AnatomyandPhysiologyoftheRetina

RelevanttoInheritedRetinalDisease

atutorialfrom

StoneRounds.org

EdwinM.Stone,M.D.,Ph.D.Editor

©TheUniversityofIowa,2018

Page 2Thisistherighteyeofahumandonorviewedfromabove.Youcantellthatit’stherighteyebecausetheopticnerveexitstheeyeonthenasalsideofcenter.

Thetransparentfrontwalloftheeyeisknownasthecorneaandthecurvedsurfaceofthisstructureprovidesabout75%oftheconverginglenspowerneededtofocusimagesonthebackoftheeye.

Theremaining25%ofthefocusingpowercomesfromtheflexiblecrystallinelens,whichactuallybecomesthickerwhenapersonlooksanearobjectsduetocontractionofacircumferentialmuscleknownastheciliarybody.Thethincoloredstructurebetweenthecorneaandthelensistheiriswhichfunctionstocontroltheamountoflightenteringtheeye.

Theposteriortwothirdsoftheeyeislinedbyaverythinlayerofnervetissue–theretina.Inlife,theretinaiscompletelytransparentandthebrownishororangecolorthatyouseewhenlookingintotheeyewithanophthalmoscopeisduealmostentirelytothebloodfilledcapillariesandpigmentedstructuresbeneathit.

Thecentralportionoftheretina-shownhereinorange-isknownasthemacula.

Thisisacolorphotographoftheretinaoftherighteyeofanormalindividual.

Theopticnerveis1.5mmindiameterandthebloodsupplyoftheinneraspectoftheretinaemanatesfromthecenterofthenerveandsweepsaroundthemacula.

Thevenulescanbedistinguishedfromthearteriolesbecauseoftheirthickercaliberanddarkerpurplecolor.Theselargecalibervesselssupplyanddraincapillaryplexusesthatlieprimarilyintheganglioncellandinnernuclearlayers.

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Theretinahasaseconddistinctbloodsupplythatliesbetweentheretinalpigmentepitheliumandthesclera.ThefinecapillariesadjacenttotheRPEareknownasthechoriocapillarisandthesevesselsprovidethetremendousamountofoxygenneededbythephotoreceptorcells.

Opticalcoherencetomographyisaclinicalimagingtechniquethatcanprovideimagesoftheretinainlivingindividualswithnear-histologiclevelsofresolution.

Computerizedimageanalysisoftheseimagescan“segment”thetomogramintolayersthatcorrespondtothedifferentanatomicallayersoftheretina.Asinthegrossanatomyofthebrainthedarkergreylayerscorrespondtocollectionsofcellbodieswhilethelightergreyareascorrespondtothedenselypackedaxonsanddendritesthatconnecttheseneuronstoeachother.

ZoominginonasmallsectionoftheretinawecanillustrateourcurrentunderstandingoftheanatomiccorrelationswiththevaryingintensitiesoftheOCTimage.Withpropercontrols,onecandetectanatomicalchangeswitharesolutionofafewmicronsusingthisinstrument.

Hereisanartist’sconceptionofthemostimportantretinallayersthatmakestherelationshipsofthesecellsalittleeasiertovisualize.

Page 4Itisimportanttonotethatthereare20timesmorerodphotoreceptorsthanconesinthehumanretina,andthattheshortwavelengthsensitiveconesaretheleastnumerousofall.

Thelowestratioofphotoreceptorstoganglioncellsoccursinthefovea–approximately5toone--whichenablestheveryhighacuityvisionassociatedwiththisregion.Intheperiphery,asingleganglioncellcanreceiveinputfrommorethan1000photoreceptors.

Hereisanotherviewofthevariationindensityofvariousretinalneuronswithincreasingeccentricityfromthefovea.

Theveryhighdensityofconephotoreceptorsinthefoveahelpsexplainwhycone-selectivediseasesareoftenassociatedwithdecreasedacuity.

Therods’peakdensityoccursmorethan3millimetersfromthefovealcenterandislikelytheexplanationfortheringscotomasthatarecharacteristicoftherod-selectivediseaseretinitispigmentosa.

Theganglioncellsarethemostnumerousinthecentral5millimetersoftheretina.Oneofthereasonsthatourinitialphotoreceptorprecursortransplantswillbeplacedinthisregionisbecauseofthegreaterchancethesetransplantswillhavetoestablishsynapticconnectionwiththerelativelynumerousinnerretinalneuronsofthisregion.

RichardYoung’senfacephotomicrographsoftheprimateretinagiveusanotherviewofhowtheretinalmosaicchangeswithincreasingeccentricity.Thefovealcenterconsistsentirelyofunusualconeswithveryslenderoutersegments.

Onemillimeterfromthefovealcenter,aringofrodscanbeseensurroundingeachconeandoutsidethemacula,theratioofrodstoconesisevenhigher.

Ifwetakeaimmunohistochemicalsectionthroughthefoveaofahumandonoreyewecanseetheabsenceofrhodopsin-containingrodsnearthefovealcenteraswellasthewayinwhichthe

Page 5ganglioncellsthataresynapticallyrelatedtothefovealconesaredisplacedlaterally.Youcantellthatthissectiondoesnotpassthroughtheabsolutecenterofthefoveabecauseifitdid,theinnernuclearlayerwouldalsobedisplacedlaterally.

