JoumaJofMolecu］arNeuro＄Cience
NeuropeptideW−inducedhypophagiai＄medjatedthroughcorticotropin−relea＄ing homurOn8
M8ロリ8dptNumbon FullTl鵬：
人相do一けPO：
Ko叩帽e：
加匹れd】叩Au廿Ⅶ爪
JOMN−D−15−00007RI
NeuropeptideW−inducedhypophagiaismediatedthroughcorticotropln−releaslng hormone−COntaEnlngneurOnS
Ori！IinalRos08rCh
Neuropeptid8W（NPW）；lmmunohistochemistry；Hypotha［amus；mOUSe SdjiShida．Ph皿．
Show8UniversitySch00lofMedicine ShInagawa−Ku，TokyoJAPAN Co汀朋POnd柚Au廿1ⅣSo￠Ond馴y
ln知m劇On：
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Co汀舶POnd】叩Autho「8So￠Ond8け In8utu廿On：
Fl帽t仙hor：
Fl帽tAuthor＄○￠On加町lnbmtbn：
Oldor01血t血：
ShowaUnivorsiけSch∞lofModicino
FumikoTak◎nOy∂
Fumiko Takenoya Lihu∈lWan⊆】
JH∈lrU如dKagoym SatoshiHirako
NobuhiroWad8 HirofumiHashimoto YoichiUota Junichi Sakagami NaokoNonaka SeijiShioda．Ph．n Ordor01Author8S00Dnd8Ⅳlnfom8tbn：
触ねd：
NeuropeptideW（NPW），WhichwasoTiginaEIyisolatedfromthBPOrCinehypothalarnus，hasbeenidentifiedastheendogenousligandforboththeNPBWRl（GPR7）and NPBWR2（GPR8）receptors・ThesereceptoTS−WhichbelongtotheorphanGproteinT
COUPledreceptor（GPCR）family．shareahighs8qUenCehomoIogywiththeopioidand
＄OmatO＄tatinreceptorfamilies．NPWandNPBWRlareIWidelydistributedintherat
CentralneⅣOuSSyStem（CNS）・Whiletheintracerebrovent（icular（i．c．v．）in5ectionof
NPWelevate＄Plasmacorticosteron81ev8fs，theintravenousadministrationofNPWin COnjunctionwithacorticotropin−releasinghormone（CRH）antagonistblocksNPW−inducedcorticosterones8Cretion．1thasbeenroportedthatNPWisinvoIvedin
reguIatingthehypotha［amus−Pituitary−adrenalcort8X（HPA）axis，andthati．c．∨．
admTnistrationofNPWdecreasesfeedingbehavior．Theaimofthepresentstudywas
toascertainifNPWrsroleinf88dingregulationismediated（OrnOt）through
CO止Ecotropin−re18a＄inghormon8（CRH）−COntainingneurons．W8foundthatNPW−
COntainingaxonterminalsmakesynapsesvnthCRH−immunoreactiveceIIbodiesand
dendriticproce＄SeSinthehypothaFamicparaventricu［arnucleus（PVN）．Thecentral infusionofNPWsfgnificantlyinducedc−FDSeXPreS＄ioninCRH−immunoreactive n8UrOnSinthemousePVN，butnotinvasopressh−OrOXytOCin−immunoreactiv8
neurons・TodetermheifNPWregulatesf8edingbehaviorthroughCRHneurons，the
Po〝eredムy亡甜0血／〃∂∩叩er㊥∂〃dPro血ズわ〃〃∂∩∂ger㊧什omノ1√／es5ys始m5C（叩Or∂亡旧〃
ResponsetoReviewerCornments
ShowaUniversitySchoolofMedicine
l−5−8，HatanOdai，Shinagawa−ku，Tbkyo142−8555，Japan
一切：十81−3−3784−8104，Fax：十81−3−3784−6815
March16，2015 JournalofMolecularNeuroscience
DearDrIllanaGozes，Editor−in−Chief
Thankyouverymuchforyourcarefu1andkindcommentsono11rmanuSCript
（JOMN−D−15−00007）entitled Neuropeptide W−induced hypophagiais mediatedthrough corticotropln−releaslng hormOne−COntaining neurons by Takenoyaetal．
AccordingtothebothReviewer＃1and＃2andtheircoInmentS，WereVisedour manuSCript．Ihaveshowntherevisedportionsbyredcolor・Istronglyhopethat therevisedmanuSCriptcouldbeacceptableforthepublicationinthejoumal
HJournalofMolecularNellrOSCience叩．
Correspondingauthor：ProfbssorSeiiiShioda，
DepartmentofAnatomySchoolofMedicineShowaUniversity
l・5−8Hatanodai，Shinagawa−ku，Tokyo142−8555，Japan
Tel：＋81−3−3784−8103fax：＋81−3−3784−6815 E−mai1：Shioda＠med．sllOWa−u・aCjp
ShowatJniversitySchoolofMedicine
l−5−8，Hatanodai，Shinagawa−ku，Tbkyo142−8555，Japan
Tel：＋81−3−3784蠣8104，Fax：＋81−3−3784−6815
Speci鎖ccomments
TbReviewer＃1
Thankyouverymuchforyourvaluableandfavorablecomments・Wethinkthatyour COlnmentSOnOurPaPerareVeryhelpfu1，andwerevisedthemanuSCriptlnaCCOrdanCeWith yoursuggestionsandcommentS．Ourresponsesaredescribedasshownbelow・
TherevisedsentencesarecoloredredinthismanuSCript．
Reviewer＃1：ThisisaneXCellentpaperprovidingstrongevidencethatNPW−inhibitionof fbedingbehaviorismediatedbyanupregulationoftheCRFsystem．Thepaperisclearly Written．IhaveJuStafbwcomments：
1．Themethodsneedtobeorganizedalittlebetter．Therearetwoheadingsentitled‖hsitu hybridizationhistochemistryfbrCRHmRNA‖．
Thankyouforyourcomment．Astoyourindication，WedeletedoneheadingT hsitu hybridizationhistochemistryforCRHmRNA一一．（Page8，1ine9）
2．Page8（inmethodssection）． lWhattimeofdaywasNPW−30administered？
WehaveadministeredNPWintheanimalsatthetimeof6p．m．inthelateaftern00nbecause thetimeistheonsetofthebegimingofdarkphase．Thereisalreadypublishedpaper（DateY，
ShiodaS，NakazatoMetal．，Endocrinology151：2200−10，2010）thatNPWstimulatesfood intakeinlightphasebutinl1ibitsindarkphase・WethinksphysiologlCalefftctofNPWis foodintakeinhibitionandthereforewemadeexperiments．
PleaselookatPage8，Line14to15．
3．Pagell．c−FosantibodyislistedasTl？Mb−5？？
Thankyouforyourcheck．Accordingtoyourcomments，WeCOrreCtedthewords・
d H？Mb−5？？Mto‖Ab−5：OncogeneResearchProducts．Cambridge．MA．USA （Page Ichange
11，1ine50）
ShowaUniversitySchoolofMedicine
l−5−8，HatanOdai，Shinagawa−ku，Tokyo142−8555，Japan
Tbl：＋8ト3−3784−8104，アax：＋81−3−3784−6815
4．Fig．3B−Whatarethefu11unitsontheY−aXis？＃cells／section，＃cells／entirePVN，Or SOmethingelse？
WehaveaddedtosentencesofFigurelegendsofFig．3thatitTscells／section．
5・Page18・Itisstated‖increasedCRHimmunOreaCtivltyWaSClearlyapparentinthe ParvOCellularpartOfthePVN（Fig．2） ・ButIthinkFig．2justshowsonetimepointofCR immunoreactivity・hsteadtheysholユIdperhapsreftrtotheincreaseCRFmRNAsignalsin
Fig．1．
Yes，theindicationofCRHchangeSismRNAleverasshownbyISHbutnotbyⅡIC．So，We havedeletedthesenlenceswhileincreasedCRHimmllnOreaCtivitywasclearlyapparentin tlleparvOCellularpartofthePVN（Fig．2）．
