Optimization of a GCaMP Calcium Indicator for Neural Activity Imaging

TLDR

Genetically encoded calcium indicators, particularly the single‑wavelength GCaMP3, enable reliable detection of multiple action potentials in vivo, and recent protein‑engineering efforts have markedly enhanced their performance. The study aimed to expand GCaMP3’s dynamic range by applying protein‑structure‑guided mutagenesis, high‑throughput screening, and in‑vitro assays, yielding the GCaMP5 family. GCaMP5s were evaluated across diverse preparations—including cultured neurons and astrocytes, mouse retina, C. elegans chemosensory neurons, Drosophila neuromuscular junctions and antennal lobes, zebrafish retina and tectum, and mouse visual cortex—to assess performance.

Abstract

Genetically encoded calcium indicators (GECIs) are powerful tools for systems neuroscience. Recent efforts in protein engineering have significantly increased the performance of GECIs. The state-of-the art single-wavelength GECI, GCaMP3, has been deployed in a number of model organisms and can reliably detect three or more action potentials in short bursts in several systems in vivo . Through protein structure determination, targeted mutagenesis, high-throughput screening, and a battery of in vitro assays, we have increased the dynamic range of GCaMP3 by severalfold, creating a family of “GCaMP5” sensors. We tested GCaMP5s in several systems: cultured neurons and astrocytes, mouse retina, and in vivo in Caenorhabditis chemosensory neurons, Drosophila larval neuromuscular junction and adult antennal lobe, zebrafish retina and tectum, and mouse visual cortex. Signal-to-noise ratio was improved by at least 2- to 3-fold. In the visual cortex, two GCaMP5 variants detected twice as many visual stimulus-responsive cells as GCaMP3. By combining in vivo imaging with electrophysiology we show that GCaMP5 fluorescence provides a more reliable measure of neuronal activity than its predecessor GCaMP3. GCaMP5 allows more sensitive detection of neural activity in vivo and may find widespread applications for cellular imaging in general.