DOI: 10.1073/pnas.0407976101. Lentivirus-based genetic manipulations of cortical neurons and their optical and electrophysiological monitoring <i>in vivo</i>

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

10.1073/pnas.0407976101

Crossref journal-article

Proceedings of the National Academy of Sciences

Proceedings of the National Academy of Sciences (341)

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 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.

Bibliography

Dittgen, T., Nimmerjahn, A., Komai, S., Licznerski, P., Waters, J., Margrie, T. W., Helmchen, F., Denk, W., Brecht, M., & Osten, P. (2004). Lentivirus-based genetic manipulations of cortical neurons and their optical and electrophysiological monitoring in vivo. Proceedings of the National Academy of Sciences, 101(52), 18206â18211.

Authors 10

Tanjew Dittgen (first)
Axel Nimmerjahn (additional)
Shoji Komai (additional)
Pawel Licznerski (additional)
Jack Waters (additional)
Troy W. Margrie (additional)
Fritjof Helmchen (additional)
Winfried Denk (additional)
Michael Brecht (additional)
Pavel Osten (additional)

References 43 Referenced 403

10.1016/S0960-9822(06)00287-9
10.1006/meth.1998.0593
10.1146/annurev.genet.36.041002.120114
10.1113/jphysiol.1962.sp006837
10.1038/nrn1058
10.1016/j.conb.2004.03.016
10.1038/nature02266
10.1146/annurev.pharmtox.43.100901.140257
10.1016/S0952-7915(00)00247-8
Ailles, L. E. & Naldini, L. (2002) Curr. Top. Microbiol. Immunol. 261, 31–52.11892252 / Curr. Top. Microbiol. Immunol. (2002)
10.1038/sj.gt.3301105
10.1128/JVI.72.10.8150-8157.1998
10.1126/science.1067081
10.1016/0378-1119(91)90434-D
10.1016/S0165-0270(96)00121-5
10.1126/science.274.5293.1678
10.1016/S0021-9258(19)74341-5
10.1074/jbc.273.41.26891
10.1126/science.272.5259.263
10.1038/nbt0997-871
10.1002/jnr.490350513
10.1073/pnas.092143499
10.1073/pnas.0437912100
Kleinfeld D. & Denk W. (1999) in Imaging Neurons: A Laboratory Manual eds. Yuste R. Lanni F. & Konnerth A. (Cold Spring Harbor Lab. Press Plainview NY) pp. 23.1–23.15.
10.1113/jphysiol.2003.044222
10.1016/0006-8993(79)90728-5
10.1016/0165-0270(88)90114-8
10.1523/JNEUROSCI.10-06-01788.1990
10.1038/35009107
10.1038/nature01273
10.1038/nature01276
10.1016/j.neuron.2003.08.012
10.1007/s00424-002-0831-z
10.1038/35078107
10.1073/pnas.232688199
10.1038/nbt739
10.1038/nm964
10.1089/104303403322611809
10.1038/ng1117
10.1073/pnas.90.17.8033
10.1016/S0896-6273(00)00084-2
10.1006/exnr.1999.7178
10.1002/jnr.10137

Dates

Type	When
Created	20 years, 8 months ago (Dec. 17, 2004, 8:17 p.m.)
Deposited	3 years, 4 months ago (April 12, 2022, 10:19 a.m.)
Indexed	2 weeks, 2 days ago (Aug. 19, 2025, 6:44 a.m.)
Issued	20 years, 8 months ago (Dec. 17, 2004)
Published	20 years, 8 months ago (Dec. 17, 2004)
Published Online	20 years, 8 months ago (Dec. 17, 2004)
Published Print	20 years, 8 months ago (Dec. 28, 2004)

Funders 0

None

BibTeX

@article{Dittgen_2004, title={Lentivirus-based genetic manipulations of cortical neurons and their optical and electrophysiological monitoring in vivo}, volume={101}, ISSN={1091-6490}, url={http://dx.doi.org/10.1073/pnas.0407976101}, DOI={10.1073/pnas.0407976101}, number={52}, journal={Proceedings of the National Academy of Sciences}, publisher={Proceedings of the National Academy of Sciences}, author={Dittgen, Tanjew and Nimmerjahn, Axel and Komai, Shoji and Licznerski, Pawel and Waters, Jack and Margrie, Troy W. and Helmchen, Fritjof and Denk, Winfried and Brecht, Michael and Osten, Pavel}, year={2004}, month=dec, pages={18206–18211} }

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