6．Fig．4．Ifpossible，allistogramShowingquant摘cationofchangeswouldbedesirable．
Asthereviwer scommentareveryreasonableandwetriedtoanalysesemlquantitativelyln OurSeCtionswithdoubleimmunollitochemistry．However，thegoodsamPlestomakeanalysis areverysmallinnumberandwethinkthatatpresentitisnotavailabletomakethisstudy．But，
insma11sampleswhichweowendinourlab．Iwillshowdatawhicharepreliminarycounted asshownbelow・ThesearedatameasearedinthesamPleswhichiareusendinFig．4．
C−Fos−CRH‥ Saline22・2％（C−Fos−POSitivecells16inCRH−pOSitive72cells NPW65．5％（c−Fos−POSitivecells38i11CRH−POSitivecells58）．
C−Fos−Oxytocin：Saline19．2％（C−Fos5／OXT28）
NPW14．5％；（C−Fos4／0ⅩT27）
C−Fos−Vasopressin：Saline5．9％（c−Fos2／AVP34）
NPW3．6％（c−Fosl／AVP28）
ShowatJniversitySchoolofMedicine
l−5−8，Hatanodai，Shinagawa−ku，Tokyo142−8555，Japan
Tヒ1：＋8ト3−3784−8104，Fax：十81−3−3784−6815
ToReYieⅥrer＃2
Reviewer＃2：Thepaperisawell−WrittenwellNdocumentedpaper，itonlyneedssomeminor revision．Theauthorsshouoldcarefu11ycheckthe負gurelegendsinordertomakesurethat everythingindicatedinthefigureisexplainedinthe負gurelegends（arrows，Sigmicifances etc），alsotherefbrencelist（somerefbrencesarenotfound）andthetextforspe11ingmistakes．
ThankyouverymuchforreviewlngandeditingourMS．Wecheckedourgrammaticalerrors andsentencesthatyouhavepointed．Wecoloredbothcorrectedsentencesandwordred．
1・Wehaveadded arrowhead‖tosentencesofFig2C，D．（Page26）
2．WehaveaddedtosentencesofFigurelegendsofFig3旦＿（Page26）
3．VVecon負rmedthatal1reftrenceexistsinthesentences．
Manuscript
CIickheretodpwnZoadManuscript：JomsTakenoyaetal（NPW−CRH）rev董se＿1・docx
CIickheretovleWLinkedReferences
1︼ 2 3 4 5 6 7 8 q′ O 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 ⊥ 2 1J 4 5 6 7 8 9 0 ⊥ 2 3 4 5 6 7 8 Qノ O 1 2 3 4 5 6 7 8 9 0 ュ 2 3 4 5 ュ ュ ⊥ ⊥ 1︼ ュ ュ 工 1t ュ 2 2 2 2 2 2 2 2 2 2 3 3 3 ■J 3 3 3 3 3 3 4 4一444 4 4−4 4 4 5 5 5 5 5 5 5 5 5 5 6 6一b 6 ′b ′b
NeuropeptideW−ind11CedhypophagiaismediatedtIlrOugh
COrticotropln−releasinghormone−COntainingneurons
Fumiko助kenoyal・2，LihuaWangl，HaruakiKageyamal・3，SatoshiHirakol，NobuhiroWadal，
HirofumiI‡asl血oto4，YoichiUeta4，JunichiSakagami1，NaokoNonakal，Se再iShiodal
1：DepartmentofAnatomy，ShowaUniversitySchoolofMedicine，Shinagawa−ku，Tokyo，Japan．
2：DepartmentofExerciseandSportsPhysiology，HoshiUniversitySchoolofPharmaCyand PharmaceuticalScience，Shinagawa−ku，Tbkyo，Japan．
3：DepartmentofNutrition，FacultyofHealthCare，KiryuUniversity，Gunma，Japan．
4：DepartmentOfPhysiology，SchoolofMedicine，UmiversityofOccupationaland Enviro皿entalHealth，Kitakyushu，Japan
Correspondingauthor：Proftssor
Se可iShioda，
DepartmentofAnatomySchoolofMedicineShowaUniversity
1−5−8Hatanodai，Shinagawa−ku，Tbkyo142−8555，Japan
Tel：＋81−3−3784−8103
Fax：＋81−3−3784−6815
E−mail：Shioda（amed．showa−u．aC．jl］
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．拍∫け爪・J
NeuropeptideW（NPW），Whichwasorigina11yisolated蝕omtheporcinehypothalamus，
hasbeenidentifiedastheendogenousligandforboththeNPBWRl（GPR7）andNPBWR2
（GPR8）receptors．Thesereceptors，WhichbelongtotheorphanGprotein−COuPledreceptor
（GPCR）fami1y，Shareahighsequencehomologywiththeopioidandsomatostatinreceptor
臨milies．NPWandNPBWRlarewidelydistributedintheratcentralnervoussystem（CNS）・
Whiletheintracerebroventricular（i．c．v．）injection ofNPW elevatesplasmacorticosterone
levels，theintravenousadministrationofNPWincoI叩nCtionwithacorticotropln−releaslng
hormOne（CRIl）antagOnist blocks NPW−induced corticosterone secretion・It has been
reportedthatNPWisinvolvedinregulatingthehypothalamus−Pituitary−adrenalcortex（HPA）
axis，andthati．c．v．administrationofNPWdecreasesf岳edingbellavior，Theaimofthepresent
studywasto ascertainifNPW sroleinfeedingregulationismediated（OrnOt）through
CRH−COntainlngneurOnS．WefbundthatNPW−COntainlngaXOnteminalsmakesymaPSeSWith
CRH−immunoreactivecellbodiesanddendriticprocessesinthehypothalamicparaventricular
nucleus（PVN）．The centralinfusion ofNPW signincantlyinduced c−Fos expressionin
CRH−immunOreaCtive neuronsin the mouse PVN，but notin vasopressin− Or
oxytocin−immunoreactiveneurons．TodetermineifNPWregulatesfbedingbehaviorthrough
ュ 2 3 4 5 6 7 8 う 0 ⊥ 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 ⊥ 2 3 4 5 6 7 8 9 0 ⊥ 2 3 4 5 6 7 8 9 0 ⊥ 2 3 4 5 6 7 8 9 0 1 2 3 4 5 ⊥ ュ l ⊥ l l l ⊥ ⊥ ュ 2 2 2 2 2 2 2 2 2 2 3 3 3 3 3 3 3 3 3 3 4 4 4 4 4■ 4 4 4 4 4 5 5 5 5 5 5 5 5 5 亡つ 6 6 6 6 6 6
CRHneurons，thefeedingbehaviorofmicewasstudiedfo1lowlngthei．c．v．administration
NPWinthe presence or absence ofpretreatment witha CRIIantagOnist．While NPW
administrationdecreasedfeedingactivity，theCRH antagonlstinhibitedthis effbct・These
resultsstronglysuggestthatNPWregulatesfヒedingbehaviorthrotlghCRHneuronsinthe
mousebrain．
Keywords：NeuropeptideW（NPW）；lmmunohistochemistry；Hypothalamus；mOuSe
Introduttion
0 Dowdetal・（0−Dowdetal．，1995）usedoligonucleotidesbasedontheopioidreceptorand
thestruCturally−relatedsomatostatinreceptortoidentifytwogenes−GPR7andGPR8which
WerePredictedto encodetwo Gprotein−COupledreceptors（GPCRs）inthehumanbrain．
GPR7八軒βWRlis expressedinthebrainandperipheralorgans oflmmanS androdents，
Whereas〃PβWR2isnotfoundinrodentgenomes（Leeetal．，1999）．h2002，neurOPePtideW
OiPW）wasisolated丘omthe porcine hypothalamuS（Shimomura etal．，2002）andits
