Opto-Current-Clamp Actuation of Cortical Neurons Using a Strategically Designed Channelrhodopsin 英文参考文献.docVIP

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Opto-Current-Clamp Actuation of Cortical Neurons Using a Strategically Designed Channelrhodopsin 英文参考文献.doc

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Opto-Current-Clamp Actuation of Cortical Neurons Using a Strategically Designed Channelrhodopsin 英文参考文献

Opto-Current-ClampActuationofCorticalNeuronsUsing aStrategicallyDesignedChannelrhodopsin LeiWen1,2,3.,HongxiaWang1,2,3.,SakiTanimoto1,2,3,RyoEgawa1,2,3,YoshiyaMatsuzaka2,4 ,Hajime Mushiake2,3,4,5,ToruIshizuka1,2,HiromuYawo1,2,3,5 * 1DepartmentofDevelopmentalBiologyandNeuroscience,TohokuUniversityGraduateSchoolofLifeSciences,Sendai,Japan,2JapanScienceandTechnologyAgency (JST),CoreResearchofEvolutionalScienceTechnology(CREST),Tokyo,Japan,3TohokuUniversityBasicandTranslationalResearchCenterforGlobalBrainScience, Sendai,Japan,4DepartmentofPhysiology,TohokuUniversityGraduateSchoolofMedicine,Sendai,Japan,5CenterforNeuroscience,TohokuUniversityGraduateSchool ofMedicine,Sendai,Japan Abstract Background:Optogeneticmanipulationofaneuronalnetworkenablesonetorevealhowhigh-orderfunctionsemergein the central nervous system. One of the Chlamydomonas rhodopsins, channelrhodopsin-1 (ChR1), has several advantages overchannelrhodopsin-2(ChR2)intermsofthephotocurrentkinetics.Improvedtemporalresolutionwouldbeexpectedby theoptogeneticsusingtheChR1variantswithenhancedphotocurrents. Methodology/Principal Findings: The photocurrent retardation of ChR1 was overcome by exchanging the sixth helix domain with its counterpart in ChR2 producing Channelrhodopsin-green receiver (ChRGR) with further reform of the molecule.WhentheChRGRphotocurrentwasmeasuredfromtheexpressingHEK293cellsunderwhole-cellpatchclamp,it waspreferentiallyactivatedbygreenlightandhasfastkineticswithminimaldesensitization.Withitskineticadvantagesthe useofChRGRwouldenableonetoinjectacurrentintoaneuronbythetimecourseaspredictedbytheintensityofthe sheddinglight(opto-currentclamp).TheChRGRwasalsoexpressedinthemotorcorticalneuronsofamouseusingSindbis pseudovirionvectors.WhenanoscillatoryLEDlightsignalwasappliedsweepingthroughfrequencies,itrobustlyevoked actionpotentialssynchronizedtotheoscillatorylightat5–10Hzinlayer5pyramidalcellsinthecorticalslice.TheChRGR- expressing neurons were also driven in vivo with monitoring local field