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Imaging neuromodulators with high spatiotemporal resolution using genetically encoded indicators

Patriarchi, Tommaso and Cho, Jounhong Ryan and Merten, Katharina and Marley, Aaron and Broussard, Gerard Joey and Liang, Ruqiang and Williams, John and Nimmerjahn, Axel and von Zastrow, Mark and Gradinaru, Viviana and Tian, Lin (2019) Imaging neuromodulators with high spatiotemporal resolution using genetically encoded indicators. Nature Protocols, 14 (12). pp. 3471-3505. ISSN 1754-2189.

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[img] Image (JPEG) (Supplementary Fig. 1: Design of GPCR-based sensors using the universal cpGFP module) - Supplemental Material
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[img] Image (JPEG) (Supplementary Fig. 2: Comparison of locomotion-related dopamine transients using dLight1.1 or dLight1.2 in behaving mice with two-photon microscopy) - Supplemental Material
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[img] Image (JPEG) (Supplementary Fig. 3: Comparison between dLight1.3b and GRAB-DA1m in neurons) - Supplemental Material
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[img] Image (JPEG) (Supplementary Fig. 4: Equipment setup for photometry recordings) - Supplemental Material
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[img] Image (JPEG) (Supplementary Fig. 5: Example raw photometry traces) - Supplemental Material
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[img] Image (JPEG) (Supplementary Fig. 6: Equipment setup of perfusion system for titrations on cells) - Supplemental Material
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Multiple aspects of neural activity, from neuronal firing to neuromodulator release and signaling, underlie brain function and ultimately shape animal behavior. The recently developed and constantly growing toolbox of genetically encoded sensors for neural activity, including calcium, voltage, neurotransmitter and neuromodulator sensors, allows precise measurement of these signaling events with high spatial and temporal resolution. Here, we describe the engineering, characterization and application of our recently developed dLight1, a suite of genetically encoded dopamine (DA) sensors based on human inert DA receptors. dLight1 offers high molecular specificity, requisite affinity and kinetics and great sensitivity for measuring DA release in vivo. The detailed workflow described in this protocol can be used to systematically characterize and validate dLight1 in increasingly intact biological systems, from cultured cells to acute brain slices to behaving mice. For tool developers, we focus on characterizing five distinct properties of dLight1: dynamic range, affinity, molecular specificity, kinetics and interaction with endogenous signaling; for end users, we provide comprehensive step-by-step instructions for how to leverage fiber photometry and two-photon imaging to measure dLight1 transients in vivo. The instructions provided in this protocol are designed to help laboratory personnel with a broad range of experience (at the graduate or post-graduate level) to develop and utilize novel neuromodulator sensors in vivo, by using dLight1 as a benchmark.

Item Type:Article
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URLURL TypeDescription ReadCube access ItemData
Patriarchi, Tommaso0000-0001-9351-3734
Cho, Jounhong Ryan0000-0001-9542-716X
Merten, Katharina0000-0002-0197-0186
Broussard, Gerard Joey0000-0003-1636-2615
Liang, Ruqiang0000-0002-3075-4554
Nimmerjahn, Axel0000-0002-0875-7855
von Zastrow, Mark0000-0003-1375-6926
Gradinaru, Viviana0000-0001-5868-348X
Tian, Lin0000-0001-7012-6926
Additional Information:© 2019 Springer Nature Limited. Received 11 March 2019; Accepted 20 August 2019; Published 15 November 2019. Data availability: All DNA plasmids and viruses mentioned in this protocol can be obtained from either the Tian laboratory at UC Davis or Addgene under a materials transfer agreement. All data present in this article are available from the authors upon request. Implemented and curated computer codes have been deposited in github ( This work was supported by NIH BRAIN Initiative grants U01NS090604, U01NS013522, DP2MH107056 and U01NS103571 (L.T.); grants DP2NS083038, R01NS085938 and P30CA014195 (A.N.); BRAIN Initiative grants U01NS013522 (J.T.W. and M.v.Z.), and NIH grant DP2NS087949 and NIH/NIA grant R01AG047664 (V.G.). K.M. is a DFG research fellow and recipient of a Catharina Foundation postdoctoral scholar award. V.G. is a Heritage Principal Investigator supported by the Heritage Medical Research Institute. Author Contributions: T.P. and L.T. wrote the manuscript with contributions from J.R.C. and V.G. (fiber photometry and optogenetics), G.J.B. (rAAV preparation and cloning), R.L. (structural modeling), A.M. and M.v.Z. (TIRF microscopy, FACS and cAMP measurements), K.M. and A.N. (in vivo two-photon imaging in behaving mice), and J.W. (ex vivo two-photon imaging). Competing interests: T.P. and L.T. are co-inventors on a patent application (WO/2018/098262A1) for the technology described in this paper. L.T. is the co-founder of Seven Biosciences.
Group:Heritage Medical Research Institute
Funding AgencyGrant Number
Catharina FoundationUNSPECIFIED
Heritage Medical Research InstituteUNSPECIFIED
Subject Keywords:Fluorescence imaging; Molecular neuroscience; Neurotransmitters; Sensors and probes; Synaptic vesicle exocytosis
Issue or Number:12
Record Number:CaltechAUTHORS:20190801-132221928
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Official Citation:Patriarchi, T., Cho, J.R., Merten, K. et al. Imaging neuromodulators with high spatiotemporal resolution using genetically encoded indicators. Nat Protoc 14, 3471–3505 (2019) doi:10.1038/s41596-019-0239-2
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:97593
Deposited By: George Porter
Deposited On:15 Nov 2019 17:40
Last Modified:26 Nov 2019 17:42

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