endogenousligandreceptorswereconsideredtobeneuropeptideB／Wreceptorl（NPBWRl，
formernameGPR7）andneuropeptideB／Wreceptor2（NPBWR2，formernameGPR8）．NPW
hastwoisoformS，designatedasneuropeptideW−23andneuropeptideW−30，andthesetwo
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PePtidesarethoughttobederived丘omtheprecursorNPWpeptidebyproteolyticprocessing
attwopairsofarginineresidlleS（positions24and25，and31and32）（Shimomuraetal・，
2002）．
GivenNPBWRsreceptorgeneexpressionpattemsinrodents，NPWandNPBmostlikely
bindtoNPBWRl，Whichmediatestheirphysiologicalfunctions（0 Dowdetal．，1995；Leeet
al．，1999；Brezillonetal．，2003；Tanaka et al．，2003）．Ithasbeen shownthatNPBWRl
knockoutmicearehyperphagic and exhibitdecreasedenergyexpenditure，SuggeStingthat
NPBandNPWplaysanimportantroleinfeedingbehavior（Shimomuraetal・，2002）・The
intracerebroventricular（i．c．v）infusionofNPWinthelightphaseinanimals exposedto a
12：12−hlight−darkscheduleincreasedfoodintakeduringthefirst2hinmalerats（Shimomura
etal．，2002），WhileLevineetal．（Levineetal．，2005）alsoreportedthatirtiectionofNPWinto
theratPVNincreasedfbodintake．TheseresultssuggestthatNPWmayhaveanorexlgenic
functionintheacllteftedingphase（Shimomuraetal．，2002）．However，aPreViousreport
demonstratedthatbothformSOfNPWsuppressfasting−inducedfoodintakeinthedarkphase，
indicatingthat the e鮎ct ofNPW onfeeding difftrs according to whetheranimals are
maintainedinalightordarkphase（Mondaletalリ2003）．Thephysiologicalsignificanceand
mechanismsunderlyingtheseefftctsarestillnotclear．
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ItshouldbenotedthatNPBWRlisexpressedinthehypothalamuS，includingthePVNand
SuPraOPticnuclei（SON），andthearcuatenucleus（ARC），andthattheseareascontainneurons
WhichregulateanteriorpituitaryhormOnerelease（Singhetal．，2004；Kitamuraetalり2006；
Skrzypskietal．，2012）．Moreover，NPBWRlisabundantlyexpressedinthecentralnucleusof
theamygdalaandbednucleusofthes廿iaterminalis，WhichisinvoIvedintheregulationof
StreSSandemotion（Leeetal．，1999；Tbnakaetal．，2003）．Fromtheseobservations，NPBWRl
maybeinvoIvedinrespondingtostressviatheHPAaxis（Mazzocchietal．，2005；Niimiand
Murao，2005）．In addition，thei．c．Ⅴ．infusion ofNPWslightly elevates plasmalevels of
growthhormOne and signincantlyincreasesplasmalevels ofcorticosterone（Bakeretal．，
2003；SamSOnetal．，2004）．Theintraperitoneal（i．p．）iniectionofNPWalsoincreasesplasma
1evels ofcorticosteroneand adrenocorticotropic hormOne（ACTH）（Hocholet al．，2007）．
TheseobservationssuggestthatNPW sactionisrelatedtotheactivationoftheHPAaxis
（Bakeretal．，2003；Brezillonetal．，2003；Samsonetalリ2004；Tayloretal．，2005；Sekietal．，
2008）
lmmunOhistochemistryexperimentshaveshownthatNPW−COntainlngneurOnSareWidely
distributedintheratbrain（Dunetal．，2003；「mkenoyaetal．，2010）．Wehaverecentlyshown
thatNPWmRNAisexpressedintheratPVN）ventromedialnucleus（VMH），ARCandlateral
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hypothalamus（LH）（Takenoyaetal．，2010）．ManyNPW−COntainingaxonterminalshavealso
been observedinthe PVN（Dun etal．，2003；Thkenoya et al．，2010）．In addition，
CRH−immunOreaCtive neurons are abundantly expressedinthe PVN．Based onthese
Observations，WehypothesizedthatNPW−COntainlngneurOnSinthePVNmayinl1ibitfood
intakeinamaTmermediatedbyCRH−COntainlngneurOnS．Tbtestthishypothesis，WeStudied
interactions between NPW− and CRH−COntainlng neurOnS by uslng mOrPhologlCal，
PhysiologicalandmolecularbiologymethodstodeterminewhetherNPWdirectlyafEbctsfood
intakeviaanactiononCRHneuronsinthePVN．
Materials and Methods
．加わM／∫
AdultmaleC57BL／6NCrmicewerepurchased倉omSankyoLabServiceCorporation，Inc
（Tokyo，Japan）．Animalshadadlibiiumaccesstostandardchowandwaterandwereho11Sed
Ona12：12−hlight−darkschedllle（1ightsonat8a．m．ando董fat8p．m．）withtheambient
temperaturemaintainedat23j＝20C．AllexperimentalprotocoIswerereviewedandapproved
bytheInstitutionalAnimalCareandUseCommitteeofShowaUniversity．
⊥ 2 3 4 5 6 7 8 9 0 ュ 2 3 4 5 ′b 7 8 9 0 ⊥ 2 3 4 5 6 7 8 qノ 0 ⊥ 2ユー・4 5 6 7 8 9 0 1t 2 3 4 5 6 7 8 9 0 ⊥ 2 3 4 5 6 7 8 9 0 ュ 2 3 4 5 ュ ュ ュ ュ ュ ⊥ ュ ⊥ ⊥ ⊥ 2 2 2 2 2 2 2 2 2 2 3 3 3 3 3 3 3 3 3 3 4 4 4 4 4 4 4−4 4 4 5 5 5 5 5 5 5 5 5 5 6 6 6 6 6 6
Cb乃77〃ねfわ乃
Animalswereanesthetizedwithani，P．iniectionofsodiumpentobarbital（So皿OPentyl）
（50mg此gbodyweight；Kyoritsu Seiyaku Corp．，Tbkyo，Japan），Placedina stereotaxic
apparatus，andimplantedwithaguidecannula（extemaldiameter，0．5mm；length7．2mm）
throughtheskullthatreachedthelateralventricleofthebrain．Stereotacticcoordinateswere
0．2mmposteriortobregma；1．1mmlateralfromthemidline；and2．2mmbelowtheouter
Surfaceoftheskull，aCCOrdingtotheatlasofFranklinandPaxinos（16）．Forallcannulations，
theincisorbarwassetat2．OmmbelowtheearbarS．Theguidecannulawassecuredbydental
CementOnthedorsalsurfaceofthesku11．Aftersurgery，aStyluswasinsertedintotheguide
Cannulatopreventocclusion．AninjectorcarLnula（extemaldiameteち0．2mm）wasdesigned
tofitflushwiththeendoftheguidecannula．Theanimalswerehousedinindividualcages
with丘■eeaCCeSStOfoodandwater．Fo1lowlngSurgery，theywereallowedtorecoverfor7days
andwerehandleddailyduringthattimetomimimizenonspecificstressrelatedtotheiIUeCtion
PrOCedure．Verification of carulula placement was performed7days after surgery by
monitoringthewaterintakeofmiceinresponsetoani．c．v．inbsionofangiotensinII（50ng／2
