Coaxiality of Foxb1- and parvalbumin-expressing neurons in the lateral hypothalamic PV1-nucleus

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Coaxiality

of

Foxb1-

and

parvalbumin-expressing

neurons

in

the

lateral

hypothalamic

PV1-nucleus

Alessandro

Bilella

a

_,

_Gonzalo

_{Alvarez-Bolado}

b

_,

_Marco

_R.

_Celio

a,∗

a_Anatomy_Unit,_Department_of_Medicine_and_Program_in_{Neuroscience,}_University_of_Fribourg,_CH-1700_Fribourg,_Switzerland b_Institute_of_Anatomy_and_Cell_Biology,_University_of_Heidelberg,_Im_Neuenheimer_Feld_307,₆₉₁₂₀_Heidelberg,_Germany

h

i

g

h

l

i

g

h

t

s

•Foxb1-expressingneuronslieinthelateralhypothalamicPV1-nucleus.

•CoaxialdistributionofFoxb1-andPV-expressingneuronsinthePV1-nucleus.

•Foxb1-expressingneuronsoutnumberPV-expressingonesinthePV1-nucleus.

•Onlyasmallproportionofthetwoneuralpopulationsco-expressbothmarkers.

In the ventrolateralhypothalamus, thePV1-nucleus is defined byits population of parvalbumin-expressing neurons. During embryogenesis, the ventrolateral hypothalamus is colonized also by Foxb1-expressingneurons.InadultFoxb1-EGFPmice,manyimmunofluorescentneuronswerefound withintheregionthatisoccupiedbythePV1-nucleus.Theyformedacloudaroundtheaxialcordofthe parvalbumin-immunopositivecells,whichtheygreatlyoutnumber(3:1).Onlyasmallproportionofthe neuronsinthePV1-nucleusco-expressedbothparvalbuminandFoxb1.Inthelightofthesefindings,a redesignationofthislateralhypothalamicstructureasthePV1-Foxb1nucleuswouldmoreaccurately reflectitsspecificbiochemicalproperties.

1. Introduction

Progress in the understanding of the lateral hypothalamic functionshasbeenhamperedbythescarcityofmarkersforthe taggingofitsspeciﬁcneuronalpopulations.Wehavepreviously described a novellateral hypothalamicentity, the PV1-nucleus

[1,2],whichisdeﬁnedbyitspopulationofparvalbumin-expressing

neurons.Itislodgedwithintheventrolateralsubareaofthelateral hypothalamus (LHVL) intermingled withthe axons of the ven-trolateralhypothalamictract(vlt)ofthemedialforebrainbundle

Abbreviations:f,fornix;LH,lateralhypothalamicarea;LHVL,ventrolateral sub-areaofthelateralhypothalamicarea;LM,lateralmammillarynucleus;MM,medial mammillarynucleus,medialpart;MP,medialmammillarynucleus,posteriorpart; Opt,optictract;PAG,periaqueductalgrey;vlt,ventrolateralhypothalamictract;3v, thirdventricle.

∗ Correspondingauthor.

E-mailaddress:marco.celio@unifr.ch(M.R.Celio).

[3,4],disposedinthehorizontalplane,andsandwichedbetween

theoptictractlaterallyandthefornixmedially[1,2].

Aroundembryonicday14.5,Foxb1-expressingneurons stem-mingfromtheperiventricularzonemigratetowardsthe ventrolat-eralhypothalamus.Foxb1isatranscriptionfactorandamember of the forkhead family of genes [5–9]. It was the aim of the presentstudytodeﬁnemoreaccuratelythenatureofthespatial relationshipexistingbetweenFoxb1-andparvalbumin-expressing neuronsintheregionofthePV1-nucleus.Usingadult Foxb1-Cre-EGFP mice, Foxb1-expressing neuronswere revealed to forma sleevearound,andtogreatlyoutnumber(3:1),theaxially orien-tatedparvalbumin-positiveones.

2. Materialsandmethods

2.1. Mice

FiveadultFoxb1-Cremice[8]wereusedinthepresentstudy. In this strain, Foxb1-expressing neurons co-express EGFP and

1

Published in 1HXURVFLHQFH/HWWHUV±

which should be cited to refer to this work.

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Cre-recombinase.Theanimalsweremaintainedona12-h light/12-hdarkcycleataconstanttemperatureof24◦Candfedadlibitum. ThestudywasapprovedbytheCantonalCommissionforAnimal Experimentation(permitnumber:201305FR).

