mice The effects of CRF and urocortins on the preference for social noveltyof Behavioural Brain Research

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Behavioural Brain Research

j o ur na l h o me p a g e :w w w . e l s e v i e r . c o m / l o c a t e / b b r

Research report

The effects of CRF and urocortins on the preference for social novelty of mice

Zsolt Bagosi

^∗

, András Czébely-Lénárt, Gergely Karasz, Krisztina Csabaﬁ, Miklós Jászberényi, Gyula Telegdy

DepartmentofPathophysiology,FacultyofMedicine,UniversityofSzeged,Hungary

h i g h l i g h t s

•MaleandfemaleCFLPmicewereinvestigatedinaCrawleysocialinteractiontest.

•CRFandUCN1decreasedthepreferenceforsocialnoveltyofmalemice.

•CRF1receptormediatestheeffectsofCRFandUCN1onmale-femaleinteraction.

•UCN2andUCN3didnotinﬂuencethepreferenceforsocialnoveltyofmalemice.

•CRF2receptordoesnotparticipatetomale-femaleinteraction.

a r t i c l e i n f o

Articlehistory:

Received7December2016

Receivedinrevisedform1February2017 Accepted6February2017

Availableonline9February2017

Keywords:

CRF Urocortins Socialnovelty Mice

a b s t r a c t

Theaimofthepresentstudywastodeterminetheroleofcorticotropin-releasingfactor(CRF),theurocortins(UCN1,UCN2andUCN3)andtheirreceptors(CRF1andCRF2)inthepreferenceforsocialnovelty ofmice.MaleCFLPmicewereadministeredintracerebroventricularly(ICV)withCRF,UCN1,UCN2 orUCN3and/orantalarminorastressin2B,selectiveantagonistsofCRF1receptorandCRF2receptor, respectively.ThemicewereinvestigatedinaCrawleysocialinteractiontestarenaconsistingofthree chambers:anunknownfemalewassetinthefirstchamberandaknownfemale,withwhichthemale wasfamiliarizedpreviouslyfor24h,wassetinthethirdchamber.Firstthetestedmalewashabituated withthemiddlechamberfor5minandthenallowedtoexploretheremainingchambersfor5min,during whichthenumberofentriesandthetimeofinteractionweremeasured.CRFdecreasedsignificantlythe numberofentriesandthetimeofinteractionwiththeunknownfemale,butnottheknownfemale.UCN 1decreasedsignificantlythenumberofentriesintothechamberoftheunknownfemale,butnotthe knownfemale,withoutchangingthetimeofinteraction.Alldecreasingeffectswerereversedbyanta- larmin,butnotastressin2B.UCN2andUCN3didn’tinfluencesignificantlyanyoftheparameters.The presentstudysuggeststhatCRFandUCN1decreasethepreferenceforsocialnoveltybyactivatingCRF1

receptor,whileUCN2andUCN3,activatingselectivelyCRF2receptor,donotparticipatetomale-female interaction.

1. Introduction

Corticotropin-releasingfactor(CRF)isa hypothalamicneuro- hormone and also an extrahypothalamic neurotransmitter that regulatestheendocrine,autonomicandbehavioral responsesto stress[1].DuringstressCRF,alongwiththesynergisticarginine-

∗Correspondingauthorat:DepartmentofPathophysiology,UniversityofSzeged, 6725,Szeged,Semmelweisstr.1,Hungary.

