The improved properties of GCaMP3 allow new types of neuroscience applications in multiple model organisms. In C. elegans
, expression of GECIs has caused behavioral phenotypes 29
, likely due to altered neural physiology caused by indicator over-expression (Supplementary Fig. 10 and Supplementary Table 3
). Neurons expressing GCaMP3 showed high SNR without detectable cytotoxicity or behavioral perturbations (Supplementary Fig. 10
). The much higher signal levels provided by GCaMP3 () may allow experiments with lower expression levels, ameliorating problems associated with calcium buffering.
, the poor expression of GCaMP2 25
has left GCaMP1.3 and 1.6 as the state-of-the-art single-FP indicators, but their poor sensitivity and non-linear response has precluded detection of low firing rates 30
. GCaMP3 showed greatly improved expression and signal levels in vivo
() with no apparent cytotoxicity or behavioral phenotype (data not shown), and could permit imaging experiments not previously possible.
In behaving mice, GCaMP3 allows the simultaneous recording of the activity of dozens of identified neurons over months (). Thus, GCaMP3 should allow tracking learning-induced changes in circuit-level activity over multiple recording sessions. Pan-neuronal expression of GCaMP3 might allow wide-field imaging of whole-cortex activity and signal propagation akin to super-resolution functional MRI 31
Long-term, high-level expression of GECIs in the mouse brain can result in nonfunctional indicators32
and abnormal physiology (Supplementary Fig. 11
). Though expression of GCaMP3 in the postnatal brain, mediated by viral gene transfer, limited such adverse effects, the proportion of nuclear-filled neurons still increased with proximity to the injection site, and slowly increased with time after injection. Therefore, optimizing the timing and magnitude of expression to balance signal levels and cytotoxicity needs further study.
Ratiometric indicators have potential advantages over single-FP GECIs, including higher baseline brightness and insensitivity to motion artifacts via wavelength ratioing. However, GCaMP3 is more photostable, likely because of reduced bleaching in the low fluorescence state (). Ratiometric measurements could be achieved with GCaMP3 by co-expression or fusion of a reference fluorescent protein. The smaller size of GCaMP3 potentially facilitates a wider array of targeted protein fusions for calcium imaging in subcellular compartments. The faster kinetics of GCaMP3 may allow more faithful detection of the number and timing of action potentials in spike trains.
GCaMP3 may not be the most suitable GECI for all applications. Single action potentials can also be detected using D3cpV 14
; indeed, the slow kinetics of D3cpV might be suitable for situations characterized by very low and sparse spike rates. Troponin-based indicators, such as TN-XXL, may cause less perturbation of endogenous calcium signaling 6
. Prudence dictates testing each of these indicators in the context of specific applications.
Although GCaMP3 is a major improvement over GCaMP2, additional avenues for protein engineering remain. Mutagenesis to ablate interactions of the CaM and M13pep domains of GCaMP, such as that done to the D2/D3/D4 indicators 13
, may improve performance at high expression levels. A single-FP indicator based on troponin C 33
might combine the benefits of a non-endogenous calcium binding protein with the high signal-to-noise and fast kinetics of GCaMP. Quantitative modeling studies 20
have suggested that increasing the fluorescence on-rate would further improve detection of the brief calcium transients associated with single APs. Expression cassettes that maintain steady, moderate levels of GCaMP3 expression for months would facilitate signal calibration and further reduce toxicity concerns.