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      ED SUM: Signaling by the neurotransmitter acetylcholine is monitored in cells and animals with a sensitive reporter. : A genetically encoded fluorescent acetylcholine indicator for in vitro and in vivo studies

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          Abstract

          The neurotransmitter acetylcholine (ACh) regulates a diverse array of physiological processes throughout the body. Despite its importance, cholinergic transmission in the majority of tissues and organs remains poorly understood owing primarily to the limitations of available ACh-monitoring techniques. We developed a family of G-protein-coupled receptor activation-based ACh (GACh) sensors with sensitivity, specificity, signal-to-noise ratio, kinetics and photostability suitable for monitoring ACh signals in vitro and in vivo. GACh sensors were validated with transfection, viral and/or transgenic expression in a dozen types of neuronal and non-neuronal cells prepared from multiple animal species. In all preparations, GACh sensors selectively responded to exogenous and/or endogenous ACh with robust fluorescence signals that were captured by epifluorescence, confocal and/or two-photon microscopy. Moreover, analysis of endogenous ACh release revealed firing pattern-dependent release and restricted volume transmission, resolving two long-standing questions about central cholinergic transmission. Thus, GACh sensors provide a user-friendly, broadly applicable tool for monitoring cholinergic transmission underlying diverse biological processes.

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          Most cited references65

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          Acetylcholine as a neuromodulator: cholinergic signaling shapes nervous system function and behavior.

          Acetylcholine in the brain alters neuronal excitability, influences synaptic transmission, induces synaptic plasticity, and coordinates firing of groups of neurons. As a result, it changes the state of neuronal networks throughout the brain and modifies their response to internal and external inputs: the classical role of a neuromodulator. Here, we identify actions of cholinergic signaling on cellular and synaptic properties of neurons in several brain areas and discuss consequences of this signaling on behaviors related to drug abuse, attention, food intake, and affect. The diverse effects of acetylcholine depend on site of release, receptor subtypes, and target neuronal population; however, a common theme is that acetylcholine potentiates behaviors that are adaptive to environmental stimuli and decreases responses to ongoing stimuli that do not require immediate action. The ability of acetylcholine to coordinate the response of neuronal networks in many brain areas makes cholinergic modulation an essential mechanism underlying complex behaviors. Copyright © 2012 Elsevier Inc. All rights reserved.
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            An optimized fluorescent probe for visualizing glutamate neurotransmission

            We describe an intensity-based glutamate-sensing fluorescent reporter (“iGluSnFR”) with signal-to-noise ratio and kinetics appropriate for in vivo imaging. We engineered iGluSnFR in vitro to maximize its fluorescence change, and validated its utility for visualizing glutamate release by neurons and astrocytes in increasingly intact neurological systems. In hippocampal culture, iGluSnFR detected single field stimulus-evoked glutamate release events. In pyramidal neurons in acute brain slices, glutamate uncaging at single spines showed that iGluSnFR responds robustly and specifically to glutamate in situ, and responses correlate with voltage changes. In mouse retina, iGluSnFR-expressing neurons showed intact light-evoked excitatory currents, and the sensor revealed tonic glutamate signaling in response to light stimuli. In worms, glutamate signals preceded and predicted post-synaptic calcium transients. In zebrafish, iGluSnFR revealed spatial organization of direction-selective synaptic activity in the optic tectum. Finally, in mouse forelimb motor cortex, iGluSnFR expression in layer V pyramidal neurons revealed task-dependent single-spine activity during running.
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              Crystal structure of the human beta2 adrenergic G-protein-coupled receptor.

              Structural analysis of G-protein-coupled receptors (GPCRs) for hormones and neurotransmitters has been hindered by their low natural abundance, inherent structural flexibility, and instability in detergent solutions. Here we report a structure of the human beta2 adrenoceptor (beta2AR), which was crystallized in a lipid environment when bound to an inverse agonist and in complex with a Fab that binds to the third intracellular loop. Diffraction data were obtained by high-brilliance microcrystallography and the structure determined at 3.4 A/3.7 A resolution. The cytoplasmic ends of the beta2AR transmembrane segments and the connecting loops are well resolved, whereas the extracellular regions of the beta2AR are not seen. The beta2AR structure differs from rhodopsin in having weaker interactions between the cytoplasmic ends of transmembrane (TM)3 and TM6, involving the conserved E/DRY sequences. These differences may be responsible for the relatively high basal activity and structural instability of the beta2AR, and contribute to the challenges in obtaining diffraction-quality crystals of non-rhodopsin GPCRs.
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                Author and article information

                Journal
                9604648
                20305
                Nat Biotechnol
                Nat. Biotechnol.
                Nature biotechnology
                1087-0156
                1546-1696
                24 July 2018
                09 July 2018
                September 2018
                09 January 2019
                : 36
                : 8
                : 726-737
                Affiliations
                [1 ]State Key Laboratory of Membrane Biology, Peking University School of Life Sciences, Beijing 100871, China
                [2 ]PKU-IDG/McGovern Institute for Brain Research, Beijing 100871, China
                [3 ]Peking-Tsinghua Center for Life Sciences, Beijing 100871, China
                [4 ]Department of Pharmacology, University of Virginia School of Medicine, Charlottesville, VA 22908
                [5 ]Center for Life Sciences, School of Life Science and Technology, Harbin Institute of Technology, Harbin 150001, China
                [6 ]Zilkha Neurogenetic Institute, Department of Physiology & Neuroscience, Keck School of Medicine, University of Southern California, Los Angeles, CA, 90033
                [7 ]Department of Neurobiology and Behavior, Stony Brook University, Stony Brook, NY 11794
                [8 ]Undergraduate Class of 2019, University of Virginia College of Arts and Sciences, Charlottesville, VA 22908
                [9 ]Department of Surgery, University of Virginia School of Medicine, Charlottesville, VA 22908
                [10 ]Department of Molecular, Cell, and Developmental Biology, University of California, Santa Cruz, CA 95064
                [11 ]School of Life Sciences, Tsinghua University, Beijing 100084, China
                [12 ]National Institute of Biological Sciences, Beijing 102206, China
                [13 ]School of Medicine, Ningbo University, Ningbo, 315010, China
                [14 ]Donders Institute for Brain, Cognition and Behavior, Radboud University Nijmegen, 6525 EN, Nijmegen, Netherlands
                [15 ]Department of Physiology, School of Basic Medicine, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China
                Author notes
                [17 ]Correspondence and requests for materials should be addressed to Julius Zhu ( jjzhu@ 123456virginia.edu ) and Yulong Li ( yulongli@ 123456pku.edu.cn ).
                Manuscript correspondence: Yulong Li ( yulongli@ 123456pku.edu.cn )
                Article
                NIHMS964311
                10.1038/nbt.4184
                6093211
                29985477
                96dd84cd-8089-4143-acdd-071453742504

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