stoffel&Cunningham,2008).Wedecidedtoexploretheimpulsivityphenotype
ofNespm/+mice further by using operant tasks to examine discrete aspects of
impulse control, namely choice impulsivity, as measured on a delayed
reinforcementtask,andimpulsiveaction,asmeasuredontheSSRTtask(Dent&
Isles, 2014b). In the delayed reinforcement task,Nespm/+ mice showed higher
levels of impulsivity, switching their choice of the larger, but increasingly
delayed, reward, more quickly than control mice. Critically though, when the
delayassociatedwith the largerrewardwasequivalent to thatassociatedwith
the smaller reward throughout the session, there was no difference in
performance between Nespm/+ and control mice. Taken together with other
control measures within the delayed-reinforcement task, which indicated that
therewerenodifferencesbetweenNespm/+ and controlmice in their ability to
learnthetaskortheirgeneralmotivationtoperformthetask,thismanipulation
demonstrates that the prime factor controlling the difference in behavior
15
betweenNespm/+ and controlmice on the delayed-reinforcement task,was the
contingency between delay and reward. Conversely, behavior of Nespm/+ and
controlmice on the SSRT task, was equivalent. All mice showed the expected
reductionstoppingasthestop-signalpresentationmovedprogressivelycloserto
theexecutionofthegoresponse,buttherewerenogenotype-relateddifferences
acrossthetaskorinthederivedSSRTmeasure.
Overall, thesedatasuggestthat lossofmaternalNesp55expressionproducesa
specific increase in impulsive choicebehavior,whilst impulsiveaction remains
unaffected.Asaconcept,thedissociationofchoiceandactionimpulsivityisnow
wellestablished.Forinstance,inbredstrainsofmicehavebeenshowntoexhibit
dissociations in terms of impulsive behaviors in a delayed reinforcement task
(Isles et al., 2004), and a measure of action impulsivity in the 5-choice serial
reaction time task (Patel et al., 2006). Furthermore, a cross-species study
examiningbehavior inanumberofdifferent tasks found that, inboth ratsand
humans, measures of impulsive choice and impulsive action did not correlate
(Broos etal., 2012). Our data add to the growing body of evidence suggesting
thatimpulsivityisnotunitary,andinsteadisamulti-facetedconstruct.
Impulsivechoiceandimpulsiveactionarenotonlydissociablebehaviorally,but
alsointermsoftheunderlyingneurobiology(Bari&Robbins,2013).Oneneural
pathwaythathas importantdissociableeffectsondiscreteaspectsof impulsive
behavioristheserotoninsystem(Barietal.,2009,Humbyetal.,2013,Talposet
al., 2006, Winstanley et al., 2004). Although excluded from the key areas of
cognitive control important for mediating impulsive behavior, such as the
frontostriatalcircuitry,Nesp55isstronglyexpressedinanumberofserotonergic
areasofthemidbrain(Baueretal.,1999,Plaggeetal.,2004),andhereweshow
that in some cells Nesp55 and 5HT are co-localised. This is particularly
interesting given the recently established role of serotonergic dorsal raphé
neurons in mediating waiting for delayed reward (Miyazaki et al., 2012a,
Miyazakietal.,2012b), independentfromanyeffectsonreward(Fonsecaetal.,
2015). One mechanism underlying the specificity of impulsive behavior
phenotypeseeninNespm/+micecouldbeviaanimbalanceinserotoninsignaling
intheDRN.
16
QuantitativePCRanalysisrevealedthatinmicelackingNesp55therewasa10-
fold reduction in midbrain expression of Tph2, which encodes tryptophan
hydroxylasetheratelimitingenzymeforbrain5HTsynthesis(Fitzpatrick,1999),
and the serotonin transporter gene, Slc6a4. Expression of Th (tyrosine
hydroxylase) was unaltered, consistent with a specific effect on the serotonin
system. Nesp55 is associated with fast anterograde axonal transport in the
peripheral nervous system and is considered a marker for the constitutive
secretory pathway (Fischer-Colbrie et al., 2002, Li et al., 2002). It is unknown
whetherNesp55influencesthetransportorreleaseofneurotransmittervesicles.
We have previously found no alteration in monoamine levels in brain tissue
taken from the prefrontal cortex and midbrain ofNespm/+ mice (Plagge et al.,
2005). However, thesewhole tissue analysesmaymask the impact of Nesp55
deficiencyonneurobiological indicesmoreclosely related to synaptic function,
suchasextracellularlevelsoftransmittersandchangesinpre-andpostsynaptic
receptor moieties, an idea maintained by the correlative changes in Slc6a4
expression. Moreover, given the known interplay between the serotonin and
dopaminesystemsininfluencingimpulsivebehaviour(Dalley&Roiser,2012),it
is not possible to rule out the action of othermonoamines inNespm/+mice as
beingcausalof thedelayed-reinforcementphenotype.Therefore,atpresentthe
exact neural mechanism(s) by which Nesp55 influences behavior remains
unclear.
