A multicolor suite for deciphering population coding of calcium and cAMP in vivo

cAMP is a universal second messenger regulated by various upstream pathways including Ca²⁺ and G-protein-coupled receptors (GPCRs). To decipher in vivo cAMP dynamics, we rationally designed cAMPinG1, a sensitive genetically encoded green cAMP indicator that outperformed its predecessors in both dynamic range and cAMP affinity. Two-photon cAMPinG1 imaging detected cAMP transients in the somata and dendritic spines of neurons in the mouse visual cortex on the order of tens of seconds. In addition, multicolor imaging with a sensitive red Ca²⁺ indicator RCaMP3 allowed simultaneous measurement of population patterns in Ca²⁺ and cAMP in hundreds of neurons. We found Ca²⁺-related cAMP responses that represented specific information, such as direction selectivity in vision and locomotion, as well as GPCR-related cAMP responses. Overall, our multicolor suite will facilitate analysis of the interaction between the Ca²⁺, GPCR and cAMP signaling at single-cell resolution both in vitro and in vivo.