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Functional labeling of neurons and their projections using the synthetic activity–dependent promoter E-SARE

Nature Methods volume 10, pages 889–895 (2013)Cite this article

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A Corrigendum to this article was published on 30 January 2014

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Abstract

Identifying the neuronal ensembles that respond to specific stimuli and mapping their projection patterns in living animals are fundamental challenges in neuroscience. To this end, we engineered a synthetic promoter, the enhanced synaptic activity–responsive element (E-SARE), that drives neuronal activity–dependent gene expression more potently than other existing immediate-early gene promoters. Expression of a drug-inducible Cre recombinase downstream of E-SARE enabled imaging of neuronal populations that respond to monocular visual stimulation and tracking of their long-distance thalamocortical projections in living mice. Targeted cell-attached recordings and calcium imaging of neurons in sensory cortices revealed that E-SARE reporter expression correlates with sensory-evoked neuronal activity at the single-cell level and is highly specific to the type of stimuli presented to the animals. This activity-dependent promoter can expand the repertoire of genetic approaches for high-resolution anatomical and functional analysis of neural circuits.

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Figure 1: Construction of the synthetic E-SARE promoter.
Figure 2: In vivo application of the E-SARE reporter virus.
Figure 3: In vivo functional labeling of long-distance axons.
Figure 4: E-SARE–driven reporter activity preferentially correlates with evoked neuronal firing.
Figure 5: Orientation-specific induction of an E-SARE–driven reporter.

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  • 19 September 2013

    In the version of this article initially published, the schema shown in Figure 5a was wrong. The error has been corrected in the HTML and PDF versions of the article.

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Acknowledgements

We thank R. Kotin (US National Institutes of Health), B. Davidson (University of Iowa) and I. Bezprozvanny (University of Texas Southwestern Medical Center) for original AAV vectors; S. Josselyn and P. Frankland for the in vivo viral injection protocol; H. Kawasaki for advice on LGN experiments; V. Ramirez-Amaya for advice on Arc detection; S. Andreandis (The State University of New York, Buffalo) for a cHS4 construct; C. Cepko (Harvard University) for an ERT2CreERT2 construct; T. Curran (University of Pennsylvania) for a c-fos promoter construct; I. Imayoshi (Kyoto University) for a loxP-flanked STOP construct; L. Looger (Howard Hughes Medical Institute, Janelia Farm) for a GCaMP5G construct; A. Miyawaki (RIKEN BSI) for a Venus construct; H. Miyoshi (RIKEN BRC) for a lentiviral construct; the Research Support Center of the Graduate School of Medical Sciences at Kyushu University for technical support; and Carl Zeiss Japan for access to an LSM780. We thank all of the members of the Bito and Ohki laboratories for support and discussion. We are particularly indebted to Y. Kondo, K. Saiki, R. Gyobu and T. Kinbara for assistance. This work was supported in part by grants-in-aid from the Japanese Ministry of Education, Culture, Sports, Science and Technology (MEXT) and Japan Society for the Promotion of Science (JSPS) (to K.K., M.N., M.K., H.O. and H.B.), the Strategic Research Program for Brain Sciences (to M.K.) as well as grants from CREST–Japan Science and Technology Agency (JST) (to K.O. and H.B.), the Strategic International Research Cooperative Program Japan-Mexico (SICPME-JST, to H.B.) and the Mitsubishi Foundation (to H.B.). T.K., K.S., M. N. and S.K. were supported by JSPS fellowships.

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Author notes

  1. Takashi Kawashima & Mio Nonaka

    Present address: Present addresses: Howard Hughes Medical Institute, Janelia Farm Research Campus, Ashburn, Virginia, USA (T.K.); Centre for Cognitive and Neural Systems, University of Edinburgh, Edinburgh, UK (M.N.).,

Authors and Affiliations

  1. Department of Neurochemistry, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan

    Takashi Kawashima, Kanzo Suzuki, Mio Nonaka, Satoshi Kamijo, Sayaka Takemoto-Kimura, Hiroyuki Okuno & Haruhiko Bito

  2. Department of Molecular Physiology, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan

    Takashi Kawashima & Kenichi Ohki

  3. Department of Neurophysiology, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan

    Kazuo Kitamura & Masanobu Kano

  4. Precursory Research for Embryonic Science and Technology (PRESTO), Japan Science and Technology Agency, Saitama, Japan

    Kazuo Kitamura & Sayaka Takemoto-Kimura

  5. Core Research for Evolutional Science and Technology (CREST), Japan Science and Technology Agency, Tokyo, Japan

    Hiroyuki Okuno, Kenichi Ohki & Haruhiko Bito

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Contributions

T.K., K.K., H.O., K.O. and H.B. conceived the experiments. T.K. performed the in vitro characterizations, design of virus vectors and image analyses. T.K., K.S. and M.N. performed virus purifications and injections. K.K. and M.K. performed electrophysiological recordings, and T.K. and K.K. performed two-photon imaging experiments in the barrel cortex. T.K. and K.O. performed two-photon imaging experiments of the visual cortex. T.K. and H.O. performed histological analyses. S.K. and S.T.-K. provided help with reagents and animal management. T.K., H.O., K.O. and H.B. wrote the paper. H.B. supervised the entire project. All authors discussed and commented on the manuscript.

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Correspondence to Haruhiko Bito.

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Kawashima, T., Kitamura, K., Suzuki, K. et al. Functional labeling of neurons and their projections using the synthetic activity–dependent promoter E-SARE. Nat Methods 10, 889–895 (2013). https://doi.org/10.1038/nmeth.2559

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