� Immunocytochemistry for LHRH Neurons in the � Arcuate Nucleus Area of the Rat

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� Immunocytochemistry for LHRH Neurons in the � Arcuate Nucleus Area of the Rat

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  ‘ThisworkwassupportedinpartbyNIHgrantsHD-15040and AA-06014 andaNIHpostdoctoralfellowshiptoW.L.D.fromTrain-ingGrantHD-07062. 83 0022-1554/84/13.00 TheJournalofHistochemistryandCytochemistryCopyright© 1984 byTheHistochemicalSociety,Inc.Vol. 32, No.1,pp.83-91,1984 PrintedinU S II  ImmunocytochemistryforLHRHNeuronsinthe ArcuateNucleusAreaoftheRat: FactorArtifact?1 GERALDP.KOZLOWSKI and W.LESDEES DepartmentofPhysiology UniversityofTexasHealthScienceCenteratDallas SouthuesternMedicalSchool Dallas Texas75235 ReceivedforpublicationMarch29,1983andinrevisedformJuly20,1983;acceptedAugust12,1983(OA83-136) Antiseratoluteinizinghormone-releasinghormone(LHRH)generatedagainsthapten-conjugatesinwhichBSA(bo-vineserumalbumin)isusedasacarriercancontainbothanti-LHRHandanti-BSAantibodies.Suchantiseracancausethefalse-positivestainingofneuronaicellbodiesandotherelementsofthemedialbasalhypothalamusowingtothepresenceofanti-BSA,whichcross-reactswithalbu-minoidsubstanceswithinthesecells.DifferentialblockingexperimentsusingeitherBSAorLHRHorbothBSAandLHRHasimmunoabsorbentsdemonstratedthatrecentre- Introduction ThepresenceofLHRHinneuronsoftheratarcuatcnucleus has beenanimportanthistoricalquestionforncurocndocri- nologists,andremainsasourceofdebate.Sincetheratisawidelyusedanimal modelforstudiesofthehypothal-amo-hypophysial-gonadalaxis,itisofprimeimportanceto determinethosebrainareashavingLHRH-containingneu-rons.Althoughuseful,otherexperimentalapproachessuchas Hal {225}szknife-cuts(Hal {225}sz,1969)andradioimmunoassayforLHRHinPalkovits-typepunchspecimensfromvariousbrainareas(Palkovitsetal.,1974)haveprovidedonlyindirectin- formationaboutthelocationofLHRH-containingneurons. EvenimmunocytochemicallocalizationstudiesforLHRHhavebeencontroversial,despitetheirpotentialforprovidingthemostdirectevidenceofLHRH-containingneurons.Studies byNaik(1974,1975a,l975b,1976)areyettobeconfirmed,andaccordingtoKelly etal.(1982),thereisconcernabout theuseoflow-titeranti-LHRHantisera.TheclaimofHoffmanetal.(1978)ofLHRHinarcuateneuronsoftheratwaslater retractedwhenitwasdiscoveredthattheanti-LHRHanti- portsofLHRH-containingneuronsinthemedialbasal hypothalamusoftherathavearisenfromfalse-positiveresultsduetothepresenceofanti-BSAwithintheanti-LHRHantiseraused.ImmunocytochemicalevidenceofLHRH-containingcellbodiesinthemedialbasalhypo- thalamus(especiallywithinthearcuatenucleus)oftherat remainsunproven. KEYWORDS: Luteinizinghormone-releasinghormone;Immu-nocytochemistry;Arcuatenucleus;Medianeminence;Rat.serumtheseauthorsusedcontainedanti-ACTHantibodies (ClaytonandHoffman,1979).PerikaryacontainingLHRHhavebeendemonstratedwithinthehumanmedialbasalhypothalamus(MBH)(Barry,1976;Bugnonetal.,1977;Paulinetal., 1977) andthatofseveralspeciesoflaboratoryanimalsincludingprimates(BarryandCarette,1975;MarshallandGoldsmith,1980;Silvermanet al.,1982),guineapigs(Barryetal.,1974;Silverman,1976), dogsandcats(BarryandDubois,1975),hamsters(Pickard andSilverman,1976),and rabbits(S {233}t {225}lOtal.,1978).Severallargedomesticanimalshavealsobeenstudiedinthisregard.Theinfundibularnucleusofthehorse(Deesetal.,1981a)hasbeenshowntocontainimmunopositivcLHRHneurons,whereas immunoreactivecellsinthesheep(Deesetal.,1981b)and cow(DeesandMcArthur,1981)havebeenreportedinboththeinfundibularandventromedialnuclei.Incontrast,theat-temptedimmunocytochemicallocalizationofLHRH-contain- ingperikaryaintherathasproducedinconsistentandcon-flictingresults.KellyandRonnekleiv(1981),Ronncklcivetal.