光遗传技术比较好的一篇nature综述.pdf

光遗传技术比较好的一篇nature综述.pdf

《光遗传技术比较好的一篇nature综述.pdf》由会员分享，可在线阅读，更多相关《光遗传技术比较好的一篇nature综述.pdf（12页珍藏版）》请在冰点文库上搜索。

298VOLUME15|NUMBER2|FEBRUARY2012natureneurOSCIenCeartICleSRetinalganglioncell（RGC）projectionstothebrainformstereo-typicmapsforeyeoforiginandretinotopiclocation,makingthemanidealmodelsystemtostudythedevelopmentandplasticityofpreciselypatternedneuralcircuits1,2.Theinitialformationofthesevisualcircuitsisthoughttobeguidedbymolecularcues3,whereastherefinementandmaintenance46oftheseconnectionsseemstobeactivitydependent7.Substantialevidencesupportsageneralroleforactivity-dependentbinocularcompetitioninretinofugalmapdevel-opment.Forinstance,arelativeincreaseinactivityinoneeyeleadstotheexpansionofthateyestargetterritoryinthedorsallateralgenicu-latenucleus（dLGN）8,9,indicatingthatthemoreactiveeyemakesandfurtherstrengthensmoretargetsynapseswhenitisatacompeti-tiveadvantage.Hebbiansynapticlearningrulesthatmaymediatetheactivity-dependentdevelopmentofvisualmapshavebeenobservedinavarietyofretinofugalsystems,includingspike-timingdependentplasticityatretinotectalsynapsesintadpolesinvivo10andbursttimingdependentplasticityatretinogeniculate11andretinocollicular12synapsesinrodentsinvitro.Theseobservationssuggestthatsynapticconnectionsarefunctionallystrengthenedwhencellsaresynchronouslyactiveandweakenedwhencellsareasynchronouslyactiveovertimewindowsthataredistinctindifferentmodelsystems13.Ithaslongbeenpostulatedthatthetimingofspontaneouswave-likeactivityinRGCs14iscriticalfortheestablishmentandmaintenanceofeye-specificsegregationthroughaHebb-basedsynapticlearningrule11,13beforetheonsetofvision.Theshortdurationofretinalwavesrelativetotheintervalbetweenwavesisthoughttoasynchronouslyactivatethetwoeyes,resultingintherefinementofeye-specificdomains15.Evidenceforthistimingmodelforbinocularcompetitionislimited,withtheonlydirectexperimentalsupportcomingfromclassiccatexperimentsinwhichartificiallyasynchronousstimulationoftheopticnervesproducedneuronsthatrespondedpredominantlytoonlyoneeye,whereasstimulationoftheoptictract,whichsynchro-nouslyactivatesRGCafferentsfrombotheyes,causedmostcellsinvisualcortextobecomefunctionallybinocular16.Similarly,alternat-ingmonocularocclusionincatsresultsinreducedcorticalbinocular-ityanddisrupteddepthdiscrimination17.However,theseexperimentswererestrictedtoaphysiologicalanalysisofbinocularityinthecortexandmanipulatedRGCactivityaftertheonsetofnormalvisualexperi-ence,wheneyesegregationinthedLGNandvisualcortexhasalreadyemerged2,18.Becauseithasbeendifficulttopreciselymanipulateneo-natalRGCactivityinmammalsinvivo,theroleoftimingintheinitialdevelopmentofvisualmapsremainsunexplored.Wechronicallymanipulatedretinalactivityinmicebeforetheonsetofvisionoverarangeoftimescalesinvivobyexpressingthelight-gatedcationchannelChannelrhodopsin-2（ChR2）19directlyinRGCsusingtransgenicandviraltransfectionmethods20,21.Light-drivenactivationofChR2-expressingRGCstriggeredpreciselytimedpost-synapticcalciumsignalsinthesuperiorcolliculus,demonstratingthatoptogenetictechniquescanreliablydriveneuronalresponseevenearlyinvisualdevelopment.Whenthetwoeyesweresynchronouslystimu-lated,wefoundthattheinitialemergenceofeye-specificdomainswasdisrupted,whereasasynchronousstimulationimprovedsegregation.Aftereye-specificdomainswerealreadyestablishedinthesuperiorcolliculusanddLGN,asynchronousstimulationhadnoeffect,butsyn-chronousstimulationcauseddomainstodesegregate.Thedisruptiveeffectofoptogeneticstimulationoneyesegregationwanedasthetimedifferencebetweenstimulationoftheeyesincreasedbeyond100ms,whichsuggestsasub-secondtimewindowforbinocularcompeti-tion.Ofnote,whensynchronousstimulationdisruptedeye-specific1DepartmentofNeurobiology,YaleUniversity,NewHaven,Connecticut,USA.2DepartmentofOphthalmology&VisualSciences,YaleUniversity,NewHaven,Connecticut,USA.CorrespondenceshouldbeaddressedtoM.C.C.（michael.crairyale.edu）.Received8September;accepted9November;publishedonline18December2011;doi:

