A genetically encoded fluorescent sensor for rapid and specificin vivodetection of norepinephrine

Abstract

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...

High-throughput evolution of near-infrared serotonin nanosensors

“…nIRHT-labeled brain dorsomedial striatum slice after addition of 100 M 5-HT and subsequent washing by aCSF. References (50)(51)(52)(53)(54)(55)(56)(57)(58)(59)(60) View/request a protocol for this paper from Bio-protocol.…”

G‐protein‐coupled receptor‐based sensors for imaging neurochemicals with high sensitivity and specificity

“…Because GPCRs are membrane proteins with an extracellular ligand‐binding pocket, it is both essential and challenging to ensure that the GPCR‐based sensors are trafficked properly to the cell surface and has the correct membrane topology. Indeed, based on our own experience when screening new sensors, the majority of recombinant GPCR‐cpFP proteins have relatively poor trafficking to the plasma membrane and are therefore unable to sense extracellular neurochemicals (Jing et al ; Feng et al ). Second, post‐translational modifications such as phosphorylation and lipid modification can potentially change the sensor's properties, affecting its ability to accurately report extracellular neurochemicals.…”

“…To develop neurochemical sensors that have a high signal‐to‐noise ratio and are suitable for in vivo applications, we recently combined the conformation‐sensitive cpFP with GPCRs to generate the GRAB (GPCR Activation‐Based) series of sensors for detecting a variety of neurochemicals, including GACh for sensing acetylcholine, GRAB DA for sensing dopamine, and GRAB NE for sensing norepinephrine (Jing et al ; Sun et al ; Feng et al ). Using a similar strategy, Lin Tian's group has developed dLight for sensing dopamine (Patriarchi et al ).…”

“…Recently, G‐protein‐coupled receptors (GPCRs) were used as the scaffold for generating genetically encoded sensors because of their high ligand selectivity and physiologically relevant ligand affinity. To date, GPCR‐based sensors have been developed for measuring the in vivo dynamics of several neurochemicals; these sensors include the GPCR‐activation‐based acetylcholine sensor (GACh sensor) for detecting acetylcholine (Jing et al ), the GPCR‐activation‐based dopamine (DA) sensor (GRAB DA sensor) (Sun et al ) and dLight (Patriarchi et al ) sensors for detecting dopamine, and the GPCR‐activation‐based norepinephrine (NE) sensor (GRAB NE sensor) for detecting norepinephrine (Feng et al ). When combined with advanced optical imaging techniques, GPCR‐based sensors can provide scalable, long‐term recordings of specific neurochemicals in a variety of model organisms.…”

Advances in Engineering and Application of Optogenetic Indicators for Neuroscience

“…Similar design approaches enabled the development a pair of NE sensors with altered ligand affinity based on the α-adrenergic receptor (α2AR) [137]. Two NE sensors, named GRAB NE1m and GRAB NE1h , are characterized by micromolar and nanomolar apparent affinity for NE, respectively, and exhibit several fold increase in fluorescence upon NE binding when measured in cultured cells (Table 3).…”