Letter

A light- and calcium-gated transcription factor for imaging and manipulating activated neurons

Received:
Accepted:
Published online:

Abstract

Activity remodels neurons, altering their molecular, structural, and electrical characteristics. To enable the selective characterization and manipulation of these neurons, we present FLARE, an engineered transcription factor that drives expression of fluorescent proteins, opsins, and other genetically encoded tools only in the subset of neurons that experienced activity during a user-defined time window. FLARE senses the coincidence of elevated cytosolic calcium and externally applied blue light, which together produce translocation of a membrane-anchored transcription factor to the nucleus to drive expression of any transgene. In cultured rat neurons, FLARE gives a light-to-dark signal ratio of 120 and a high- to low-calcium signal ratio of 10 after 10 min of stimulation. Opsin expression permitted functional manipulation of FLARE-marked neurons. In adult mice, FLARE also gave light- and motor-activity-dependent transcription in the cortex. Due to its modular design, minute-scale temporal resolution, and minimal dark-state leak, FLARE should be useful for the study of activity-dependent processes in neurons and other cells that signal with calcium.

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Acknowledgements

We thank J. Einstein and A. Draycott for neuron cultures. J. Einstein also assisted with cloning and some imaging assays. F. Zhang, S. Konermann, and M. Brigham provided AAV vectors, AAV protocols, and guided us on the setup of our light stimulation device. G. Liu built the LED light box. M. Heidenreich advised us on preparation of concentrated AAVs. T.J. Wardill and L. Looger provided electrode information. H. Wang and M. Djuristic assisted with electrical stimulation setup. P. Han assisted with statistical analysis of the in vivo data. FACS experiments were performed at the Koch Institute Flow Cytometry Core (MIT). The TEVp gene was a gift from the Waugh laboratory (National Cancer Institute). GCaMP5f was a gift from L. Looger, Janelia Research Campus. A.Y.T. received funding from MIT and Stanford. K.M.T. is a New York Stem Cell Foundation Robertson Investigator and McKnight Scholar and this work was supported by funding from the JPB Foundation, the PIIF and PIIF Engineering Award, R01-MH102441-01 (NIMH), and NIH Director's New Innovator Award DP2-DK-102256-01 (NIDDK). G.A.M. was supported by a fellowship from the Charles A. King Trust Postdoctoral Research Fellowship Program, Bank of America, N.A., Co-Trustees.

Author information

Affiliations

  1. Departments of Genetics, Biology, and Chemistry, Stanford University, Stanford, California, USA.

    • Wenjing Wang
    • , Mateo I Sanchez
    •  & Alice Y Ting
  2. Department of Chemistry, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA.

    • Wenjing Wang
    • , Tanyaporn Pattarabanjird
    • , Mateo I Sanchez
    •  & Alice Y Ting
  3. Picower Institute for Learning and Memory and Department of Brain and Cognitive Sciences, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA.

    • Craig P Wildes
    • , Gordon F Glober
    • , Gillian A Matthews
    •  & Kay M Tye

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Contributions

W.W. performed all experiments except those noted. W.W. and T.P. together performed the LOV directed evolution. M.I.S. generated AAV viruses and measured eLOV recovery kinetics. C.P.W. and K.M.T. designed the in vivo experiments. C.P.W., G.F.G., and G.A.M. performed the in vivo experiments. W.W. and M.I.S. analyzed the in vivo data. W.W. and A.Y.T. designed the research, analyzed the data, and wrote the paper. All authors edited the paper.

Competing interests

A.Y.T. and W.W. have filed a patent application covering some aspects of this work.

Corresponding authors

Correspondence to Kay M Tye or Alice Y Ting.

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