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キーワード:
GFP; calcium; neural activity; fluorescence; genetic probes; in vivo imaging
要旨:
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 (Delta F) in vivo and the relationship to the underlying neural activity and changes in intracellular calcium concentration (Delta[Ca2+](i)) has not been given. We used two-photon microscopy, microinjection of synthetic Ca2+ dyes and in vivo calibration of Oregon-Green-BAPTA-1 (OGB-1) to estimate [Ca2+](i) at rest and Delta[Ca2+](i) at different action potential frequencies in presynaptic motoneuron boutons of transgenic Drosophila larvae. We calibrated Delta F of eight different GECIs in vivo to neural activity, Delta[Ca2+](i), and Delta F of purified GECI protein at similar Delta[Ca2+](i) in vitro. Yellow Cameleon 3.60 (YC3.60), YC2.60, D3cpv, and TN-XL exhibited twofold higher maximum Delta F compared with YC3.3 and TN-L15 in vivo. Maximum Delta F of GCaMP2 and GCaMP1.6 were almost identical. Small Delta[Ca2+](i) were reported best by YC3.60, D3cpv, and YC2.60. The kinetics of Delta[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 GECIs were blind for Delta[Ca2+](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 Delta 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.
