A multicolor suite for deciphering population coding in calcium and cAMPin vivo

SUMMARY cAMP is a pivotal second messenger regulated by various upstream pathways including Ca 2+ and G protein-coupled receptors (GPCRs). To decipher in vivo cAMP dynamics, we rationally designed cAMPinG1, an ultrasensitive 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 highly sensitive new red Ca 2+ indicator RCaMP3 allowed simultaneous measurement of population patterns in Ca 2+ and cAMP in hundreds of neurons. We identified Ca 2+ -induced cAMP responses that represented specific information, such as direction selectivity in vision and locomotion, as well as GPCR-induced cAMP responses. Overall, our multicolor suite revealed that information encoded in Ca 2+ and GPCRs signaling is integrated and stored as cAMP transients for longer periods in vivo . Highlights Developing an ultrasensitive cAMP indicator, cAMPinG1, for visualizing cAMP transients in somata and dendritic spines in vivo . Developing a highly sensitive red Ca 2+ indicator, RCaMP3, for visualizing Ca 2+ transients in large neuronal population. Dual-color Ca 2+ and cAMP imaging for dissecting Ca 2+ -induced and GPCR-induced cAMP responses. Single-cell, single-timepoint cAMP imaging for GRCR biology and drug screening.