2.2. Immunohistochemistry

Afterbeinganaesthetized(Pentobarbital;20␮l/10gperbody weight), themice wereperfused first withphysiological (0.9%) saline and then with phosphate (0.1M, pH7.3)-buffered 4% paraformaldehyde.Thebrainswereremoved,immersedovernight inthesamechemicalfixative,andthentransferredtoachilled(4◦C) 30%solutionofphosphate(0.1M)-bufferedsucrose,for cryopro-tection.Horizontal,40␮m-thickcryosectionswerepreparedusing afreezingmicrotome(FrigomobilReichert-Jung,Vienna,Austria) andcollectedin0.1Mphosphatebuffer(pH7.3).Free-floating sec-tionswereexposedfor1dayatroomtemperature(RT)toarabbit anti-EGFPantiserum[(LifeTechnologies,Japan,Ltd),diluted1:1000 inTBS+0.2%Triton-X100+10%bovineserum].Thesectionswere rinsedinTBSandthenexposedfor2hatroomtemperaturetothe anti-rabbitbiotinylatedsecondaryantibody[(VectorLaboratories, Burlingame,CA,USA),diluted1:200inTBS+10%bovineserum]. AfterrinsingonceinTBSandtwiceinTris–HCl(pH8.2),the sec-tionswereexposedfor2htoAlexa488-conjugatedstreptavidin [(JacksonImmunoResearchLaboratories,Inc.,USA)diluted1:200 in Tris–HCl(pH8.2)]. Allsectionswerethenexposed for1 day atroomtemperaturetoa primarymonoclonalantibodyagainst parvalbuminPV235[(Swant,Marly,Switzerland)diluted1:1000in TBS+10%bovineserum]andthentoananti-murineCy3antibody [(JacksonImmunoResearchLaboratories,Inc.,USA)diluted1:200 inTris–HCl(pH8.2)].Cellnucleiwererevealedbycounterstaining withDAPI(diluted1:2000inphosphate-bufferedsaline)for5min atroomtemperature.

ThesectionsweremountedandevaluatedineitheraLeica6000 epiﬂuorescencemicroscope[equippedwithaHamamatsu C4742-95camera],adigitalslidescanner(Nanozoomer,Hamamatsu)]or aLeicaTCSSP5confocallasermicroscope.

Imageswerepost-processedforbrightnessandcontrastusing Adobe Photoshop CS5. The image-stacks were prepared with ImageJ1.44psoftwareand theﬁgures collatedusingtheAdobe IllustratorCS6.

2.3. Cellcounting

TheFoxb1-EGFP-and theparvalbumin-immunopositivecells were counted using the optical fractionator method [10]. This taskwasexecutedwithaZeissPhotomicroscope(equippedwith a Hamamatsu Orca0G5 camera)usingStereoinvestigator 10.52 software(MBF.Bioscience).Countsweremadeoneverysecond sec-tionusinga70␮m×70␮mcountingframe,whichwasplacedat 100␮mintervalsalongtheX-andY-axes.Thethicknessofeach sectionwassetat30␮m.

3. Results

Inhorizontalsectionsthroughthehypothalamus, parvalbumin-immunoreactivesitesarelimitedtothelateralmammillarynucleus (LM),to a clusterof cells (theCircular nucleus)in theanterior region(notshown),andtothePV1-nucleusintheventrolateral area(Fig.1A). ThePV1-nucleus is formedby a slender cordof parvalbumin-immunopositiveneurons(boxedinFig.1A),which areintermingledwithaxonsofthemedialforebrainbundle.Since inthemiceusedinthisstudyFoxb1-expressingneuronsexpress also EGFP (Enhanced Green Fluorescent Protein) as a reporter, weusedanantibodyagainstEGFPtodetectthem. Foxb1-EGFP-immunopositiveneuronsarecommonlyencounteredatthelevel

Fig. 1.Low-magniﬁcation images ofthesame horizontalsectionthrough the hypothalamusofaFoxb1-EGFP-expressingmouserevealingimmunoﬂuorescence forparvalbumin(A)andEGFP(B).(A)Parvalbumin-immunopositiveneuronsare encounteredinthelateralmammillarynucleus(LM),andinthelateral hypo-thalamus,close totheoptictract,aswellasinthePV1-nucleus(boxed).(B) Foxb1-EGFP-immunopositiveneuronscolonizenucleiofthemammillarycomplex (e.g.,MMandMP).Immunoreactivityisdetectedinsinglecellsofthe periventric-ularzoneinthethirdventricleandinalargecontigentofneuronsinthelateral hypothalamus,correspondinginlocationtothePV1-nucleus(boxed).Scalebar 50␮m.

ofthemammillarybodiesandinthelateralhypothalamicregion (Fig.1B).Ventrolaterally,Foxb1-expressingneuronsformabroad, elongated structure, which is also located in the medial fore-brainbundle.Itextendsfromtheinterstitialnucleusofthestria medullaristothemedialmammillarynucleus,andisborderedby theoptictractlaterallyandbythefornixmedially(Fig.1B).Inthe regionofthePV1-nucleus,Foxb1-expressingneuronsare numer-ous; more numerous indeed thanthe PV-immunopositiveones (Fig.1B).

