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.

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.

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 red fluorescent GPCR-activation–based DA (GRAB DA ) sensors and optimized versions of green fluorescent GRAB DA sensors. In response to extracellular DA, both the red and green GRAB DA sensors exhibit a large increase in fluorescence, with subcellular resolution, subsecond kinetics, and nanomolar to submicromolar affinity. Moreover, the GRAB DA sensors resolve evoked DA release in mouse brain slices, detect evoked compartmental DA release from a single neuron in live flies, and report optogenetically elicited nigrostriatal DA release as well as mesoaccumbens dopaminergic activity during sexual behavior in freely behaving mice. Co-expressing red GRAB DA with either green GRAB DA or the calcium indicator GCaMP6s allows simultaneously tracking neuronal activity and dopaminergic signaling in distinct circuits in vivo .

To achieve simultaneous measurement of multiple cellular events in molecularly defined groups of neurons in vivo, we designed a spectrometer-based fiber photometry system that allows for spectral unmixing of multiple fluorescence signals recorded from deep brain structures in behaving animals. Using green and red Ca indicators differentially expressed in striatal direct- and indirect-pathway neurons, we were able to simultaneously monitor the neural activity in these two pathways in freely moving animals. We found that the activities were highly synchronized between the direct and indirect pathways within one hemisphere and were desynchronized between the two hemispheres. We further analyzed the relationship between the movement patterns and the magnitude of activation in direct- and indirect-pathway neurons and found that the striatal direct and indirect pathways coordinately control the dynamics and fate of movement. VIDEO ABSTRACT.

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Committee (18JC1410100) to J.D.; the NIH grants R01MH101377 and R21HD090563 and 48 an Irma T. Hirschl Career Scientist Award to D.L.; and the Intramural Research Program of 49 the NIH/NIEHS of the United States (1ZIAES103310) to G.C.50We thank Yi Rao for sharing the two-photon microscope and Xiaoguang Lei for the platform 51 support of the Opera Phenix high-content screening system at PKU-CLS. We thank the 52 Core Facilities at the School of Life Sciences, Peking University for technical assistance. 53We thank Bryan L. Roth and Nevin A. Lambert for sharing stable cell lines and plasmids. 54We thank Yue Sun, Sunlei Pan, Lun Yang, Haohong Li for inputs on sensors' 55 characterization and application. We thank Yanhua Huang, Liqun Luo and Mickey London 56 for valuable feedback of the manuscript. 57 58 Author Contributions 59 Y. L conceived and supervised the project. J.F., M.J., H.Wang, A.D., and Z.W. performed 60 experiments related to sensor development, optimization, and characterization in culture 61 HEK cells, culture neurons and brain slices. Y.Z., P.Z. and J.J.Z designed and performed 62 experiments using Sindbis virus in slices. C.Z., W.C., and J.D. designed and performed 63 experiments on transgenic fish. Abstract 73Norepinephrine (NE) and epinephrine (Epi), two key biogenic monoamine 74 neurotransmitters, are involved in a wide range of physiological processes. However, their 75 precise dynamics and regulation remain poorly characterized, in part due to limitations of 76 available techniques for measuring these molecules in vivo. Here, we developed a family 77 of GPCR Activation-Based NE/Epi (GRABNE) sensors with a 230% peak ΔF/F0 response 78 to NE, good photostability, nanomolar-to-micromolar sensitivities, sub-second rapid 79 kinetics, high specificity to NE vs. dopamine. Viral-or transgenic-mediated expression of 80 GRABNE sensors were able to detect electrical-stimulation evoked NE release in the locus 81 coeruleus (LC) of mouse brain slices, looming-evoked NE release in the midbrain of live 82 zebrafish, as well as optogenetically and behaviorally triggered NE release in the LC and 83 hypothalamus of freely moving mice. Thus, GRABNE sensors are a robust tool for rapid and 84 specific monitoring of in vivo NE/Epi transmission in both physiological and pathological 85 processes. 86 87 5 Introduction 88Both norepinephrine (NE) and epinephrine (Epi) are key monoamine neurotransmitters in 89 the central nervous systems and peripheral organs of vertebrate organisms. These 90 transmitters play an important role in a plethora of physiological processes, allowing the 91 organism to cope with its ever-changing internal and external environment. In the brain, 92 NE is synthesized primarily in the locus coeruleus (LC), a small yet powerful nucleus 93 located in the pons. Noradrenergic LC neurons project throughout the brain and exert a 94 wide range of effects, including processing sensory information (Berridge and Waterhouse, 95 2003), regulating the sleep-wake/arousal state (Berridge et al., 2012), and mediating...