Our data provide a novelmode of action for genomic imprinting in the brain,
addingtothelistofbehaviorssensitivetoimprintedgenefunction(Daviesetal.,
2015). Imprinted genes are thought to have evolved as a consequence of
conflictingphenotypic “interests”betweenmaternal andpaternal genes,which
causesanescalatingarmsraceinrelationtoallelicexpression,eventuallyleading
tosilencingofoneorotherparentalallele(Moore&Haig,1991,Wilkins&Haig,
2003). Inadditiontotheallelic level,parentalconflictmayalsobemanifestat
thegeneandtissuelevels(Daviesetal.,2005,Wilkinsetal.,2016).Evidenceof
thisparentalconflictisseenintheoppositeactionofimprintedgenesduringin
utero growth and early post-natal life (Haig, 2004) but as yet there are no
behaviouralexamples.Ourfindingsmayaddsomesupporttothetheorythat,as
aconsequenceofintra-genomicconflict,imprintedgenesexpressedintheadult
17
brain could lead to a “parliamentof themind”with regard todecision-making
(Haig, 1997) in that there are opposing parental interests pulling impulsive
choice in different directions. However, whilst here we show that loss of a
maternallyexpressedgene,Nesp,causesanincreaseinimpulsivechoices,there
is, currently at least, no paternally expressed imprinted gene that has been
shown to influence impulsive responding. A potential candidate here isGrb10,
which shows overlapping expression with Nesp (Dent & Isles, 2014a), and
influencessocialdominance(Garfieldetal.,2011),abehavioraldomainthathas
beenlinkedtoimpulsivity(Davisetal.,2009).Finally,anextensionofthisidea
andourexperimental findings is that, an imbalance inexpressionof imprinted
genes,suchasNesp,maynotonlycontributetofacetsof impulsivitywithinthe
normal range, but also to pathological conditions, such as gambling and drug
addiction, where response control becomes maladaptive. It is therefore, of
particularinterestthataNESPdeletionpolymorphismhasbeenassociatedwith
theoccurrenceofneuropsychiatricillness,includingaddictivebehaviour(Kimet
al.,2000).
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ACKNOWLEDGEMENTS
CLD,KLandARIperformedtheexperiments;TH,JFW,LSWandARIdesignedthe
study;APandRF-Csuppliedreagents;CLD,TH,LSWandARIwrote thepaper.
Thisworkwas funded by a Leverhulme Trust project grant (F/00 407/BF) to
ARI and JFW, which supported CLD. The work was also funded by the MRC
Centre forNeuropsychiatricGenetics andGenomics (G0801418),ofwhichARI,
THandLSWaremembers,andwhichalsosupportedKLviaaPhDstudentship.
Theauthorshavenoconflictsofinteresttodeclare.
21
Figure1.Nespm-/p+miceshowincreasedimpulsivechoicebehaviorinthe
delayed-reinforcementtask.BehaviorofbothNespm/+andcontrolmice
changedacrosssessionblocks(blk)withincreasingdelay,suchthatchoicebias
movedawayfromtheresponseleadingtothelargerewardtowardsthesmall
rewardwithincreasingdelay(a).However,thereweresystematicdifferences
betweenthegroupsintheirbehavior,suchthatNespm/+animals(N=11)
switchedtheirchoicetothesmall,lessdelayedrewardmorequicklythancontrol
mice(N=7).Whenthedelayassociatedwiththelargeandsmallrewardswas
equal(1s)throughoutthesession(b),choicebiaswasconsistentlyhigh(large
rewardchosenapproximately90%ofthetime).Moreover,underthese
conditionstherewerenodifferencesinchoicebiasbetweenNespm/+andcontrol
mice.Datashowsmean±SEMofthreeconsecutivestablesessions;*represents
P
22
Figure2.NodifferencebetweenNespm/+andcontrolmiceperformancein
thestop-signal(SSRT)task.BothcontrolandNespm/+miceshowedan
equivalentabilitytoperformtheSSRTtaskshowingtheexpectedchangein
percentagecorrectrespondingduringa‘stop’trial(a)asthepositionofthestop-
signalwasaltered,buttherewerenodifferencesbetweenNespm/+(N=17)and
controlmice(N=12).Nespm/+andcontrolmicealsoshowedequivalentSSRTsat
50%correctstopping(b).Therewerenogenotypedifferencesforthe‘Go’
responseforbothcohortsofmice,intermsofpercentagecorrectresponding(c)
orresponsespeed(d).Datashowsmean±SEM;**representsP
23
Figure3.Co-localisationofNesp55with5HTinthebrain.Sectionswere
