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mGRASP enables mapping mammalian synaptic connectivity with light microscopy

Abstract

The GFP reconstitution across synaptic partners (GRASP) technique, based on functional complementation between two nonfluorescent GFP fragments, can be used to detect the location of synapses quickly, accurately and with high spatial resolution. The method has been previously applied in the nematode and the fruit fly but requires substantial modification for use in the mammalian brain. We developed mammalian GRASP (mGRASP) by optimizing transmembrane split-GFP carriers for mammalian synapses. Using in silico protein design, we engineered chimeric synaptic mGRASP fragments that were efficiently delivered to synaptic locations and reconstituted GFP fluorescence in vivo. Furthermore, by integrating molecular and cellular approaches with a computational strategy for the three-dimensional reconstruction of neurons, we applied mGRASP to both long-range circuits and local microcircuits in the mouse hippocampus and thalamocortical regions, analyzing synaptic distribution in single neurons and in dendritic compartments.

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Figure 1: Synaptic mGRASP components and gene-delivery strategy.
Figure 2: Synaptic expression of mGRASP components.
Figure 3: Reconstitution of mGRASP in hippocampal CA3-CA1 connectivity.
Figure 4: mGRASP detects actual synapses with high specificity.
Figure 5: Distribution of excitatory and inhibitory synapses revealed by mGRASP.

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Acknowledgements

We thank A. Losonczy for valuable discussions and preliminary physiological experiments, R. Sprengel for valuable discussions and help with the 2A-peptide, C. Bargmann (Rockefeller University) the ace-4-CD4spGFP1-10 and rig-3p-CD4spGFP11 expression constructs11, K. Swartz for simulation of molecular length, B.V. Zemelman (University of Texas at Austin) for the sst-Cre and GAD-Cre mouse lines and Y.-X. Wang for help with the immuno-silver-gold study. This work was supported by Howard Hughes Medical Institute, US National Institute on Deafness and other Communication Disorders intramural research program, as well as the World Class Institute Program of the National Research Foundation of Korea funded by the Ministry of Education, Science and Technology of Korea.

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Authors and Affiliations

Authors

Contributions

J.K. designed mGRASP components and performed molecular biology, animal surgery, imaging and data analysis. T.Z. and E.M. developed the image stitching and neuron tracing programs. Y.Y. and H.P. developed the mGRASP puncta detecting program. R.S.P. performed electron microscopy experiments. J.K. and J.C.M. wrote the manuscript.

Corresponding authors

Correspondence to Jinhyun Kim or Jeffrey C Magee.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–8, Supplementary Notes 1–3 (PDF 8543 kb)

Supplementary Video 1

Schematic illustration of mGRASP in the synapse and reconstitution of mGRASP in hippocampal CA3-CA1 connectivity. Confocal z-stack images show that discrete puncta of reconstituted mGRASP fluorescence are visible along dTomato-labeled CA1 basal dendrites in locations where blue CA3 axons and red CA1 dendrites intersect. (MOV 3524 kb)

Supplementary Video 2

High-magnification of reconstitution of mGRASP in hippocampal CA3-CA1 connectivity. Cropped confocal z-stack images show strong mGRASP fluorescence signals in the spine heads of CA1 dendrites. (MOV 5310 kb)

Supplementary Software

Programs for image stitching and 3D neuron tracing (neuTube) and for mGRASP puncta detection (puncta detector). (ZIP 49394 kb)

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Kim, J., Zhao, T., Petralia, R. et al. mGRASP enables mapping mammalian synaptic connectivity with light microscopy. Nat Methods 9, 96–102 (2012). https://doi.org/10.1038/nmeth.1784

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