光遗传学技术的发展和应用.pdf
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光遗传学技术的发展和应用.pdf
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NE34CH17-DeisserothARI21May201115:
22TheDevelopmentandApplicationofOptogeneticsLiefFenno,1,2OferYizhar,1andKarlDeisseroth1,3,41DepartmentofBioengineering,2NeuroscienceProgram,3DepartmentsofPsychiatryandBehavioralSciences,4HowardHughesMedicalInstitute,StanfordUniversity,Stanford,California94305;email:
deisserostanford.eduAnnu.Rev.Neurosci.2011.34:
389412TheAnnualReviewofNeuroscienceisonlineatneuro.annualreviews.orgThisarticlesdoi:
10.1146/annurev-neuro-061010-113817Copyrightc?
2011byAnnualReviews.Allrightsreserved0147-006X/11/0721-0389$20.00Keywordschannelrhodopsin,halorhodopsin,bacteriorhodopsin,electrophysiologyAbstractGeneticallyencoded,single-componentoptogenetictoolshavemadeasignificantimpactonneuroscience,enablingspecificmodulationofselectedcellswithincomplexneuraltissues.Astheoptogenetictoolboxcontentsgrowanddiversify,theopportunitiesforneurosciencecon-tinuetogrow.Inthisreview,weoutlinethedevelopmentofcurrentlyavailablesingle-componentoptogenetictoolsandsummarizetheappli-cationofvariousoptogenetictoolsindiversemodelorganisms.389Annu.Rev.Neurosci.2011.34:
389-412.Downloadedfromwww.annualreviews.orgbyShanghaiInformationCenterforLifeScienceson04/21/12.Forpersonaluseonly.ClickhereforquicklinkstoAnnualReviewscontentonline,including:
OtherarticlesinthisvolumeTopcitedarticlesTopdownloadedarticlesOurcomprehensivesearchFurtherANNUALREVIEWSNE34CH17-DeisserothARI21May201115:
22ContentsINTRODUCTION.390EARLYEFFORTSTOWARDOPTICALCONTROL.390MICROBIALOPSINS.391OPTOGENETICTOOLSFORNEURONALEXCITATION.393OPTOGENETICTOOLSFORNEURONALINHIBITION.396OPTOGENETICTOOLSFORBIOCHEMICALCONTROL.397DELIVERINGOPTOGENETICTOOLSINTONEURONALSYSTEMS.398TRANSGENICANIMALS.399DEVELOPMENTALANDLAYER-SPECIFICTARGETING.399CIRCUITTARGETING.401LIGHTDELIVERYANDREADOUTHARDWAREFOROPTOGENETICS.401OPTOGENETICSINDIVERSEANIMALMODELS.402Caenorhabditiselegans.402Fly.402Zebrafish.403Mouse.404Rat.405Primate.405OUTLOOK.405INTRODUCTIONIndescribingunrealizedprerequisitesforas-semblingageneraltheoryofthemind,FrancisCrickobservedthattheabilitytomanipulateindividualcomponentsofthebrainwouldbeneeded,requiring“amethodbywhichallneuronsofjustonetypecouldbeinactivated,leavingtheothersmoreorlessunaltered”(Crick1979,p.222).Extracellularelectricalmanipulationdoesnotreadilyachievetruein-activation,andevenelectricalexcitation,whileallowingfortemporalprecisioninstimulatingwithinagivenvolume,lacksspecificityforcelltype.However,pharmacologicalandgeneticmanipulationscanbespecifictocellswithcertainexpressionprofiles(inthebestcase)butlacktemporalprecisiononthetimescaleofneuralcodingandsignaling.Becausenopriortechniquehasachievedbothhigh-temporalandcellularprecisionwithinintactmammalianneuraltissue,therehasbeenstrongpressuretodevelopanewclassoftechnology.Asaresultoftheseefforts,neu-ronsnowmaybecontrolledwithoptogeneticsforfast,specificexcitationorinhibitionwithinsystemsascomplexasfreelymovingmammalsforexample,withmicrobialopsinmethods,light-inducedinwardcationcurrentsmaybeusedtodepolarizetheneuronalmembraneandpositivelymodulatefiringofactionpotentials,whileopticalpumpingofchlorideionscaninduceoutwardcurrentsandmembranehyperpolarization,therebyinhibitingspiking(Figure1).Theseoptogenetictoolsofmicro-bialorigin(Figure1)maybereadilytargetedtosubpopulationsofneuronswithinhetero-geneoustissueandfunctiononatemporalscalecommensuratewithphysiologicalratesofspikingorcriticalmomentsinbehavioraltests,withfastdeactivationuponcessationoflight.Withtheseproperties,microbe-derivedoptogenetictoolsfulfillthecriterionsetforthbyCrickin1979(Deisseroth2010,2011).EARLYEFFORTSTOWARDOPTICALCONTROLThemicrobialopsinapproachisheirtoalongtraditionofusinglightasaninterventioninbiology.Withchromophore-assistedlaserinactivation,lightcanbeusedtoinhibittargetedproteinsbydestroyingthemwhatageneticistwouldcall“lossoffunction”(Schmuckeretal.1994);conversely,laserscanbeusedtostimulateneuronsdirectlyinawaythatcouldbeadapted(inprinciple)tocontrolfluorescentlylabeled,geneticallytargetedcellswhatageneticistwouldcall“gainoffunction”(Fork1971,Hiraseetal.2002).Next,variouscascadesofgenes,andcombinationsofgeneswithchemicals,weretestedasmulticomponent390FennoYizharDeisserothAnnu.Rev.Neurosci.2011.34:
389-412.Downloadedfromwww.annualreviews.orgbyShanghaiInformationCenterforLifeScienceson04/21/12.Forpersonaluseonly.NE34CH17-DeisserothARI21May201115:
22Na+Na+Na+Na+Na+Na+K+H+H+H+H+H+H+K+K+K+K+K+Na+Na+Na+Na+Ca2+Ca2+Ca2+Na+Na+ChRClClClClClClClClClClClClClClClClHRIP3DAGcAMPcAMPGsGqGiOptoXRFigure1Optogenetictoolfamilies.Channelrhodopsinsconductcationsanddepolarizeneuronsuponillumination(left).Halorhodopsinsconductchlorideionsintothecytoplasmuponyellowlightillumination(center).OptoXRsarerhodopsin-GPCR(Gproteincoupledreceptor)chimerasthatrespondtogreen(500nm)lightwithactivationofthebiologicalfunctionsdictatedbytheintracellularloopsusedinthehybrid(right).strategiesforopticalcontrol;rhodopsinandarrestingenesfromDrosophilaphotoreceptorswerecombinedtolight-sensitizeneurons(Zemelmanetal.2002);ligand-gatedchannels,combinedwithultraviolet(UV)-lightpho-tolysisofcagedagonists,weredevelopedforDrosophilaexperiments(Lima&Miesenbock2005,Zemelmanetal.2003);andUVlightisomerizablechemicalslinkedtogeneticallyencodedchannelswereemployedinculturedcellsandinzebrafish(Banghartetal.2004,Szobotaetal.2007,Volgrafetal.2006).Theseeffortshavebeenreviewed(Gorostiza&Isacoff2008,Miesenbock&Kevrekidis2005)andwhileelegant,havethusfarbeenfoundtobelimitedtovariousextentsinspeed,targeting,tissuepenetration,and/orapplicabilitybecauseoftheirmulticomponentnature.Here,wereviewdevelopmentandapplicationeffortsfocusedonthedistinctsingle-componentopto-genetictools,suchasmicrobialopsins,overthepastsixyearssincetheywerefirstimplemented.MICROBIALOPSINSSpeciesfrommultiplebranchesofthean-imalkingdomhaveevolvedmechanismstosenseelectromagneticradiationintheirenvironments.Likewisemanymicrobes,intheabsenceofcomplexeyestructuresemployedbymetazoans,havedevelopedlight-activatedproteinsforavarietyofpurposes.Forsome,thisservesasamechanismofhomeostasistore-mainatacertaindepthintheocean(Bejaetal.2000,2001);forothers,thishelpsmaintainosmoticbalanceinahighlysalineenvironment(Stoeckenius1985).Theseandotherdiverserolesare,inmanycases,fulfilledbyafamilyofseven-transmembrane,light-responsiveproteinsencodedbyopsingenes.Opsingenesaredividedintotwodistinctsuperfamilies:
