Fast calcium sensor proteins for monitoring neural activity

Abstract

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.

Chronic imaging of movement-related Purkinje cell calcium activity in awake behaving mice

“…To date, only morphological studies have been reported in chronically imaged cerebellum (Allegra Mascaro et al 2013; Carrillo et al 2013aCarrillo et al , 2013bKuhn et al 2012). Even in cases where acute imaging of a GECI was performed, functional imaging occurred within 14 days of surgery (Badura et al 2014;Kuhn et al 2012;Najafi et al 2014). In this report, we chronically imaged Ca 2ϩ activity in PCs for up to 7 wk.…”

“…Genetically encoded Ca 2ϩ indicators (GECIs) provide a solution to problems found with injectable dyes but until recently have not been used in PCs (acutely, Badura et al 2014;Kuhn et al 2012;Najafi et al 2014). The inability to maintain a clear window long enough to image, particularly after viral injections, could account for this scarcity.…”

Calcium signaling in human pluripotent stem cells

“…As a summary, calcium sensitive small molecular dyes have several advantages over GECIs, including faster responses, near-linear responses, and higher sensitivities [22,23]. In contrast, GECIs can be better targeted into specific cell types or specific cell compartments, allow long term measurements, and have better signal to noise ratios at least in in vivo experiments [22].…”

“…In contrast, GECIs can be better targeted into specific cell types or specific cell compartments, allow long term measurements, and have better signal to noise ratios at least in in vivo experiments [22]. A recent development of GECIs has resulted in a new class of indicator proteins, including GCaMP6, with higher sensitivity and detection reliability even for individual action potentials in neurons [24].…”

Imaging, manipulation and optogenetics in zebrafish

“…• GCamp6f protein for calcium imaging [78,107]. It is the fastest genetically-encoded calcium indicator (for cytoplasmic free calcium) in neurons which makes it very attractive for fast acquisitions.…”