Dierk Reiff’s lab has done another head-to-head in vivo showdown between various GECIs and a synthetic dye. Their paper, Fluorescence changes of genetic calcium indicators and OGB-1 correlated with neural activity and calcium in vivo and in vitro, is very interesting and deserves a full write-up. I will present a detailed analysis of the paper in a future update. For now, check the abstract.
Recent advance in the design of genetically encoded calcium indicators (GECIs) has further increased their potential fordirect measurements of activity in intact neural circuits. However, a quantitative analysis of their fluorescence changes (F) in vivo and the relationship to the underlying neural activity and changes in intracellular calcium concentration ([Ca2+]i) has not been given. We used two-photon microscopy, microinjection of synthetic Ca2+ dyes and in vivocalibration of Oregon-Green-BAPTA-1 (OGB-1) to estimate [Ca2+]i at rest and [Ca2+]i at different action potential frequencies in presynaptic motoneuron boutons of transgenic Drosophila larvae. We calibrated F of eight different GECIs in vivo to neural activity, [Ca2+]i, and F of purified GECI protein at similar [Ca2+] in vitro. Yellow Cameleon 3.60 (YC3.60), YC2.60, D3cpv, and TN-XL exhibited twofold higher maximum F compared with YC3.3 and TN-L15 in vivo. Maximum F of GCaMP2 and GCaMP1.6 were almost identical. Small [Ca2+]i were reported best by YC3.60, D3cpv, and YC2.60. The kinetics of [Ca2+]i was massively distorted by all GECIs, with YC2.60 showing the slowest kinetics, whereas TN-XL exhibited the fastest decay. Single spikes were only reported by OGB-1; all GECIswere blind for [Ca2+]i associated with single action potentials. YC3.60 and D3cpv tentatively reported spike doublets. In vivo, the KD(dissociation constant) of all GECIs was shifted toward lower values, the Hill coefficient was changed, and the maximum F was reduced. The latter could be attributed to resting [Ca2+]i and the optical filters of the equipment. These results suggest increased sensitivity of new GECIs but still slow on rates for calcium binding.