ドL）（SigmaChemicals，StLouis，MO，USA）．Thosemicethatdidnotdrinkwaterwitl血5
minoftheangiotensinIIinJeCtionwereexcludedfromthestudy．Animalswereallowedto
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recoverfor7daysa氏erthisiTgeCtiontoru1eouttheactionofanybioavailableang10tenSinII
OnSllbsequentexperimentalprocedures．
助曙加Jp川Ceゐ作∫α〃dα（加If〃≠∫frαJわ〃qr〃P肝
Animalswereanesthetizedwithpentobad）italsodium（50mg／kgbodywti．p．injection）and
thenplacedinastereotaxic丘ame．Two stainlesssteelanchoringscrewswere丘Ⅹedtothe
Skull，andthe cannula was securedinplaceby acrylic dentalcement．Theanimals were
thenretumedtotheircagesandallowedtorecoverforatleast7days．Theywerethenhandled
every day and housedin cagesbefore tlle Start Of the experiments．Fori．c．v．
administrationofNPWor vehicle，a Stainless steel叫eCtOr WaSintroducedthrough the
Cannula at a depthofl．O mm beyond theend ofthe guide．The totalvolume of
11月eCtedsolutionofNPWandsalineintothelateralventriclewas2LLl．NPWwaspurchased
丘omthePeptideInstitute（MinohCo，Japan）anddissoIvedinpyrogen−freesteri1eO．9％saline
SOlution（OtsukaPharCo．Japan）．WeadministeredNPWintheanimalsatthetimeofonsetof
darkphaseat6p．m．
The animals used forin silu hybridization histochemistryfor CRHmRNA were
decapitated6hours af［erthei．c．v．administrationofNPW−30（2．8nmolノmouse）orvehicle
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（n＝5ineachgroup）to conscious mice．Brains were rapidly removedand placed on
POWdereddryiceforinsituhybridizationhistochemistryforCRHmRNA．
血∫gf〃砂占rgゐαgわ乃旭加ゐe∽ねfワカrC凡打〃1尺M．
1nsituhybridizationhistochemistrywasperformedon鉦ozen12−Pm−thickcoronalbrain
SeCtions cuton a cryostat at−200C，thawed，and mounted ongelatin／chrome alum−COated
Slides．Braintissuewas stored atT80OCbefbre cutting．Sections containlngthePVNwere
Chosenfromplate18intheatlas．TbnsetsoftwosectionscontainingthePVNwereusedfrom
each mouse to measure the denslty Ofautoradiography．Slides were warmed to room
temperature，allowedtodryforlOmin，andthenfixedin4％血rmaldehydeinPBSfor5min．
TheywerethenwashedtwotimesinPBSandincubatedinO．9％NaCIcontainlngO．25％
aceticanhydride（VOl／vol）andO．1Mtriethan01amineatroomtemperatureforlOmin．The
SeCtionsweredehydratedusingaseriesof70％（1min），80％（1min），95％（2min），andlOO％
（1min）ethan01sol11tionsconsecutivelyanddelipidatedinlOO％chloroformfbr5min．The
Slideswerethenpartial1yre】1ydratedfirstinlOO％（1min）andthenin95％（1min）ethanol
andallowedbrieflytoair−dry．
Hybridizationwasperfbrmedat370Covernlghtin45−Plofbu脆rsolutionconsistingof
2
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5∈‡59 60 61 62 63 64
50％fbrmamide and4xsaline sodium citrate（SSC；1xSSC＝150mMNaCland15mM
SOdiumcitrate），COntaining500pghnlshearedsalmonspermI）NA（Sigma，St．Lollis，MO），
250Llg／mlbaker syeasttotalR凡A匹OCheMolecularBiochemicals，Mannhelm，Germany），
1xDenhardt s solution，and lO％dextran Sulfhte（500，000 MW；Sigma）．The
hybridizationwas performed under a Nesconlm（Bando ChemicalIMD，Osaka，Japan）
COVerSlip・A35S−3 −end−1abeleddeoxyoligonucleotidethatwascomplementarytotranSCripts
COding fbr
CRH（5 −CAGTTTCCTGTTGCTGTGAGCTTGCTGAGCTAACTGCTCTGCCCTGGC−3
）was used as a speci6c probe．The specificity of tlle PrObe has beendescribed
previously（Harbuzetal．，1993）．AtotaloflxlO6cpm／slideforCRHanti−SenSetranSCripts WaSuSed．Af［erhybridization，thesectionswerewashedforlhinfourseparatelxSSCrinses
at550C and fbran0therhourin two changes oflxSSC at room temperature．All
illdependentexperimentalsections were treated simultaneously tomimimizethevariable
e鮫ctsofhybridizationandwashstringency．HybridizedsectionscontainlngthePVNwere
apposedtoautoradiographyfilm（Hyperfi1m；Amersham，Buckinghamshire，UK）for5daysto
detectCRHtranscrlPtS．Theautoradiographicimageswerequanti董iedusingaMCIDimaglng
analyzer（ImagingResearch，St．Catherines，Ontario，Canada）．Theimageswerecapturedbya
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Chargecoupleddevicecamera（DAGE−MTI，MichiganCity，IN）withx40magni点cation．The
mean optical density（OD）of autoradiographs wasmeasured by comparison with
SimultaneOuSlyexposed14Cmicroscalesamples（Amersham）．TlleStandardcurvewasRtted
bytheODofthe14Cmicroscaleonthesame創m．
C功∫叩erf∽e〃お
Two nmolofNPW30（PeptideInstitute，hc．）orsalinewasi．c．Ⅴ．administeredto ad
libitum鈷dmice（n＝4／group）beforeonsetofthedarkphase（1945h）．Ninetyminafterthe
lIt）eCtion，mice were perfusedand fixedwith4％paraformaldehyde／0．01M PBS under
aneSthesia・Brains were removed and20pm−thick鮎e−nOating sections wereprepared・
Sectionswereblockedfor60mininphosphate−bu飴redsaline（PBS）contaiI血glO％normal
horseserum（VectorLaboratories，Inc．，Burlingame，CA，USA）．Then，inordertodetectc−Fos
immunoreactivity，thesectionswereincubatedovemightwithanti−C−Fosantiserum（1：20000，
Ab−5；OncogeneResearchProducts，Cambridge，MA，USA）at4OC，WaShedthreetimeswith
PBSforlOmin，andincub．atedwithbiotinylatedanti−ral）bitIgG（1：400；DAKO，CarPinteria，
CA，USA）for2hatroomtemperature．ThesignalswereamplifiedbyABCkit（Vector
Laboratories，Inc・），andvisualizedusingaperoxidasesubstrate（diaminobenzidine；DAB）kit
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（Vector Laboratories，Inc．）following washes withPBS．A鮎r dehydration，the mounted
SeCtions were coverslipped with malinol（Muto Pure Chemicals Ltd．，Tokyo，Japan）．
C−Fos−1ikeimmunoreactivity（c−Fos−LI）wasdetectedwithanOPticalmicroscope（PROVIS，
