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 genetically-encoded GPCR-Activation-Based-DA (GRABDA) sensors that enable these measurements. In response to extracellular DA, GRABDA sensors exhibit large fluorescence increases (ΔF/F0 ~90%) with subcellular resolution, sub-second kinetics, nanomolar to sub-micromolar affinities, and excellent molecular specificity. GRABDA sensors can resolve a-single-electrical-stimulus evoked DA release in mouse brain slices, and detect endogenous DA release in living flies, fish, and mice. In freely-behaving mice, GRABDA sensors readily report optogenetically elicited nigrostriatal DA release and depict dynamic mesoaccumbens DA signaling during Pavlovian conditioning or during sexual behaviors. Thus, GRABDA sensors enable spatiotemporally precise measurements of DA dynamics in a variety of model organisms while exhibiting complex behaviors.

Summary Vesicular secretion of neurotransmitter is essential for neuronal communication. Kiss-and-run is a mode of membrane fusion and retrieval without the full collapse of the vesicle into the plasma membrane and de novo regeneration. The significance of kiss-and-run during efficient neurotransmission has remained in doubt. We developed an approach for loading individual synaptic vesicles with single quantum dots. Their size and pH-dependent photoluminescence change allowed us to distinguish kiss-and-run from full-collapse fusion and to track single vesicles through multiple rounds of kiss-and-run and reuse, without perturbing vesicle cycling. Kiss-and-run dominated at the beginning of stimulus trains, reflecting the preference of vesicles with high release probability. Its incidence was increased by rapid firing, a response appropriate to meet the dynamic demands of neurotransmission.

Norepinephrine (NE) is a key biogenic monoamine neurotransmitter involved in a wide range of physiological processes. However, its precise dynamics and regulation remain poorly characterized, in part due to limitations of available techniques for measuring NE in vivo. Here, we developed a family of GPCR activation-based NE (GRAB NE ) sensors with a 230% peak DF/F 0 response to NE, good photostability, nanomolar-to-micromolar sensitivities, sub-second kinetics, and high specificity. Viral-or transgenic-mediated expression of GRAB NE sensors was able to detect electrical-stimulation-evoked NE release in the locus coeruleus (LC) of mouse brain slices, looming-evoked NE release in the midbrain of live zebrafish, as well as optogenetically and behaviorally triggered NE release in the LC and hypothalamus of freely moving mice. Thus, GRAB NE sensors are robust tools for rapid and specific monitoring of in vivo NE transmission in both physiological and pathological processes.