E-mailaddress:bagosi.zsolt@med.u-szeged.hu(Z.Bagosi).

vasopressin(AVP),isreleasedfromtheparaventricularnucleusof thehypothalamus(PVN)and,gettingintothecirculationthrough themedianeminence,stimulatesthereleaseoftheadrenocorti- cotropichormone(ACTH)intheanteriorpituitary[1].ACTH,inturn stimulatesthesynthesisofglucocorticoidsintheadrenalcortex resultinginelevationoftheconcentrationoftheplasmaglucocorti- coids[2].Theelevationoftheplasmaglucocorticoidconcentration not only reﬂects the activation of the hypothalamic-pituitary- adrenal (HPA)axis,but also exerts negativefeedback effect on thehypothalamicCRFandthepituitaryACTHrelease,inhibiting theHPAaxis[3].ICVinjectionofCRFand UCNImaystimulate http://dx.doi.org/10.1016/j.bbr.2017.02.009

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ACTHsecretionintheanteriorpituitarythatpeaksin15minand maydecreasetheactivesocialinteraction.ACTH,inturnstimulates thesynthesisofglucocorticoidsintheadrenalcortexresultingin elevationoftheconcentrationoftheplasmaglucocorticoids[2].

Theelevationoftheplasmaglucocorticoidconcentrationnotonly reﬂectstheactivationofthehypothalamic-pituitary-adrenal(HPA) axis,butalsoexertsnegativefeedbackeffectonthehypothalamic CRFandthepituitaryACTHrelease,inhibitingtheHPAaxis.

SinceCRFwasﬁrstlyisolated[1],agrowingfamilyofCRF-like peptideshavebeendiscovered.Todaythemammalianmembers ofthisfamilyincludefourligands:CRF,urocortin1(UCN1)[4], urocortin2 (UCN2),alsoknown asstresscopin-relatedpeptide (SRP)[5],andurocortin3(UCN3),alsoknownasstresscopin(SCP) [6],two receptors(CRF₁ and CRF₂)[7]and onebindingprotein (CRF-BP)[8].Thenameurocortinderivesfromtheﬁshhomologue urotensin(63%sequenceidentity)andthemammaliananalogue corticotropin(45%sequenceidentity)[9],astheurocortinsshare commonaminoacidicelements(45–33%),buthavedifferentphar- macologicalpropertiescomparedtoCRF.CRFbindspreferentially toCRF₁receptor,whileUCN1attachesequipotentlytobothCRF receptors(CRF1andCRF2),andUCN2andUCN3bindselectively toCRF₂receptor[10].ThesereceptorsbelongtotheclassBsubtype ofGprotein-coupledreceptors(GPCRs)and,likeallGPCRs,con- sistofanamino-terminalextracellularregion,acarboxyl-terminal intracellulartailandseven,transmembranesegments,connected byalternatingintracellularandextracellularloops[11].Thereis nearly70%identitybetweenCRF₁andCRF₂receptorsattheamino acidlevelwiththetransmembraneandintracellulardomainsofthe CRFreceptorspresentingthehighesthomology(over80%identity) [11].Inaddition,CRFandUCN1canbefoundattachedtoCRF-BP [10],whichwasfoundinthebrainandthepituitaryandisthought toinhibittheactionsofCRFandUCN1[8].

Asregardstheanatomicaldistributionoftheligands,theyare represented both in the brain and the peripheral tissues [12].

CRFissynthesizedmainlyinthehypothalamus(PVN),thecentral nucleusoftheamygdalaandthehindbrainregionsintheCNS,and expressedinthegut,skin,andadrenalglandintheperiphery.UCN 1expressionhasbeendescribedpredominantly intheEdinger- Westphalnucleusinthebrainandexpressedinthegastrointestinal tract,testis, cardiac myocytes, thymus, skin, and spleen in the periphery.UCN2expressionhasbeendetectedinthehypotha- lamus(e.g.arcuatenucleus),thebrainstemandthespinalcordin thecentralnervoussystem(CNS),andintheheart,thebloodcells andtheadrenalglandintheperiphery.UCN3expressionhasbeen observedintheamygdala(e.g.medialnucleus)intheCNS,andin thegastrointestinaltractandthepancreasintheperiphery[13].