01ymPuS，Tbkyo，Japan）．Asacontrol，theproceduredescribedabovewasperformedwiththe
Prlmaryantibodystepomitted．Nospecificimmunoreactivitywasobservedinthesecontrol
SeCtlOnS．
加〟あね∫∽m〃〃q伽0柁∫Ce乃Ce∫fα∫刀加g〟∫わIgの痛−CタbぷαJ7dα乃fゴーC足花α〃fゴーVα∫甲柁∫∫f〝α〃d
α〃Jゴー叩血血即戒加肋
TwonmolofNPW300rSalinewereadministeredi．c．v．toadlibitumftdmice（n＝4／group）
beforetheonsetofdarkphase（1900h−2000h）．Ninetyminaftertheirtjection，aneSthetized
micewereperfusedand丘xedwith4％paraformaldehyde／0．01MPBS．Brainswereremoved
and5一皿−thickparaffinsectionswereprepared．Brainsections鉦omsaline−andNPW−treated
animalswerecarefu11ymatchedunderamicroscopeaccordingtotheshapeofbrainstruCtureS
inimmunOhistochemicallystainedsections．A丑erbeingdeparafnmizedandrehydrated，the
SeCtionswereblockedinPBScontaiminglO％normalhorseserum（VectorLaboratories，hc．）
andthenincubatedwithgoatanti−C−Fosantibody（1：1000，SantaCruZBioteclmology，hc．）
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⊥ 2 つ︺一4−﹁⊃ ′0 7 8 q′ 0 ュ つ一3 4 5 ′b 7 nO qノ 0 1﹂ 2 3 4 5 6 7 ︵8 q′ O 1 2 3 4−5 ′b 7 8 9 0 1︼ 2 3 4 E﹂ 6 7 8 9 0 1t 2 3 4 5 6 7 8 9 0 ュ 2 3 4 5 1■ ュ ー ュ ⊥ ⊥ ュ ⊥ ⊥ ュ 2 2 2 2 2 2 つq 2 つつ血 3 3 3 3 3 3 3 3 3 つ﹂ 4 4 4 4 4 4 4−4 4 4 5 5 5 5 5 5 ■b 5 5 5一b 6 6 ′b 6 6
OVemightat40C，fbllowedbyincubationwithAlexaFluor488−COqugateddonkeyanti−gOat
IgG（1：400，Invitorgen，Carlsbad，CA）forl．5hatroomtemperature．Next，thesectionswere
incubatedwithrabbitantiTCOrticotropinreleasingfactor（CRF）antibody（1：500，Advanced
TargetingSystems，SanDiego，CA），guineapiganti−VaSOPreSSinantibody（1：500，Peminsula
Laboratories LLC，San Carlos，CA）or rabbit anti−OXytOCin antibody（1：400，Incstar
Corporation，Stillwater，MN）ovemightat40C．A氏erwashingwithPBS，thesectionswere
incubatedwithAlexaFluor568−1abeledgoatanti−rabbitIgGorAlexaFluor546−labeledgoat
anti−guineapigIgG（1：400，Invitrogen）forl．5hatroomtemperature．Doubleimmunostained
SeCtionswereobservedwiththeaidofaconfocallasermicroscope（AIsi，NikonCo．，Tokyo，
Japan）．
e〟α〃f折cα∫わ乃げc一凡∫−Jg鮎血′ 〟〃0柁αC伽卸α〝（仇）rCR〃」J7鮎7∽∽〟乃0柁αC如砂
Brainsectionsfrom saline−andNPW−treatedmimals werecarefu11ymatchedundera
microscopeaccordingtotheappearanceofbrainstruCtureSinimmunohistochemicallystained
SeCtions・Sections were selectedataround O．50mmposteriortothebregma．Theareain
WhichCRHimmunoreactivitywaspresentwasde丘nedasthePVN．lmages丘omtheseiected
twosectionspermouse（n＝4／group）werecapturedusingaconfocallasermicroscope（AISi，
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Nikon，Tbkyo，Japan）．haddition，thenumberofc−Fos−POSitiveorCRH−POSitivecellsinthe
PVNwascollntedintwosections倉omeachgroup．
EJecfro乃〃lgCrO∫CWOあ∫e川αfわ乃∫
Forelectronmicroscopyexperiments，Vibratome−CutSeCtions（40−50mthickness）thathadbeen
丘xedasdescribedabovewereincubatedinnormalhorseseruln（1：20）fbr20min，andthenin
mouseanti−NPWantiserum（dilutedtol：2000）for2hatroomtemperature，fo1lowedbyovemight
incubationat4OC．Thefb1lowlngday，SeCtionswereincubatedwithbiotinylatedanti−mOuSeIgG
（Lametal．201l．）forlhandthenwithABCfor45minatroomtemperature（VectastainElite
ABCkit，VectorLaboratories）．Thesectionswerethentreatedforabout3mininthedarkwith
diaminobenzidine（DAB）in O．05M TrisLHCl（pH7．6）buf臨r containing O．005％hydrogen
PerOXide．A氏erthe DABreaction，SOmeOfthe sectionswerefurthertreatedwithsilver−gOld
intensification（Guanetal．2001）．Thesesectionswerepost一触edwith1％OsO4inO．1MPB（pH
7．4）fbrlhat40C，dehydratedinagradedethan01seriesandthenembeddedinamixtureof
Epon−Araldite．Ultrathin sections werethen cutand examined byuslng an HitachiH−7000
electronmicroscope．Thespeci王icityoftheNPWantiserumuSedinthisstudyhasbeenreported
elsewhere（Dateetal．2010；Takenoyaetal．2010）．
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ュ 2 3 4 5 6 7 8 9 0 ⊥ 2 3 4 S 6 7 8 9 0 ュ 2 3 4 5 6 7 8 9 0 ⊥ 2 3 4 5 6 7 8 9 0 ⊥ 2 3 4 5 6 7 8 9 0 ⊥ 2 3 4 5 6 7 8 9 0 ュ 2 3 4 5 ⊥ ュ ⊥ ⊥ ュ ⊥ ⊥ ⊥ ュ ⊥ 2 2 2 2 2 2 2 2 2 2 3 3 3 コ コ 3 3 3 3 3 4 4 4 4 4 4 4−4 4 4 5 5 5 5 5 5 5 5 5 5 6 6 6 6 6 6
ダeed7乃gゐeろαγわr
Cannulatedmicewereindividuallyhousedincages（dimensions：240mmwide，290mm
long and300mm height）and habituated to fteding behavior measuring equipment
（ACTIMO−100，Shinfactory Co．，Ltd．，Fuknoka，Japan）for approximatelylweek．Food
intake，Waterintal（eWerereCOrdedatl−minintervals．Foodintakewascalculatedaccordingto
theweightchangeofthechowbox．Waterintakewasmeasuredusingadropsensor．Saline（2
LLL）wasi．c．v．administeredtoadlibitum氏dmice（n＝3／group）beforeonsetofdarkthephase
（1945h），andfoodintake，WaterintakeandlocomotoractivityweresimultaneOuSlyrecorded
for24h．Bodyweightwasmeasured24haftertheiqection．Onedaylater，thesameanimals
Werei．c．v．infusedwith NPW30at a dose of2nmol（2pLoflnlnOl／pL）．Similar to
proceduresfo1lowed afterthe salinelrUeCtion，foodintake，Waterintake andlocomotor
activitywererecordedfbr24h．Bodyweightwasmeasured24haftertheNPW30irtiection．
聯cね府相加α加e乃fw油CR仔α〃ぬgo乃ね′画一ゐeJ∫cαJ−C足印oJl♪od油αおα乃dゐ0卸weな加
汗仁＼丁リー二さJさJざざ∴．f招∫、・．・
Animals（11−Week−01dmaleC57BL／6Jmice）thatpassedtheangiotensinⅠIdrinkingwater
test（n＝6）werepretreatedwiththeCRHantagonista−helical−CRH（1nmol）insalineirtiected
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6：≧
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i・C・V．intoeachlateralventricleat1830h．Thirtyminuteslater（1900h），NPW（1nmol）was