microbialopsins(typeI)andan-imalopsins(typeII).Opsinproteinsfrombothfamiliesrequireretinal,avitaminArelatedorganiccofactorthatservesastheantennaforphotons;whenretinalisbound,thefunctionalopsinproteinsaretermedrhodopsins.Retinalcovalentlyattachestoaconservedlysineresidueofhelix7byformingaprotonatedretinalSchiffbase(RSBH+).Theionicenvi-ronmentoftheRSB,definedbytheresiduesofthebindingpocket,dictatesthespectralandkineticcharacteristicsofeachindividualprotein.Uponabsorptionofaphoton,retinalisomerizesandtriggersasequenceofconfor-mationalchangeswithintheopsinpartner.www.annualreviews.orgTheDevelopmentandApplicationofOptogenetics391Annu.Rev.Neurosci.2011.34:
389-412.Downloadedfromwww.annualreviews.orgbyShanghaiInformationCenterforLifeScienceson04/21/12.Forpersonaluseonly.NE34CH17-DeisserothARI21May201115:
22Thephotoisomerizedretinalisthetriggerforsubsequentstructuralrearrangementsandactivitiesperformedbytheseproteins.Althoughbothopsinfamiliesencodeseven-transmembranestructures,sequenceho-mologybetweenthetwofamiliesisextremelylow;homologywithinfamilies,however,ishigh(25%80%residuesimilarity)(Manetal.2003).WhereastypeIopsingenesarefoundinprokaryotes,algae,andfungi(Spudich2006),typeIIopsingenesarepresentonlyinhighereukaryotesandareresponsiblemainlyforvision(butalsoplayrolesincircadianrhythmandpigmentregulation)(Sakmar2002,Shichida&Yamashita2003).TypeIIopsingenesencodeGproteincoupledreceptors(GPCRs)and,inthedark,bindretinalinthe11-cisconfiguration.Uponillumination,retinalisomerizestotheall-transconfigurationandinitiatesthereactionsthatunderliethevisualphototransductionsecondmessengercascade.Afterphotoisomerization,theretinal-proteinlinkageishydrolyzed;freeall-transretinalthendiffusesoutoftheproteinandisreplacedbyafresh11-cisretinalmoleculeforanotherroundofsignaling(Hofmannetal.2009).Incontrast,typeIopsinsmoretypicallyencodeproteinsthatutilizeretinalintheall-transconfiguration,whichphotoisomerizesuponphotonabsorptiontothe13-ciscon-figuration.UnlikethesituationwithtypeIIrhodopsins,theactivatedretinalmoleculeintypeIrhodopsinsdoesnotdissociatefromitsopsinproteinbutthermallyrevertstotheall-transstatewhilemaintainingacovalentbondtoitsproteinpartner(Hauptsetal.1997).TypeIopsinsencodeseveraldistinctsubfami-liesofprotein,discussedinmoredetailbelow.Thecentraloperatingprincipleoftheseele-gantmolecularmachinesestablishedforthisbroadfamilyofopsinssincebacteriorhodopsin(BR)in1971(Oesterhelt&Stoeckenius1971)andnowincludinghalorhodopsinsandchan-nelrhodopsins(Figure1)istheirunitaryna-ture.Theycombinethetwotasksoflightsensationandionfluxintoasingleprotein(withboundsmallorganiccofactor),encodedbyasinglegene.In2005,oneofthesemicro-bialopsinswasbroughttoneuroscienceasthefirstsingle-componentoptogenetictool(Boy-denetal.2005),andtheothermicrobialopsinsubfamiliesfol
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