Asregardstheanatomical localizationofthereceptors,CRF1

receptorisrepresentedmoreanundantlyintheCNS,whereasCRF₂ receptorisdistributedpredominantlyintheperiphery[12].Inthe CNS,CRF1receptorisexpressedinthecerebralcortex,thecerebel- lumandtheanteriorpituitary,butalsofoundintheamygdala,the striatumandthehypothalamus[14].CRF₂receptorislimitedcen- trallytosubcorticalregions:theamygdala,thehippocampusand thehypothalamusandscatteredallovertheperiphery:theheart, thegastro-intestinaltract,thelung,theskeletalmusclesandthe vessels[14].

Besidestheirprincipalroleinthemodulationofstressresponses [15–18],regulationoffoodintakeandsatiety[19],gastrointesti- nalmotility[20],vasodilationandcardioprotection[21],CRFand theCRF-relatedpeptideshavebeenimplicatedinsocialinteraction [22–24],althoughtheirintimaterolewasnotinvestigatedthor- oughly[25,26].Hence,theaimofourstudywastodeterminethe roleofCRF,theurocortins(UCN1,UCN2andUCN3)andtheir receptors(CRF₁ andCRF₂)inthepreferenceforsocialnoveltyof mice.

2. Materialsandmethods 2.1. Animals

MaleandfemaleCFLPmiceweighing24–30gwereused.The totalnumberofmicewas504,ofwhich168malesand336females (dividedin168partnerfemalesand168strangerfemales).CFLP micewereusedbecauseweintendedtotestanoutbredstrainof mice,ratherthananinbredstrain,suchasC57/BL6mice,which, accordingtopreviousstudies,failtoexhibitpreferenceforsocial noveltyinthethree-chamberapparatus[27].Femalemicewere usedaspartnersorstrangersinsteadofthemalemice,becausewe aimedtoinvestigatethepreferenceforsocialnoveltyfollowingpair bondformation(male-femaleinteraction),andnotsocialaffiliation (male–maleinteraction).Four-fivemaleswerehousedtogetherin theirhomecagesandseparatedfromthestrangerfemales,butthen kepttogetherwiththepartnerfemalesfor24hbeforetheexperi- mentsstarted.Duringtheexperimentstheanimalswerekeptand handledinaccordancewiththeinstructionsoftheUniversityof SzegedEthicalComitteefortheProtectionofAnimalsinResearch, atconstantroomtemperature(23^◦C)onastandardillumination schedule,with12-hlightand12-hdarkperiods(lightsonfrom6:00 a.m.).Commercialfoodandtapwaterwereavailableadlibitum.All experimentswereperformedinaccordancewiththeARRIVEguide- linesandtheU.K.Animals(ScientificProcedures)Act,1986and associatedguidelines,EUDirective2010/63/EUforanimalexperi- ments.

2.2. Surgery

The mice were implanted with a stainless steel Luer cannula of 10mm lengthand 0.4mm diameteraimed atthe right lateralcerebralventricleunderanesthesiawith60mg/kgEuthana- sol (CEVA-Phylaxia,Hungary).The stereotaxiccoordinates were 0.5mmposteriorand0.5mmlateraltothebregma,and3mmdeep fromtheduralsurface,adaptedfromtheatlasdescribingthemouse braininstereotaxiccoordinates[28].Cannulasweresecuredtothe skullwithdentalcementandacrylate.Thepolyethylenetubeofthe injectorhaving0.8mmoutsidediameterand0.4mminsidediam- eterwasﬁttedcloselyintothecannulawithasharppointingtip projectingbeyondthecannula.Themicewereallowedfor5days torecoverafterthesurgeryandthepermeabilityofthecanullawas testedwithmethylene-blueaftertheexperiments.Thehitandmiss ratiowas168–143,whichmeans85%forrightpositioningofthe cannula.

