Fluorescence changes of genetic calcium indicators and OGB-1 correlated with neural activity and calcium in vivo and in vitro.

Recent advance in the design of genetically encoded calcium indicators (GECIs) has further increased their potential for direct measurements of activity in intact neural circuits. However, a quantitative analysis of their fluorescence changes (DeltaF) in vivo and the relationship to the underlying neural activity and changes in intracellular calcium concentration (Delta[Ca(2+)](i)) has not been given. We used two-photon microscopy, microinjection of synthetic Ca(2+) dyes and in vivo calibration of Oregon-Green-BAPTA-1 (OGB-1) to estimate [Ca(2+)](i) at rest and Delta[Ca(2+)](i) at different action potential frequencies in presynaptic motoneuron boutons of transgenic Drosophila larvae. We calibrated DeltaF of eight different GECIs in vivo to neural activity, Delta[Ca(2+)](i), and DeltaF of purified GECI protein at similar Delta[Ca(2+)] in vitro. Yellow Cameleon 3.60 (YC3.60), YC2.60, D3cpv, and TN-XL exhibited twofold higher maximum DeltaF compared with YC3.3 and TN-L15 in vivo. Maximum DeltaF of GCaMP2 and GCaMP1.6 were almost identical. Small Delta[Ca(2+)](i) were reported best by YC3.60, D3cpv, and YC2.60. The kinetics of Delta[Ca(2+)](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 GECIs were blind for Delta[Ca(2+)](i) associated with single action potentials. YC3.60 and D3cpv tentatively reported spike doublets. In vivo, the K(D) (dissociation constant) of all GECIs was shifted toward lower values, the Hill coefficient was changed, and the maximum DeltaF was reduced. The latter could be attributed to resting [Ca(2+)](i) and the optical filters of the equipment. These results suggest increased sensitivity of new GECIs but still slow on rates for calcium binding.