Absolute measurement of fast and slow neuronal signals with fluorescence lifetime photometry at high temporal resolution

The concentrations of extracellular and intracellular signaling molecules, such as dopamine and cAMP, change over both fast and slow timescales and impact downstream pathways in a cell-type specific manner. Fluorescence sensors currently used to monitor such signals in vivo are typically optimized to detect fast, relative changes in concentration of the target molecule. They are less well suited to detect slowly-changing signals and rarely provide absolute measurements of either fast and slow signaling components. Here, we developed a system for fluorescence lifetime photometry at high temporal resolution (FLIPR) that utilizes frequency-domain analog processing to measure the absolute fluorescence lifetime of genetically-encoded sensors at high speed but with long-term stability and picosecond precision in freely moving mice. We applied FLIPR to investigate dopamine signaling in two functionally distinct regions in the striatum, the nucleus accumbens core (NAC) and the tail of striatum (TOS). We observed higher tonic dopamine levels at baseline in the TOS compared to the NAC and detected differential and dynamic responses in phasic and tonic dopamine to appetitive and aversive stimuli. Thus, FLIPR enables simple monitoring of fast and slow time-scale neuronal signaling in absolute units, revealing previously unappreciated spatial and temporal variation even in well-studied signaling systems.