2.3. Treatment

Fourexperimentswereperformedwithfourdifferentgroups.In experimentI,thefirstgroupwastreatedwith2␮lsalinesolution, thesecondonewith5␮g/2␮lCRF,thethirdonewith0.1␮g/2␮l of the selective CRF₁ receptor antagonist antalarmin and the fourthonewith1␮g/2␮loftheselectiveCRF₂receptorantagonist astressin2B,30minbeforethesocialinteractiontest.Inexperi- mentII,thefirstgroupwastreatedwith2␮lsaline,theremaining groups weretreated with5␮g/2␮l CRF,30minbeforethetest and pretreatedin orderwith2␮lsaline, 0.1␮g/2␮lantalarmin or1␮g/2␮lastressin2B,60minbeforethetest.InexperimentIII, thefirstgroupwastreatedwith2␮lsaline,thesecond,thethird andthefourthoneswith5␮g/2␮lUCN1,5␮g/2␮lUCN2and 5␮g/2␮lUCN3,respectively,30minbeforethetest.Inexperiment IV,thefirstgroupwastreatedwith2␮lsaline,theremaininggroups weretreatedwith5␮g/2␮lUCN1,30minbeforethetestandpre- treatedinorderwith2␮lsaline,0.1␮g/2␮lantalarminor1␮g/2␮l astressin2B60minbeforethetest.Thesaline(0.9%NaCl)solution wasprovidedbyBiogalLtd.,Hungary,CRF,UCN1,UCN2andUCN

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3werepurchasedfromBachemLtd.,Switzerland,andantalarmin andastressin2BwerepurchasedfromSigmaAldrichInc.,USA.The ICVinfusionofthesubstanceswasperformedwithahand-held microinjector(HormuthLtd.,Germany)andthemice,beingprevi- ouslyhandleddailytominimizetheeffectsofnonspeciﬁcstress, werealsohand-heldduringinfusion.

2.4. Socialinteractiontest

ThirtyminutesaftertheICVinjectionofthepeptidesmicewere investigatedinasocialinteractiontestarenadescribedoriginallyby Crawleyandcolleaguesandusedtotestthesociabilityandthepref- erenceforsocialnoveltyofmice.[29].Inthepresentexperiments weusedamodiﬁedversionofthistestthatwasmeanttoinvesti- gatethepreferenceforsocialnoveltyfollowingpairbondformation (male-femaleinteraction), but not social afﬁliation (male–male interaction).Theprincipleofthetestisbasedontheassessment thatawildtypemousewouldvisitandspendmoretimewiththe strangerfemaleoverthepartnerfemale,indicativeforanintact socialmemoryandanaturalpreferenceforsocialnovelty.

Theapparatusisarectangular,three-chamberboxmadefrom clearPlexiglass.Eachchamberisof19×45×25cm,withanopen middlesection,whichallowsfreeaccesstoeachchamber.Theright andleftchamberscouldbeisolatedfromthemiddleonebyusing twodividingPlexiglasswalls.Twoidentical,wirecup-likecellsof 10×17cmwithremovablelidsthatlargeenoughtoholdasin- glemousewereplacedverticallyinsidetheapparatus,oneineach sidechamber.Eachcelliscomprisedofmetalwirestoallowfor airexchangebetweentheinteriorandexteriorofthecylinderbut smallenoughtopreventdirectphysicalinteractionsbetweenan animalontheinsidewithoneontheoutside.

Inthepresentexperimentsanunknownfemalemouse(stranger female)wassetintheﬁrstchamberandaknownfemalemouse (partner female), withwhich themale was familiarizedprevi- ouslyfor24h,wassetinthethirdchamber.Firstthetestedmale mousewashabituatedwiththemiddlechamberfor5minandthen allowedtoexploretheremainingchambersfor5min,duringwhich thenumberofentriesandthetimeofinteractionweremeasured.

