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 (ΔF)in vivoand the relationship to the underlying neural activity and changes in intracellular calcium concentration (Δ[Ca2+]i) has not been given. We used two-photon microscopy, microinjection of synthetic Ca2+dyes andin vivocalibration of Oregon-Green-BAPTA-1 (OGB-1) to estimate [Ca2+]iat rest and Δ[Ca2+]iat different action potential frequencies in presynaptic motoneuron boutons of transgenicDrosophilalarvae. We calibrated ΔFof eight different GECIsin vivoto neural activity, Δ[Ca2+]i, and ΔFof purified GECI protein at similar Δ[Ca2+]in vitro. Yellow Cameleon 3.60 (YC3.60), YC2.60, D3cpv, and TN-XL exhibited twofold higher maximum ΔFcompared with YC3.3 and TN-L15in vivo. Maximum ΔFof GCaMP2 and GCaMP1.6 were almost identical. Small Δ[Ca2+]iwere reported best by YC3.60, D3cpv, and YC2.60. The kinetics of Δ[Ca2+]iwas 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 Δ[Ca2+]iassociated with single action potentials. YC3.60 and D3cpv tentatively reported spike doublets.In vivo, theKD(dissociation constant) of all GECIs was shifted toward lower values, the Hill coefficient was changed, and the maximum ΔFwas reduced. The latter could be attributed to resting [Ca2+]iand the optical filters of the equipment. These results suggest increased sensitivity of new GECIs but still slow on rates for calcium binding.