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Review
. 2016 Aug 26;19(9):1142-53.
doi: 10.1038/nn.4359.

Genetically Encoded Indicators of Neuronal Activity

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Free PMC article
Review

Genetically Encoded Indicators of Neuronal Activity

Michael Z Lin et al. Nat Neurosci. .
Free PMC article

Abstract

Experimental efforts to understand how the brain represents, stores and processes information require high-fidelity recordings of multiple different forms of neural activity within functional circuits. Thus, creating improved technologies for large-scale recordings of neural activity in the live brain is a crucial goal in neuroscience. Over the past two decades, the combination of optical microscopy and genetically encoded fluorescent indicators has become a widespread means of recording neural activity in nonmammalian and mammalian nervous systems, transforming brain research in the process. In this review, we describe and assess different classes of fluorescent protein indicators of neural activity. We first discuss general considerations in optical imaging and then present salient characteristics of representative indicators. Our focus is on how indicator characteristics relate to their use in living animals and on likely areas of future progress.

Figures

Figure 1
Figure 1
Genetically encoded pH indicators (GEPIs). Superecliptic pHluorin (SEP) is shown as an example. pH-dependent fluorescent proteins in the low pH of synaptic vesicles have a protonated chromophore (above) and absorb primarily at ~400 nm (below). Fusion of the synaptic vesicle induces near-instantaneous loss of the proton from the chromophore, shifting its absorbance peak to ~490 nm and allowing excitation by 488-nm light (blue sinusoidal arrow), with resulting green emission (green sinusoidal arrow). Times shown are half-rise and half-decay times. Half-reuptake time is from ref. .
Figure 2
Figure 2
Genetically encoded transmitter indicators (GETIs). iGluSnFR reports glutamate with increased fluorescence (above). A glutamate-induced conformational change in the glutamate-binding domain from a bacterial glutamate transporter (Glt1) induces loss of the proton from the chromophore, shifting its absorbance peak to ~490 nm (below) and allowing excitation by 488-nm light (blue sinusoidal arrow), with resulting green emission (green sinusoidal arrow). Binding time was measured in vitro for an increase in glutamate concentration from 0 to 4.6 µM (ref. 46). The indicated unbinding time is an upper limit deduced from live cell experiments.
Figure 3
Figure 3
Genetically encoded voltage indicators (GEVIs). (a) ArcLight-family GEVIs respond to depolarization by reduced green fluorescence (rightward green sinusoidal arrow) from superecliptic pHluorin (SEP) upon blue-light excitation (blue sinusoidal arrow). The mechanism is not fully known but is believed to involve voltage-dependent dimerization leading to protonation of the SEP chromophore. Kinetics are shown for ArcLightQ239 and Bongwoori as measured at 33 °C (ref. 123), with the slash (/) separating ArcLightQ239 and Bongwoori values. Among the ArcLight variants, these have the largest amplitude and fastest signaling kinetics, respectively. (b) GEVIs of the ASAP family also report depolarization by dimming of a circularly permuted GFP (cpGFP). The mechanism presumably involves coupling of VSD movement to chromophore protonation, similar to that in iGluSnFR and single-fluorophore GECIs. Kinetics were measured at 22 °C (ref. 25). (c) FlicR reports depolarization with increased red fluorescence (red sinusoidal arrow) from a circularly permuted red fluorescent protein (cpRFP) upon green excitation (leftward green sinusoidal arrow), presumably as a result of chromophore deprotonation. Kinetics shown were measured at 37 °C (ref. 59). (d) Opsins report depolarization with increased red fluorescence (red sinusoidal arrow) upon excitation by ~600-nm light (orange sinusoidal arrow), but this emission is weak (quantum yield < 0.01). Kinetics are shown for QuasAr2 measured at 34 °C (ref. 65). (e) Opsin–fluorescent protein fusions report depolarization with a dimming of fluorescence, due to absorbance shift in the opsin leading to increased FRET. In the case of Ace2N-mNeonGreen, emission is yellow-green (yellow-green sinusoidal arrow) and excitation is cyan (cyan sinusoidal arrow). Kinetics are shown for Ace2N-mNeonGreen measured at 22 °C (ref. 5).
Figure 4
Figure 4
Genetically encoded calcium indicators (GECIs). GECIs respond to calcium with increased fluorescence. Events following a glutamate release event (top row) and a single AP (middle row) that opens voltage-gated calcium channels (VGCC) are shown, with open channels in the first time point and closed channels before the second time point. Calcium-induced binding of calmodulin (CaM) to a peptide from smooth-muscle myosin light-chain kinase (RS20) causes repositioning of Arg377 in GCaMPs and Lys80 in R-GECOs, inducing loss of a proton from the chromophore and an absorbance shift (bottom row). GCaMPs can detect calcium transients induced by synaptic activation (top row) and action potentials (middle row), with increased green emission (rightward green sinusoidal arrow) upon blue excitation (blue sinusoidal arrow). R-GECOs can report APs with red emission (red sinusoidal arrow) upon green excitation (leftward green sinusoidal arrow). Spine calcium kinetics are from ref. . Dendrite and soma kinetics are from ref. . With repeated neurotransmitter release or prolonged depolarization, calcium rise and decay times will be longer. Times for calcium half-binding or half-unbinding are for GCaMP6f-RS09 and jRGECO1a, separated by a slash (/), as these are respectively the fastest green and red GECIs tested in neurons. GCaMP6f values were used for jRGECO1a, as they show similar in cellulo kinetics, but only GCaMP6f in vitro kinetics were measured. Half-binding times for GCaMP6f and GCaMP6f-RS09 were calculated by normalizing the half-binding time of GCaMP3-RS06 (ref. 91) after a 200-nM step at 25 °C (ref. 114) by the kon values of GCaMP6f and GCaMP6f-RS09 relative to GCaMP3-RS06 at 25 °C (ref. 91). Times at 37 °C may be similar, as GCaMPs show little temperature dependence in binding rates. Unbinding times shown measured at 37 °C for GCaMP6fRS09 and GCaMP6f. Note that observed rise times of fluorescence transients in cells will be mostly determined by calcium decay kinetics.

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