HistamineReceptorsinMammalian - PDF document

HistamineReceptorsinMammalian MATTHEWJ.GASTINGER,ALISTAIRJ.BARBER,NOGAVARDI,DAVIDW.MARSHAKGraduateSchoolofBiomedicalSciences,TheUniversityofTexasHealthScienceCenterHouston,Houston,Texas77225 Mammalianretinasareinnervatedbyhistaminergicaxonsthatoriginatefromperikaryaintheposteriorhypothalamus.Toidentifythetargetsoftheseretinopetalaxons,welocalized Vertebrateretinasreceiveinputfromthebrainviareti-nopetalaxons,andthispathwayhasimportantmodula-toryeffectsonretinalneuronsinbirdsand“sh,thegroupsthathavebeenstudiedmostintensively.Thefunctionsofretinopetalaxonsinmammalianretinasarenotwellun-derstood,however(Uchiyama,1989).Histaminehasbeenlocalizedtoretinopetalaxonsintheguineapig(Airaksi- Grantsponsor:NationalEyeInstitute;Grantnumber:EY06472;Grantnumber:EY11105;Grantnumber:EY12610;Grantnumber:CoreGrantEY10608;Grantsponsor:JuvenileDiabetesResearchFoundation;Grantsponsor:PennsylvaniaLionSightConservationandEyeResearchFoun-dation;Grantsponsor:AmericanDiabetesAssociation. 3(HR3).Amongthese,onlyHR1hasbeencharacterizedpreviouslyinmammalianretinas(Nowak,1993;Sawaietal.,1988).However,thereisindirectevidencethatHR2andHR3arealsopresentinmammalianretinas.Hista-mineinhibitsaforskolin-inducedincreaseincAMPintherabbitretinaviaHR2(Nowak,1991;Nowaketal.,1989).HistaminealsoincreasesaGABA-mediatedchloridecurrentinamacrinecellsfromratretinalslicesviaproteinkinaseA(PKA;FeigenspanandBormann,1994),thesig-nalingpathwaytypicallyusedbyHR2(Gantzetal.,1991).ElectricallyevokeddopaminereleasefromthepigretinaisinhibitedbyhistamineviaHR3(Schlickeretal.,1990).Toidentifythetargetsofhistaminergicretinopetalaxons,welocalizedhistaminereceptorsinmonkeyandratreti-nasbyusingbothlightandelectronmicroscopicimmuno-labelingtechniques.MATERIALSANDMETHODSAdultbaboon(Papioanubis)eyeswereobtainedfromtheBiologicalMaterialsDistributionProgramattheSouthwestFoundationforBiomedicalResearch(SanAn-tonio,TX).Theseanimalsweresedatedwithketamine(15mg/kg),exsanguinatedviathefemoralvein,andkilledwithanoverdoseofsodiumpentobarbital(100mg/kg),followingprotocolsapprovedbytheInstitutionalAnimalCareandUseCommitteeandconformingtoNIHguide-lines.Theeyeswerequicklyremovedandhemisected;thevitreouswasremovedwith“neforceps.Macaque()eyes“xedforimmunohistochemistrywerepur-chasedfromCovanceResearchProducts(Alice,TX).AdultSprague-DawleyalbinorateyeswereobtainedfromresearchersattheUniversityofTexasMedicalSchoolatHouston.Thesewerecontrolsorsham-operatedratsthatwereeithereuthanizedbyCOinhalation,overdosedwithketamine/xylazine,orelsedecapitatedwithoutanesthesia.Inallcases,theprotocolsforeuthanasiawereapprovedbytheAnimalWelfareCommitteefortheUni-versityofTexasHealthScienceCenteratHoustonandconformedtoNIHguidelines.Alleyeswereremovedandhemisectedunderroomillumination.LightmicroscopyEyecupswereimmersion“xedin4%paraformaldehydein0.1Msodiumphosphatebuffer(PB),pH7.4,for2hoursatroomtemperatureorovernightat4°C.Theretinaswereisolatedfromtheretinalpigmentepithelium,andthevitreouswasremoved.Somepieceswereembeddedin4%agaroseinphosphate-bufferedsalinewith0.3%sodiumazide(PBSa),and100mverticalVibratome(OxfordLaboratories,St.Louis,MO)sectionswerecut.Someotherpieceswerecryoprotectedin30%sucroseinPBSa,embeddedinTissue-Tekoptimalcuttingtemperaturecompound(SakuraFinetekInc.,Torrance,CA),frozen,andcutinto30-msectionswithacryostat(InternationalEquipmentCorp.,NeedhamHeights,MA).Somepieceswerealsoprocessedas”atmounts.Allthesectionswereincubatedin1:100donkeyseruminPBSawith0.3%Tri-tonX-100at4°Covernightorfor3hoursatroomtemper-ature.Allsectionswereincubatedinprimaryantibodywith0.3%Tritonfor3…6days;”atmountswereincubatedfor8…10daysat4°C.Therabbitpolyclonalantibodieswereeitherneatantiseraoraf“nitypuri“edanddilutedinPBSa:HR1C(1:2,000…1:5,000,AB5652P;Chemicon,Te-mecula,CA),HR1CL(1:500…1:2,000,AB5654P;Chemi-con),HR2(1:2,000,AB5656P;Chemicon),HR3C(1:5,000,AB5660P;Chemicon),HR3C(1:1,000…1:5,000H3R31;ADI,SanAntonio,TX),HR3N(1:1,000…1:5,000H3R32;ADI),andhumanmetabotropicglutamatereceptor6(mGluR6;1:1,000;Vardietal.,2000).Af“nity-puri“edsecondaryantibodies,biotinylateddonkeyanti-rabbit,andCy3-conjugatedstreptavidin(1:100;JacksonImmu-noresearch,WestGrove,PA)wereappliedinsuccessionfor1…2daysat4°C.Forfrozensections,secondaryanti-bodieswereappliedfor1…3hoursatroomtemperature.DoublelabelingSomesectionsand”atmountswereincubatedinasecondprimaryantibody:goatanti-cholineacetyltrans-ferase(ChAT1:200,AB144P;Chemicon),mouseanti-receptor(GABA-chain(MAB339,clonebd24;Chemicon),mouseanti-humanmGluR6(1:100;Vardietal.,2000),ormouseanti-tyrosinehydroxylase(TH,1:10,000;T2928Sigma,St.Louis,MO).Af“nity-puri“edsecondaryantibodiesconjugatedtoAlexa488,donkeyanti-goatordonkeyanti-mouse(1:200;MolecularProbes,Eugene,OR),wereappliedfor1dayat4°C.TissuewasmountedonslidesandcoverslippedwithVectashield(Vec-torLaboratories,Burlingame,CA).ConfocalanalysisAllimageswereacquiredwithaZeissconfocallaserscanningmicroscope(LSM410;CarlZeiss,Thornwood,NY)withakrypton-argonlaser.Byusinglaseremission“ltersof590…610nmforCy3and515…540nmforAlexa488,imageswereacquiredwitha63oil-immersionob-jectiveasaseriesofopticalsections(0.5-mstepsize).Eachmarkerwasassignedapseudocolor(redorgreen).Theimageswereanalyzedassinglesectionsandstacksofopticalsectionsprojectedalongthex-axis.AllimageswereprocessedinAdobePhotoshop(AdobeSystems,SanJose,CA)toenhancebrightnessandcontrast;allretinalverti-calsectionswereorientedwiththephotoreceptorsup-ward.Thealignmentofthelaserswasveri“edbyimagingmbeadsthatwereexcitedbythesamewavelengthsasthoseusedinourexperiments.Thedouble-labeledverticalsectionswereanalyzedintheLSMimagebrowser(CarlZeiss)andPhotoshop.Rodspheruleswhoseinvaginatingprocesseswereorientedparalleltotheplaneofsectionwereanalyzedwithasignalaveragingprogram(Lietal.,2002).Imagesfromsingleopticalsectionsorsmallreconstructedstackswereim-portedintothesignal-averagingprogram,andnineto13rodspherule-relatedpunctaperimagewereselectedwitha1.9-m-squarebox(3030pixels).EachselectionboxwascenteredonthemGluR6signalandencompassedtheentirespherule.A33median“lterwasappliedtothedataforsmoothing.Thenormalizedmeanintensityvs.pixelpositionfortheselectedareaswascalculatedandexportedtoanExcelworksheet(MicrosoftCorp.,Red-mond,WA).SurfaceplotsofthedataweregeneratedinExcelandcopiedintoPhotoshopforanalysis.Inallcases,thegraphsrepresentnormalizedpixelintensitiesfrom100…255;pixelintensitieslessthan100wereconsideredback-ground.Todeterminethedistancebetweenthecentersofthetwosignals,thesurfaceplotswereoverlaid,andthepeaktopeakseparationwasmeasured.AllvaluesarereportedasmeanHISTAMINERECEPTORSINMAMMALIANRETINAS ElectronmicroscopyEyecupswere“xedin4%paraformaldehyde,0.5%glu-taraldehyde,or4%paraformaldehyde,0.1%glutaralde-hydein0.1MPB(pH7.4)for2hoursatroomtempera-ture,followedby4%paraformaldehyde0.1MPB(pH10)at4°Covernight.Thetissuewastreatedwith1%sodiumborohydrideinPBSfor1hour,followedbyanascendinganddescendingseriesofgradedethanolsolutionsinPBS(Marshaketal.,1990).Retinaswerecutintosmallpiecesandincubatedin1:5,000rabbitanti-HR3C(AB5660;Chemicon)for8…10daysat4°C.Thepieceswereincu-batedfor2daysinbiotinylatedgoatanti-rabbitIgG(1:100;VectorLaboratories),andthebiotinwasvisualizedwithaVectastainavidin-biotin-peroxidasekit(1:100,PK-4000;VectorLaboratories)overnightat4°C,followedbydiaminobenzidine(0.5mg/ml)withhydrogenperoxide(0.005%,30…45minutes).Theretinawasthenpost“xedin1%osmiumtetroxidein0.1MPBandembeddedinepon.Sections80…100nminthicknesswerecollectedonformvar-coatedgrids.Thesectionswerestainedwith2%uranylacetatein50%methanolfor60minutes,followedby0.2%leadcitratefor1…2minutes.Labeledprocesseswereviewedat20,000…50,000inaJEOL(Peabody,MA)100CXelectronmicroscope(EM).Imageswereacquiredwithaside-mountedAMTAdvantageHR1000CCDcam-erasystem(AdvancedMicroscopyTechniquesCorp.,Dan-vers,MA)andprocessedinAdobePhotoshop.PeptidecontrolexperimentsTheimmunogenfortheHR1Cantibody(AB5652P;Chemicon)andantiserum(H1R11-S;ADI)wasCNENFKK-TFKKILHIRS,apeptide(H1R11P;ADI)correspondingtoasequencenearthecytoplasmicC-terminusoftheratHR1(Fujimotoetal.,1993).TheimmunogenfortheHR1CLan-tibody(AB5654P;Chemicon)andantiserum(H1R12-S;ADI)was(C)PSFSEIKLRPENPKGDAKK,apeptide(H1R12P;ADI)correspondingtoasequencewithinthethirdcytoplas-micloopofthehumanHR1(Moguilevskyetal.,1994);cys-teinewasaddedforcouplingtothecarrierproteinandwasnotpartoftheoriginalsequence.TheimmunogenforHR3N(H3R32;ADI)was(C)SGALAGDAAAAGGARGFS,apep-tide(H3R32P;ADI)correspondingtoasequencewithintheN-terminusofthehumanHR3(Lovenbergetal.,1999,2000);cysteinewasaddedforcouplingtothecarrierpro-teinandwasnotpartoftheoriginalsequence.Theim-munogenfortheHR3Caf“nity-puri“edpolyclonalanti-body[AB5660P(Chemicon)andH3R32A(ADI)]wasCPQKLKVQPHGSLEQCWK,apeptide(H3R31P;ADI)correspondingtoasequencewithintheextracellularN-terminusofthehumanHR3(Lovenbergetal.,1999,2000);cysteinewasaddedforcouplingtothecarrierpro-teinandwasnotpartoftheoriginalsequence.AregionoftheHR3Cpeptide,LKVQPH,ishomologoustoaregioninprotocadherin2A,anunrelatedpeptidethatisexpressedinthedevelopingretina;thissequencewassynthesized(98.4%HPLCpuri“ed;Chemicon).TheHR1peptidecontrolexperimentswerepeformedonverticalsectionsofratretinapreparedasdescribedabovebyincubatingthepeptides(20l)intheantiserafor1.5hoursat37°C,thenmadetovolumewithPBS.FortheHR3peptidecontrolexperiments,macaqueretinaswereprocessedwithgradedethanolsolutionsasintheEMstudy,toenhancethepermeationoftheantibodiesthroughthetissue.TheHR3antibodieswereincubatedinthepeptides(20l)for1hourat4°C.Inallinstances,thepreincubationwiththeimmunogenblockedalllabel-ingintheretinalsections.TotestwhethertheHR3Cantibodyrecognizedtheshorthomologoussequenceofprotocadherin2A,thesynthesizedpeptide(1mg/ml)wasincubatedwiththedilutedantibody;thelabelingwasnotblockedandappearedthesameaswithuntreatedanti-LightandelectronmicroscopiclocalizationofHR3inmonkeyretinasHR3-immunoreactive(-IR)punctawererestrictedtotheouterplexiformlayer(OPL;Fig.1A).AntibodiesdirectedagainsttwodifferentregionsofHR3wereused,andtheresultswerethesamewithboth(Fig.2).Becausetheresultswerethesameinbaboonandmacaqueretinas,theyareconsideredtogetherhere.SmallHR3-IRpuncta,approximately0.3mindiameter,werearrangedinasinglerow(Figs.1,2A).Thesepunctawereaggregatedinshortbands,approximately5…6minlength,beneatheachconepedicle;nolabelingwasfoundinperikaryaintheINL.IntheEM,reactionproductwasfoundattheapexofON-bipolarcelldendrites,whichwereidenti“edbytheircentralpositionatribbonsynapses(Fig.1).Inmostdendrites,theentiretipwas“lledwithreactionproduct,and,insomecases,reactionproductalsoextendedvit-reallyortowardtheganglioncelllayer(GCL).AllconepediclesanalyzedhadmultiplelabeledON-bipolarcelldendrites.Noreactionproductwasfoundinconepedicles,indendritesofhorizontalcells,orindendritesofOFF-bipolarcells.NumerouslargerHR3-IRpuncta(0.7…1.2m)werefoundscleradto,ortowardthephotoreceptorouterseg-ments,andbetweentheconepedicles;theywereassoci-atedwithrodspherules(Fig.2).Analysisofserialopticalsectionsrevealedthatsomeofthelargepunctawerecom-posedoftwocloselyassociatedsmallerpunctawithintherodspherules(Fig.2B,inset).Primaterodspheruleshavefromtwotosevencentralelements,consistingofrodbi-polarcelldendrites,andtwopairsoflateralelements,consistingofH1horizontalcellaxonterminals(Migdaleetal.,2003).IntheEM,reactionproductwasfoundontheapexofrodbipolarcelldendritesalongthebifurcationridge,theregionofthesynapticcleftbeneaththehorizon-talcellprocesseswherethesynapticcleftbifurcates(Fig.1C).Reactionproductalsoextendedatleast500nmvit-reallyfromthebifurcationridge.Nootherelementsasso-ciatedwiththerodspheruleswerelabeled,but,inafewsections,HR3-IRpunctawereobservedintheneuropil,2mvitreadtotherowofconepedicles.TocomparethedistributionofHR3andtheglutamatereceptorsofON-bipolarcelldendrites,retinalsectionswerelabeledwithanti-mGluR6andanti-HR3(Figs.3,4).Inprimates,mGluR6isfoundonallON-bipolarcellden-drites,atbothrodspherulesandconepedicles(Vardietal.,2000).IntheOPL,allHR3-IRpunctaassociatedwiththerodspheruleswerecloselyassociatedwithmGluR6-IRpuncta,butthetwoproteinsdidnothavepreciselythesamedistribution(Fig.4).TheHR3-IRpunctawerescleradrelativetothemGluR6-IRpuncta(Fig.4A).ThisresultisshowngraphicallyinFigure4E…G,asaplotofthenormalizedmeanintensityvs.positionfortheHR3andM.J.GASTINGERETAL. mGluR6signals.Ananalysisofsevendifferentdouble-labeledsectionsfromtwoanimalsshowedthattheHR3signalwasshifted0.286mwithrespecttothepeakofthemGluR6signal.TheHR3immunoreactivityatthebasesofconepedicleswasalsoscleradrelativetomGluR6immunoreactivity(Fig.3A…C),butindividualpunctawerenotanalyzedquantitatively.TheHR3-IRpunctawerefurthercharacterizedbydou-blelabelingsectionswithamonoclonalantibodytoreceptor.IntheOPL,GABAR-IRpuncta,arrangedinbandsapproximately5minlengthand2thick,werelabeledasdescribedpreviously(Haverkampetal.,2000;VardiandSterling,1994),exceptthattherewasnolabelingassociatedwithrodspherules.HR3-IRpunctawerescleradtotheGABAR-IRband(Fig.5A…C).peakoftheHR3signalwasshiftedrelativetothepeakoftheGABARsignal,andtheGABARsignalwasvitreadtothemGluR6signal(Fig.5D…F).ItwasnotpossibletostudytheregionaldistributionofHR3inmonkeyretinas,becausewhole-mountprepara-tionscouldnotbelabeled.WewereunabletodetectHR3inratretinas,eventhoughoneoftheantiserawasraisedagainstthecytoplasmic,C-terminusoftheratHR3.LocalizationofHR1inratretinasInratretinas,thedistributionofHR1wasanalyzedbyusingneatantiseraoraf“nitypuri“edIgGfromtwoan-tiseradirectedagainstdifferentregionsofthemolecule,andthepatternoflabelingwasidentical.HR1CandHR1CLbothlabeledlargeprocessesinstratum1(S1)oftheIPLandlargecellbodiesintheinnernuclearlayer(INL),approximately15mindiameter.Smallerpro-cesseswerealsolabeledinS4oftheIPLandintheOPL.HR1CandHR1CLbothlabeledallpartsofdopaminergicamacrinecells,includingsomas,dendrites,andaxons(Fig.6).ThereweresmallerHR1C-IRpunctainS1oftheIPLthatwerenotassociatedwiththeTH-IRprocessesandalsoafewHR1C-IRpunctainS2oftheIPL.The Fig.1.HR3localizationinON-bipolarcelldendritesofmacaqueandbaboonretinas.AverticalsectionofmacaqueretinalabeledwithHR3C.Punctaassociatedwithseveralconepedicles(arrows)androdspherules(arrowheads)werelabeled.Nolabelingwasdetectedinanyotherpartoftheretina.ElectronmicroscopiclocalizationofHR3receptorsinON-conebipolarcelldendritesinababoonretina.ReactionproductwasfoundatthetipsoftheON-bipolarcellden-dritesandalongtheelectron-denseportionoftheplasmamembrane.ElectronmicroscopiclocalizationofHR3Cintworodbipolarcelldendritesofamacaqueretina.Reactionproductwasfoundalongthemembraneoftherodbipolarcelldendritesandextendedtothetip(arrow).ScalebarsminA;500nmforB,C. Fig.2.HR3intheOPLofmacaque(A,B)andbaboon(C)retinas.ThepatternoflabelingintheOPLwasidenticalwiththetwoanti-bodiesraisedagainstHR3,HR3C()andHR3N().Clustersofsmall,HR3C-immunoreactive(IR)punctaformedabandatthebasesofconepedicles.Numerouslargerpunctascleradtotheconepedicleswereassociatedwithrodspherules.Someofthelargepunctawerecomposedoftwosmallerpunctaincloseapposition(usinganantibodytoHR3C,anidenticalpatternoflabelingwasfoundinbaboonretina.ScalebarsminA…C;1mininset.HISTAMINERECEPTORSINMAMMALIANRETINAS HR1C-IRpunctaintheIPLwerenotassociatedwithChAT-IRdendrites(notshown).The“rstmajor“ndingwasthatHR3receptorswerelocalizedtothetipsofON-bipolarcelldendritesinmon-keys.Thesewouldbeactivatedbyhistaminereleasedtonicallyduringtheday.ThereceptorsarestrategicallypositionedtomodulatetransmissionfromphotoreceptorsandhorizontalcellstoON-bipolarcells,becausetheyareclosertothesitesofglutamatereleasefromthephotore-ceptorsthaneithermGluR6orGABAreceptors.Thisisthe“rstdirectevidenceofHR3atpostsynapticmem-branes,anunexpectedresult,inthatHR3aregenerallydescribedaspresynapticreceptors.ActingatHR3autore-ceptors,histamineinhibitsitsownrelease(Arrangetal.,1983).HR3agonistsinhibitthereleaseofotherneuro-transmittersinthebrain(Brownetal.,2001)andretina(Schlickeretal.