A fast genetically encoded fluorescent sensor for faithful in vivo acetylcholine detection in mice, fish, worms and flies

Creators: Borden, Philip M.; Shivange, Amol V.; Lester, Henry A.; Fan, Chengcheng; Rees, Douglas C.

Abstract

Here we design and optimize a genetically encoded fluorescent indicator, iAChSnFR, for the ubiquitous neurotransmitter acetylcholine, based on a bacterial periplasmic binding protein. iAChSnFR shows large fluorescence changes, rapid rise and decay kinetics, and insensitivity to most cholinergic drugs. iAChSnFR revealed large transients in a variety of slice and in vivo preparations in mouse, fish, fly and worm. iAChSnFR will be useful for the study of acetylcholine in all organisms.

Additional Information

The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license. bioRxiv preprint first posted online Feb. 8, 2020. We would like to thank Deepika Walpita (Janelia) for rat neuronal culture, and Kim Ritola (Janelia) for making AAV virus. We thank Jens T. Kaiser at the Caltech Molecular Observatory and the beamline staff at SSRL 12-2 for their support during data collection. DD and HK thank Dr. Martin Fuhrmann (DZNE, Bonn, Germany) for help with virus injections. This work was supported by the Howard Hughes Medical Institute. JJZ is supported by NIH NS104670, PZ by an Alzheimer's Association research fellowship, CPF by NIH NS95809, PKZ by a 2019 International Science and Engineering Fair award, WSZ by a 2019 Harrison undergraduate research award, and GW by Epilepsy Foundation postdoctoral fellowship No. 310443. JJZ is the Radboud Professor and Sir Yue-Kong Pao Chair Professor. HAL and AVS are supported by NIH DA037161, NIH DA043829, California Tobacco-Related Disease Research Program 23XT-0007 and 27IP-0057, and NARSAD. JSD is supported by R01-GM095674. JFC is supported by NIH DA022340, and AF by a STAR-PREP fellowship. LL is supported by a National Natural Science Foundation of China grant NSFC81771284. We gratefully acknowledge the Gordon and Betty Moore Foundation and the Beckman Institute for their generous support of the Molecular Observatory at Caltech. Use of the Stanford Synchrotron Radiation Lightsource, SLAC National Accelerator Laboratory, is supported by the U.S. Department of Energy, Office of Science, Office of Basic Energy Sciences under Contract No. DE-AC02-76SF00515. The SSRL Structural Molecular Biology Program is supported by the DOE Office of Biological and Environmental Research, and by the NIH, NIGMS (P41GM103393). BSK is supported by NIH (NS111583, DA047444, MH104069). DD and HK are supported by DFG (SPP1757, SFB1089, DI853/3-5) and BONFOR to DD. WBG is supported by NIH R01 NS047325. ES is supported by JSPS KAKENHI #15KK0340. Author contributions: P.M.B., P.Z., H.A.L., J.J.Z., and L.L.L. led the project. P.M.B., A.V.S., J.S.M., H.A.L., and L.L.L. performed protein engineering. P.M.B., A.V.S., and J.S.M. performed biochemical characterization. P.M.B., J.S.M., C.F., D.C.R. acquired and refined crystal structures. P.M.B., A.A., and R.P. performed spectroscopy. P.M.B. and J.S.M. performed HEK293 and cultured neuron experiments. P.Z. and Y.Z. screened sensor variants in brain slice. P.Z., P.K.Z., Y.Z., W.S.Z., G.W., and B.X. acquired and analyzed nanoscopic imaging data. P.Z., Y.Z., L.G., G.-X.Z., K.G., and L.L. made Sindbis and lentivirus constructs and live virus. P.Z. and Y.Z. performed other slice characterization. H.K. and D.D. performed hippocampal stratum radiatum slice characterization. E.S. and B.S.K. performed hippocampal astrocyte slice characterization. Y.C., A.G.Y., and C.P.F. performed striatal slice characterization. J.S.D. acquired and analyzed the C. elegans data. C.D. and V.J. acquired and analyzed the Drosophila data. M.T. and M.K. acquired and analyzed the zebrafish data. O.N. and K.P. did SLAP imaging and experiments on the effects of anesthesia. J.C. and W.-B.G. did motor cortex experiments. A.F. and J.F.C. did hippocampal reward experiments. P.M.B., H.A.L., J.J.Z., and L.L.L. wrote the paper, with help from all authors. Code availability: All analysis code used in this study is available upon request. Data availability: All data from this study is available upon request. Accession codes: All constructs have been deposited at Addgene (#137950-137959). Sequences have been deposited in Genbank. PDB accession code for iAChSnFR0.4 is 6URU. PDB accession code for the binding protein-only component of iAChSnFR is 6V1R. AAV virus is available from Addgene. The authors declare no competing financial interests.

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