On March 25, 2019, a research paper entitled "A genetically encoded fluorescent sensor for rapid and specific in vivo detection of norepinephrine" was published online by "Neuron". This work was done by Yulong Li’s research group at the School of Life Sciences of Peking University, Peking University-Tsinghua Joint Center for Life Sciences, PKU-IDG/McGovern Institute for Brain Research, and Chinese Institute for Brain Research in Beijing. In this study, Yulong Li’s research group successfully, for the first time, developed a novel, genetically encoded norepinephrine fluorescent sensor, and applied it to detect the dynamics of endogenous norepinephrine in living zebrafish and mice. This sensor enables specifically discrimination of structurally similar norepinephrine and dopamine by up-to 1000-fold and will become a useful tool for studying norepinephrine-related neuronal circuits.
Norepinephrine (NE), as a key biogenic monoamine neurotransmitter, involves in a wide range of physiological processes in central nervous system and sympathetic nervous system, including modulation of sensory information, regulation of attention, control of sleep & awake, and learning & memory. Impaired NE secretion or signaling is linked to a series of mental illnesses and neurodegenerative diseases. It is therefore particularly critical to be able to detect the dynamics of NE with good spatiotemporal resolution in a complex neural circuit, in order to fully understand the regulation or its alteration of NE in physiological or pathophysiological conditions. Unfortunately, currently available techniques are limited in terms of their sensitivity, specificity, spatiotemporal resolution and non-invasiveness, hampering our ability to understand NE’s dynamics in vivo.
To overcome the above technical bottlenecks, in this study, Yulong Li’s research group developed and optimized a family of G protein-coupled Receptor Activation Based NE (GRABNE) sensors. By inserting an environmental-sensitive fluorescent protein (cpEGFP) at a specific location of a human adrenergic receptor, the conformational change induced by norepinephrine is converted to the fluorescent signal, and combined with existing imaging techniques, real-time monitoring of endogenous norepinephrine dynamics can be achieved. After optimization, they developed two versions of GRABNE sensors with EC50 values for NE of ~1 μM and ~100 nM (namely NE1m and NE1h), suitable for detecting synaptic or volume NE transmission with sensitivity, rapid kinetics and high specificity.
G protein-coupled Receptor Activation Based NE (GRABNE) sensors
Taking advantages of the genetically encoded sensors, GRABNE enables expression in cultured cells, brain slices, zebrafish and mice by means of transfection, virus injection and transgenic animals. By constructing the transgenic zebrafish line pan-neuronally expressing GRABNE1m, the time-locked specific NE release induced by looming stimulation in the midbrain can be successfully detected. Single-cell-resolution monitoring of NE release evoked by repeated visual stimuli could be achieved by sparsely labeled norepinephrine sensors. By virus-mediated expression, optogenetic activation of LC-NE neurons reliably induced increases in GRABNE1m fluorescence by fiber photometry recording in freely moving mice. During forced swimming and tail suspension tests, both of which were stressful, a significant increase in GRABNE1m fluorescence in hypothalamus of freely moving mice was observed, suggesting the modulation of NE dynamics during behaviors.
Yulong Li, a principle investigator at School of Life Sciences in Peking University, is the corresponding author of this paper. Jiesi Feng in his research group is the first author; Miao Jing, Huan Wang, Yajun Zhang, Ao Dong, Zhaofa Wu, and Hao Wu contributes to this study. Collaborators of this work include: Jiulin Du’s research group at the Shanghai Institute of Neuroscience of the Chinese Academy of Sciences; Dayu Lin’s research group at New York University; Guohong Cui’s research group at the National Institutes of Health; J. Julius Zhu’s research group at University of Virginia School of Medicine; S. Andrew Hires research group at the University of Southern California. This work was supported by the State Key Laboratory of Membrane Biology at Peking University School of Life Sciences, Peking University-Tsinghua Joint Center for Life Sciences, National Basic Research Program of China (973 Program), the General Program of National Natural Science Foundation of China, and the NIH BRAIN Initiative grant.