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Genetically timed, activity-sensor and rainbow transsynaptic viral tools

Abstract

We developed retrograde, transsynaptic pseudorabies viruses (PRVs) with genetically encoded activity sensors that optically report the activity of connected neurons among spatially intermingled neurons in the brain. Next we engineered PRVs to express two differentially colored fluorescent proteins in a time-shifted manner to define a time period early after infection to investigate neural activity. Finally we used multiple-colored PRVs to differentiate and dissect the complex architecture of brain regions.

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Figure 1: As1-PRV08 reports neural activity.
Figure 2: Timer PRVs define a time window early after infection.
Figure 3: Multiple, colored PRVs reveal spatial organization of parallel circuits.

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Acknowledgements

We thank S. Djaffer for technical assistance. The following researchers provided materials: I. Provencio (University of Virginia, Charlottesville), melanopsin antibody; D.W. Piston (University of Maryland School of Medicine, Baltimore), vector encoding mCerulean; P. Caroni (Friedrich Miescher Insitute, Basel), vector encoding MARCKS-GFP and pThy1-mEGFP vector; R.E. Campbell (University of Alberta, Edmonton), vector encoding mTFP1; R.Y. Tsien (University of California, San Diego), vectors encoding mRFP1 and mCherry; R.H. Scheller (Genentech), vector encoding VAMP-GFP; O. Griesbeck (Max Planck Institute of Neurobiology, Martinsried), vector encoding TN-L15. F. Rijli commented on the manuscript. This study was supported by Novartis Research Foundation funds, US Office of Naval Research Naval International Cooperative Opportunities in Science and Technology Program grant, Marie Curie Excellence grant and Human Frontiers Science Program Young Investigator grant to B.R.; Hungarian National Fund for Scientific Research and Human Frontiers Science Program Young Investigator grant to Z.B.; Swiss National Center of Competence in Research in Genetics fellowship to V.B.; and Human Frontiers Science Program fellowship to G.B.A.

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Authors

Contributions

K.B., E.P., D.T., J.S.T., I.F.T., V.B., P.S.L. and B.G.S. engineered the plasmids. Z.B. made the PRVs. K.B. performed PRV injections, time-lapse, two-photon and confocal imaging, and electrophysiological experiments as well as immunohistochemistry and data analysis. G.B.A. helped with two-photon imaging and electrophysiological experiments. D.B. wrote the data acquisition and analysis software for two-photon microscopy. T.J.V. helped with Ti-PRV injections. J.D. built the two-photon microscope. B.R., Z.B., K.B. and G.B.A. designed the experiments.

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Correspondence to Botond Roska.

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Competing interests

K.B., Z.B. and B.R. are authors on a European patent application 08 152 609.7 that covers the work described in the manuscript.

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Supplementary Figures 1–3, Supplementary Tables 1–4, Supplementary Results, Supplementary Methods (PDF 1111 kb)

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Boldogkői, Z., Balint, K., Awatramani, G. et al. Genetically timed, activity-sensor and rainbow transsynaptic viral tools. Nat Methods 6, 127–130 (2009). https://doi.org/10.1038/nmeth.1292

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