Parvalbumin-immunopositiveneuronsaresmallandbipolarin therostralportionofthePV1-nucleus,andlargeandmultipolarin thecaudalone[2],whereasFoxb1-expressingneuronsare charac-terizedbysmall,bipolarperikarya.

The results of the cell-counting analysis reveal the num-ber of Foxb1-EGFP-expressingneurons tobe3-fold higherthan that of the parvalbumin-expressing ones (Table 1). Hence, the parvalbumin-expressing cells of the eponymous nucleus are a minoritypopulation.Lessthan10%ofthetotalnumberofneurons

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Table1

Meantotalnumberofparvalbumin-andEGFP-expressingneuronsinthelateralhypothalamusofadultmice,obtainedbytheopticalfractionatormethod. Marker Region Meantotalnumber Coefﬁcientoferror Countingframearea

(XY)(␮m2₎

Samplinggridarea (XY)(␮m2₎

PV235 LH 290,55 0.15≤CE≥0.29 4900 10000

EGFP LH 962,72 0.14≤CE≥0.18 4900 10000

Doublelabelling LH 79,91 0.18≤CE≥0.36 4900 10000

Themeantotalnumberofparvalbumin(PV)andEGFP-immunopositiveneurons,aswellasthemeantotalnumberofcellsthatco-expressedthetwomarkers,wereestimated onsectionsthroughthelateralhypothalamusof5miceusingtheopticalfractionatormethod.Theprecisionofthenumericalestimatesisdescribedbythecoefﬁcientof error,whichembracescountingnoise,systematicuniformrandomsamplingandvariancesinsectionthickness.

Fig.2.High-magniﬁcationconfocalimagesrevealingFoxb1-EGFP-andparvalbumin-immunopositiveneuronsinahorizontalsectionthroughthelateralhypothalamus. (A)Thecellbodiesandtheinterminglingdendritesofthebi-andmultipolarparvalbumin-immunopositiveneuronsarerevealedintheircompletenessafterexposuretoa speciﬁcantibody.(B)OnlythenucleusandperinuclearregionoftheneuronsisgranularlystainedwiththeantibodyagainstEGFPinthisFoxb1-EGFP-expressingmouse. Thecontoursofthecellsthemselvesarenotvisible.(C)Inafewcases,neuronsexpressingbothEGFPandparvalbuminareencountered,asrevealedafterthemergerofthe imagesinAandB(arrows).

manifestdouble stainingforboth parvalbuminand Foxb1-EGFP

(Table1andFig.2).

4. Discussion

Giventhefunctionalimportanceofthelateralhypothalamus andthedearthofspeciﬁcmarkersforthisregion,thediscovery of novelidentiﬁable neuronal populationsthereinis tobe her-aldedwithinterest.Withintheadultventrolateralhypothalamus, thelocationoftheFoxb1-expressingpopulationofneurons[11]

coincideswiththatoccupiedbythePV1-nucleus[1,2].Thislocus partially perpetratesthe LHVL2-region,as deﬁned ina seminal

publicationbyNieuwenhuysandhisco-workers[3].Onthebasisof ourobservationthatacompactandwell-deﬁnedmantleof Foxb1-expressingcellssurroundtheparvalbumin-expressingneuronsof theeponymousnucleus[1,2],weproposethatthisbehenceforth referredtoasthePV1-Foxb1-nucleus.

OurdatarevealthattheFoxb1-promoterremainsactiveeven afterthecompletionofembryogenesis,insofarastheFoxb1-driven expressionofEGFPwasdetectedintheadultmicethatcomprised ourstudypopulation.ThepatternofFoxb1-EGFP-expressioninthe adultmurinediencephaloncorrespondstothatofFoxb1-expression duringembryogenesis[5].ThenumberofFoxb1-expressing neu-rons in this lateral hypothalamicregion exceeds bya factor of

3

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3 that of the parvalbumin-expressing ones, albeit that in this murinestrain,thelatterare2-foldlessnumerousthaninwild-type (C56bl/6)mice(unpublisheddata). Theparvalbumin-expressing neuronsconstitutetheaxisofthePV1-nucleus,whichismantled byagroupofFoxb1-expressingones,therebygivingthestructure anappearanceofcoaxiality.

