Discovery of how the complex nervous system coordinates during behaviors remains a central question in the field of neuroscience, as well as whether abnormalities in neuronal communication are linked with diseases. Neurotransmitters are bridging molecules that mediate the chemical communication between neurons. The first identified neurotransmitter, acetylcholine (ACh), is widely understood to be an essential component in the regulation of multiple physiological processes, ranging from development, to sensation, motor control, cardiovascular function, and higher brain cognitive functions such as learning and memory [1-3]. Due to the complex nature of the nervous system and the functional diversity of ACh, questions persist as to when ACh release is triggered during behaviors, where it is released, and how it is sensed by different cells, the resolution of which will further our understanding of nervous system function.
On September 28th, 2020, in close collaboration with Dr. Yulong Li’s lab at Peking University, the Miao Jing Lab at CIBR published a research paper entitled "An Optimized Acetylcholine Sensor for Monitoring In Vivo Cholinergic Activity" in Nature Methods, which reports the development of a next generation ACh sensor and its application in tracking cholinergic signals in various model organisms in vivo. This ACh sensor is engineered to use a G-Protein Coupled Receptor (GPCR) backbone that can convert the conformational change of GPCR induced by ACh binding into a sensitive fluorescent signal through coupling with a circular permutated GFP. In their previous work, an initial version of the ACh sensor was developed that could specifically report ACh dynamics . Based on their success, the team further optimized the ACh sensor performance using site-directed random mutagenesis, resulting in the most recent version (ACh3.0), which exhibited a greater than 3-fold increase in sensitivity. More importantly, through rational engineering, the downstream signal coupling was removed in the new ACh sensor, making it as an isolated ACh detector that does not interfere with cellular physiology. The latest ACh sensor retains the fast response kinetics, physiologically relevant affinity, and precise molecular specificity for ACh detection, making it highly suitable for probing cholinergic signals in vivo.
Figure 1. Optimization and in vitro characterization of next generation GRAB-ACh sensors
Following in vitro characterization and optimization of ACh3.0, Jing and co-authors applied ACh3.0 in multiple model organisms, demonstrating successful in vivo detection of ACh release and dynamics. In Drosophila, this extremely sensitive ACh3.0 sensor enabled the detection of compartment-specific releases of ACh in the olfactory mushroom body while exposing live flies to various stimuli, thus suggesting that Cholinergic signaling in different regions may have distinct physiological functions. In mice, the ACh3.0 sensor also made it possible to detect ACh dynamics and precise spatiotemporal distribution of ACh during behaviors. The improved ACh3.0 sensor is sufficiently sensitive to track the rapid and transient ACh release evoked by a brief foot-shock stimulation, while also having the stability necessary to report long-term ACh dynamics during full sleep-wake cycles, therefore making it an ideal tool for future and ongoing exploration of ACh function.
Figure 2. Successful application of the improved sensor in Drosophila and mice
Dr. Miao Jing, a research fellow at CIBR, serves as the first author as well as co-corresponding author with Dr. Yulong Li (principal investigator at Peking University). Other collaborators include the Min Xu Lab from the Shanghai Institute for Biological Science, Chinese Academy of Science; the Haohong Li Lab from Huazhong University of Science and Technology; the Liangyi Chen Lab and Heping Cheng Lab from the Institute of Molecular Medicine at Peking University; Marco Prado, Lisa Saksida, Vania Prado, and Tim Bussey from Western University in Canada; and Andrew Hires Lab from the University of Southern California in the USA. This work is supported by the State Key Laboratory of Membrane Biology at Peking University, National Natural Science Foundation of China, US BRAIN Initiative, and the scientific research programs of CIBR.
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