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

A Corrigendum to this article was published on 30 January 2014

This article has been updated

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.

Change history

  • 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.

References

  1. Guzowski, J.F., McNaughton, B.L., Barnes, C.A. & Worley, P.F. Environment-specific expression of the immediate-early gene Arc in hippocampal neuronal ensembles. Nat. Neurosci. 2, 1120–1124 (1999).

    Article  CAS  Google Scholar 

  2. Ramírez-Amaya, V. et al. Spatial exploration-induced Arc mRNA and protein expression: evidence for selective, network-specific reactivation. J. Neurosci. 25, 1761–1768 (2005).

    Article  Google Scholar 

  3. Tse, D. et al. Schema-dependent gene activation and memory encoding in neocortex. Science 333, 891–895 (2011).

    Article  CAS  Google Scholar 

  4. Schilling, K., Luk, D., Morgan, J.I. & Curran, T. Regulation of a fos-lacZ fusion gene: a paradigm for quantitative analysis of stimulus-transcription coupling. Proc. Natl. Acad. Sci. USA 88, 5665–5669 (1991).

    Article  CAS  Google Scholar 

  5. Barth, A.L., Gerkin, R.C. & Dean, K.L. Alteration of neuronal firing properties after in vivo experience in a FosGFP transgenic mouse. J. Neurosci. 24, 6466–6475 (2004).

    Article  CAS  Google Scholar 

  6. Kawashima, T. et al. Synaptic activity-responsive element in the Arc/Arg3.1 promoter essential for synapse-to-nucleus signaling in activated neurons. Proc. Natl. Acad. Sci. USA 106, 316–321 (2009).

    Article  CAS  Google Scholar 

  7. Grinevich, V. et al. Fluorescent Arc/Arg3.1 indicator mice: a versatile tool to study brain activity changes in vitro and in vivo . J. Neurosci. Methods 184, 25–36 (2009).

    Article  CAS  Google Scholar 

  8. Eguchi, M. & Yamaguchi, S. In vivo and in vitro visualization of gene expression dynamics over extensive areas of the brain. Neuroimage 44, 1274–1283 (2009).

    Article  Google Scholar 

  9. Okuno, H. et al. Inverse synaptic tagging of inactive synapses via dynamic interaction of Arc/Arg3.1 with CaMKIIβ. Cell 149, 886–898 (2012).

    Article  CAS  Google Scholar 

  10. Koya, E. et al. Targeted disruption of cocaine-activated nucleus accumbens neurons prevents context-specific sensitization. Nat. Neurosci. 12, 1069–1073 (2009).

    Article  CAS  Google Scholar 

  11. Liu, X. et al. Optogenetic stimulation of a hippocampal engram activates fear memory recall. Nature 484, 381–385 (2012).

    Article  CAS  Google Scholar 

  12. Garner, A.R. et al. Generation of a synthetic memory trace. Science 335, 1513–1516 (2012).

    Article  CAS  Google Scholar 

  13. Matsuda, T. & Cepko, C.L. Controlled expression of transgenes introduced by in vivo electroporation. Proc. Natl. Acad. Sci. USA 104, 1027–1032 (2007).

    Article  CAS  Google Scholar 

  14. Montero, V.M., Brugge, J.F. & Beitel, R.E. Relation of the visual field to the lateral geniculate body of the albino rat. J. Neurophysiol. 31, 221–236 (1968).

    Article  CAS  Google Scholar 

  15. Tagawa, Y., Kanold, P.O., Majdan, M. & Shatz, C.J. Multiple periods of functional ocular dominance plasticity in mouse visual cortex. Nat. Neurosci. 8, 380–388 (2005).

    Article  CAS  Google Scholar 

  16. Mrsic-Flogel, T.D. et al. Homeostatic regulation of eye-specific responses in visual cortex during ocular dominance plasticity. Neuron 54, 961–972 (2007).

    Article  CAS  Google Scholar 

  17. O'Connor, D.H., Peron, S.P., Huber, D. & Svoboda, K. Neural activity in barrel cortex underlying vibrissa-based object localization in mice. Neuron 67, 1048–1061 (2010).

    Article  CAS  Google Scholar 

  18. Kitamura, K., Judkewitz, B., Kano, M., Denk, W. & Hausser, M. Targeted patch-clamp recordings and single-cell electroporation of unlabeled neurons in vivo . Nat. Methods 5, 61–67 (2008).

    Article  CAS  Google Scholar 

  19. Margrie, T.W. et al. Targeted whole-cell recordings in the mammalian brain in vivo . Neuron 39, 911–918 (2003).

    Article  CAS  Google Scholar 

  20. Akerboom, J. et al. Optimization of a GCaMP calcium indicator for neural activity imaging. J. Neurosci. 32, 13819–13840 (2012).