infusedi．c．vinto eachmouse．Cumulativefoodintake，bodyweightand ablood−glucose
1evelsweremeasuredafter24h．
、†い∫＝さい・／〃㍉小し、
Di飴rencesbetweengroups（means土Sem）wereanalyzedbyANOVÅandposthocFisher s
test．Pvalueslessthan0．05wereconsideredtoindicatesignificance（two−tailedtests）．
Res111ts
、＼Jリlィ…川J山血ぷ一廿〃…Jヾ〟仙J＼、Jりl、…〟．＼二Il・．＼丁り・ ・ヽヽ山JI山一加Jり、、＼
Atthelightmicroscopiclevel，NPW−COntainingnervePrOCeSSeSandtheirterminalscould
beidentifiedinthemouse PVN（Fig．1A）．1nsituhybridizationhistochemistryonbrains
excised90minafterthei．c．v，administrationofsalineorNPWshowedincreasedCRHmRNA
expressioninNPW−infused mice compared to control（Fig．1B，C）．Average CRH probe
bindingactivity（arbitraryunits）wasalsoincreasedinNPW−treatedanimalscomparedto
COntrOl（Saline−treated）animals（N＝5，P＜0，01）（Fig．1D）．
16
ュ 2 3 4 5 6 7 8 9 0 ュ 2 3 4 5 6 7 8 q一︵∪ ⊥ 2 3 4 5 6 7 8 9 0 ⊥ 2 3 4 5 6 7 8 9 0 ュ 2 3 4 5 6 7 8 9 0 ュ 2 3 4 5 6 7 8 9 0 ュ 2 つ一4 5 1 ュ ュ ュ ュ ュ ュ ⊥ ⊥ ⊥ 2 2 2 2 2 2 2 2 2 2 3 3 3 1J 3 3 3 3 3 3 4 4 4 4 4 4 4 4 4 4 5 5 5 5 5 5 5 5 5 5 6 6 6 6 6 6
九お川C〟〃Jl∂g加■ggJ‡∧p隣〟J‡〟CRガニc〃〃ぬ加加gJ‡e〟r〃那加班gタI′Ⅳ
Light microscopeimages ofNPW−and CRHTCOntainlngneurOnSinthe PVN clearly
ShowedNPW−COntainlngaXOnterminalsinclosecontactwithCRH−COntainlngCellbodies
and dendritic process（Fig．2A，B）． At the electron microscopelevel，
dollble−immunOlabelingshowedNPW−COntainlngaXOnterminalsinthePVNmakingsynaptic
COntaCtSwithCRH−COntainingdendriticprocesses（Fig．2C，D）．Thesesynapseswerefbund
tobeofboththesymmetrical（Fig．2C）andasymetrical（Fig．2B）type．
い∩一．ヾl叩一・l・．ヾ∫山ノ目′IJhリリ1一＝一J．…、＼、■け山・一J一丁川血八什−JJJり〃−！ノー＼1一軒
Thei．c．v．admimistrationofNPWinducedanincreaseinthelevelofc−Fosexpressionin
thePVN．We examinedthenumberofc−Fbs−immunoreactiveneuronsinthePVN90min
aftertllei・C・V・infusionofsalineorNPW，andfollndthat，COmParedtocontrol（saline）group
（Fig・3A），NPWinfusionledtoasignificantincreaseinc−FosexpressioninthePVN（Fig．
3B）・C−Fos−immunoreactiveneuronswerebothfoundinthemagnocellularandparvocellular
divisionsofthePVN（Fig．3B）．Smallnumbersofc−Fosirrmnoreactiveneuronswerealso
ObservedintheSONofbothcontrol（Fig．3C）andNPW−treated（Fig．3D）animals．0vera11，
anaverageof46・3j＝7・4and163．3j＝24．3c−Fos−immunopositiveneuronswereseeninthePVN
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incontrolandNPW−injectedmice，reSPeCtively，WhereasintheSONanaVerageOf12．7土4．4
and46．7j＝17．6，reSPeCtively，WereObserved（Fig．3E）．
〃仙JJ山川川J＝／7…り・ハl・川l・l・ノiり・l・−n一．ヾ ■仙J（、〟Jト、−・．1二l仙イI卜 川・l・廿…Jり・ハヽ山 一／恥
山川＝J…r仙・丁目Jん●＼ ＝ Jィ肌＝J坤・ JJんJI−小・−n一、ヾ−J丑一用仙J…・（1ⅣJトJ砧一山川＝仙肌・仙心吊Jムーヾ
Tbidentify the target neurons of NPW，We Subsequently performed double
immunofluorescence experiments for cTFos and CRH−，OXytOCin or vasopressin
immunoreactivityinthemousebrain（Fig．4）．Asshownabove，COmParedtosaline−irtjected
brains，thei．c．v．infusionofNPWledtoasignificantincreaseinthenumberofc−Fos−1ike
immunoreactiveneuronsinthePVN（Fig．3）．Delete：WhileincreasedCRHimmunoreactivity
WaSClearlyapparentintheparvocellularpartofthePVN（Fig．2）．Wefoundthatthei．c．Ⅴ．
lqeCtionofNPWsigni丘cantlyelevatedthenuniberofneuronsco−eXpreSSlngC−FosandCRH
inthePVNcomparedtocontrol（Fig．4A，B）lncontrast，doubleirrmno且uorescencestudies
employingantibodiesagainstc−Fosandoxytocin（Fig．4C，D）orc−Fosandvasopressin（Fig．
4E，F）showed that a few number of neurons inthe PVN displayed overlapping
immunoreactivity．
F／汀l・J・イ人l・㍑山／iハん＝イ、ヽ7川■り〃ノi仙J山川人l・一＝t川川小机七山
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WeexaminedtheefEbctofNPWonfoodintakeinthedarkphase．Here，NPWadministered
i．c．v．sigmificantly reducedfoodintake，body weightand waterintake compared to
Saline−treatedanimals（＊P＜0．05Vehiclevs．NPWicv；Student sl−teSt）（Fig．5）．
左肺・l・J・・ハ′（WJJ …叫￥小〃山〃〃＼J川一川小山山侶／ノi・・一−／山川Al・．ん一吋…丞巾l…lJ砧仙JgJ仇・〟仙・
Jピl・どJ
Wenextexaminedwhethertheanorexlgenice董艶ctofNPWwasmediatedviaanaCtionon
CRH neurons．Body weightandfoodintake was compared tothe previous day−slevel．
AnimalswerepretreatedwiththeCRHantagonistα−helical−CRH（1nmol）orsalineinjected
intoeachlateralventricleinthedarkphase．Thirtyminuteslater，NPW（2nmol）wasinfused
（seemethods）．FoodintakeandbodyweightinthegrouppretreatedwiththeCRHantagOnist
Wereincreased compared withthe saline pretreated group（＊P＜0．05 saline＋NPW vs．
α−h−CRH＋NPW；Student s t−teSt）．hcontrast，bloodglucoselevelswerenotsignincantly
difEtrentbetweenthetwogroups（Fig6）．
DiscllSSion
NPW，WhichwasidentifiedasanendogenousligandfbrtheGPR7andGPR8receptors，lS
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expressedinthehypothalamicreglOnOfthemousebrain．AsGPR7knockoutmicehavebeen
Showntoexhibithyperphagicbehavioranddecreasedenergyexpenditure，thiscouldsuggest
thatNPWactsasamodulatorofftedingactivityPreviousreportshaveshownthatthecentral
administration ofNPWincreases fbodinlake duringthe Brst2hrsin thelight−Phase
（Shimomuraet al．，2002），and Levine etal．（Levine et al．，2005）similarlyreportedthat
1qeCtionofNPWintothePVNofratsalsoincreasedfoodintake．Incontrast，Mondaletal．
（Mondaletal・，2003）reportedthatNPWsuppressesfoodintakeinthedark−Phase（fbeding
Phase），reducesfhsting−inducedfbodintake，andthatsuppressesbodyweightgain．Inthe
PreSelltStudy，WeCOn茄rmedthatthei．c．v．1nJeCtionofNPWdecreasesfoodintakeinthedark−
Orfteding−Phase（Fig．5），WhichperhapsindicatesthatendogenollSNPWexertsbothcatabolic
