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Using mammalian GFP reconstitution across synaptic partners (mGRASP) to map synaptic connectivity in the mouse brain
Linqing Feng1,3, Osung Kwon13, Bokyoung Lee1,2, Won Chan Oh1 & Jinhyun Kim1,2
1Center for Functional Connectomics, Korea Institute of Science and Technology (KIST), Seoul, Korea. 2Neuroscience program, University of Science and Technology, Daejeon, Korea. 3These authors contributed equally to this work. Correspondence should be addressed to J.K. ([email protected]).
Published online 18 September 2014; http://www.nature.com/doifinder/10.1038/nprot.2014.166
Web End =doi:10.1038/nprot.2014.166
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 56 weeks, including time for incubation and virus expression.
2014 Nature America, Inc. All rights reserved.
INTRODUCTION
Knowledge of synaptic connectivity in complex neuronal circuits is crucial for understanding brain functions. Mapping synaptic connectivity in the mammalian brain is a challenging task because the processes of densely packed neurons are thin (<1 m in diameter) yet long (sometimes >1 mm in length), and the structures of synapses are only nanometers in scale (~20 nm). Mapping these structures requires advanced neuronal labeling, imaging and reconstructing techniques that provide high resolution at multiple scales14. Serial reconstruction approaches by electron microscopy (EM) offer high resolution to find and characterize synapses; however, despite recent advances, these approaches remain practical only for very small volumes5,6. Recently, to circumvent the time- and labor-intensive EM-based approaches and the low resolution of light microscopy (LM), fluorescence-based approaches together with sophisticated genetic and optical methods have been developed, but their pitfalls, which include ambiguous results and cytotoxicity, remain problematic1,711.
mGRASP is a genetically controlled, molecular engineering method for detecting mammalian synapses at the level of LM12,13. It is based on two complementary nonfluorescent...