,1990),andtheseeffectshavealsobeenattributedtopresynapticreceptors.However,thedistri-butionofHR3hasbeendescribedinthebrainusingau-toradiography(Pollardetal.,1993)andimmunohisto-chemistry(Chazotetal.,2001),andbothstudiesprovideindirectevidenceforHR3onpostsynapticmembranes.Thesecondmajor“ndingwasthatdopaminergicneu-ronsinratretinasexpressHR1.Inrats,thesereceptorswouldbeactivatedbytonicreleaseofhistamineatnight.Histaminestronglyinhibitsthereleaseofdopaminefromguineapigretinas(WeberandSchlicker,2001),andthesameislikelytobetrueinrats.Becausedopamineplaysamajorroleinlightadaptation(Witkovsky,2004),this Fig.3.HR3C-IRandmGluR6-IRpunctaarecloselyassociated(verticalsectionofamacaqueretina).HR3C-IR(red)punctaatthebasesoftwoconepediclesandsurroundingrodspherulesintheOPL.Inthesamesection,mGluR6-IR(green)punctahadasimilarCompositeofAandBshowingthatallHR3-IRpunctawereassociatedwithmGluR6-IRpuncta.NotethescleradshiftintheHR3-IRpunctarelativetothemGluR6-IRpuncta.ThisseriesoffouropticalsectionsfromCshowshowtheHR3-IRpunctaonden-dritesorientedperpendiculartotheplaneofsection(whiteboxes)weremoreprominentinsection1(top),whereasthemGluR6-IRpunctaweremoreprominentinsection4.Punctainsections2and3wereyellow,indicatingoverlapbetweenthetwosignals.ScalebarsminC(appliestoA…C);5minD(appliestoallpanels). Fig.4.HR3-IRpunctaarescleradtoMGluR6-IRpunctainrodbipolarcells.AsingleverticalsectionofmacaqueretinainwhichtherodbipolarcelldendriteswereparalleltotheplaneofsectionwithHR3C-IR(red)andmGluR6-IR(green)puncta.Whiteboxesindicateexamplesoftherod-spherulesselectedforsignalaveraginganalysis.NormalizedmeanintensityoftheHR3CandmGluR6signalsfrompunctainrodspherules.Composite“gureshowingboththeredandthegreensignals;yellowindicatesoverlap.Thenormal-izedmeanpixelintensitiesoftheredHR3CandgreenmGluR6signalsareplottedas”atcontourgraphs.SuperimpositionofthetwocontourgraphsshowingthattheHR3signalwasscleradrelativetothemGluR6signal.Measurementsweremadefromthepeakofthered(whiteasterisk)tothepeakofthegreen(blackasterisk)signals.ScalebarsminA;0.5minE(appliestoB…G).M.J.GASTINGERETAL. reductionindopaminereleaseatnightmightfacilitatedarkadaptation.Dopaminergicneuronsarealsothetar-getsofretinopetalaxonsin“shretinas(ZuckerandDowl-ing,1987).Inmammalianretinas,dopaminergicneuronsexpressreceptorsforotherneuromodulators,somatosta-tin(Cristianietal.,2000),andsubstanceP(Casinietal.,2002;Catalanietal.,2004).Itispossiblethatthereareregionaldifferencesinhis-taminereceptordistributionthatwereundetectedinthisstudy.Histamine-IRaxonsaredistributeddifferentlyinthenasalandtemporalretinaofmacaques,butthesedifferencesareintheprimaryaxonsandtheircollaterals.Theseaxonsinitiallyruntowardthetemporalretinaintheoptic“berlayer,encirclethefovea,andthenreturntotheopticnerve.Axonterminalsaredistributedmoreuni-formly;afewprimaryaxonshavebranchesthatsupplytherestoftheretina,includingthenasalretinaandtheparafovea(Gastingeretal.,1999).Ratretinasaretypi-callysuppliedbyonlyonehistamine-IRaxon.Althoughthesebranchextensively,thereareregionsoftheretinathatareapparentlynotinnervated(Gastingeretal.,Histamineclearlyexertssomeofitseffectsinmonkeyandratretinasviavolumetransmission,anephapticin-teractioninwhichtheneurotransmitterdiffusesthroughtheextracellularspaceoveradistancemuchgreaterthanthewidthofthesynapticcleftbeforereachingitsrecep-tors.Histamine-IRretinopetalaxonsterminateexclu-sivelyintheIPL(Gastingeretal.,1999,2001),butsomehistaminereceptorswerefoundintheOPL.Inthisre-spect,histamineactsasatypicalretinalneuromodulator.Therearereceptorsforneuropeptides(Bagnolietal.,2003),dopamine(Witkovsky,2004),serotonin(PootanakitandBrunken,2001),andacetylcholine(Yamadaetal.,2003)intheOPLeventhoughtheprocessesoftheneuronscontainingthosemodulatorsareverysparseorabsentItispossiblethatretinopetalaxonsalsomakesynapses,becauseHR1waslocalizedintheIPLofrats.EMimmu-nohistochemicalstudieswillberequiredtodeterminewhetherthesearesitesofsynapticcontacts.Formamma-lianbrains,somegroupshavefoundafewsynapsesinhistaminergicvaricosities(Inagakietal.,1987;Takagietal.,1986)butothershavefoundnosynapses(Hayashietal.,1984;Uhlrichetal.,1993;Wilsonetal.,1999).HR3inmonkeyretinasIncones,HR3waslocalizedatthetipsoftheON-bipolarcelldendritesandtypicallyextendedonlyasfarasthemouthoftheinvaginations.Inmacaqueconepedicles,mGluR6islocalizedalongtheON-bipolarcellmembranes,atleast200nmfromthetipsofthedendrites(VardiandSterling,1994).InpreviousEMstudies,GABARwaslocalizedtothebasesoftheconepedicles,whereitwasfoundonbothON-andOFF-conebipolarcelldendrites(Haverkampetal.,2000;VardiandSterling,1994).These Fig.5.LocalizationofGABA-chain(GABAmGluR6,andHR3intheOPLofamacaque.HR3C-IR(red)punctaareassociatedwiththreeconepedicles.Arrowheadsindicatelabelinginrodspherules.ThereareGABAR-IR(green)punctaatthesamethreeconepedicles,butnonewasassociatedwithrodspherules(ar-CompositeimageofAandBshowingthatHR3C-IRpunctaarescleradrelativetoGABAR-IRpuncta.TherearemGluR6-IR(red)punctaatthebasesoftwoconepediclesandseveralrodspherules(arrowheads).Inthesamesection,GABApuncta(green)areassociatedwiththesameconepedicles.positeimageofEandFshowingthatmGluR6-IRpunctaarescleradtotheGABAR-IRpuncta.ScalebarsminC(appliestoA…C),F(appliestoD…F).HISTAMINERECEPTORSINMAMMALIANRETINAS resultsaresummarizedinFigure7A.Inrodspherules,HR3andmGluR6werebothlocalizedtorodbipolarcelldendrites,but,unlikemGluR6,HR3wasfoundatthetipsofbipolarcelldendrites(Fig.7B).ThefunctionofHR3hereisunknown;however,else-whereinthecentralnervoussystem,HR3activateseitherorG(ClarkandHill,1996).ThealphasubunitofG)hasbeenlocalizedtoON-bipolarcelldendritesinmacaques(Vardi,1998)andshowntobecoupledtomGluR6(Wengetal.,1997).AswithHR3,GisfoundatthetipsofON-bipolarcelldendrites(Vardi,1998).Thediametersofthelabeledpunctacannotbemeasuredac-curatelybylightmicroscopy,buttheresultsofthesignalaveraginganalysissuggestthatthedistributionsofmGluR6andHR3overlaptosomeextent.AcomparisonofourEMresultswiththosepublishedpreviouslyalsosug-geststhattheyoverlap(VardiandSterling,1994).IfHR3activatesGassociatedwithmGluR6,nonselectivecationchannelswouldclose,hyperpolarizingON-bipolarcells(Nawy,2000).Voltage-gatedpotassium(Kv)channelsinbipolarcelldendritesmightalsobemodulatedbyHR3.AswithHR3,theKv1.2subunitshavebeenlocalizedbyEMtothetipsofON-bipolarcelldendrites(YazullaandStudholme,1998).Othersubunits,Kv1.1andKv1.3,havebeenlocal-izedtoON-bipolarcelldendritesbylightmicroscopyaswell(Klumppetal.,1995).Somevoltage-gatedpotassiumchannelsinON-bipolarcellsareclosedbycAMP-dependentPKAphosphorylation(FanandYazulla,1999).BecauseHR3isknowntoinhibitadenylatecyclase(Dru- Fig.6.DopaminergicamacrinecellsinratretinaexpressHR1.ThreestacksofopticalsectionsshowingredHR1C-IRpuncta(A,D,G),greenTH-IRamacrinecells(B,E,H),orboth(C,F,I).arefromS4oftheIPL,fromS1,andfromtheOPL.Adopaminergiccellbodyandnearlyallofitsprocesses(arrowheads),includingthepri-marydendrite(arrow),weredoublylabeled.OtherHR1C-IRpunctaintheIPLarenotassociatedwithdopaminergiccells.ScalebarminI(appliestoA…I).M.J.GASTINGERETAL. teletal.,2001),histamineactingviaHR3onmacaqueON-bipolarcellswouldopenmoreKchannels,hyperpolarizingtheON-bipolarcells.OtherpossibletargetsofHR3aretheexcitatoryaminoacidtransporters(EAAT)coupledtochloridechannels,whicharealsofoundinON-bipolarcellsof“shretinas(GrantandDowling,1996).Oneofthese,EAAT5,isex-pressedathighlevelsinhumanretinas(Arrizaetal.,1997)andhasbeenlocalizedtorodspherulesinmacaqueretinasbylightmicroscopy(Powetal.,2000).ItispossiblethatEAAT5orarelatedmoleculeisalsopresentinthetipsofrodbipolarcelldendrites.BecausePKAstimulatesEAATs(Lortetetal.,1999),HR3activationwouldbeexpectedtodecreasethesignalingthroughthispathway.HistaminemightalsoactbymodulatingtheresponsesofON-bipolarcellstoGABAreleasedfromhorizontalcells.HR3mightstimulatethecation-coupledchloridecotransporter,Na-K-Clcotransporter(NKCC),whichhasalsobeenlocalizedtotheapexofON-bipolarcelldendrites(Vardietal.,2002).