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

Neuron. 2018 Aug 22;99(4):665-679.e5. doi: 10.1016/j.neuron.2018.07.020. Epub 2018 Aug 9.


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. VIDEO ABSTRACT.

Keywords: A Kinase Activity Reporter (AKAR); Forster resonance energy transfer (FRET); cAMP dependent kinase (PKA); dopamine; enforced locomotion; in vivo imaging; neuromodulation; norepinephrine; two-photon fluorescence lifetime imaging (2pFLIM); wakefulness.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Animals
  • Animals, Newborn
  • Cyclic AMP-Dependent Protein Kinases / analysis
  • Cyclic AMP-Dependent Protein Kinases / metabolism*
  • Enzyme Activation / drug effects
  • Enzyme Activation / physiology
  • Female
  • Hippocampus / chemistry
  • Hippocampus / drug effects
  • Hippocampus / enzymology*
  • Male
  • Mice
  • Mice, Inbred C57BL
  • Microscopy, Fluorescence, Multiphoton / methods*
  • Neurotransmitter Agents / pharmacology*
  • Norepinephrine / pharmacology
  • Organ Culture Techniques
  • Wakefulness / physiology*


  • Neurotransmitter Agents
  • Cyclic AMP-Dependent Protein Kinases
  • Norepinephrine