Serotonin (5-HT) is a phylogenetically conserved monoamine neurotransmitter modulating a variety of processes in the brain. To directly visualize the dynamics of 5-HT, we developed a genetically encoded G PC R - A ctivation- B ased 5-HT (GRAB _5-HT ) sensor with high sensitivity, selectivity, and spatiotemporal resolution. GRAB _5-HT , detected 5-HT release in multiple physiological and pathological conditions in both flies and mice, and thus provides new insights into the dynamics and mechanisms of 5-HT signaling.

The ability to directly measure acetylcholine (ACh) release is an essential first step towards understanding its physiological function. Here we optimized the GRAB _ACh ( G PC R - A ctivation– B ased- ACh ) sensor with significantly improved sensitivity and minimal downstream coupling. Using this sensor, we measured in-vivo cholinergic activity in both Drosophila and mice, revealing compartmental ACh signals in fly olfactory center and single-trial ACh dynamics in multiple regions of the mice brain under a variety of different behaviors

The monoamine neuromodulator dopamine (DA) plays a critical role in the brain, and the ability to directly measure dopaminergic activity is essential for understanding its physiological functions. We therefore developed the first red fluorescent GPCR-activation–based DA (GRAB _DA ) sensors and optimized versions of green fluorescent GRAB _DA sensors following our previous studies. In response to extracellular DA, both the red and green GRAB _DA sensors have a large increase in fluorescence (ΔF/F ₀ values of 150% and 340%, respectively), with subcellular resolution, subsecond kinetics, and nanomolar to submicromolar affinity. Moreover, both the red and green GRAB _DA sensors readily resolve evoked DA release in mouse brain slices, detect compartmental DA release in live flies with single-cell resolution, and report optogenetically elicited nigrostriatal DA release as well as mesoaccumbens dopaminergic activity during sexual behavior in freely behaving mice. Importantly, co-expressing red GRAB _DA with either green GRAB _DA or the calcium indicator GCaMP6s provides a robust tool for simultaneously tracking neuronal activity and dopaminergic signaling in distinct circuits in vivo .