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.

Microglia are the primary immune cells in the brain. Under physiological conditions, they typically stay in a "resting" state, with ramified processes continuously extending to and retracting from surrounding neural tissues. Whether and how such highly dynamic resting microglia functionally interact with surrounding neurons are still unclear. Using in vivo time-lapse imaging of both microglial morphology and neuronal activity in the optic tectum of larval zebrafish, we found that neuronal activity steers resting microglial processes and facilitates their contact with highly active neurons. This process requires the activation of pannexin-1 hemichannels on neurons. Reciprocally, such resting microglia-neuron contact reduces both spontaneous and visually evoked activities of contacted neurons. Our findings reveal an instructive role for neuronal activity in resting microglial motility and suggest the function for microglia in homeostatic regulation of neuronal activity in the healthy brain.

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.

SUMMARY Reduced nicotinamide adenine dinucleotide phosphate (NADPH) is essential for for biosynthetic reactions and antioxidant functions; however, detection of NADPH metabolism in living cells remains technically challenging. We develop and characterize ratiometric, pH-resistant, genetically encoded fluorescent indicators for NADPH (iNap sensors) with various affinities and wide dynamic range. The iNap sensors permitted quantification of cytosolic and mitochondrial NADPH pools that were controlled by cytosolic NAD+ kinase levels, and revealed cellular NADPH dynamics under oxidative stress depending on glucose availability. We find that mammalian cells have a strong tendency to maintain physiological NADPH homeostasis, which is regulated by glucose-6-phosphate dehydrogenase (G6PD) and AMP kinase (AMPK). Moreover, using the iNap sensors we monitor NADPH fluctuations during the activation of macrophage cells or wound response in vivo. These data demonstrate that the iNap sensors will be valuable tools for monitoring NADPH dynamics in live-cells, and gaining new insights into cell metabolism.