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

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

doi:10.1371/journal.pone.0077728

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.

Highlights

Fluorescent protein-based sensors of neuronal activity are beginning to revolutionize neurophysiology [1]
We evaluated the capabilities of this novel screening platform by expressing and imaging of variants of the green fluorescent genetically-encoded calcium indicators (GECIs), GCaMP3 [5]
Optimization of calcium indicator proteins has been slowed by development cycles that relied on testing in non-neuronal assays and validating performance in neuronal systems

Summary

Introduction

Fluorescent protein-based sensors of neuronal activity are beginning to revolutionize neurophysiology [1]. Protein sensors can be targeted to specific neuron types using gene regulatory elements [10]. They can be delivered to cells of interest in a non-invasive manner [11,12]. Previous efforts in engineering sensors have tested candidates in non-neuronal assays, including assays using purified proteins and tissue culture cells [5,18,19,20]. Other efforts have tested sensors in lower throughput in vitro and in vivo systems, such as rat neuronal slice cultures, fly neurons, and fish neurons [7,21,22,23,24]

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