Green Fluorescent-Calmodulin Proteins (GCaMPs) have been the reporters of choice for visualizing neuronal network activity in vivo. With the GCaMP6 generation sufficient brightness was achieved for single action potential (AP) detection1. However, the Ca2+-response kinetics of GCaMP6 probes remained limiting to separating individual APs. The aim was to accelerate the Ca2+ response kinetics of GCaMP6 probes by point mutations in the Ca2+.CaM.RS20 complex2. A series of probes retaining high fluorescence dynamic ranges was generated with in vitro dissociation constants (Kd) for Ca2+ in the μM range (0.1-3.3 μM) and Hill coefficients of 1.7 to 4.2. Two highlighted probes termed GCaMP6fast and GCaMP6bright had in vitro half times (t1/2) at 37°C for Ca2+ rise of 1.3 ms and 3.3 ± 0.2 ms and decay t1/2-s of 2.8 ± 0.1 and 3.5 ± 0.1 ms, respectively, compared to the 10 ± 1 ms rise and 63 ± 6 ms decay times for GCaMP6f. Fluorescence changes on Ca2+ association were highly cooperative and characterized by a rate limiting conformational change. In vivo Ca2+ responses associated with AP firing patterns were tested in cultured hippocampal slices by two-photon imaging at 28°C. The kinetic performance of GCaMP6fast and GCaMP6bright was compared with that of GCaMP6f. Ca2+ decay kinetics were determined by monitoring fluorescence changes evoked by 5 APs fired at 100 Hz. t1/2 values for GCaMP6fast and GCaMP6bright were 58 ms and 126 ms, respectively. With an up to 7-fold faster in vivo decay kinetics than GCaMP6f, GCaMP6fast and GCaMP6bright are promising tools for monitoring brain activity.1Podor et al., Neurophotonics, Epub 2015 Apr 30.2Helassa et al., Scientific Reports, accepted.This work was funded by Wellcome Trust (094385/Z/10/Z to KT), BBSRC (BB/M02556X/1 to KT), CIHR (MOP-123514 to AF) and NSERC (RGPIN-170421 to AF).