(1981),Fraseretal.(1982),Kellyetal.(1982),andRon- nekleivandKelly(1982)havereportednumerousperikarya intheratmedianeminence(ME)andarcuatenucleus,andin theareajustlateraltothearcuatenucleus.KawanoandDai- koku(1981)localizedLHRH-positivecellbodiesadjacentto thelateralborderoftheME,butdeniedtheexistenceofLHRH-containingperikaryainthearcuatenucleus.Thepres-  by guest on May 30, 2015 jhc.sagepub.comDownloaded from   Figure1   Sectionoftherostralmedianeminenceoftheratusingthe Kelch 12anti-LHRHantiserum.Withunabsorbedantiserumthere arestainedcellbodies(arrows)intheregionofthemedianeminence,andbilateralstainingforLHRHfibersinthezonaexterna(ZE).III-thirdventricle.Originalmagnificationx180.Bar =55 p.m. 84KOZLOWSKI,DEESentstudywasdesignedtocontinuetheinvestigationandchar-acterizationofpresumedLHRH-containingperikaryaintheMBHoftherat. MaterialsandMethods MaleSprague-Dawleyratsweighing200-250gwereanesthetizedwithsodiumpentobarbital,flushedviacardiacperfusionwithphysi-ologicsaline,andfixedwith10%phosphate-bufferedformalin.AblockcontainingthehypothalamusandmedianeminencewasisolatedfromeachbrainandpostfixedinBouin’ssolutionfor24hr.Transverse10-p.mparaffinsectionswerecutfromtheblock,mountedonglassslides,anddriedovernightat40 {176}C.ehydratedsectionswerestainedforLHRHwiththeindirectimmunoperoxidasetechniqueofStern-bergeretal.(1970),usingtheperoxidase-antiperoxidasecomplex (PAP).Additionally,thebrainsofanimalsfixedbyperfusionwithZamboni’sfixative(seeKozlowskiandNilaver,1983)weresectioned withaVibratome(OxfordLabs,CA)andimmunostainedforLHRHasdescribedbyKozlowskietal.(1980).Thestainingprocedurein-volvedthesequentialapplicationofthefollowingreagentspreparedinphosphate-bufferedsaline(PBS),pH 7.4, containing0.1%gelatin:primaryrabbitantiserumtoLHRH(Arnel 104,Kelch 12,Koz-lowski 705,Nett-Niswender 42,Nilaver 3,White-Porter 1)usedatdilutionsof1:700for24hrat4 {176}C;heepanti-rabbit IgG (MilesLaboratories,Elkhart,IN)usedatadilutionof1:200for30mmatroomtemperature;rabbitPAPcomplex(Miles)usedata1:200dilutionfor30mmatroomtemperature;and7.5mg3,3’-diamino-benzidinctetrahydrochloride(Sigma,StLouis,MO)per50mlgel-PBSwith50p.1of3%hydrogenperoxide,usedfor5-10mmatroom temperatureonanautomaticshaker.TheslidescontainingthesectionswerethoroughlywashedwithPBS,pH 7.4,betweenthestepsoftheprocedure.Thespecificityofstainingwasverifiedthroughtheabsorptionof1.0mlofeachoftheprimaryantisera,diluted1:100with120ggsyntheticLHRH(Beckman),24hrbeforeuse.Theanti-BSAactivitypresentintheantiserageneratedagainstBSAconjugates(Kelch 12, Arnel 104,andWP-1)wasremovedbytheadditionof500mgofBSAto1.0mlofa 1:100dilutionofeachantiserum24hrbeforeuse.Addingasmuchas1.0mgofBSApermilliliterofantiserumeliminatedalbuminoidstainingwithoutaffectingspecificLHRHstain-ing.TheLHRHantiseraweretestedforcross-reactivitywithotherpeptides;1mlofeachantiserum(diluted1:100)waspreabsorbedfor24hrat4 {176}Cith120mgofoxytocin,arginine-vasopressin,thyro-tropin-releasinghormone,andadrenocorticotrophichormone(ACTH).Finally,stainingwaseliminatedwhenPBSwassubstitutedforeithertheprimaryantiserum,sheepanti-rabbitIgG,orthePAPcomplex. Results Usingadjacentsectionsthroughtherostral,middle,andcaudal medianeminenceofthcrat,wewereabletodemonstratefalse-positivestainingofneuronsinthemedianeminenceand me- dialbasalhypothalamus,alongwithtrue-positivestainingfor immunoreactiveLHRHfibersinthemedianeminence.The false-positivestainingwascausedbythepresenceofanti-BSAantibodiesinrabbitantiseratoLHRH,generatedagainsthap- ten-conjugateswhenBSAwasusedasacarriertoenhancetheirimmunogenicity.ThoseantiseratoLHRHthatcontainedanti-BSAantibodiesincludedtheArnel 104,Kelch 12,Nett-Niswender 42,andWhite-Porter 1(WP-1antisera). NotonlydidtheseantiserademonstrateLHRH-positivefibersinthemedianeminence,butalsocellbodiesofthemedialbasalhypothalamus(Figures1,2a,3a,4a,and5a).Thisfalse-positiveresultwaseliminatedwitheitherliquid-orsolid- phaseimmunoabsorptionoftheantiserawithBSA(Figures2c,3c,4b,andSc),withoutaffectingthestainingoffibers. ImmunoabsorptionwithLHRHalonedidnotaffectthestain-ingofmedialbasalcellsofthehypothalamus,buteffectively abolishedthestainingofLHRH-containingfibersinthetub- eroinfundibularsulcusregionofthemedianeminence(Figures 2bandSb),andlikewiseabolishedthestainingofLHRH- containingcellbodiesandfibersinotherbrainareas.Liquid- phaseimmunoabsorptionwithbothBSAandLHRHelimi- natedallimmunopositivestaining(Figures2d, 3d,andSd). Withthepresenceofanti-BSAinanti-LHRHantisera,thereisartifactualstainingoffibersandcellsofthemagnocellularsystem(Figures3a,3b,and4a)aswellasofependymalsur- faces(Figures2aandb,3aandb,and4a),connectivetissue,andbloodvessels.Thesestructuresandcellsofthemedial basalhypothalamusarepresumedtocontainalbuminoidsub- stancesabletocross-reactwithanti-BSAantibodies,causing afalse-positivestainingreaction.Whenanti-BSAisnotpresentinanti-LHRHantisera,asisthecasebothforantiseragen- cratedagainstthyroglobulinasaconjugate(Kozlowski 705orNilaver 3)andantiserageneratedagainstBSAasacon- jugatebutabsorbedwithBSA(WP-I,Nett-Niswcnder 42, Arnel 104,Kelch 12),theobservedcytoarchitecturalchar-acteristicsofLHRH-containingcellbodies(Figures 7and8) aresimilar,anddifferinappearancefromthosecontaining albuminoidsubstances(Figure6).Whenanti-BSAwasre- movedfromtheantiseratoLHRH,cellsintheregionofthearcuatenucleusnevershowedimmunopositivity(Figures2c and d,3candd,4bandScandd).Althoughnotdemonstrated here,Arnel 104andNett-Niswender 42antiseratoLHRH  by guest on May 30, 2015 jhc.sagepub.comDownloaded from   B . 11’   V ‘a  = 0 a 4 . IMMUNOCYTOCHEMISTRYFORLHRHNEURONS85   hadreactiviticstoalbuminoidsthatweresimilartothoseshownfortheKelch 12andWP-1antisera.Bycontrast,withanti- LHRHantiserainwhichthyroglobulinwasusedasacarrier (Kozlowski 705andNilaver 3),therewasnofalseimmuno-stainingofcellbodiesinthemedianeminenceormedialbasalhypothalamusoftherat.However,inthecaseofantiserum  705 therewasoneinstanceofasinglepositivelyLHRH-stainingcellbodyinthearcuatenucleus(Figure8). Discussion ArecentreviewbyFlerk {243}etal.