Rational design of a high-affinity, fast, red calcium indicator R-CaMP2

An improved genetically encoded red calcium sensor enables the monitoring of neuronal activity in cell culture and in vivo . R-CaMP2 has fast kinetics and a high affinity to calcium and can follow action potentials up to 40 Hz. Fluorescent Ca 2+ reporters are widely used as readouts of neuronal activities. Here we designed R-CaMP2, a high-affinity red genetically encoded calcium indicator (GECI) with a Hill coefficient near 1. Use of the calmodulin-binding sequence of CaMKK-α and CaMKK-β in lieu of an M13 sequence resulted in threefold faster rise and decay times of Ca 2+ transients than R-CaMP1.07. These features allowed resolving single action potentials (APs) and recording fast AP trains up to 20–40 Hz in cortical slices. Somatic and synaptic activities of a cortical neuronal ensemble in vivo were imaged with similar efficacy as with previously reported sensitive green GECIs. Combining green and red GECIs, we successfully achieved dual-color monitoring of neuronal activities of distinct cell types, both in the mouse cortex and in freely moving Caenorhabditis elegans . Dual imaging using R-CaMP2 and green GECIs provides a powerful means to interrogate orthogonal and hierarchical neuronal ensembles in vivo .