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Nat Protoc. 2014 Oct;9(10):2425-37. doi: 10.1038/nprot.2014.166. Epub 2014 Sep 18.

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

Author information

1
1] Center for Functional Connectomics, Korea Institute of Science and Technology (KIST), Seoul, Korea. [2].
2
1] Center for Functional Connectomics, Korea Institute of Science and Technology (KIST), Seoul, Korea. [2] Neuroscience program, University of Science and Technology, Daejeon, Korea. [3].
3
1] Center for Functional Connectomics, Korea Institute of Science and Technology (KIST), Seoul, Korea. [2] Neuroscience program, University of Science and Technology, Daejeon, Korea.
4
Center for Functional Connectomics, Korea Institute of Science and Technology (KIST), Seoul, Korea.

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.

PMID:
25232938
DOI:
10.1038/nprot.2014.166
[Indexed for MEDLINE]

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