Fordescriptivepurposes,themaculahasbeendividedintosixconcentriczonesandthedimensionoftheouterboundaryofeachofthesezonesisshownhere.Clinically,thefoveolaandfovealavascularzoneareessentiallysynonymous.Thefoveaisthesamediameterastheopticnervehead–1.5millimeters.Theprefix“para”means“nextto”andtheterm“parafovea”ismostappropriatelyusedforfindingsthatlieinazonegreaterthanonebutabitlessthantwodiskdiameterscenteredonthefoveallightreflex.Theprefix“peri”means“around”andtheterm“perifovea”isusedforaringonediskdiameterinthicknessattheoutermostedgeofthemacula.

Thereasonthatweareinterestedintheunevendistributionsofthevariouscelltypesintheretinaisthatitislikelythattheregionaldifferencesinretinalanatomyplayanimportantroleinthediseasemechanismsofmanyoftheconditionswe’reinterestedinandanunderstandingofthesemechanismswillbeimportantinthesuccessfultreatmentoftheseconditions.

Oneothertypeofregionalspecializationweshouldtouchonbrieflyinvolvestheinnermostlayersoftheretina–theganglioncellsandtheiraxons.

Hogan’sclassicdrawingshowshowtheaxonsofganglioncellsnasaltothefoveaapproachtheopticnerveheadfairlydirectlywhilethosetemporaltothefoveasweepaboveandbelowthemaculatominimizetheamountoftissuelyingbetweentheincomingphotonsandthemacularphotoreceptors.

Oneofthepracticaleffectsofthisanatomicspecializationisthatganglioncellsthatarequiteclosetoeachotherinthetemporalretinacansendtheiraxonstotheopticnervebyquitedifferentroutes.Thisistheanatomicalexplanationforthe“nasalstep”observedinthevisualfieldsofmanyglaucomapatients.

Page 6Thelinealongwhichthissuperior/inferiorroutedecisionismadeisknownasthehorizontalraphe.

Becauseofthelowratioofphotoreceptorstoganglioncellsinthemacula,therearealargenumberofaxonsthatoriginatethereandgodirectlytotheopticnerve.Theseaxonsarerequiredforhighacuityvisionandareknownasthepapillomacularbundle.

Now,let’sspendafewminutesreviewingsomeofthephysiologyofthemoreimportantcelltypesintheretina.Thebasicwiringoftheretinaconsistsofjustthreeneuronsthephotoreceptorcellthebipolarcellandtheganglioncell.Theaxonsofthephotoreceptorsandbipolarcellstravelonlyafewdozenmicronsbeforesynapsingonthenextcellbuttheaxonsoftheganglioncellstravelover90millimeters–threeandahalfinches--togettothelateralgeniculatenucleusofthethalamus.

Allthreeoftheseneuronsuseglutamateastheirneurotransmitter.

Thephotoreceptorsandbipolarcellshavegradedinputsandoutputsbuttheganglioncellsconverttheirgradedinputsintotrainsofactionpotentialsthattraveldownmyelinatedneurons.Tomaintainthetransparencyofthenervefiberlayer,themyelindoesn’tbeginuntiltheaxonsexittheeye.

Somepeoplewonderwhyevolutionplacedthelightdetectingphotoreceptorsclosesttothescleraandalloftheotherneuronsbetweenthemandtheincominglight.

Thereasonisbecauseoftheessentialfunctionsperformedbytheretinalpigmentepithelium.TheRPEistheretina’sprimarysourceofthelightsensitivechromophore11-cisretinal.

Aswe’llseeinmoredetailinjustaminute,theprocessthatrecycles11-cis-retinalfromtheall-trans-retinalreleasedfromthephotoreceptorsisknownasthevisualcycleandittakesplaceoveraperiodoftensofminutes.Thisistheprocessthatyouexperiencewhenyoureyes“getusedtothedark”inamovietheatreorwalkingonatrailatnightonacampingtrip.

Theprocessthatconvertsastreamofphotonstoanincreaseinphotoreceptormembranepotentialisknownasphototransduction.

Thisprocessisfourordersofmagnitudefasterthanthevisualcycle–lessthan1/30thofasecondinconephotoreceptors.

Let’slookatthemechanismofphototransductioninabitmoredetail.

Page 7Todothiswe’ll“magup”ontheoutersegmentofarodphotoreceptor,showingaflattenedlipid-bilayerdiskcontainingatransmembraneproteinknownasrhodopsin.

Rhodopsinisa“G-protein-coupledreceptor”thatmediatesthefirststepofphototransduction.

ThelightsensitiveformofrhodopsincontainsamoleculeofvitaminAknownas11-cis-retinalcovalentlylinkedtoitatlysine296.

When11-cisretinalabsorbsaphoton,itisomerizestoalltransretinal.Thatisomerizationcausesaconformationalchangeinrhodopsinwhichsetsofftheremainderofthephototransductioncascade.