andanabolicfunctionsdependingonwhethertheanimalistreatedinthedarkorlightphase．
NPW−1ikeimrrrunoreactivlty WaS particularly abundantin the PVN and SON of the
hypothalamuS（Tbkenoya et al．2010）．In addition，NPW mRNAis expressedinthe
hypothalamus，Pituitaryglandandadrenalgland（1払Ilal（aetal．2003；Kitamuraetal．2006；Seki
etal．2008；Tbkenoya2010）．Baker et al．reported thati．c．v．infusion ofNPW pelevated
PrOlactinandcorticosteroneintherat（Shimomuraetal．，2002；Bakeretalり2003），While
NPWsignincantlyaltersprolactin，grOWthhormoneandACTHreleasefromdispersedrat
20
ュ 2 3 4 5 6 7 8 9 0 ⊥ 2 3 4 5 6 7 n0 9 ︵U ュ 2 3 4 5 6 7 ■H▼ ︵プ 0 ュ 2 ■J 4 5 6 7 8 qノ ︵∪ ュ 2 3 4 5 6 7 0︶ 9 0 ⊥ 2 34 5 6 7 8 9 0 ュ 2 3 4 5 ⊥ ⊥ 1▲ ュ ュ 1︻ ュ ュ ⊥ ⊥ つ一2 2 つ一2 2 2 2 つ〃 つ﹈ 3 3 3 3 3 3 3 3 つ﹂ つ︺ 4 4 4 4︻ 4 4 4 4 4︻ 4 5 5 5 5 5 5 ■⊃ 5 5 5 ′b 6 ′○ ′0 6 6
anteriorpituitaryce11sinvitro（Bakeretal．，2003）．haddition，Yogoetal．，havereportedthat
i．c，V．in魚ISion ofNPW stimulates ACTH secretionfromthe pituitary gland，andthat
Pretreatmentwithavasopressinreceptorantagonistdoesnotinhibitthise飴ct（Yogoetal．，
2012）・ThesedatasuggestthatAVPisnotinvoIvedintheNPW−mediatedincreaseinplasma
ACTH・Rather，ithasbeensuggestedthatNPWactivatestheHPAaxisandplaysanimportant
roleinthehypothalamicresponsetostress（Niimietal．2005；Tayloretal．2005；Becketal．
2010）．Tbthis end，hypothalamic CRH−COntaining neuronsarelocalizedinthe medial
ParvOCe11ularpartofthePVNandmodulatetheHPAaxisresponsetothestress（Ⅵ11eetalリ
1981）・GPR7mRNAhasbeenpreviouslydetectedintheparvOCellularpartOfthePVN（Lee
etal．，1999），andithasbeenshownthatNPⅥLimmunOreaCtiveneuronalcellbodiesandfibers
are presentin that brainregion（Dun et al．，2003；Takenoya，2010）．Moreover，both
NPW−immunOreaCtivecellbodiesandNPBWRlhavebeendetectedintheparvOCe11ularpart
OfthePVN（Kitamuraetal．，2006）．
Furthertotheabove，CRHplaysanimportantrOleinthestressresponseandafftctsfbeding
behavior（Drescheretal．，1994）．SeveralstudieshaveassessedtheinhibitoryroleofCRHon
foodintake（Araseetal・，1989；Eralmetal・，1990）．IthasbeenproposedthatCRHis
released缶omthenerveterminalsintheARC，therebyinhibitingNPY／AGRPneurons；Onthis
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basis，ltWaSSuggeStedthatCRHisnormal1yresponsibleforstimulatlngftedingbehaviorand
SupPreSSing energy expenditure a鮎r acute stress（Richard et al．，2002）．Furtherto this，
Pretreatmentwith（エーh−CRH，administeredintravenously，attenuatedthe abilityofNPWto
increasetheplasmaconcentrationofcorticosterone（Tayloretalリ2005），aSterOidhormOne
intimatelylnVOIvedinthestressresponse．
Inthepresentstudy，WefoundthatinfusionofNPWintothemousebraininduceda
Signi負cantincreasein c−Fos expressioninthe PVNand SON．0therinvestlgatOrS have
reportedsimilarreSults（Levineetal．，2005；NiimiandMurao，2005；Kawasakietal．，2006）．
While c−Fosimmunoreactive neurons were previously reported to be distributedinthe
ParVOCellularandmagnocellularpartsofthePVN（Mourietal．，1993），thepresentstudy
COnfirmed血isandclearlyshowedthatmanyc−Fosimmunoreactiveneuronswereco−hbeled
WithCRIlneurons．hthisway，doubleimmunostainlngeXPerimentsshowedco−1abelingof
neuronswithanti−C−Fosandanti−CRHantibodiesintheparvOCe11ularpartofthePVN，but
lesssowithanti−AVアandanti−OXTantibodies．
In additiontoNPW，NPB alsobinds to GPR7and GPR8．NPB−regulatedfoodintakeis
mediatedbyCRF activity（Aikawa etal．，2008），implyingthatftedingregulationbythe
NPW／NPBsystemsiscloselyrelatedtoCRFactivity．Theseresultssuggestthattheroleof
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⊥ 2 3 4 5 6 7 8 9 0 ⊥ 2 3 4 5 6 7 8 9 0 ⊥ 2 3 4 5 6 7 8 9 0 ⊥ 2 3 4 5 6 7 8 9 0 ⊥ 2 3 4 5 6 7 8 9 0 ュ 2 つJ4 5 6 7 8 9 0 ⊥ 2 3 4 5 ⊥ ュ ュ ー ュ ュ ⊥ ュ ュ 1 2 2 2 2 2 2 2 2 2 2 3 3 3 3 3 3 3 3 3 3 4 4 4 4 4−4 4 4 4 4 5 5 亡コ 5 5 5 5 5 5 5 6 6 6 6 6 6
NPWinftedingregulationcouldbepartlythroughtheactionofCRH，andthatthemain
targetofNPWcouldbeCRH−COntainingneurons．Kawasakietal（Kawasakietal．，2006）
reportedthati．c．vinfusionofNPWinthelightphasedemonstratedthecoexistenceofc−Fos
andmagnocellularAVP／OXTneuronsinthePVNand SONintherat．h addition，they
reportedthatcentraladministrationofNPW30causesasignificantincreaseinplasmaAVP
andOXTlevelsinconsciousrats（Kawasakietal．，2006）．Howeverinthepresentstudy，We
foundtllataSma11numberoftheseneuronsdidnotrespondtoNPWadmimistrationinmicein
thedarkphase．Thediscrepancyintheseresultsmayduetothedi脆rentanimalstrainsused
andwhethertheNPWinfusiontakesplaceinthedarkorlightphase．
AtanultrastruCturallevel，OurimmunOhistochemicalstudyenabledustoobservethatmany
NPW−iIrLmunOreaCtive丘berswereincloseappositionwithCRH−immunOreaCtivece11bodies
anddendriticprocessesinthePVN．Moreover，NPW−POSitivenerveteminalswerefoundto
makeaxo−SOmaticandaxo−dendriticsymaPtlCCOntaCtSwithCRH−COntainlngneurOnSinthis
brainreglOn．WepreviouslyreportedtherelationshipbetweenNPW−COntainlngneurOnSand
0ther peptide−COntainmg neuronsinthelateralhypothalamus ofthe brain，along with
NPW−COntainlngnervefibersbeingindirectcontactwithorexin−Ormelanin−COnCentrating hormOne−COntainingneuronsintherat（Thkenoyaetal．，2005）．NPW，COntaimingneuronshave
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10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64
alsobeens】10WntOPrq］eCttOPOMC−andNPY−COntainlngneurOnSintheARC；inthisway，
loosepatchextracellularreCOrdingsshowedthattreatmentwithNPWinhibitstheBringof
