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GCaMP is a genetically encoded Calcium imaging (GECI) initially developed in 2001 by Junichi Nakai. It is a synthetic fusion of green fluorescent protein (GFP), calmodulin (CaM), and M13, a peptide sequence from myosin light-chain kinase. When bound to Ca2+, GCaMP fluoresces green with a peak excitation wavelength of 480 nm and a peak emission wavelength of 510 nm. It is used in biological research to measure intracellular Ca2+ levels both in vitro and in vivo using Viral vector Transfection or Transgene cell and animal lines. (2010). 9780123748416 ISBN 9780123748416 The genetic sequence encoding GCaMP can be inserted under the control of promoters exclusive to certain cell types, allowing for cell-type specific expression of GCaMP. Since Ca2+ is a second messenger that contributes to many cellular mechanisms and signaling pathways, GCaMP allows researchers to quantify the activity of Ca2+-based mechanisms and study the role of Ca2+ ions in biological processes of interest.
In the absence of Ca2+, the GFP chromophore is exposed to water and exists in a protonated state with minimal fluorescence intensity. Upon Ca2+ binding, the CaM domain undergoes a conformational change and tightly binds to the M13 domain alpha helix, preventing water molecules from accessing the chromophore. As a result, the chromophore rapidly deprotonates and converts into an anionic form that fluoresces brightly, similar to native GFP.
In 2006, Tallini et al. subsequently reported the improvement of GCaMP1 to GCaMP2, which exhibited brighter fluorescence than GCaMP1 and greater stability at body temperatures. Tallini et al. expressed GCaMP2 in cardiomyocytes in mouse embryos to perform the first in vivo GCaMP imaging of Ca2+ in mammals.
Further modifications of GCaMP, including GCaMP3, GCaMP5, GCaMP6, and jGCaMP7, have been developed to progressively improve the signal, sensitivity, and dynamic range of Ca2+ detection, with recent versions exhibiting fluorescence similar to native GFP.
In 2018, Yang et al. reported the development of GCaMP-X, generated by the addition of a calmodulin-binding motif. Since the GCaMP calmodulin domain, when unbound, disrupts L-type calcium channel gating, the added calmodulin-binding motif prevents GCaMP-X from interfering with calcium-dependent signaling mechanisms.
In 2020, Zhang et al. reported the development of jGCaMP8, including sensitive, medium, and fast variants, which exhibit faster kinetics and greater sensitivity than the corresponding jGCaMP7 variants.
Red fluorescent indicators have also been developed: jRCaMP1a and jRCaMP1b use a circular permutation of the red fluorescent protein mRuby instead of GFP, while jRGECO1a is based on the red fluorescent protein mApple. Since the blue light used to excite GCaMP is scattered by tissue and the emitted green light is absorbed by blood, red fluorescent indicators provide more penetration and imaging depth in vivo than GCaMP. Use of red fluorescent indicators also avoids the photodamage caused by blue excitation light. Moreover, red fluorescent indicators allow for concurrent use of optogenetics, which is difficult with GCaMP because the excitation wavelengths of GCaMP overlap with those of Channelrhodopsin (ChR2). Simultaneous use of red and green GECIs can provide two-color visualization of different subcellular regions or cell populations.
GCaMP has played a vital role in establishing large-scale neural recordings in animals to investigate how activity patterns in neuronal networks influence behavior. For example, Nguyen et al. (2016) used GCaMP in whole-brain imaging during free movement of C. elegans to identify neurons and groups of neurons whose activity correlated with specific locomotor behaviors.
Muto et al. (2003) expressed GCaMP in zebrafish embryos to measure and map the coordinated activity of spinal to different parts of the brain during the onset, propagation, and recovery of seizures induced by pentylenetetrazol. GCaMP expression in zebrafish brains has also been used to study activation of neural circuits in cognitive processes like prey capture, impulse control, and attention.
Additionally, researchers have used GCaMP to observe neuronal activity in mice by expressing it under control of the Thy1 promoter, which is found in excitatory Pyramidal cell. For instance, integration of neurons into circuits during motor learning has been tracked by using GCaMP to observe synchronized fluctuation patterns in Ca2+ levels. GCaMP has also been used to observe Ca2+ dynamics in subcellular compartments of mouse neurons: Cichon and Gan (2015) used GCaMP to show that neurons in the mouse motor cortex exhibit NMDA-driven increases in Ca2+ that are independent for each dendritic spine, thus showing that individual dendritic spines regulate synaptic plasticity. Finally, GCaMP has been used to identify activity patterns in specific regions of the mouse brain. For instance, Jones et al. (2018) used GCaMP6 in mice to measure neuronal activity in the suprachiasmatic nucleus (SCN), the mammalian Circadian rhythm pacemaker, and showed that SCN neurons that produced vasoactive intestinal peptide (VIP) exhibited daily activity rhythms in vivo that correlated with VIP release.
GCaMP has also been combined with Optical fiber photometry to measure population-level Ca2+ changes within subpopulations of neurons in freely moving animals. For instance, Clarkson et al. (2017) used this method to show that neurons in the arcuate nucleus of the hypothalamus synchronize to increases in Ca2+ immediately prior to pulses of luteinizing hormone (LH). While GCaMP imaging with fiber photometry cannot track changes in Ca2+ levels within individual neurons, it provides greater temporal resolution for large-scale changes.
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