Thetestedmalemousewashabituatedforthemiddlecompart- mentfor5min,butnottheothercompartments.Thepartnerand thestrangerfemalemiceweretransferredintheirhomecagesinto thebehavioralroom30minbeforetheﬁrsttrial,buttheywerenot habituatedpreviouslytothecompartments.Thepartnerandthe strangermiceweresystematicallyalternatedbetweentheleftor rightcompartmentacrossthetrialstopreventsidepreference.All trialswereperformedbetween9:00a.m.and13:00p.m.General roomlightingwas650lx.Thepersonwhomadetheobservation wasatleast2mawayfromtheapparatus.Aftereachtrial,allcham- berswerecleanedwith70%ethanolandthenwithClidox1:5:1to preventolfactorycuebiasandtoensureproperdisinfection[30].

2.5. Statisticalanalysis

Statisticalanalysis of theresultswas performedby analysis ofvariance(ANOVA, GraphPadPrismSoftware).Thedifferences betweengroups were tested by two-way ANOVA, followed by Tukey post-hoc comparison test. A probability level of 0.05 or lesswasacceptedasindicatingastatisticallysigniﬁcantdifference (Tables1–4).

3. Results

CRF decreased signiﬁcantly the number of entries (F(3,28)=5.489; p<0.001) and the time spent in interaction (F(3,28)=4.641; p<0.001) with the stranger female, but not that with thepartner female (F(3,28)=0.03286; p=0.9918 and

Table1

TheeffectsofCRF,antalarminandastressin2Bonthepreferenceforsocialnovelty ofmice.

Numberofentries

ANOVAtable SS DF MS F(DFn,DFd) pvalue

Interaction 92.05 6 15.34 F(6,108):3.582 p=0.0028 Novelty 182.2 2 91.08 F(2,108):21.27 p<0.0001 Treatment 175.4 3 58.48 F(3,108):13.65 p<0.0001

Residual 462.5 108 4.282

Timeofinteraction

Interaction 61947 6 10325 F(6,108):2.711 p=0.0172 Novelty 189754 2 94877 F(2,108):24.91 p<0.0001 Treatment 134586 3 44862 F(3,108):11.78 p<0.0001

Residual 411337 108 3809

Abbreviations:SSsumofsquares;DFtotaldegreesoffreedom;MSmeansquare;F (DFnDFd)Fdistribution(degreesoffreedomnumeratordegreesoffreedomdenom- inator);Pvalueprobabilityvalue.

Summaryofthestatisticaldatafromthesocialinteractiontest(two-wayANOVA withfactorA=noveltyandfactorB=treatmentwasperformed).

Table2

TheeffectsofCRF,CRFwithantalarminandCRFwithastressin2Bonthepreference forsocialnoveltyofmice.

Numberofentries

Interaction 58.89 6 9.816 F(6,84)=3.905 p=0.0017

Novelty 50.29 2 25.14 F(2,84):10 p=0.0001

Treatment 98.72 3 32.91 F(3,84):13.09 p<0.0001

Residual 211.1 84 2.513

Timeofinteraction

Interaction 43738 6 7290 F(6,84)=1.915 p=0.0877 Novelty 117742 2 58871 F(2,84):15.47 p<0.0001 Treatment 123125 3 41402 F(3,84):10.78 p<0.0001

Residual 319728 84 3806

Table3

TheeffectsofUCN1,UCN2andUCN3onthepreferenceforsocialnoveltyofmice.

Numberofentries

Interaction 18.2 6 3.034 F(6,105):0.789 p=0.5801 Novelty 235.9 2 118 F(2,105):30.7 p<0.0001 Treatment 129.8 3 43.28 F(3,105):11.26 p<0.0001

Residual 403.4 105 3.842

Timeofinteraction

Interaction 2509 6 418.2 F(6,105):0.1446 p=0.9897 Novelty 268522 2 134261 F(2,105):46.43 p<0.0001 Treatment 4298 3 1433 F(3,105):0.495 p<0.6862

Residual 303621 105 2892

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0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

into the partner's area into the stranger's area total entries

nu m ber of en tr ie s

control (10) CRF (10) antalarmin (10) astressin 2B (10)

*

0 50 100 150 200 250 300

with the partner with the stranger total time

tim e s pe nt (s )

control (10) CRF (10) antalarmin (10) astressin 2B (10)

*

Fig.1.TheeffectsofCRF,antalarminandastressin2Bonthepreferenceforsocialnoveltyofmice.Thenumberofanimalsusedineachgrouphasbeenindicatedinbrackets.

