부산대학교 도서관

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 vivo and the relationship to the underlying neural activity and changes in intracellular calcium concentration (Δ[Ca2+]i ) has not been given.Weused 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 andΔ[Ca2+]i at different action potential frequencies in presynaptic motoneuron boutons of transgenic Drosophila larvae.Wecalibrated ΔF of eight different GECIs in vivo to neural activity, Δ[Ca2+]i , and ΔF of purified GECI protein at similar Δ[Ca2Δ] in vitro. Yellow Cameleon 3.60 (YC3.60), YC2.60, D3cpv, and TN-XL exhibited twofold higher maximum ΔF compared with YC3.3 and TN-L15 in vivo. Maximum ΔF of GCaMP2 and GCaMP1.6 were almost identical. Small Δ[Ca2+]i were reported best by YC3.60, D3cpv, and YC2.60. The kinetics ofΔ[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 forΔ[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 maximumΔF 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. [ABSTRACT FROM AUTHOR]