dual-labelledwithanti-bodiesagainstNesp55and5HT,imageswerethen
mergedtogaugecellularco-localisation.Whitearrowsdepictareasofco-
localisation.Therewasevidenceofco-localisationintheregionsofthemidbrain
(a-c),forexamplethelocuscoeruleus.However,therewasverylittleco-
localisationobservedinthehypothalamus(d-e).Allimagesareatx40
magnification
24
Figure4.Altered5HT-relatedgeneexpressioninNespm/+mice.Gene
expressionanalysisofmidbrainmacro-dissectionsfromNespm/+andcontrol
mice(N=4forbothgroups)showedsignificant10-foldreductionsinTph2and
Slc6a4expression,butnodifferenceinThexpression.Datashowsmean±SEM;*
representsP
25
ImpulsivechoicesinmicelackingimprintedNesp55-
supplementarymaterial
Dent CLa, Humby Tb, Lewis Ka, Plagge Ac, Fischer-Colbrie Rd, Wilkinson LSa,b,
WilkinsJFf&IslesARa
aBehavioural Genetics Group, MRC Centre for Neuropsychiatric Genetics and
Genomics,NeuroscienceandMentalHealthResearchInstitute,CardiffUniversity,
HadynEllisBuilding,MaindyRoad,Cardiff,UnitedKingdom
bBehavioural Genetics Group, School of Psychology, Cardiff University, Tower
Building,Cardiff,UnitedKingdom
cDepartment of Cellular and Molecular Physiology, Institute of Translational
Medicine,UniversityofLiverpool,Liverpool,UnitedKingdom
dDepartmentofPharmacology,InnsbruckMedicalUniversity,Innsbruck,Austria
eRoninInstitute,Montclair,NJ07043,USA
Authorforcorrespondence:AnthonyR.Isles,[email protected]
SUPPLEMENTARYMATERIALSANDMETHODSAnimals
Loci closely linked to a targeted mutation may cause concern with regard to
phenotypicdifferencesbetweenknock-outsandcontrols,sincetheymostlikely
retaintheoriginal129inbredstrainEScellalleles,evenafterseveralgenerations
ofbackcrossingtoadifferentstrain.Foranalysisofmicecarryingahomozygous
mutation of a biallelically expressed gene, this may imply comparing an
associated homozygous 129/Sv genetic background with wild-type C57BL/6J
counterparts.However,theknockoutanalysisofmonoallelicallyexpressedgenes
providestheopportunityforamoredirectcomparisonofheterozygousgroups.
In the case of maternally expressed Nesp, heterozygous deficient mice
(Nespm/+)can be compared with heterozygous Nesp-expressing mice (Nesp+/p)
obtained fromreciprocalcrosses toC57BL/6J. Inbothof thesegroups, the loci
26
surrounding the targeted mutation will comprise compound C57BL/6J and
129/Svalleles,andthus,anypotentialstrainvariationsshouldmanifestequally
in both groups. In contrast, thewild-type littermates of the two heterozygous
groupsarehomozygousC57BL/6Jforthesegenomicregions,couldconfoundthe
interpretation of behavioral assays (Gerlai, 1996), particularly given known
differences in impulsive behavior between inbred strains (Isles et al., 2004).
Thus,weconsidered itmostappropriate tocompareNespm/+withNesp+/pmice
ascontrols.
Immunohistochemistry
For the ImmunohistochemistryanalysisaVectastain®EliteABCKit (PK-6100)
was used on brain sections fromNespm/+ (lacking maternal Nesp55), Nesp+/P
(control, carrying paternal knockout), and wild-type (Nesp+/+) mice. Sections
werefirstwashedinTBS(4x10mins).Theywerethenincubatedinaperoxidase
block on a stirrer for 30minutes (0.6%hydrogenPeroxidase inTBS) to block
endogenousperoxidase.SectionswerewashedinTBSagain(3x10mininTBS),
andthenincubatedinTBSTwith3%normalgoatserum(NGS)(S-1000,vector
labs)onastirrerfor30minutesatroomtemperature.FollowingNGSblocking,
sectionswereincubatedinprimaryantibody(anti-Nesp55)dilutedat1:1000in
TBSTwith3%NGS.Thiswasstirredfor10minutesandthencoveredandstored
overnightat4˚c.The followingdaythesectionswerewashed3x10minutes in
TBSTwith3%NGS. Sectionswere then incubated for1hour in the secondary
antibody diluted 1:200 in TBSTwith 3%NGS at room temperature. Following
thesecondaryantibodyincubationsectionswerewashed3x10minutesinTBST,
andthenallowedtoincubateintheABCcomplex(madeasperkitspecifications)
at room temperature, on a stirrer for 1 hour. Sections were then washed as
before(3x10minutesinTBST),thenwashed2x10minutesin0.05MTrisbuffer.
