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Using mammalian GFP reconstitution across synaptic partners (mGRASP) to map synaptic connectivity in the mouse brain

Nature Protocols volume 9, pages 24252437 (2014) | Download Citation

Abstract

Many types of questions in neuroscience require the detection and mapping of synapses in the complex mammalian brain. A tool, mammalian GFP reconstitution across synaptic partners (mGRASP), offers a relatively easy, quick and economical approach to this technically challenging task. Here we describe in step-by-step detail the protocols for virus production, gene delivery, brain specimen preparation, fluorescence imaging and image analysis, calibrated substantially and specifically to make mGRASP-assisted circuit mapping (mGRASPing) practical in the mouse brain. The protocol includes troubleshooting suggestions and solutions to common problems. The mGRASP method is suitable for mapping mammalian synaptic connectivity at multiple scales: microscale for synapse-by-synapse or neuron-by-neuron analysis, and mesoscale for revealing local and long-range circuits. The entire protocol takes 5–6 weeks, including time for incubation and virus expression.

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Acknowledgements

We thank M. Son for technical support. This work was supported by the KIST Institutional Program (project no. 2E24210) and by the World Class Institute (WCI) Program of the Nation Research Foundation (NRF) of Korea (NRF grant no. WCI 2009-003).

Author information

Author notes

    • Linqing Feng
    •  & Osung Kwon

    These authors contributed equally to this work.

Affiliations

  1. Center for Functional Connectomics, Korea Institute of Science and Technology (KIST), Seoul, Korea.

    • Linqing Feng
    • , Osung Kwon
    • , Bokyoung Lee
    • , Won Chan Oh
    •  & Jinhyun Kim
  2. Neuroscience program, University of Science and Technology, Daejeon, Korea.

    • Osung Kwon
    • , Bokyoung Lee
    •  & Jinhyun Kim

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Contributions

O.K., B.L., W.C.O. and J.K. performed the experiments, and L.F. made MATLAB functions for analysis. L.F., O.K. and J.K. drew figures. B.L. and W.C.O. wrote the stereotaxic and specimen preparation sections. L.F. wrote the synapse mapping section. J.K. wrote the paper.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Jinhyun Kim.

Supplementary information

PDF files

  1. 1.

    Supplementary Methods

    AAV production and titer determination.

Videos

  1. 1.

    Procedures for in utero electroporation.

  2. 2.

    Procedures for in vivo stereotaxic injection.

Zip files

  1. 1.

    Supplementary Data

    The zipped mGRASP detection package named 'puncta'.

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DOI

https://doi.org/10.1038/nprot.2014.166

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