Lentivirus-based genetic manipulations of cortical neurons and their optical and electrophysiological monitoring in vivo.
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.
Dittgen
Tanjew
T
Department of Molecular Neurobiology, Max Planck Institute for Medical Research, Jahnstrasse 29, 69120 Heidelberg, Germany.
Nimmerjahn
Axel
A
Komai
Shoji
S
Licznerski
Pawel
P
Waters
Jack
J
Margrie
Troy W
TW
Helmchen
Fritjof
F
Denk
Winfried
W
Brecht
Michael
M
Osten
Pavel
P
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
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