Optogenetic Manipulation of Cerebellar Purkinje Cell Activity In Vivo 英文参考文献.docVIP

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Optogenetic Manipulation of Cerebellar Purkinje Cell Activity In Vivo 英文参考文献.doc

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Optogenetic Manipulation of Cerebellar Purkinje Cell Activity In Vivo 英文参考文献

OptogeneticManipulationofCerebellarPurkinjeCell ActivityInVivo TadashiTsubota1,YoheiOhashi1,KeitaTamura1,AyanaSato1,2,YasushiMiyashita1,2 * 1DepartmentofPhysiology,TheUniversityofTokyoSchoolofMedicine,Bunkyo-ku,Tokyo,Japan,2DepartmentofPhysics,TheUniversityofTokyoSchoolofScience, Bunkyo-ku,Tokyo,Japan Abstract Purkinje cells (PCs) are the sole output neurons of the cerebellar cortex. Although their anatomical connections and physiologicalresponsepropertieshavebeenextensivelystudied,thecausalroleoftheiractivityinbehavioral,cognitiveand autonomic functions is still unclear because PC activity cannot be selectively controlled. Here we developed a novel techniqueusingoptogeneticsforselectiveandrapidlyreversiblemanipulationofPCactivityinvivo.Weinjectedintorat cerebellar cortex lentiviruses expressing either the light-activated cationic channel channelrhodopsin-2 (ChR2) or light- drivenchloridepumphalorhodopsin(eNpHR)underthecontrolofthePC-specificL7promoter.Transgeneexpressionwas observed in most PCs (ChR2, 92.6%; eNpHR, 95.3%), as determined by immunohistochemical analysis. In vivo electrophysiological recordings showed that all light-responsive PCs in ChR2-transduced rats increased frequency of simplespikeinresponsetobluelaserillumination.Similarly,mostlight-responsivePCs(93.8%)ineNpHR-transducedrats decreased frequency of simple spike in response to orange laser illumination. We then applied these techniques to characterizetherolesofratcerebellaruvula,oneofthecardiovascularregulatoryregionsinthecerebellum,inrestingblood pressure(BP)regulationinanesthetizedrats.ChR2-mediatedphotostimulationandeNpHR-mediatedphotoinhibitionofthe uvulahadoppositeeffectsonrestingBP,inducingdepressorandpressorresponses,respectively.Incontrast,manipulation ofPCactivitywithintheneighboringlobuleVIIIhadnoeffectonBP.BlueandorangelaserilluminationontoPBS-injected lobuleIXdidn’taffectBP,indicatingtheobservedeffectsonBPwereactuallyduetoPCactivationandinhibition.These results clearly demonstrat