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Neurons in the nervous system can change their functional properties over time. At present, there are no techniques that allow reliable monitoring of changes within identified neurons over repeated experimental sessions. We increased the signal strength of troponin C-based calcium biosensors in the low-calcium regime by mutagenesis and domain rearrangement within the troponin C calcium binding moiety to generate the indicator TN-XXL. Using in vivo two-photon ratiometric imaging, we show that TN-XXL exhibits enhanced fluorescence changes in neurons of flies and mice. TN-XXL could be used to obtain tuning curves of orientation-selective neurons in mouse visual cortex measured repeatedly over days and weeks. Thus, the genetically encoded calcium indicator TN-XXL allows repeated imaging of response properties from individual, identified neurons in vivo, which will be crucial for gaining new insights into cellular mechanisms of plasticity, regeneration and disease.

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.

Genetically encoded calcium biosensors have become valuable tools in cell biology and neuroscience, but some aspects such as signal strength and response kinetics still need improvement. Here we report the generation of a FRET-based calcium biosensor employing troponin C as calcium-binding moiety that is fast, is stable in imaging experiments, and shows a significantly enhanced fluorescence change. These improvements were achieved by engineering magnesium and calcium-binding properties within the C-terminal lobe of troponin C and by the incorporation of circularly permuted variants of the green fluorescent protein. This sensor named TN-XL shows a maximum fractional fluorescence change of 400% in its emission ratio and linear response properties over an expanded calcium regime. When imaged in vivo at presynaptic motoneuron terminals of transgenic fruit flies, TN-XL exhibits highly reproducible fluorescence signals with the fastest rise and decay times of all calcium biosensors known so far.