Valuesarepresentedasmeans±SEM;statisticallysigniﬁcantdifferencewasacceptedforp<0.05andindicatedwith*forCRForantalarminvs.control.

Table4

TheeffectsofUCN1,UCN1withantalarminandUCN1withastressin2Bonthe preferenceforsocialnoveltyofmice.

Numberofentries

Interaction 22.99 6 3.832 F(6,84):1.965 p=0.0799 Novelty 157.1 2 78.54 F(2,84):40.26 p<0.0001 Treatment 156.5 3 52.17 F(3,84):26.74 p<0.0001

Residual 163.9 84 1.951

Table4(Continued) Timeofinteraction

Interaction 35465 6 5911 F(6,84):2.187 p:0.0521 Novelty 322153 2 161077 F(2,84):59.59 p<0.0001 Treatment 25498 3 8499 F(3,84):3.144 p=0.0294

Residual 227046 84 2703

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0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

into the partner's area into the stran ger's area total entries

nu m ber of en tr ie s

control (10) CRF (10) CRF + antalarmin (6) CRF + astressin 2B (6)

*

#

0 50 100 150 200 250 300

with the partner with the stranger total time

tim e s pe nt (s )

control (10) CRF (10) CRF + antalarmin (6) CRF + astressin 2B (6)

*

#

Fig.2. TheeffectsofCRF,CRFwithantalarminandCRFwithastressin2Bonthepreferenceforsocialnoveltyofmice.Thenumberofanimalsusedineachgrouphasbeen indicatedinbrackets.Valuesarepresentedasmeans±SEM;statisticallysigniﬁcantdifferencewasacceptedforp<0.05andindicatedwith*forCRFvs.controland#forCRF withantalarminvs.CRF.

F(3,28)=0.4039,p=0.7513),comparedtothecontrols(Fig.1).In addition,thetotalnumberofentries(F(3.28)=16.04;p<0.001)and thetotal timeof interaction(F(3.28)=8.259;p<0.05)decreased signiﬁcantly(Fig.1).Antalarmin and astressin2Badministered alonewereineffective(p<0.05)(Fig.1).Thedecreasingeffectsof CRFwereblockedbyantalarmin(p<0.05),but notastressin2B (p<0.05)(Fig.2).

UCN1decreasedsigniﬁcantlythenumberofentriesintothe chamberoftheunknownfemale(F(3,35)=6.277;p<0.001), but notintothatwiththepartnerfemale(F(3,35)=1.614;p=0.2036),

withoutchanging thetimesof interactionspentwiththepart- ner female (F(3,35)=0.144; p=0.09328) or the strangerfemale (F(3,35)=0.2763; p=0.8421), compared to the control (Fig. 3).

Thetotalnumberofentries(F(3,35)=4.827;p<0.001)decreased significantly, in contrast with the total time of interaction (F(3,35)=0.5033;p=0.6825)thatwasnotinfluencedconsiderably byUCN1(Fig.3).UCN2andUCN3didnotinfluenceremarkably anyoftheparametersmeasured(p>0.05)(Fig.3).Thedecreasing effectsofUCN1werereversedbyantalarmin(p<0.05),butnot astressin2B(p>0.05)(Fig.4).

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0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

into the partner's area into the strang er's area total entries

nu m ber of en tr ie s

control (10) urocortin 1 (10) urocortin 2 (10) urocortin 3 (9)

*

0 50 100 150 200 250 300

with the partner with the stranger total time

tim e s pe nt ( s)

control (10) urocortin 1 (10) urocortin 2 (10) urocortin 3 (9)

Fig.3. TheeffectsofUCN1,UCN2andUCN3onthepreferenceforsocialnoveltyofmice.Thenumberofanimalsusedineachgrouphasbeenindicatedinbrackets.Values arepresentedasmeans±SEM;statisticallysigniﬁcantdifferencewasacceptedforp<0.05andindicatedwith*forUCNvs.control.