Sections were then incubated in DAB solution for 35 seconds at room
temperature; theywere then immediatelywashed in coldPBS inorder to stop
thereaction.FinallysectionswerethenwashedinTBSTfor2minutesandleftin
a new change of TBST solution overnight. The following day sections were
mountedon topolysinecoatedslides,andallowedtodryover-night.Themounted
slides were then dehydrated through a process of incubation in a rising
27
concentration of alcohol followed by xylene, then cover-slipped and sealed using
DPX (Raymond Lamb DPX), andallowed todryover-night.All experimentswere
carriedoutalongsidenegativecontrols.
CalculatingtheStopSignalReactionTime
Correct go reaction timeswere determined directly, however the stop-signal reaction
time(SSRT)hadtobederivedfromthedistributionofcorrectgoreactiontimesandthe
proportionofcorrectlystoppedtrials.SSRTsinthetaskwereestimatedemployingthe
standard procedure described in Logan et al. (1984), using data from where the
proportion of correct stop responses was ~50%. For each subject, data from the
sessionsinwhichthestop-signalpositionswerevariedrelativetotheindividualisedgo
reactiontime,wererankedbytheproportionofcorrectstopresponses,anddatafrom
sessionsinwhichthisvaluewasbetween40%and60%(i.e.50%±10%)wereaveraged.
The latency of stopping as defined by the SSRTwas derived from the distribution of
correct go reaction times and the proportion of correctly stopped trials as previously
described (Eagle & Robbins, 2003, Logan, 1994). Hence, for each of the sessions
determined above, the correct go reaction timeswere rank ordered from smallest to
largest and the nth value found, where n is the rank order position based on the
proportionoffailingtostopcorrectlyinstoptrialswascorrectedfortheoccurrenceof
omittedgotrials.Althoughomittedgotrialswerearareoccurrence,thiscorrectionwas
implemented based on the rationale that they could alter the observed inhibition
functionandaffectdeterminationofthenthcorrectgoreactiontimevalueandhencethe
final SSRT (Eagle & Robbins, 2003, Solanto et al., 2001, Tannock et al., 1989). To
determine the SSRT, the time the stop-signal was presented (i.e. ‘mean correct go
reaction time’ x ‘%meanstop-signalposition’)was subtracted from thenth correct go
reactiontimevalue.
SUPPLEMENTARYRESULTS
Initial validation of the Nesp55 anti-body was carried out by completing
immunohistochemistrystainingonbrainsectionsfromWT,Nespm/+andNesp+/p
adultmice(FigureS1).ThestainingrevealedthattheNespanti-bodysuccessfully
targetstheNesp55protein,showingcellspecificstaininginbothWTandNesp+/p
(wherebymaternalNespispresentinthebrain);inthehypothalamus,ponsand
mid-brain regions, consistent with the discrete regions previously described
28
(Plaggeetal.,2005);andshowingnostaining intheNespm/+ sections(whereby
maternalNesp55hasbeendeleted).
Additionalmeasuresinthedelayedreinforcementtask
Inadditiontochoice,anumberofothermeasuresofbasicbehaviorwithin the
delayed reinforcement task were obtained. These relate to general motoric
competence andmotivation within the task. As expected,Nespm/+ and control
micedemonstratedageneralmaineffectofDELAYonmanyof thesemeasures
(Figure S2) as follows: start (F1.7,27.5=22.06, P
29
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30
FigureS1
FigureS1ImmunohistochemistrystainingforNespincoronalsectionsoftheadultmouse
brain.(a-c)ShowsstaininginWT,Nesp+/p(usedascontrols)andNespm/+adultmice;showingthe
presenceofdiscreteexpressionofNesp55 in thehypothalamus inbothWT(a)andNesp+/p(b)
mice,butnotNespm/+mice(c).(d-f)WTmousebrainprobedusinganti-Nesp55antibodyshows
distinct staining in the hypothalamus, specifically the DM, dorsomedial hypothalamic nucleus,
Arc, arcuatehypothalamicnucleus, and theLH, lateral hypothalamic area (d); in themidbrain,
specifically the EWn, Edinger-Westphal nucleus (e); and in the pons, specifically the LC, Locus
Coeruleus(f).
31
FigureS2
FigureS2AdditionalbehavioralmeasuresofperformanceofNespm/+andcontrol
mice in the delayed reinforcement task. a Start latency; b Choice latency; c Non-
startedtrials;dPanelpushes.Alldatarepresentmeans±s.e.m.
32
FigureS3
FigureS3AdditionalbehavioralmeasuresofperformanceofNespm/+andcontrol
miceintheSSRTtask.aNumberoftrialsinasessionwithindividualisedSSRT(when
thestopcueispresented50%intotheindividual’sGo-reactiontime); b Initiationand
magazinelatencyinasessionwithindividualisedSSRT.Alldatarepresentmeans±s.e.m.