The monoamine neuromodulator dopamine (DA) plays a critical role in the brain, and the 21 ability to directly measure dopaminergic activity is essential for understanding its 22 physiological functions. We therefore developed the first red fluorescent GPCR-activation-23 based DA (GRABDA) sensors and optimized versions of green fluorescent GRABDA sensors 24 following our previous studies. In response to extracellular DA, both the red and green 25 GRABDA sensors have a large increase in fluorescence (ΔF/F0 values of 150% and 340%, 26 respectively), with subcellular resolution, subsecond kinetics, and nanomolar to 27 submicromolar affinity. Moreover, both the red and green GRABDA sensors readily resolve 28 evoked DA release in mouse brain slices, detect compartmental DA release in live flies with 29 single-cell resolution, and report optogenetically elicited nigrostriatal DA release as well as 30 mesoaccumbens dopaminergic activity during sexual behavior in freely behaving mice. 31 Importantly, co-expressing red GRABDA with either green GRABDA or the calcium indicator 32GCaMP6s provides a robust tool for simultaneously tracking neuronal activity and 33 dopaminergic signaling in distinct circuits in vivo. 35Dopamine (DA) is an essential monoamine neuromodulator produced primarily in the midbrain and 36 released throughout the central nervous system. A multitude of brain functions are regulated by DA, 37including motor control, motivation, learning and memory, and emotional control 1-9 . Consistent with 38these key physiological roles, altered DA signaling has been implicated in a variety of brain 39 disorders, including Parkinson's disease, addiction, schizophrenia, attention-deficit/hyperactivity 40 disorder, and posttraumatic stress disorder 10-20 . Thus, tools that can sense changes in DA 41 concentration with high spatiotemporal resolution, high specificity, and high sensitivity will greatly 42 facilitate our study of the diverse functions that the dopaminergic system plays under both 43 physiological and pathological conditions. 44Previous techniques for measuring DA dynamics, including microdialysis, electrochemical 45 probes, reporter cells, and gene expression-based assays, lack sufficient spatiotemporal resolution 46 and/or molecular specificity [21][22][23][24][25][26][27][28][29][30] . Recently, our group 31 and Patriarchi et al. 32 independently 47 developed two series of genetically encoded, G-protein-coupled receptor (GPCR)-based DA 48 sensors called GRABDA and dLight, respectively. Taking advantage of naturally occurring DA 49 receptors, these sensors convert a ligand-stabilized conformational change in the DA receptor into 50 an optical response via a conformation-sensitive fluorescent protein inserted in the receptor's third 51 intracellular loop. Our first-generation DA receptor-based sensors called GRABDA1m and 52 GRABDA1h were used to detect cell type-specific DA dynamics in several organisms, including 53 Drosophila, zebrafish, mice, and zebra finches 31,33-35 . Here, we employed semi-rational en...

Neuromodulation of neural networks, whereby a selected circuit is regulated by a particular modulator, plays a critical role in learning and memory. Among neuromodulators, acetylcholine (ACh) plays a critical role in hippocampus-dependent memory and has been shown to modulate neuronal circuits in the hippocampus. However, it has remained unknown how ACh modulates hippocampal output. Here, using in vitro and in vivo approaches, we show that ACh, by activating oriens lacunosum moleculare (OLM) interneurons and therefore augmenting the negative-feedback regulation to the CA1 pyramidal neurons, suppresses the circuit from the hippocampal area CA1 to the deep-layer entorhinal cortex (EC). We also demonstrate, using mouse behavior studies, that the ablation of OLM interneurons specifically impairs hippocampus-dependent but not hippocampus-independent learning. These data suggest that ACh plays an important role in regulating hippocampal output to the EC by activating OLM interneurons, which is critical for the formation of hippocampus-dependent memory.