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.

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Biomimetic superhydrophobic surfaces display many excellent underwater functionalities, which attribute to the slippery air mattress trapped in the structures on the surface. However, the air mattress is easy to collapse due to various disturbances, leading to the fully wetted Wenzel state, while the water filling the microstructures is difficult to be repelled to completely recover the air mattress even on superhydrophobic surfaces like lotus leaves. Beyond superhydrophobicity, here we find that the floating fern, Salvinia molesta, has the superrepellent capability to efficiently replace the water in the microstructures with air and robustly recover the continuous air mattress. The hierarchical structures on the leaf surface are demonstrated to be crucial to the recovery. The interconnected wedge-shaped grooves between epidermal cells are key to the spontaneous spreading of air over the entire leaf governed by a gas wicking effect to form a thin air film, which provides a base for the later growth of the air mattress in thickness synchronously along the hairy structures. Inspired by nature, biomimetic artificial Salvinia surfaces are fabricated using 3D printing technology, which successfully achieves a complete recovery of a continuous air mattress to exactly imitate the superrepellent capability of Salvinia leaves. This finding will benefit the design principles of water-repellent materials and expand their underwater applications, especially in extreme environments.

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...

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Cell-cell communication via gap junctions regulates a wide range of physiological processes by enabling the direct intercellular electrical and chemical coupling. However, the in vivo distribution and function of gap junctions remain poorly understood, partly due to the lack of non-invasive tools with both cell-type specificity and high spatiotemporal resolution. Here, we developed PARIS (pairing actuators and receivers to optically isolate gap junctions), a new fully genetically encoded tool for measuring the cell-specific gap junctional coupling (GJC). PARIS successfully enabled monitoring of GJC in several cultured cell lines under physiologically relevant conditions and in distinct genetically defined neurons in Drosophila brain, with ~10 s temporal resolution and sub-cellular spatial resolution. These results demonstrate that PARIS is a robust, highly sensitive tool for mapping functional gap junctions and study their regulation in both health and disease.

The purinergic signaling molecule adenosine (Ado) modulates many physiological and pathological brain functions， but its spatiotemporal release dynamics in the brain remains largely unknown. We developed a genetically encoded GPCR-Activation-Based Ado sensor (GRABAdo) in which Ado-induced changes in the human A2A receptor are reflected by fluorescence increases. This GRABAdo revealed that neuronal activity-induced extracellular Ado elevation was due to direct Ado release from somatodendritic regions of the neuron, requiring calcium influx through L-type calcium channels, rather than the degradation of extracellular ATP. The Ado release was slow (~30 s) and depended on equilibrative nucleoside transporters (ENTs) rather than conventional vesicular release mechanisms. Thus, GRABAdo reveals an activity-dependent slow Ado release from somatodendritic region of the neuron, potentially serving modulating functions as a retrograde signal. Main textExtracellular adenosine (Ado) plays an important role in a wide range of physiological processes (1-3), including the sleep-wake cycle, learning and memory, cardiovascular function, and immune responses. Moreover, impaired adenosinergic signaling has been implicated in a variety of diseases and conditions (2, 4) such as pain, migraine, epilepsy, stroke, drug addiction, and neurodegeneration (e.g., Parkinson's disease). In the brain, Ado acts as a neuromodulator or a homeostatic modulator at the synaptic level, through the activation of distinct G proteincoupled Ado receptors (5). Although Ado's function has been extensively studied, the mechanisms underlying Ado release in the brain remains poorly understood, the difficulty lies largely in the lack of sensitive methods for directly detecting Ado in vivo with both cell-type specificity and high spatiotemporal resolution. Recently, a group of genetically encoded GPCR-Activation-Based (GRAB) sensors were developed for measuring the dynamics of several neuromodulators-including acetylcholine, dopamine, and norepinephrine-and had been used under various in vivo conditions (6-9). Using a similar strategy, we designed a genetically encoded GRAB sensor for Ado ( Fig. 1A) and used this novel tool to examine the mechanism underlying a neuronal activity-dependent Ado release. Development and characterization of GRAB sensors for adenosineTo develop the genetically encoded GRAB sensor for Ado, we first screened candidate GPCR scaffolds by inserting cpEGFP in the receptor flanked by short linker peptides at both the N-and C-terminus (fig. S1A); we then selected an A2A receptor (A2AR)-based chimera (GRABAdo0.1) for further optimization, based on its membrane trafficking and high fluorescence response upon Ado application ( fig. S1B). By systematically optimizing the length and amino acid composition of the linkers between the A2AR and the cpEGFP, we identified the protein with the largest fluorescence response (fig. S1C), and named it GRABAdo1.0 (hereafter referred to as Ado1.0). When expressed in HEK293T cells, Ado1.0 trafficked to the cell...

The purinergic signaling molecule adenosine (Ado) modulates many physiological and pathological functions in the brain. However, the exact source of extracellular Ado remains controversial. Here, utilizing a newly optimized genetically encoded GPCR-Activation-Based Ado fluorescent sensor (GRAB Ado ), we discovered that the neuronal activity–induced extracellular Ado elevation is due to direct Ado release from somatodendritic compartments of neurons, rather than from the axonal terminals, in the hippocampus. Pharmacological and genetic manipulations reveal that the Ado release depends on equilibrative nucleoside transporters but not the conventional vesicular release mechanisms. Compared with the fast-vesicular glutamate release, the Ado release is slow (~40 s) and requires calcium influx through L-type calcium channels. Thus, this study reveals an activity-dependent second-to-minute local Ado release from the somatodendritic compartments of neurons, potentially serving modulatory functions as a retrograde signal.