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 .

Dopamine (DA) is a central monoamine neurotransmitter involved in many physiological and pathological processes. A longstanding yet largely unmet goal is to measure DA changes reliably and specifically with high spatiotemporal precision, particularly in animals executing complex behaviors. Here we report the development of novel genetically-encoded GPCR-Activation-Based-DA (GRAB _DA ) sensors that enable these measurements. In response to extracellular DA rises, GRAB _DA sensors exhibit large fluorescence increases (ΔF/F ₀ ∼90%) with sub-second kinetics, nanomolar to sub-micromolar affinities, and excellent molecular specificity. Importantly, GRABDA sensors can resolve a single-electrical-stimulus evoked DA release in mouse brain slices, and detect endogenous DA release in the intact brains of flies, fish, and mice. In freely-behaving mice, GRABDA sensors readily report optogenetically-elicited nigrostriatal DA release and depict dynamic mesoaccumbens DA changes during Pavlovian conditioning or during sexual behaviors. Thus, GRAB _DA sensors enable spatiotemporal precise measurements of DA dynamics in a variety of model organisms while exhibiting complex behaviors.