Inskeletalmuscle,bothmitogen-activatedproteinkinaseandG-coupledmechanismsincreaseNKCCactivity(Gosmanovetal.,2002;Wongetal.,2001),andHR3canactivatebothofthosesignalingpath-ways(Druteletal.,2001).Thus,histaminemightincreasethechloridein”ux,makingtheequilibriumpotentialforchloride(E)morepositive.Histaminewouldthenincreasetheef“cacyofGABA-mediatedinputfromhorizontalcells.However,thereisonlyasmalldifferencebetweenEattheON-bipolarcelldendritesandEtheaxonterminalsinrats(BillupsandAttwell,2002).ThecloseproximityofHR3andGABAreceptorssuggeststhathistaminemaymodulatetheactivityofiono-tropicGABAreceptorsofON-bipolarcelldendrites.Inslicepreparationsfromferretretinas,GABAappliedtodendritesproducesverysimilarresponsesmediatedbyreceptorsinalltypesofbipolarcells(Shieldsetal.,2000).However,thefunctionofthesereceptorsvariesindifferenttypesofmousebipolarcells(Du¨beletal.,2004),andtheeffectsonmacaquebipolarcellswouldbeexpectedtobevariableaswell.Forexample,theGABAantagonistpicrotoxinhasverylittleeffectonthesurroundresponsesofparasolganglioncellsand,presumably,thebipolarcellsthatprovidetheirinputinmacaqueretinas(McMahonetal.,2004).HR1inratretinasTheprimarytargetsofhistaminergicretinopetalaxonsinratsareHR1ondopaminergicamacrinecells,andthereisevidencesuggestingthattheyareinhibitory.Inratretinas,dopaminereleaseislowestatnightandhighestduringtheday,eveninconstantdarknessandinanimalswithoutphotoreceptors,adultRCS/N-ratshomozy-gousforthe(retinaldystrophy)alleleofphotorecep-tors(Doyleetal.,2002b).Theserhythmsmightbemain-tainedbymelatoninreleasefromthepinealgland(Doyleetal.,2002a)orbyendogenousoscillationsinthedopami-nergicamacrinecellsthemselves(Gustincichetal.,2004;Witkovsky,2004),buthistaminergicretinopetalaxons,whichremainedintactintheseexperiments,mightalsoplayanimportantrole.AlthoughHR1receptorstypicallymediateexcitatoryeffects,therearealsoknowninhibitoryeffects(Brownetal.,2001).HistaminedirectlyinhibitsCA1hippocampalneuronsviaHR1(Selbachetal.,1997),possiblyasaresultofCareleasedfromintracellularstoresactivatingacalcium-dependentpotassiumconduc-tance,asobservedinculturedglialcells(Weigeretal.,1997).Thiscurrenthasbeendescribedpreviouslyindo-paminergicamacrinecells(Feigenspanetal.,1998). Fig.7.SummarydiagramshowingthedistributionofHR3,mGluR6,andGABARinmonkeyOPL.Attheconepedicle,HR3(red)arelocalizedtothetipsofON-bipolarcelldendrites,andmGluR6(green)arevitreadtoHR3.TheoverlapbetweenmGluR6andHR3isrepresentedbyyellow.GABAR(hatching)arelocalizedtobothON-andOFF-bipolarcelldendrites.Arrowsindicatethemouthoftheinvagination.Inrodspherules,HR3extendstothetipsofrodbipolarcelldendritesalongthebifurcationridge(blue),andmGluR6islocatedvitreadtoHR3.Basedonpreviouselectronmicroscopicstudies,GABARarealsoshownonrodbipolarcelldendrites.H,horizontalcell.HISTAMINERECEPTORSINMAMMALIANRETINAS Oneofthemoststrikingresultsinthisstudywasthatthelocalizationofhistaminereceptorswassodifferentinratandmonkeyretinas.Thiswasunexpected,inthatthemorphologyoftheretinopetalaxonsthemselvesisquitesimilarinthetwospecies.Inmonkeys,dendritesofON-bipolarcellscontainedhistaminereceptors,andthesewereHR3.Actingatthesereceptors,histaminewouldbeexpectedtoin”uencetheentireneuralcircuitthatdetectsincrementsinlightintensity.Inrats,themajortargetsweredopaminergicamacrinecells,whichexpressHR1.Theeffectsofhistaminewouldalsobegreatlyampli“edthroughthispathwaybecausedopaminein”uencessomanytypesofneuronsintheretina.Onepossibleexpla-nationforthespeciesdifferencesisthattheyre”ectdif-ferencesinthephoticenvironmentsthatprevailwhentheanimalsaremostactive.Thiscouldbefurthertestedbydeterminingthedistributionofhistaminereceptorsinavarietyofdiurnalandnocturnalanimals.WearegratefultoMrs.LillemorKrosbyandMs.An-dreaBordtforexcellenttechnicalassistanceandtoDrs.StephenMills,BradyTrexler,andSamuelWuforvalu-ablediscussions.WealsothankDrs.KarenRice,JerilynPecotte,andGeneHubbardattheSouthwestFoundationforBiomedicalResearchinSanAntonioforprovidingmonkeyeyesandDr.PramodDashatUTMedicalSchoolatHoustonforprovidingrateyes.LITERATURECITEDAiraksinenMS,PanulaP.1988.Thehistaminergicsystemintheguineapigcentralnervoussystem:animmunocytochemicalmappingstudyusinganantiserumagainsthistamine.JCompNeurol273:163…186.ArrangJM,GarbargM,SchwartzJC.1983.Auto-inhibitionofbrainhis-taminereleasemediatedbyanovelclass(H3)ofhistaminereceptor.Nature302:832…837.ArrizaJL,EliasofS,KavanaughMP,AmaraSG.1997.Excitatoryaminoacidtransporter5,aretinalglutamatetransportercoupledtoachlo-rideconductance.ProcNatAcadlSc94:4155…4160.BagnoliP,DalMonteM,CasiniG.2003.Expressionofneuropeptidesandtheirreceptorsinthedevelopingretinaofmammals.HistolHis-topathol18:1219…1242.BillupsD,AttwellD.2002.ControlofintracellularchlorideconcentrationandGABAresponsepolarityinratretinalONbipolarcells.JPhysiolBrownRE,StevensDR,HaasHL.2001.Thephysiologyofbrainhistamine.ProgNeurobiol63:637…672.CasiniG,SabatiniA,CatalaniE,WillemsD,BoscoL,BrechaNC.2002.Expressionoftheneurokinin1receptorintherabbitretina.Neuro-science115:1309…1321.CatalaniE,GangitanoC,BoscoL,CasiniG.2004.Expressionoftheneurokinin1receptorinthemouseretina.Neuroscience128:519…530.ChazotPL,HannV,WilsonC,LeesG,ThompsonCL.2001.Immunologicalidenti“cationofthemammalianH3histaminereceptorinthemousebrain.Neuroreport12:259…262.ClarkEA,HillSJ.1996.SensitivityofhistamineH3receptoragonist-stimulated[S]GTPgamma[S]bindingtopertussistoxin.EurJPharmacol296:223…225.CristianiR,FontanesiG,CasiniG,PetrucciC,ViolletC,BagnoliP.2000.Expressionofsomatostatinsubtype1receptorintherabbitretina.InvestOphthalVisSci41:3191…3199.DoyleSE,GraceMS,McIvorW,MenakerM.2002a.Circadianrhythmsofdopamineinmouseretina:theroleofmelatonin.VisNeurosci19:593…DoyleSE,McIvorWE,MenakerM.2002b.Circadianrhythmicityindopa-minecontentofmammalianretina:roleofthephotoreceptors.JNeu-rochem83:211…219.DrutelG,PeitsaroN,KarlstedtK,WielandK,SmitMJ,TimmermanH,PanulaP,LeursR.2001.Identi“cationofratH3receptorisoformswithdifferentbrainexpressionandsignalingproperties.MolPharmacol59:1…8.¨belJ,HaverkampS,KunerT,EulerT.2004.ChlorideimaginginON-typebipolarcellsofaClomeleonindicatormouseline[abstract].InvestOphthalVisSci45:1324.FanSF,YazullaS.1999.Suppressionofvoltage-dependentKcurrentsinretinalbipolarcellsbyascorbate.VisNeurosci16:141…148.FeigenspanA,BormannJ.1994.FacilitationofGABAergicsignalingintheretinabyreceptorsstimulatingadenylatecyclase.ProcNatAcadlSciUSA91:10893…10897.FeigenspanA,GustincichS,BeanBP,RaviolaE.1998.Spontaneousactivityofsolitarydopaminergiccellsoftheretina.JNeurosci18:6776…6789.FujimotoK,HorioY,SugamaK,ItoS,LiuYQ,FukuiH.1993.GenomiccloningoftherathistamineH1receptor.BiochemBiophysResCom-mun190:294…301.GantzI,MunzertG,TashiroT,SchafferM,WangL,DelValleJ,YamadaT.1991.MolecularcloningofthehumanhistamineH2receptor.Bio-chemBiophysResCommun178:1386…1392.GastingerMJ,OBrienJJ,LarsenNB,MarshakDW.1999.Histamineimmunoreactiveaxonsinthemacaqueretina.InvestOphthalVisSci40:487…495.GastingerMJ,BarberAJ,KhinSA,McRillCS,GardnerTW,MarshakDW.2001.Abnormalcentrifugalaxonsinstreptozotocin-diabeticratreti-nas.InvestOphthalVisSci42:2679…2685.GosmanovAR,WongJA,ThomasonDB.2002.DualityofGprotein-coupledmechanismsforbeta-adrenergicactivationofNKCCactivityinskeletalmuscle.AmJPhysiolCellPhysiol283:C1025…1032.GrantGB,DowlingJE.1996.Onbipolarcellresponsesintheteleostretinaaregeneratedbytwodistinctmechanisms.JNeurophysiol76:3842…GustincichS,ContiniM,GariboldiM,PuopoloM,KadotaK,BonoH,LeMieuxJ,WalshP,CarninciP,HayashizakiY,OkazakiY,RaviolaE.2004.Genediscoveryingeneticallylabeledsingledopaminergicneu-ronsoftheretina.ProcNatlAcadSc101:5069…5074.HaverkampS,Gru¨nertU,Wa¨ssleH.2000.Theconepedicle,acomplexsynapseintheretina.Neuron27:85…95.HayashiH,TakagiH,TakedaN,KubotaY,TohyamaM,WatanabeT,WadaH.1984.Finestructureofhistaminergicneuronsinthecaudalmagnocellularnucleusoftheratasdemonstratedbyimmunocyto-chemistryusinghistidinedecarboxylasea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