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.

Keywords: GCaMP; calcium indicator proteins; in vivo imaging; kinetics.

Fig. 1

Engineering a faster GCaMP. (a) Crystal structure of calcium-bound GCaMP (PDB: 3EVR) showing the barrel of the circularly permuted EGFP attached to a calmodulin domain (with calcium binding sites indicated by arrows) and the calmodulin-binding RS20 domain. (b) A functional model of GCaMP molecular dynamics. Intramolecular associations between the cpEGFP (pale green and green), calmodulin C-lobe (high affinity loops III and IV; dark yellow) and N-lobe (low affinity loops I and II; yellow) and RS20 protein (red) require calcium. A large calcium step leads to fast activation of the cpEGFP starting with N-lobe binding, whereas small calcium transients slower activation starting with the C-lobe.

Fig. 2

Combining Fast-GCaMP3 mutations and GCaMP6f protein. (a) Schematic representation of the RS06 and RS09 mutations in the GCaMP6f sequence. The crystal structure on the left shows GCaMP in the folded state. (b) Emission spectra for Fast-GCaMP6f-RS06, Fast-GCaMP6f-RS09 and GCaMP6f.

Fig. 3

Fast-GCaMP6f variants retain the brightness of GCaMP6f. (a) ${\mathrm{Ca}}^{2+}$ titration curves of the small-molecule dye Oregon green BAPTA-1, and various GCaMP variants. Solid curves represent fits to the Hill equation. (b) Bar graphs depict high-calcium brightness ($F_{\max }$) and minimum low-calcium brightness ($F_{\min }$) of Fast-GCaMP6f-RS06, Fast-GCaMP6f-RS09, and GCaMP6f relative to GCaMP3 measured at 25°C (pH 7.20). (c) Dynamic range and maximum brightness of Fast-GCaMP6f-RS06, Fast-GCaMP6f-RS09, and GCaMP6f relative to the previously described Fast-GCaMP3 series.

Fig. 4

Purified Fast-GCaMP6f variants show rapid off-responses to decreases in calcium. (a) Schematic of a stopped-flow fluorimeter. (b) The fluorescence decay responses of OGB1 and various GCaMP variants to a downward step in ${[{\mathrm{Ca}}^{2+}]}_{\text{free}}$ from $10\mu \mathrm{M}$ to less than 10 nM at 37°C. Traces are scaled to the maximum fluorescence intensity at ${[{\mathrm{Ca}}^{2+}]}_{\text{free}}=10\mu \mathrm{M}$. (c) Relationship between $K_D$ and $t_{1/2\text{decay}}$ at 25°C of Fast-GCaMP6f-RS06, Fast-GCaMP6f-RS09, and GCaMP6f. GCaMP3, OGB, Fast-GCaMPs (both EF and RS variants), Twitch probes (Twitch-1 and Twitch 4; values adapted from Ref. , TN-XL and TN-XXL are shown for comparison (values adapted from Refs.  and 57). TN-XXL decay times were measured in Drosophila. Kinetics of all other probes were measured using stopped-flow fluorimetery.

Fig. 5

Fast-GCaMP6f-RS06 and Fast-GCaMP6f-RS09 show fast responses in mammalian brain slices. (a) A two-photon image of a patched L2/3 pyramidal neuron expressing GCaMP6f. The outline depicts the patch electrode position. The white line represents the dendritic scan location. (b) Response onsets of the fluorescent responses to action potentials elicited by a depolarizing current step for the two new variants and that of the GCaMP3 and GCaM6f (for GCaMP6f, $n=3$; GCaMP3, $n=3$; Fast-GCaMP6f-RS06, $n=4$; Fast-GCaMP6f-RS09, $n=3$). Line segments indicate means. (c) Decay times for the same neurons; $p$-value = 0.003 for comparison between the Fast-GCaMP6f-RS09 and the $\mathrm{GCaMP}6\mathrm{f}$. (d) $\mathrm{\Delta }\mathrm{F}/\mathrm{F}$ of GCaMP6f, Fast-GCaMP6f-RS09 and Fast-GCaMP6f-RS09.

Fig. 6

Fast-GCaMP6f-RS09 gives faster-decaying complex spike responses. (a) Left, average fields of view and relative fluorescence traces for GCaMP6f (top) and Fast-GCaMP6f-RS09 (bottom) expressing Purkinje cells. Right, purple and green traces represent ${\mathrm{Ca}}^{2+}$-mediated fluorescence change over time and the black tick marks represent detected complex spikes (CSs). (b) Averaged CS-evoked calcium transients for GCaMP6f (purple) and Fast-GCaMP6f-RS09 (green) scaled to match their peak amplitudes. (c) Box plots comparing the $\mathrm{\Delta }\mathrm{F}/\mathrm{F}$ of GCaMP6f and Fast-GCaMP6f-RS09. (d) Box plots comparing the decay times of GCaMP6f and Fast-GCaMP6f-RS09. In (c) and (d), the median is indicated by the horizontal black thick mark, and green (GCaMP6f) and purple (Fast-GCaMP6f-RS09) boxes represent the SEM.