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A Neuron-Based Screening Platform for Optimizing Genetically-Encoded Calcium Indicators

Author

Listed:
  • Trevor J Wardill
  • Tsai-Wen Chen
  • Eric R Schreiter
  • Jeremy P Hasseman
  • Getahun Tsegaye
  • Benjamin F Fosque
  • Reza Behnam
  • Brenda C Shields
  • Melissa Ramirez
  • Bruce E Kimmel
  • Rex A Kerr
  • Vivek Jayaraman
  • Loren L Looger
  • Karel Svoboda
  • Douglas S Kim

Abstract

Fluorescent protein-based sensors for detecting neuronal activity have been developed largely based on non-neuronal screening systems. However, the dynamics of neuronal state variables (e.g., voltage, calcium, etc.) are typically very rapid compared to those of non-excitable cells. We developed an electrical stimulation and fluorescence imaging platform based on dissociated rat primary neuronal cultures. We describe its use in testing genetically-encoded calcium indicators (GECIs). Efficient neuronal GECI expression was achieved using lentiviruses containing a neuronal-selective gene promoter. Action potentials (APs) and thus neuronal calcium levels were quantitatively controlled by electrical field stimulation, and fluorescence images were recorded. Images were segmented to extract fluorescence signals corresponding to individual GECI-expressing neurons, which improved sensitivity over full-field measurements. We demonstrate the superiority of screening GECIs in neurons compared with solution measurements. Neuronal screening was useful for efficient identification of variants with both improved response kinetics and high signal amplitudes. This platform can be used to screen many types of sensors with cellular resolution under realistic conditions where neuronal state variables are in relevant ranges with respect to timing and amplitude.

Suggested Citation

  • Trevor J Wardill & Tsai-Wen Chen & Eric R Schreiter & Jeremy P Hasseman & Getahun Tsegaye & Benjamin F Fosque & Reza Behnam & Brenda C Shields & Melissa Ramirez & Bruce E Kimmel & Rex A Kerr & Vivek J, 2013. "A Neuron-Based Screening Platform for Optimizing Genetically-Encoded Calcium Indicators," PLOS ONE, Public Library of Science, vol. 8(10), pages 1-1, October.
  • Handle: RePEc:plo:pone00:0077728
    DOI: 10.1371/journal.pone.0077728
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    References listed on IDEAS

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    1. D. Huber & D. A. Gutnisky & S. Peron & D. H. O’Connor & J. S. Wiegert & L. Tian & T. G. Oertner & L. L. Looger & K. Svoboda, 2012. "Multiple dynamic representations in the motor cortex during sensorimotor learning," Nature, Nature, vol. 484(7395), pages 473-478, April.
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