title: Lentivirus-based genetic manipulations of cortical neurons and their optical and electrophysiological monitoring in vivo.
abstract: It is becoming increasingly clear that single cortical neurons encode complex and behaviorally relevant signals, but efficient means to study gene functions in small networks and single neurons in vivo are still lacking. Here, we establish a method for genetic manipulation and subsequent phenotypic analysis of individual cortical neurons in vivo. First, lentiviral vectors are used for neuron-specific gene delivery from alpha-calcium/calmodulin-dependent protein kinase II or Synapsin I promoters, optionally in combination with gene knockdown by means of U6 promoter-driven expression of short-interfering RNAs. Second, the phenotypic analysis at the level of single cortical cells is carried out by using two-photon microscopy-based techniques: high-resolution two-photon time-lapse imaging is used to monitor structural dynamics of dendritic spines and axonal projections, whereas cellular response properties are analyzed electrophysiologically by two-photon microscopy directed whole-cell recordings. This approach is ideally suited for analysis of gene functions in individual neurons in the intact brain.
authors.affiliation: Department of Molecular Neurobiology, Max Planck Institute for Medical Research, Jahnstrasse 29, 69120 Heidelberg, Germany.
authors.forename: Tanjew
authors.initials: T
authors.lastname: Dittgen
authors.forename: Axel
authors.initials: A
authors.lastname: Nimmerjahn
authors.forename: Shoji
authors.initials: S
authors.lastname: Komai
authors.forename: Pawel
authors.initials: P
authors.lastname: Licznerski
authors.forename: Jack
authors.initials: J
authors.lastname: Waters
authors.forename: Troy W
authors.initials: TW
authors.lastname: Margrie
authors.forename: Fritjof
authors.initials: F
authors.lastname: Helmchen
authors.forename: Winfried
authors.initials: W
authors.lastname: Denk
authors.forename: Michael
authors.initials: M
authors.lastname: Brecht
authors.forename: Pavel
authors.initials: P
authors.lastname: Osten
mesh_terms: D000818:Animals; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000235:genetics; D004594:Electrophysiology / Q000379:methods*; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006624:Hippocampus / Q000378:metabolism; D007091:Image Processing, Computer-Assisted; D016086:Lentivirus / Q000235:genetics*; D051379:Mice; D008810:Mice, Inbred C57BL; D008853:Microscopy; D009474:Neurons / Q000378:metabolism*; D010641:Phenotype; D017785:Photons; D011401:Promoter Regions, Genetic; D034741:RNA, Small Interfering / Q000378:metabolism; D012342:RNA, Small Nuclear / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D016704:Synapsins / Q000235:genetics
chemical_list: D034741:RNA, Small Interfering; D012342:RNA, Small Nuclear; D011994:Recombinant Proteins; D016704:Synapsins; C405486:U6 small nuclear RNA; D049452:Green Fluorescent Proteins; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D017871:Calcium-Calmodulin-Dependent Protein Kinases