Key questions in the neuroscience field are to discover how the complex nervous system coordinate during behaviors, and whether abnormal in the neuronal communication are linked with diseases. Neurotransmitters are the bridging molecules that mediate the chemical communication between neurons. The first identified neurotransmitter, acetylcholine (ACh) has been widely accepted to be critically involved in the regulation of multiple physiological processes, ranging from development, sensation, motor control, cardiovascular function and higher brain cognitive function like learning and memory [1-3]. Due to the complex nature of the nervous system and the functional diversity of ACh, it will be important to further dissect when the ACh will be released during behaviors, where are they released, and how they are sensed by different cells.
On September 28th, 2020, in close collaboration with Dr. Yulong Li’s lab at Peking University, Miao JING Lab at CIBR published a research paper entitled An optimized acetylcholine sensor for monitoring in vivo cholinergic activity on Nature Methods, which reports the development of next-generation ACh sensor and its application in tracking cholinergic signals in various model organisms in vivo. This ACh sensor is engineered based on the G-Protein Coupled Receptor (GPCR) as the backbone, which could covert the ACh-induced conformational change of GPCR into a sensitive fluorescent signal through coupling with a circular permutated GFP. In previous work from the author, they have successfully engineered the first version of ACh sensor that could specifically report ACh dynamics . Based on their initial success, the author further optimized the performance of ACh sensors by site-directed random mutagenesis, and reached to the new version (ACh3.0) that achieved more than 3-fold improvement in the signal sensitivity. More importantly, through rational engineer, the author removed the downstream signal coupling of the new ACh sensor, making it as an isolated ACh detector without interfering with cellular physiology. New ACh sensor also maintains the fast response kinetics, physiological relevant affinity and precise molecular specificity in detecting ACh, suitable for probing the cholinergic signals in vivo.
Figure 1. Optimization and in vitro characterization of next-generation GRABACh sensors.
After the optimization and characterization of ACh3.0 in vitro, the author applied ACh3.0 in multiple model organisms and successfully detected the ACh release and dynamics in vivo. In Drosophila, the sensitive ACh3.0 sensor enabled the detection of compartment-specific releases of ACh in the olfactory mushroom body, when treating the living fly with various stimuli, suggesting that the cholinergic signals in different regions may have distinct physiological functions. In mice, the ACh3.0 sensor also made it possible to detect the ACh dynamics and its precise spatiotemporal distribution during behaviors. The improved ACh3.0 sensor has the sensitivity to track fast ACh transient evoked by brief foot-shock stimulation, and also have the stability to report the long-term ACh dynamics during the whole sleep-awake cycles, making it an ideal tool for exploring the ACh function in the future.
Figure 2. Successful application of the improved sensor in Drosophila and mice
Dr. Miao JING from CIBR serves as the first author, Dr. Miao Jing and Dr. Yulong Li are co-corresponding authors of this paper. Other collaborators include Min XU Lab from Shanghai Institutes for Biological Science, Chinese Academy of Science; Haohong LI Lab from Huazhong University of Science and Technology; Liangyi CHEN Lab and Heping CHENG Lab from Institute of Molecular Medicine at Peking University; Marco Prado, Lisa Saksida, Vania Prado and Tim Bussey Lab from Western University in Canada; and Andrew Hires Lab from University of Southern California in the US. This work is supported by State Key Laboratory of Membrane Biology at Peking University, National Natural Science Foundation of China, US BRAIN Initiative, and scientific research programs of CIBR.
1. Dale, H.H., Feldberg, W. & Vogt, M. Release of acetylcholine at voluntary motor nerve endings. The Journal of Physiology 86, 353-380 (1936).
2. Winkler, J., Suhr, S.T., Gage, F.H., Thal, L.J. & Fisher, L.J. Essential role of neocortical acetylcholine in spatial memory. Nature 375, 484-487 (1995).
3. Brezenoff, H.E. in Federation proceedings, Vol. 43 17 (1984).
4. Jing, M. et al. A genetically encoded fluorescent acetylcholine indicator for in vitro and in vivo studies. Nature biotechnology 36, 726-737 (2018).