4. Discussion

PreviousstudiesonthepossibleroleofCRFandCRF-relatedpep- tidesinsocialbehaviorofdifferentspecieshavebeenreviewedin tworecentstudies[25,26].Despitethattheprimaryfocusinthese studieshasbeenontheeffectsofsocialstressors,suchassocial defeatandsocialisolationontheCRFsystem[26],therehavebeen alsoinsightsontheroleofCRFsysteminprosocialandafﬁliative behaviors,suchasparentalcare,maternaldefense,sexualbehav- iorandpairbonding [25].Theaimofthepresent studywasto investigatetheeffectsofthesepeptidesonthepreferenceforsocial noveltyfollowingpairbondformationinmice.

OurresultsdemonstratethatcentraladministrationofCRFand UCN1inducesadecreaseofthepreferenceforsocialnoveltyvia CRF₁receptor,whichmayreﬂecttheanxiogenicactionoftheCRF₁ receptor agonists[31,32].ButCRF₁ receptoragonists mayexert theiranxiogenicactionthroughbothcentralandperipheraleffects [31,32].Thecentraleffectcanbemediatedbydifferentextrahy- pothalamicbrainregionsandneuronpopulationsinvolvedinstress reaction,suchasthebasolateralnucleusof theamygdala(BLA) [33].PreviousstudiesreferredthatinjectionofCRFandUCN1into theBLAreducedthetimesofsocialinteractioninmaleWistarrats andthatthiseffectwascompletelyabolishedbyadministrationof selectiveCRF1andnon-selectiveCRFreceptorantagonists[34–36].

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0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

into the partner's area into the stranger's area total entries

number of entries

control (10) urocortin 1 (10) urocortin 1 +antalarmin (6) urocortin 1 + astressin 2B (6)

*

#

0 50 100 150 200 250 300

with the partner with the stranger total time

time spent (s)

control (10) urocortin 1 (10) urocortin 1 + antalarmin (6) urocortin 1 + astressin 2B (6)

Fig.4.TheeffectsofUCN1,UCN1withantalarminandUCN1withastressin2Bonthepreferenceforsocialnoveltyofmice.Thenumberofanimalsusedineachgrouphas beenindicatedinbrackets.Valuesarepresentedasmeans±SEM;statisticallysigniﬁcantdifferencewasacceptedforp<0.05andindicatedwith*forUCN1vs.controland

#forUCN1withantalarminvs.UCN1.

UCN1provedevenmorepotentthanCRFinreducingthetimeof interaction,whichisconsistentwithitshigherafﬁnityfortheCRF₁ receptor[34–36].TheperipheraleffectoftheCRF₁ agonistscan bemediatedbyactivationoftheHPAaxis.Actually,ICVinjection ofthesamedose(5␮g/2␮l)ofCRForUCNIthatwasusedinthe presentexperimentsledtoelevationofplasmaACTHandcorticos- teroneconcentrationswithin30mininourpreviousexperiments [15].ThereforetheimpactoftheHPAaxisactivationonthebehav- iorofthemiceobservedinthethree-chambersocialinteraction testcannotbeexcluded.Inaddition,a previousstudyusingICV injectionofCRFinmaleListerrats,suggestedthattheanxiogenic effectofCRFobservedinaclassicalsocialinteractiontest[31],can becausedbythereleaseofpituitaryACTH,sincethishormonealso decreasedsocialinteractionwithoutreducinglocomotoractivity [31,32],butisunlikelytobeduebythereleaseofadrenalcorticos-

terone,sincethishormonedidnotdecreasesocialinteraction,and insomedosesincreasedit[31,32].