    Article  CAS  Google Scholar 

  21. Zariwala, H.A. et al. A Cre-dependent GCaMP3 reporter mouse for neuronal imaging in vivo . J. Neurosci. 32, 3131–3141 (2012).

    Article  CAS  Google Scholar 

  22. Ohki, K., Chung, S., Ch'ng, Y.H., Kara, P. & Reid, R.C. Functional imaging with cellular resolution reveals precise micro-architecture in visual cortex. Nature 433, 597–603 (2005).

    Article  CAS  Google Scholar 

  23. Melnikov, A. et al. Systematic dissection and optimization of inducible enhancers in human cells using a massively parallel reporter assay. Nat. Biotechnol. 30, 271–277 (2012).

    Article  CAS  Google Scholar 

  24. Patwardhan, R.P. et al. Massively parallel functional dissection of mammalian enhancers in vivo . Nat. Biotechnol. 30, 265–270 (2012).

    Article  CAS  Google Scholar 

  25. Böer, U. et al. CRE/CREB-driven up-regulation of gene expression by chronic social stress in CRE-luciferase transgenic mice: reversal by antidepressant treatment. PLoS ONE 2, e431 (2007).

    Article  Google Scholar 

  26. Yassin, L. et al. An embedded subnetwork of highly active neurons in the neocortex. Neuron 68, 1043–1050 (2010).

    Article  CAS  Google Scholar 

  27. Bock, D.D. et al. Network anatomy and in vivo physiology of visual cortical neurons. Nature 471, 177–182 (2011).

    Article  CAS  Google Scholar 

  28. Briggman, K.L., Helmstaedter, M. & Denk, W. Wiring specificity in the direction-selectivity circuit of the retina. Nature 471, 183–188 (2011).

    Article  CAS  Google Scholar 

  29. Hama, H. et al. Scale: a chemical approach for fluorescence imaging and reconstruction of transparent mouse brain. Nat. Neurosci. 14, 1481–1488 (2011).

    Article  CAS  Google Scholar 

  30. Chung, K. et al. Structural and molecular interrogation of intact biological systems. Nature 497, 332–337 (2013).

    Article  CAS  Google Scholar 

  31. Tian, J. & Andreadis, S.T. Independent and high-level dual-gene expression in adult stem-progenitor cells from a single lentiviral vector. Gene Ther. 16, 874–884 (2009).

    Article  CAS  Google Scholar 

  32. Miyoshi, H., Blomer, U., Takahashi, M., Gage, F.H. & Verma, I.M. Development of a self-inactivating lentivirus vector. J. Virol. 72, 8150–8157 (1998).

    CAS  PubMed  PubMed Central  Google Scholar 

  33. Nagai, T. et al. A variant of yellow fluorescent protein with fast and efficient maturation for cell-biological applications. Nat. Biotechnol. 20, 87–90 (2002).

    Article  CAS  Google Scholar 

  34. Li, X. et al. Generation of destabilized green fluorescent protein as a transcription reporter. J. Biol. Chem. 273, 34970–34975 (1998).

    Article  CAS  Google Scholar 

  35. Thiel, G., Greengard, P. & Südhof, T.C. Characterization of tissue-specific transcription by the human synapsin I gene promoter. Proc. Natl. Acad. Sci. USA 88, 3431–3435 (1991).

    Article  CAS  Google Scholar 

  36. Imayoshi, I. et al. Roles of continuous neurogenesis in the structural and functional integrity of the adult forebrain. Nat. Neurosci. 11, 1153–1161 (2008).

    Article  CAS  Google Scholar 

  37. Ageta-Ishihara, N. et al. Control of cortical axon elongation by a GABA-driven Ca2+/calmodulin-dependent protein kinase cascade. J. Neurosci. 29, 13720–13729 (2009).

    Article  CAS  Google Scholar 

  38. Bito, H., Deisseroth, K. & Tsien, R.W. CREB phosphorylation and dephosphorylation: a Ca2+- and stimulus duration-dependent switch for hippocampal gene expression. Cell 87, 1203–1214 (1996).

    Article  CAS  Google Scholar 

  39. Smith, R.H., Levy, J.R. & Kotin, R.M. A simplified baculovirus-AAV expression vector system coupled with one-step affinity purification yields high-titer rAAV stocks from insect cells. Mol. Ther. 17, 1888–1896 (2009).

    Article  CAS  Google Scholar 

  40. Kalatsky, V.A. & Stryker, M.P. New paradigm for optical imaging: temporally encoded maps of intrinsic signal. Neuron 38, 529–545 (2003).

    Article  CAS  Google Scholar 

  41. Isomura, Y., Harukuni, R., Takekawa, T., Aizawa, H. & Fukai, T. Microcircuitry coordination of cortical motor information in self-initiation of voluntary movements. Nat. Neurosci. 12, 1586–1593 (2009).

    Article  CAS  Google Scholar 

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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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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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