A Highly Sensitive A-Kinase Activity Reporter for Imaging Neuromodulatory Events in Awake Mice

Neuromodulation imposes powerful control over brain function, and cAMP-dependent protein kinase (PKA) is a central downstream mediator of multiple neuromodulators. Although genetically encoded PKA sensors have been developed, single-cell imaging of PKA activity in living mice has not been established. Here, we used two-photon fluorescence lifetime imaging microscopy (2pFLIM) to visualize genetically encoded PKA sensors in response to the neuromodulators norepinephrine and dopamine. We screened available PKA sensors for 2pFLIM and further developed a variant (named tAKARα) with increased sensitivity and a broadened dynamic range. This sensor allowed detection of PKA activation by norepinephrine at physiologically relevant concentrations and kinetics, and by optogenetically released dopamine. In vivo longitudinal 2pFLIM imaging of tAKARα tracked bidirectional PKA activities in individual neurons in awake mice and revealed neuromodulatory PKA events that were associated with wakefulness, pharmacological manipulation, and locomotion. This new sensor combined with 2pFLIM will enable interrogation of neuromodulation-induced PKA signaling in awake animals. [Display omitted] •An improved PKA sensor (called tAKARα) with enhanced sensitivity and dynamic range•tAKARα tracks physiologically relevant PKA signaling activated by neuromodulators•2pFLIM of tAKARα enables single-cell imaging of PKA activity in behaving mice•Wakefulness and locomotion are associated with cell-specific PKA dynamics It remains challenging to visualize neuromodulation, a major mode of neuronal communication, in vivo. Ma et al. demonstrate that two-photon fluorescence lifetime imaging of an improved genetically encoded sensor enables monitoring of neuromodulatory PKA signaling with single-cell resolution in behaving mice.