10.1038/nn.3007VisualmapdevelopmentdependsonthetemporalpatternofbinocularactivityinmiceJiayiZhang1,JamesBAckman1,Hong-PingXu1&MichaelCCrair1,2Binocularcompetitionisthoughttodriveeye-specificsegregationinthedevelopingvisualsystem,potentiallythroughHebbiansynapticlearningrulesthataresensitivetocorrelationsinafferentactivity.Alteringretinalactivitycandisrupteye-specificsegregation,butlittleisknownaboutthetemporalfeaturesofbinocularactivitythatmodulatevisualmapdevelopment.Weusedoptogenetictechniquestodirectlymanipulateretinalactivityinvivoandidentifiedacriticalperiodbeforeeyeopeninginmicewhenspecificbinocularfeaturesofretinalactivitydrivevisualmapdevelopment.Synchronousactivationofbotheyesdisruptedsegregation,whereasasynchronousstimulationenhancedsegregation.Theoptogeneticstimulusappliedwasspatiallyhomogenous;accordingly,retinotopyofipsilateralprojectionswasmarkedlyperturbed,butcontralateralretinotopywasunaffectedorevenimproved.Theseresultsprovidedirectevidencethatthesynchronyandprecisetemporalpatternofbinocularretinalactivityduringacriticalperiodindevelopmentregulateseye-specificsegregationandretinotopyinthedevelopingvisualsystem.npg2012NatureAmerica,Inc.Allrightsreserved.natureneurOSCIenCeVOLUME15|NUMBER2|FEBRUARY2012299artICleSsegregation,retinotopywasalsomarkedlyperturbed,butonlyforipsilateralRGCs.Bothsynchronousandasynchronousstimulationslightlyimprovedtheretinotopyofcontralateralaxons.Finally,alloftheseeffectswerelimitedtoacriticalperiodindevelopmentthatendsaroundthetimeofeyeopening.TheseresultsdemonstratetheimportanceofprecisetemporalsynchronyofbinocularRGCactivityintheanatomicaldevelopmentandmaintenanceofvisualmaps.RESULTSPrecisecontrolofRGCneuronalactivityRGCsinmiceyoungerthanpostnatalday（P）10donotrespondtolightthroughtheconventionalrod-orcone-drivenpathway22.IntrinsicallyphotosensitiveRGCs（ipRGCs）haveaslowandsluggishresponsetolightfrombirth,buttheyconstituteonlyasmallfractionofRGCs23.Toexogenouslyandpreciselymanipulateretinalactivityinneonatalmice,wefirstusedaTg（Thy1-COP4/EYFP）18Gfng（Thy1-ChR2）transgenicmouselinethathasRGC-specificexpressionofChR2andyellowfluorescentprotein21,24.ChR2inthesemiceisexpressedinaheterogeneouspopulationofRGCsdistributeduniformlyacrosstheentireretina24startingataroundP8（Fig.1a）.ChR2-eYFPexpressingRGCsconstituted25.43.5%ofallthebrn3b-positiveRGCsatP9（n=8）.Asbrn3blabels80%ofallRGCs25,20%ofRGCsexpressedChR2-eYFPinThy1-ChR2miceatP9.InvitrowholecellrecordingshowedthatYFP-positiveRGCsshowedsustainedspikingactivityinresponseto470-nmlightstimuliwitharangeofintensitiesabove0.1625mWmm2（Fig.1b）.Multielectrodearrayrecordingsrevealedthatspikeratesweresimilarfor1-sand200-msstimuli（6.40.2Hzfor1s（n=70cells）and4.70.3Hzfor200ms（n=57cells）at0.51mWmm2;Fig.1c,d）,whichindicatesthatthenumberoflight-triggeredspikeswasroughlyproportionaltothedurationofthestimuli.ThefractionofallspontaneouslyactiveRGCsthatwerelightresponsivewasalsosimilaracrossawiderangeofstimulusdura-tions（68.65.5%,55.81.4%and74.47.4%for1-s,200-msand5-msstimuli,respectively;Fig.1d）.Thelight-drivenactivitywasnotdependentonsynapticinput（SupplementaryFig.1a）,confirmingitcamedirectlyfromChR2-expressingRGCs.Weoccasionallyobservedtonicfiringthatlastedmuchlongerthanthedurationofthestimuli（SupplementaryFig.1b）;theseeventswereprobablyresponsesfromipRGCs23,26andwereexcludedfromtheanalysis.Finally,multiunitrecordingsshowedthatthe470-nmlightstimuliappliedoutsidetheeyecoulddriveneuronalresponseinthesuperiorcolliculusinvivowithhightemporalprecision（Fig.1e,f）.TheseresultsconfirmedthatoptogenetictechniquescanbeusedtomanipulateRGCactivitybeforetheonsetofnormalvisioninmiceinvivo.Eventhoughyoungmicedonotrespondtolightthroughconven-tionalretinalpathways,synapticconnectionsbetweenRGCsandneu-ronsinthesuperiorcolliculusexistatbirthandmaturethroughoutthefirsttwopostnatalweeks12.InThy1-ChR2mice,weexaminedthespatialandtemporalresponseofneuronsinthesuperiorcolliculustooptoge-neticactivationofRGCsusinglight-triggeredcalciumsignalsfromsuperiorcolliculusneuronslabeledwiththecalciumindicatorOregonGreenBAPTA-1-AM.（OGB1-AM）invivoatP9P10（Fig.2a,b）.Synchronousstimulationofbotheyes（1sduration）triggeredcalciumsignalsinbothhemispheresofthesuperiorcolliculussimultaneously（Fig.2c,d）.About30%oftheregionsofinterest（ROIs）showeda40m500m123456781234567810s10s12345678ChR2eYFPbrn3bb100ms20mVSpikesper0.2s0.5mVd4323020100Count1012345Time（s）500ms40Vef020406080Fractionlightresponsivechannel（%）1-sstimac1s5ms200ms200-msstim5-msstimMEAgridlayoutMEAgridlayoutSCChR2eYFPFigure1ChR2-expressingretinalganglioncells（RGCs）areactivatedwithhightemporalprecisionwithbluelightbothinvitroandinvivo.（a）Left:

ChR2-expressingRGCsinThy1-ChR2transgenicmicearedistributedacrosstheentireretina.Right:

brn3b-positiveRGCsinred,ChR2-expressingcellsingreen.WhitearrowsshowexamplesofRGCsexpressingbothbrn3bandChR2.（b）WholecellrecordingofaChR2RGCinresponseto300mslightstimuliatintensitiesof0.65,0.325and0.1625mWmm2（fromtoptobottom;mercurylampfiltered）.Bluebars