Let’sreturntothediagramoftheoutersegmentdiskandcellmembranetoseethemainstepsinthecascade.

Inthedark,acyclicnucleotidegatedcationchannelisopenandsodiumionsflowinfreely,therebyloweringthemembranepotential.

WhenlightstrikesavitaminAmoleculeboundtorhodopsin,itisomerizesthevitaminAtoall-trans,whichcausesaconformationalchangeintheprotein.

Thisconformationalchangedissociatesrhodopsin’strimericGprotein,knownas

Page 8transducin.ThealphasubunitoftransducinactivatesaphosphodiesterasewhichcleavesthecyclicGMPthathasbeenkeepingthecationchannelopen.Whenthechannelcloses,therestingmembranepotentialrises–diminishingthereleaseofneurotransmitterfromthesynapticterminalsofthephotoreceptoraxon.

Sotoreviewthemainproteinsinthephototransductioncascade:lightfallingonthe11-cisretinalboundtorhodopsincausesittochangeconformation,thisreleasesalphatransducinwhichactivatesphosphodiesterase,loweringcyclicGMP,closingthecationchannel,elevatingthemembranepotential,andreducingneurotransmitterrelease.

Theevolutionarypurposeofthiselaboratecascadeislow-noiseamplification–asinglephotonfallingonarodphotoreceptorcanincreaseitsmembranepotentialbyamillivolt–thisrepresentsatleastaonemillionfoldamplificationoftheinputsignal.

Theby-productofphototransductionisall-trans-retinal.Forthismoleculetobeabletodetectanotherphoton,itneedstobere-isomerizedto11-cis-retinal.

Thisprocessisknownasthevisualcycleandasyouwillsee,someofthestepstakeplaceoutsidethephotoreceptors.Also,theprocessisabitdifferentforrodsandcones.

Theprocessbeginswhenall-trans-retinalisreleasedfromrhodopsinintothelipidbilayeroftheoutersegmentdisks.There,thealdehydegroupoftheretinalisexposedtotheaminogroupsofthepolarheadsofsomeofthemembranelipidsandareversiblecovalentbondformscreatingamoleculeknownasN-retinylidenephosphatidylethanolamineorN-ret-PE.

TheproteinencodedbythegeneABCA4isanATP-bindingcassettetransporterthatflipsN-Ret-PEtotheouterleafletofthediskmembrane.

There,thealltransretinalismetbyadehydrogenase--RDH8--thatremovesitfromthephospholipidandconvertsittoalessreactivealcohol.

Page 9Itisthisall-trans-retinolformofvitaminAthatleavesthephotoreceptorfortransporttotheretinalpigmentepitheliumorMuellercellsboundtoaproteinknownastheinterphotoreceptorretinoidbindingproteinorIRBP.

Aftercrossingtheplasmamembraneoftheretinalpigmentepitheliumthealltransretinolmovestotheendoplasmicreticulumwhereitisesterifiedtoalltrans-retinylesterbythelecithinretinolacyltransferase,commonlyreferredtoasLRAT.

Theseall-trans-retinylestersareinturnacteduponbyRPE65,whichde-esterifiestheretinolandre-isomerizesittothelightsensitive11-cisisomerofvitaminA.

The11-cisretinolisthenoxidizedto11-cisretinalbymembraneboundretinoldehydrogenasessuchasRDH5.The11-cis-retinalthenmovestothecellsurface,crossestheplasmamembraneandisreturnedtothephotoreceptorcellbyIRBP.

Thevisualcycleiscompletewhenthe11-cisretinalreturnstothebindingpocketofrhodopsin,formsacovalentbondwithlysine296,andrestoresrhodopsintothelightsensitivestate.

Tosummarizethekeystepsofthevisualcycle:

Lightisomerizes11-cisretinaltoall-trans-retinal,whichthenbindstothediskmembraneasN-retPE.

ABCA4flipstheN-retPEtothecytoplasmicleafletofthediskmembrane.

RDH8reducesthe11-cis-retinaltoall-trans-retinol.

IRBPcarriesall-trans-retinoltotheretinalpigmentepitheliumwhereRPE65re-isomerizesitto11-cis-retinolandRHD5oxidizesitto11-cis-retinal.

IRBPthenreturnsthe11-cis-retinaltothephotoreceptor.

AutosomalrecessiveStargardtdisease-iscausedbyadeficiencyoftheATP-bindingcassettetransporterA4,whichaswehavejustseenusuallyflipsN-retinylidenephosphatidylethanolaminefromtheinnertotheouterleafletoftheoutersegmentdiskmembrane.

Page 10BecauseofthedysfunctionofABCA4,theN-ret-PEgraduallyaccumulatesontheinnerleafletuntiltheconcentrationreachesapointthatasecond-retinylidenemoleculebindstoasingleethanolaminecreatinganirreversibleandinsolublebis-retinoidknownasA2PE.

A2-PEisdirectlytoxictothephotoreceptorsbutalsoaccumulateswithinandbeneaththeretinalpigmentepitheliumintheformoftheophthalmoscopicallyvisiblepisciformflecksthatarecharacteristicofStargardtdisease.