The holy grail of cellular imaging in neuroscience is a fluorescent indicator that reliably reports action potentials in vivo. The GECIs (Genetically-Encoded Calcium Indicators) are a class of fluorescent reporter based off the fusion of one or two fluorescent proteins to a calcium binding domain. These types of indicators are one of the most promising avenues of research towards the goal of an AP detector. In a recent paper in Nature Methods, Oliver Griesbeck’s group reports the in vivo performance of one of their most recent GECI prototypes. Should we break out the champagne yet? Let’s find out.
The approach the Griesbeck group used was to bracket the calcium sensing protein Troponin C found in skeletal muscle with the enhanced fluorescent proteins Cerulean and Citrine. This sensor has enhanced kinetics compared to cameleons and GCaMP, and also seems to lack some of the problems these other sensors have had with mammalian in vivo expression. Transgenic mice expressing their sensor CerTN-L15 were generated.
The authors report :
To analyze functional properties of CerTN-L15, we first tested its Ca2+ sensitivity in acute cortical slices. For this purpose, we performed simultaneous two-photon imaging and whole-cell patch clamp recordings from labeled layer 2/3 pyramidal cells… Brief trains of 2–3 action potentials evoked clear changes in the ratio of Citrine/Cerulean fluorescence in single trials (Fig. 2b), whereas fluorescence changes caused by single action potentials were not reliably detected.
When they moved in vivo :
We used iontophoretic glutamate applications to probe for functional responses of CerTN-L15. When analyzing neurons in layer 2/3 of the visual cortex in vivo, we had to use stimulation pulse durations, which were two times longer compared to those used in cortical slices (see Supplementary Methods). The resulting amplitudes (R/R) of the Ca2+ transients were also twice as large (63.7 5.1%, n = 11 in vivo compared to 33.2 2.2%, n = 6 in vitro).
And finally :
In conclusion, the new FCIP mouse line provides important improvements for the analysis of Ca2+ signaling in the intact brain. These include (i) a homogeneous and bright staining of the entire cytosol of individual neurons down to secondary and tertiary dendrites, (ii) the full functionality of the sensor protein allowing measurement of small suprathreshold depolarizations consisting of as few as 2–3 action potentials, (iii) the linear response properties of CerTN-L15 within a physiologically relevant activity range, and (iv) the possibility of in vivo Ca2+ imaging with single-cell and even subcellular resolution.
In other words, they are getting close, but they aren’t quite there yet. It might be surprising that they can see 2APs fairly easily but cannot reliably discern 1AP. This may be due to their chosen stimulation protocol. They continuously depolarize the cell until 2APs are generated, a process that takes 35ms. Single APs have much less than 50% of that duration of depolarization. If significant calcium enters the cell during the sustained depolarization, this may overestimate the calcium signal from a naturally occurring 2AP burst. Perhaps a better model would be to give 2 closely spaced, shorter depolarizations to drive 2AP spiking.
Interestingly, CerTN-L15 is not the ‘best’ sensor that has come out of the Griesbeck group. TN-XL was shown to have 3-fold improvement in the maximal ratio change over TN-L15. How well does TN-XL express in vivo? Does having a circularly-permuted Citrine perturb the folding? Does TN-XL maintain its impressive performance advantage? Brain Windows eagerly awaits the answers to these questions.
It must also be noted that there have also been significant improvements in both cameleons and GCaMPs. At this point, it is unclear which GECI strategy will achieve ‘holy grail’ status first, but the day of reckoning appears one step closer now.