Lentivirus-based genetic manipulations of cortical neurons and their optical and electrophysiological monitoring in vivo

Proceedings of the National Academy of Sciences of the United States of America - Tập 101 Số 52 - Trang 18206-18211 - 2004
Tanjew Dittgen1, Axel Nimmerjahn1, Shoji Komai1, Pawel Licznerski1, Jack Waters1, Troy W. Margrie1, Fritjof Helmchen1, Winfried Denk1, Michael Brecht1, Pavel Osten1
1Departments of Molecular Neurobiology, Cell Physiology, and Biomedical Optics, Max Planck Institute for Medical Research, Jahnstrasse 29, 69120 Heidelberg, Germany; and Department of Physiology, Wolfson Institute for Biomedical Research, University College London, Gower Street, London WC1E 6BT, United Kingdom

Tóm tắt

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 α-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 microscopydirected whole-cell recordings. This approach is ideally suited for analysis of gene functions in individual neurons in the intact brain.

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Proceedings of the National Academy of Sciences of the United States of America

Tập 101 Số 52

18206-18211

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