Our resultsalso demonstrate that central administration of UCN2and UCN3doesnotalter thepreference forsocial novelty,andgenerallythesocialinteractionofmalemicewithfemale mice.However,previousstudiesclaimedthatmale,butnotfemale, UCN2knock-outmiceexhibitedmorepassivesocialinteractions and reduced aggressivenesstonovel conspecifics[22] and that bothmaleandfemaleUCN3andCRF₂receptorknock-outmice expressedanenhancedsocialmemoryandincreasedpreference forsocialnovelty,whencomparedtowild-typemice[23].These studiessuggestthatCRF₂agonistswouldmodulatesomeaspects ofsocialbehavior.Incontrast,amorerecentstudystatedthatmice deficientinUCN3orCRF₂ receptor localizedspecificallyinthe medialnucleusoftheamygdala(MEA)showeddecreasedprefer- enceforsocialnovelty[37].Moreover,pharmacologicalactivation

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oftheCRF₂receptorsoroptogeneticactivationofUCN3neurons intheMEAprovedtheopposite,anincreasedpreferenceforsocial noveltyofmice.ThisstudysuggeststhatUCN3fromtheMEAwould modulatetheabilityofmicetocopewithsocialchallengesviaCRF₂ receptors[37].

Ourexperimentswereoriginallyinspiredbythreestudiesusing fourdifferentvolespecies (prairie,pine,montane and meadow voles),which,based ontheircloserelationship,butremarkably differentsocialbehavior(prairieandpinevolesaremonogamous, whilemontaneandmeadowvolesarepromiscuous),areconsid- eredemergingmodelorganismsforunderstandingthesocialbrain [38].Intheﬁrststudyusingreceptorautoradiography,amarked differencein thebrain distributionof CRF₁ and CRF₂ receptors between monogamous and promiscuous species (e.g., a higher expressionofCRF2receptorandalowerexpressionofCRF1recep- torinthenucleusaccumbens(NACC)oftheprairieandpinevoles), andmaleandfemalesex(e.g.,higherlevelsofCRF₂receptorbind- ingintheencapsulatedbednucleusofstriaterminalis(BNST)in malevoles)wasdemonstrated.Inthesecondstudyusinginsitu hybridizationandimmunocytochemistry,nodifferencebetween thebrain distribution of CRF (the NACC, the BNST, thecentral nucleusoftheamygdalaetc.)andUCN1(theEdinger-Westphal nucleus)wasdemonstratedbetweenmonogamousandpromiscu- ousvoles[39].Inthethirdstudy,microinjectionofCRFandUCN1 intotheNACCinducedanaccelerationofthepartnerpreferencefor- mationinmonogamousprairievoles,butnotinnon-monogamous meadowvoles,thatwaseffectivelyblockedbyintraaccumbalinjec- tionofbothselectiveCRF1andselectiveCRF2receptorantagonists [40].Takingintoconsiderationthaturocortinergicﬁberswerenot presentintheNACC,thisstudysuggeststhatthereleaseofCRFin theNACCfacilitatespartnerpreferenceinthemonogamousspecies activatingbothCRFreceptors[40].Thelackofthisfacilitationeffect inthenon-monogamousspeciescanbeexplainedbythelackof thecorrespondentreceptorsintheirbrain,despiteofthesimilar distributionoftheirligands[39,41].

Inconclusion,thepresentstudysuggeststhatCRFandUCN1 decreasethepreferenceforsocialnoveltybyactivatingCRF1recep- tors,whileUCN2andUCN3,activatingselectivelyCRF2receptors, donotparticipatetomale-femaleinteractioninthepromiscuous CFLPmice.Thisconclusioncanbeunderlinedbythehigherexpres- sionofCRF1 receptorsandlowerexpressionofCRF2receptorsin thebrainsofnon-monogamousspecieswhichhavebeenpreviously demonstrated.

Acknowledgements

ThisstudywassupportedbytheNeuroscienceResearchGroup oftheHungarianAcademyof SciencesandtheHungarianBrain ResearchProgram.

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