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 (APs) 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 several-fold, 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 2–3-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.

The use of genetically encodable calcium indicator proteins to monitor neuronal activity is hampered by slow response times and a narrow Ca2+-sensitive range. Here we identify three performance-limiting features of GCaMP3, a popular genetically encodable calcium indicator protein. First, we find that affinity is regulated by the calmodulin domain’s Ca2+-chelating residues. Second, we find that off-responses to Ca2+ are rate-limited by dissociation of the RS20 domain from calmodulin’s hydrophobic pocket. Third, we find that on-responses are limited by fast binding to the N-lobe at high Ca2+ and by slow binding to the C-lobe at lower Ca2+. We develop Fast-GCaMPs, which have up to 20-fold accelerated off-responses and show that they have a 200-fold range of KD, allowing coexpression of multiple variants to span an expanded range of Ca2+ concentrations. Finally, we show that Fast-GCaMPs track natural song in Drosophila auditory neurons and generate rapid responses in mammalian neurons, supporting the utility of our approach.

A major goal of the BRAIN Initiative is the development of technologies to monitor neuronal network activity during active information processing. Toward this goal, genetically encoded calcium indicator proteins have become widely used for reporting activity in preparations ranging from invertebrates to awake mammals. However, slow response times, the narrow sensitivity range of Ca2+ and in some cases, poor signal-to-noise ratio still limit their usefulness. Here, we review recent improvements in the field of neural activity-sensitive probe design with a focus on the GCaMP family of calcium indicator proteins. In this context, we present our newly developed Fast-GCaMPs, which have up to 4-fold accelerated off-responses compared with the next-fastest GCaMP, GCaMP6f. Fast-GCaMPs were designed by destabilizing the association of the hydrophobic pocket of calcium-bound calmodulin with the RS20 binding domain, an intramolecular interaction that protects the green fluorescent protein chromophore. Fast-GCaMP6f-RS06 and Fast-GCaMP6f-RS09 have rapid off-responses in stopped-flow fluorimetry, in neocortical brain slices, and in the intact cerebellum in vivo. Fast-GCaMP6f variants should be useful for tracking action potentials closely spaced in time, and for following neural activity in fast-changing compartments, such as axons and dendrites. Finally, we discuss strategies that may allow tracking of a wider range of neuronal firing rates and improve spike detection.

Measurements of neuronal activity across brain areas are important for understanding the neural correlates of cognitive and motor processes like attention, decisionmaking, and action selection. However, techniques that allow cellular resolution measurements are expensive and require a high degree of technical expertise, which limits their broad use.Widefield imaging of genetically encoded indicators is a high throughput, cost effective, and flexible approach to measure activity of specific cell populations with high temporal resolution and a cortex-wide field of view. Here we outline our protocol for assembling a widefield setup, a surgical preparation to image through the intact skull, and imaging neural activity chronically in behaving, transgenic mice that express a calcium indicator in specific subpopulations of cortical neurons. Further, we highlight a processing pipeline that leverages novel, cloud-based methods to analyze large-scale imaging datasets. The protocol targets labs that are seeking to build macroscopes, optimize surgical procedures for long-term chronic imaging, and/or analyze cortex-wide neuronal recordings.