(1980)summarizedhisand hiscolleagues’workthroughouttheyears,andstated,ascon-clusions,thattheMBHdoesnotcontainLHRH-synthcsizing nervecellsandthatallLHRH-containingcellelementsdis- appearfromthisregionifitissurgicallydisconnectedfromtherestofthebrain.Thereviewfurtherstatedthatamongthenearly2000ratbrainstheauthorshadstudiedoverthepreceding5years(comprisingspecimensrepresentingdiffer-entphysiologicstatesandexperimentalinterventionsinboth sexes)withsixdifferentantibodiestosyntheticLHRH,nota singlecellwithimmunorcactiveLHRH-contentcould bede-tcctedinthetubcral(arcuate-ventromedial)regionoftherat brain.Exceptforoneexample,ourlaboratory,alsousingavarietyofantiseraovertheyears,hasalsobeenunabletodetectsuchLHRH-containingcells.   _ Figure2.AdjacentsectionsoftherostralmedianeminenceoftheratusingunabsorbedKelch 12anti-LHRHantiserum(a)andthesameantiserumabsorbedwith:LHRH(b),BSA(c),andbothBSAand LHRH(d).Whenanti-BSAispresent(a,b)intheantiserum,there isstainingofcellbodiesofthemedialbasalhypothalamus(arrows),whichdisappearswhentheserumisabsorbedwithBSA(c,d).StainingforLHRH-containingfibers(arrowheads)inthezonaexternaofthemedianeminence,seenwithunabsorbedantiserum(a),persistswhen BSAisusedasanabsorbant(c),butdisappearswhenLHRHisused asanabsorbant(b,d).Originalmagnificationx80.Bar =125 p.m.Nearly10yearsago,we(KozlowskiandZimmerman,1974;Zimmermanetal.,1980)becamesuspiciousofstainingofthe magnocellularsystemwhenusingtheNett-Niswcndcr 42anti-LHRHantiseruminstudiesofthcsheepandmousebrain.However,priortoreportingourwork(Zimmermanetal., 1974), werecognizedtheproblemthatanti-BSAantibodies couldposewhenstainingforLHRH.Subsequently,weun- dertooktopublicizethisexperience(Kozlowskietal.,1975;Kozlowskietal.,1976;Zimmermanetal.,1980;Kozlowski andNilaver,1983).InanswertothequestionofwhytheyhavebeenabletofindnumerousLHRH-positivcneurons,Kellyetal.(1982)statedthattheyweresuccessfulbecause1)certainuniquecharacteristicsoftheWP-lantiserummayfacilitatetherec-ognitionofLHRH,2)alimitedfixationtimewasused(75 mm ratherthan90 mm or1hr,whichtheyfounddiminished  by guest on May 30, 2015 jhc.sagepub.comDownloaded from   CD 86KOZLOWSKI,DEES iz ’..   : Figure3.AdjacentsectionsofthemiddlemedianeminenceoftheratusingunabsorbedKelch 12anti-LHRHantiserum(a)andthesameantiserumabsorbedwith:ACTH(b),BSA(c),andbothBSAandLHRH(d).ThereisnoeffectonstainingwhenACTHisusedasanabsorbant.Whenanti-BSAispresent(a,b)intheantiserum,artifactualstainingofalbuminoidsubstancesappearsinneurons(ar- rows)andfibers(doublearrows)ofthezonainternaofthemedian eminenceaswellasinependymalsurfaces(arrowheads),connectivetissue,andbloodvessels.Originalmagnificationx70.Bar =60 p.m.thestaining),and3)thesagittalsectioningofspccimcnsaffordsagreateropportunitytovisualizetherostrocaudallyorientedneurons.Inordertoprovidefurthersupportfortheirresults,theycited“physiological”evidencefortheexistenceofMBHneuronsusingothertechniquesincludingdeafferentationstudiesinwhichthecontentofLHRHisnottotallyeliminatedwithintheMBHisland,asaresultofHal {225}szknifecuts(Brownsteinetal.,1977;Kordonetal.,1982),andthefindingthatdcc-trochemicalstimulationofthedeafferentedMBHresultedinsignificantlyincreased peripherallevelsoflutcinizinghormone (Carrilloetal.   