Inverse-response Ca²⁺ indicators for optogenetic visualization of neuronal inhibition

We have developed a series of yellow genetically encoded Ca²⁺ indicators for optical imaging (Y-GECOs) with inverted responses to Ca²⁺ and apparent dissociation constants (K_d') ranging from 25 to 2400 nM. To demonstrate the utility of this affinity series of Ca²⁺ indicators, we expressed the four highest affinity variants (K_d's = 25, 63, 121, and 190 nM) in the Drosophila medulla intrinsic neuron Mi1. Hyperpolarization of Mi1 by optogenetic stimulation of the laminar monopolar neuron L1 produced a decrease in intracellular Ca²⁺ in layers 8-10, and a corresponding increase in Y-GECO fluorescence. These experiments revealed that lower K_d' was associated with greater increases in fluorescence, but longer delays to reach the maximum signal change due to slower off-rate kinetics.