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Related Experiment Video

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A neuron-based screening platform for optimizing genetically-encoded calcium indicators.

Trevor J Wardill1, Tsai-Wen Chen, Eric R Schreiter

  • 1Genetically-Encoded Neuronal Indicator and Effector Project, Janelia Farm Research Campus, Howard Hughes Medical Institute, Ashburn, Virginia, United States of America.

Plos One
|October 25, 2013
PubMed
Summary
This summary is machine-generated.

Developing novel fluorescent sensors for neuronal activity requires testing in actual neurons. Our platform enables efficient screening of genetically-encoded calcium indicators (GECIs) in primary neuronal cultures, improving sensor development.

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Area of Science:

  • Neuroscience
  • Biotechnology
  • Molecular Imaging

Background:

  • Fluorescent protein sensors are crucial for monitoring neuronal activity.
  • Current screening methods often use non-neuronal systems, which don't reflect neuronal dynamics.
  • Rapid cellular dynamics in neurons necessitate specialized screening platforms.

Purpose of the Study:

  • To develop and validate an electrical stimulation and fluorescence imaging platform for screening neuronal sensors.
  • To demonstrate the efficacy of screening genetically-encoded calcium indicators (GECIs) in primary neuronal cultures.
  • To compare neuronal screening with traditional solution-based measurements.

Main Methods:

  • Established a platform using dissociated rat primary neuronal cultures with electrical field stimulation.
  • Utilized lentiviruses with neuronal-selective promoters for efficient GECI expression.
  • Employed image segmentation to isolate single-neuron fluorescence signals for analysis.

Main Results:

  • Achieved efficient GECI expression and controlled neuronal activity (action potentials) via electrical stimulation.
  • Demonstrated superior performance of neuronal screening over solution measurements for GECIs.
  • Identified GECI variants with improved response kinetics and signal amplitude through neuronal screening.

Conclusions:

  • Screening fluorescent sensors in neuronal cultures provides a more realistic and effective evaluation.
  • The developed platform facilitates the identification of advanced sensors for neuronal activity.
  • This approach is applicable to various sensor types requiring cellular resolution under physiological conditions.