AsmallproportionoftheFoxb1-immunoreactiveneurons co-expressparvalbumin,therebyimplyingthatthesecellsstemfrom the caudal periventricular zone [11]. The origin of the other parvalbumin-expressingcellshasnotasyetbeenelucidated.Itis conceivablethatthePV1-Foxb1nucleusis,initsentirety,builtup ofcellsthathavemigratedfromthelateralmammillaryregion,but thatsomeoftheselaterlosetheircapacitytoexpressFoxb1.

Theparvalbumin-immunopositiveneuronsofthePV1-nucleus projecttoacircumscribedlongitudinalcolumnofneurons,whichis locatedinthemidbrain,ventraltotheaqueduct[12].Thequestion thatnowarisesiswhetherthemuchlargerpopulationof Foxb1-EGFP-positiveneuronslikewise projectstothis column,or toa functionallyadjoiningoneinthePAG.Todate,neithertheinputs totheparvalbumin-expressingneuronsnorthosetothe Foxb1-expressingoneshavebeenidentiﬁed.

Theavailability ofFoxb1-Creand parvalbumin-Cremicewill permitnotonlythetargetedapplicationoftracersanda deﬁni-tionoftheconnectionsofthetwopopulationsofneurons,butalso theperformanceofspeciﬁcablationandactivationexperiments.

Conﬂictofintereststatement

Theauthorsdeclarethattheyhavenoconﬂictofinterest.

Acknowledgements

Wewouldliketothankallmembersofthehistologylaboratory intheAnatomyUnitatFribourgUniversityfortheircontinuous support.

References

[1]M.R.Celio,CalbindinD-28kandparvalbuminintheratnervoussystem, Neu-roscience35(2)(1990)375–475.

[2]Z. Meszar, F. Girard, C.B. Saper, M.R. Celio, The lateral hypothalamic parvalbumin-immunoreactive(PV1)nucleusinrodents,J.Comp.Neurol.520 (4)(2012)798–815.

[3]L.M.Geeraedts,R.Nieuwenhuys,J.G.Veening,Medialforebrainbundleof therat: IV.Cytoarchitecture ofthe caudal(lateralhypothalamic)part of themedial forebrainbundlebednucleus,J.Comp.Neurol.294(4)(1990) 537–568.

[4]L.W.Swanson, BrainMapsIII: StructureoftheRatBrain:AnAtlaswith PrintedandElectronicTemplatesforData,Models,andSchematics,3rdrev. ed.,Elsevier/AcademicPress,Amsterdam/Boston,2004,p.215,1foldedleafof plates.

[5]G.Alvarez-Bolado,F.Cecconi,R.Wehr,P.Gruss,Theforkheadtranscription factorFkh5/Mf3isadevelopmentalmarkergeneforsuperiorcolliculuslayers andderivativesofthehindbrainsomaticafferentzone,BrainRes.Dev.Brain Res.112(2)(1999)205–215.

[6]S.L.Ang,A.Wierda,D.Wong,K.A.Stevens,S.Cascio,J.Rossant,K.S.Zaret, Theformationandmaintenanceofthedeﬁnitiveendodermlineageinthe mouse:involvementofHNF3/forkheadproteins,Development119(4)(1993) 1301–1315.

[7]K.H. Kaestner, K.H. Lee, J. Schlöndorff, H. Hiemisch, A.P. Monaghan, G. Schütz, Six members of the mouse forkhead gene family are devel-opmentally regulated, Proc. Natl. Acad. Sci. U.S.A. 90 (16) (1993) 7628–7631.

[8]T.Zhao,X.Zhou,N.Szabó,M.Leitges,G.Alvarez-Bolado,Foxb1-drivenCre expressioninsomitesandtheneuroepitheliumofdiencephalon,brainstem, andspinalcord,Genesis45(12)(2007)781–787.

[9]G.Alvarez-Bolado,X.Zhou,A.K.Voss,T.Thomas,P.Gruss,Wingedhelix tran-scriptionfactorFoxb1isessentialforaccessofmammillothalamicaxonstothe thalamus,Development127(5)(2000)1029–1038.

[10]M.J. West, L. Slomianka, H.J. Gundersen, Unbiased stereological estima-tion of the total number of neurons in thesubdivisions of the rat hippocampus using the optical fractionator, Anat. Rec. 231 (4) (1991) 482–497.

[11]G.Alvarez-Bolado,X.Zhou,F.Cecconi,P.Gruss,ExpressionofFoxb1revealstwo strategiesfortheformationofnucleiinthedevelopingventraldiencephalon, Dev.Neurosci.22(3)(2000)197–206.

[12]M.R.Celio,A.Babalian,Q.H.Ha,S.Eichenberger,L.Clément,C.Marti,C.B. Saper,Efferentconnectionsof theparvalbumin-positive(PV1) nucleus in the lateral hypothalamus of rodents, J. Comp. Neurol. 521 (14) (2013) 3133–3153.

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