1980).However,otherstudieshaveshownthatdeafferentationcanresultinthecompletedisappearanceof LHRH-containingelementswithintheMBH(forareviewseeFlerk {243}etal.,1980).Thesedifferentresultsareprobablyduetothedeafferentationtechnique.Gibbs(1973)wasthefirsttoadvocatetheapplicationofanextradownwardforcetothe stereotaxicknifeinordertoachievecompletedeafferentation. HoffmanandGibbs(1982)demonstratedthatfordeaffer-entationofthemedianeminencetobesuccessful,theknifemustpassthroughtheopticchiasmtocutthesubchiasmaticLHRHtract,andmustextendfarenoughcaudallyandlaterallytocutaLHRHtractarisingfromcaudalcellsofthelateral preopticandlateralanteriorhypothalamus. OurresultsindicatethatLHRH-containingneuronsarcquitedifferentinappearancefromthosecontainingalbumi- noidsubstances.Whenviewedinalongitudinalplane, twotypesofLHRH-containingneuronsarcseen(Krisch,1980): type1cells,whicharcbipolarwithasmoothsurface,andtype   cells,whichareunipolarwitharoughsurface(Figures 7 and8).Bothtypesofcellhaveonlyasmallrimofcytoplasm surroundingthenucleus.IncontradistinctiontoLHRH-con- tamingneurons,thealbuminoid-containingneuronsthatweobserved(Figures1and6)closelyresembledthosereported byKellyetal.(1982)tocontainLHRH.Mostoftheseal-  by guest on May 30, 2015 jhc.sagepub.comDownloaded from   A  1 1#{149} \   B . .-   ... IMMUNOCYTOCHEMISTRYFORLHRHNEURONS 87Figure4.Adjacentsectionsofthecaudalme-dianeminenceoftheratusingunabsorbed Kelch#12anti-LHRHantiserum(a)orthe sameantiserumabsorbedwithBSA(b).TheintensityofLHRHstaining(arrows)remainsunaffectedwhentheabsorbedantiserumisused.Theglobular-stainingstructuresofthemagnocellularsystem(arrowheads)seenwiththeunabsorbedantiserum(a)disappearwhen theantiserumisabsorbedwithBSA(b).Orig- inalmagnificationx100.Bar =100 p.m.   buminoid-containingcellsappearspheroidormultangular,withonlyafewbeingfusiform.Itisuncommontofindprocesses emanatingfromthesecells,butwhenfibersarepresenttheyarenotbeaded.AmajorproblemininterpretationhasbeenthefactthatmanybonafideLHRHfibersinterminglewith neuronsofthearcuatenucleus,givingbeadedfiberstheap- pearanceofemanatingfromthecellbodiesofthenucleus.However,bycarefullyfocusingatdifferentplaneswithinthe section,onecanobservethatinreality,the beadedLHRHfibersonlypassclosetotheneurons.Inthisregard,thecx- amplesofratarcuateneuronsgivingrisetobeadedfibers,as shownbyKellyeta .(1982),shouldberegardedwithsomeskepticism.Followingimmunoabsorptionofanti-BSA,theWP-1anti-LHRHantiserumisindeedusefulforimmunostaining LHRH-containingcellbodiesinotherareasofthebrain.AnotherissuebroughttolightbyKellyetal.(1982)con- cernstheinterpretationofthegranularnatureofthefinalLHRH-staining-rcactionproduct.Theyequatcthegranularappearanceofthestainasseenwithlight-microscopicim- munocytochcmistrywithneurosecretorygranulesasseenwith electron-microscopicimmunocytochemistryasreportedbyKozlowskietal.(1980).Likewise,Kellyetal.saidthattheycoulddistinguishLHRH-containingneuronsfrom/3-cndor- phin-containingneuronsonthebasisofwhetherornotthe “granules”wereaggregatedorevenlydistributedthroughoutthesoma.Infact,thegranulestowhichtheyreferareaggre-gatesofinsolublepolymersofreduceddiaminobenzidine,which areinherenttothenatureofthefinalLHRH-staining-reactionproductregardlessofthegranularoragranularnatureofthesubstancelocalized.TheultrastructuralcorrelateofthefinalreactionproductseenlightmicroscopicimmunocytochemistryforLHRHisstainedpeptideassociatedwiththeroughen-doplasmicreticulum,Golgicomplex,neurosecretorygranules,neurotubules,andtheaxonalendoplasmicreticulum(Koz- lowskiandHostetter,1978;Kozlowskietal.,1980).There-fore,onemustbecautiouswhenrelatingtheimmunostaining  by guest on May 30, 2015 jhc.sagepub.comDownloaded from 
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