NPY−COntainlngneurOnSanddecreasesthefrequencyofspontaneOuSinhibitorypostsynaptlC
CurrentSinPOMC−COntainingneuronsintheARC（Dateetal．，2010）．Thus，NPWmayinhibit
foodintakeandbodyweightgainthroughatleasttwopathways，Onethatactsviainhibition
OfNPY−COntainlngneurOnSandstimulationofPOMC−COntainlngneurOnS，andtheotherthat
actsbystimulatingaCRH−mediatedpathway．
Incontrasttothis，Ybgoetal（Yogoetal．，2012）reportedthatpretreatmentwithaCRF
receptor antagonistinl1ibited theincreasein plasma ACTHlevelsinduced by thei．c．v．
administrationofNPW，SuggeStingthatcentral1yappliedNPWactivatestheHPAaxisby
activatinghypothalamicCRF．
WealsodemonstratedherethattheCRHantagonistα一h−CRHaffectsNPW−inducedfood
intakeandbodyweight，butnotbloodglucoselevels．Tbkentogether，theseresultsindicate
thatNPWmay，viatheHPAaxis，mediatestressresponsesandcontrolfE，edingregulation，
albeitinacomplexma11nerglVenthatbloodglucoselevelsremainunafftcted．Thisfitswith
numerous reports showing that stress affbctsfoodintakein diffbrent ways，Withboth
increasedanddecreasedf岳edingbehaviordescribedinhumansandanimalsandinresponseto
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⊥ 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 ⊥ 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 ⊥ 2 3 4 5 6 7 8 9 0 1 2 つ一．4 5 1 1 工 l l ⊥ l ⊥ ⊥ 1 2 2 2 2 2 2 2 2 2 2 3 3 3 3 3 3 3 3 3 へj 4 4 4 4 4 4 4 4 4 4 5 5 5 亡つ 5 5 5 5 5 5 6 ′b 6 6 6 6
acuteorchronicstress（Martietal．，1994；Pecoraroetal．，2004；SchulzandLaessle，2012）．
Tbsummarize，WehaveshownthatNPWregulatesfeedingbehaviorviaanaCtiononCRH
neurons bydirect synapticinnervationthatresultsindecreased fbodintake andreduced
Weight．Itremainsunknown，however，WhyNPWdoesnotaffbctglucosemetabolism，thus
1eavingthisissueasanimportanttOpicforfutureresearch．
Ackmowledgment
ThisstudywassupportedinpartbygrantSfortheGrallt−in−AidforScientificResearch（C）
andGrant−in−AidforYoungScientists（B）丘omtheMimistryofEducation，Culture，Sports，
ScienceandTeclm0logyofJapan（toFT26350912，HK24590241，SH90644261）．Thisstudy
WaS SuPPOrtedin part by Grant−in−Aidfor chal1englng Exploratory Researchforthe
Grant−in−AidforScientificResearchfromtheMinistryofEducation，Culture，Sports，Science
andTechnologyofJapanOfJapan（toSS22126004）．Thestudywasalsosupportedinpartby
Grant−in−AidforChal1engingExploratoryResearchofJapan（toSS25670767）．
Figure legends
Fig．1．A：NPWLCOntainlngnervePrOCeSSeSareeVidentinthehypothalamicPVNofacontrol
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8
9
10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 2（I 29 30 31 32 33 34 35 36 37
3〔i
39 40 41 42 43 44 45 46 47
4∈‡49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64
（Saline−treated）mousebrain．B，C：EfEbctsofi．c．v，administrationofNPW（2nmol）orsaline
OnCRHmRNAexpressionasmeasuredbyinsituhybridizationhistochemistry．Compared
Withsaline−treatedanimals，CRHmRNAisexpressedmorestronglyinthePVNa氏erinfusion
OfNPW．D：SemiquantitativeanalysisofCRHmRNAexpressioninducedbyinfusionof
NPWversussaline；NPWhasasignificanteffectonexpression．
Fig．2．LightandelectronmicroscopICimagesofinteractionbetweenNPW−and
CRH−COntainlngneurOnSinthePVN．A，B：NPW−COntainlngaXOnterminalsmakingdirect
COntaCtwithCRH−COntainingneuronalcellbodiesanddendriticprocesses（arrOWS）．Scale
bar＝20um（A）andlOum（B），C，D：Synaptic（arrowhead）interactionbetween
NPW−COntaimingaxonteminals（N）withCRH−COntainingdendriticprocesses（C）．
Fig．3．E脆ctsofi．c．v．infusionofNPW（2nmol）orsalineonc−FosexpressioninthePVN
（A，B）andSON（C，D）．c−FosimmunOreaCtivityisstronglydetectedinthePVNaRerinfusion
OfNPWbutlessintheSON．A，C：Salineinfusion．B，D：NPWinfusion．E：NPWinfusion
SlgnificantlylnCreaSedthenumberofc−Fos−immunopositivecellsinthePVNandSON，but
moresointhePVN．Dataareexpressedasthemeanj＝SEM．Numberofc−Foswascounted
26
⊥ 2 3 4 5 6 7 8 9 0 ⊥ 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 ⊥ 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 ⊥ 2 3 4 5 ⊥ ⊥ ュ ュ ュ ⊥ ュ ⊥ ⊥ ュ 2 2 2 2 2 2 2 2 2 2 3 3 3 3 3 3 3 3 3 3 4 4 4 4 4 4 4 4 4 4 5 5 5 5 5 5 5 5 5 5 6 6 6 6 6 6
bynumberofce11insection（Cells／section）
Fig．4A，B：RepresentativeiInmunOnuOreSCenCePhotomicrographsshowingc−Fosexpression
inCRH−1ikeimmunopositiveneuronswithinthePVNfo1lowlngl．C．V．11月eCtionofNPW．
Dualimmunostainingofc−Fos（green）andCRH（red）inneuronsofmiceinfusedwithvehicle
（A）orNPW（B）infusion．c−Fos−eXpreSSingCRHneuronsinthePVNofNPW−irdected（2
nmol）mice．Scalebaris20pm．C，D，E，F．：Representativeimmunonuorescence
PhotomicrographsshowingcTFosexpressioninoxytocin−（OXT；C，D）andvasopressin（AVf；
E，F）−1ikeimmunopositiveneuronswithinthePVNfo1lowingi．c．v．irtjectionofNPW．
Fig．5．Foodintake，Waterintakeandbodyweight24ha鮎ri．c．v．administrationofNPW30
（2nmol）at1945h．ControlmiceweregivenO．9％saline（n＝4／group）DataareeXPreSSedas
themean土SEM．＊p＜0．05vssalinegroup．
Fig，6．B飴ctofα−h−CRHonNPW−inducedfoodintake，bodyweightandbloodglucose
glucoselevelsinthedarkphase．AnimalswerepretreatedwiththeCRHantagOnlstα−h−CRH
（1nmol）orsaline，and30minuteslateri．c．v，infusedwithNPW（2nmol）（n＝6）．Dataare
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10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64
expressedasthemeanj＝SEM．＊p＜0．05vssaline十NアWgroup．
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