Article | Published:

mGRASP enables mapping mammalian synaptic connectivity with light microscopy

Nature Methods volume 9, pages 96102 (2012) | Download Citation

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.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Accessions

NCBI Reference Sequence

References

  1. 1.

    & Single-channel currents recorded from membrane of denervated frog muscle fibres. Nature 260, 799–802 (1976).

  2. 2.

    Large-scale recording of neuronal ensembles. Nat. Neurosci. 7, 446–451 (2004).

  3. 3.

    et al. Network anatomy and in vivo physiology of visual cortical neurons. Nature 471, 177–182 (2011).

  4. 4.

    Viewing the brain through the master hand of Ramon y Cajal. Nat. Rev. Neurosci. 4, 71–77 (2003).

  5. 5.

    et al. Ultrastructural analysis of hippocampal neuropil from the connectomics perspective. Neuron 67, 1009–1020 (2010).

  6. 6.

    & Serial block-face scanning electron microscopy to reconstruct three-dimensional tissue nanostructure. PLoS Biol. 2, e329 (2004).

  7. 7.

    , , & Serial section scanning electron microscopy of adult brain tissue using focused ion beam milling. J. Neurosci. 28, 2959–2964 (2008).

  8. 8.

    & Array tomography: a new tool for imaging the molecular architecture and ultrastructure of neural circuits. Neuron 55, 25–36 (2007).

  9. 9.

    et al. Transgenic strategies for combinatorial expression of fluorescent proteins in the nervous system. Nature 450, 56–62 (2007).

  10. 10.

    et al. Monosynaptic restriction of transsynaptic tracing from single, genetically targeted neurons. Neuron 53, 639–647 (2007).

  11. 11.

    et al. GFP reconstitution across synaptic partners (GRASP) defines cell contacts and synapses in living nervous systems. Neuron 57, 353–363 (2008).

  12. 12.

    & Motor control in a Drosophila taste circuit. Neuron 61, 373–384 (2009).

  13. 13.

    & The architecture of the active zone in the presynaptic nerve terminal. Physiology 19, 262–270 (2004).

  14. 14.

    et al. Polarized targeting of neurexins to synapses is regulated by their C-terminal sequences. J. Neurosci. 28, 12969–12981 (2008).

  15. 15.

    & Neuroligins and neurexins: linking cell adhesion, synapse formation and cognitive function. Trends Neurosci. 29, 21–29 (2006).

  16. 16.

    et al. Neurexins physically and functionally interact with GABA(A) receptors. Neuron 66, 403–416 (2010).

  17. 17.

    et al. Faithful expression of multiple proteins via 2A-peptide self-processing: a versatile and reliable method for manipulating brain circuits. J. Neurosci. 29, 8621–8629 (2009).

  18. 18.

    , , & Long-term, selective gene expression in developing and adult hippocampal pyramidal neurons using focal in utero electroporation. J. Neurosci. 27, 5007–5011 (2007).

  19. 19.

    et al. Codon-improved Cre recombinase (iCre) expression in the mouse. Genesis 32, 19–26 (2002).

  20. 20.

    & Cre recombinase-mediated gene deletion in layer 4 of murine sensory cortical areas. Genesis 46, 289–293 (2008).

  21. 21.

    , , & Local circuit interactions between oriens/alveus interneurons and CA1 pyramidal cells in hippocampal slices: electrophysiology and morphology. J. Neurosci. 7, 1979–1993 (1987).

  22. 22.

    & Neuronal diversity and temporal dynamics: the unity of hippocampal circuit operations. Science 321, 53–57 (2008).

  23. 23.

    , , & Total number and distribution of inhibitory and excitatory synapses on hippocampal CA1 pyramidal cells. Neuroscience 102, 527–540 (2001).

  24. 24.

    et al. Autism and abnormal development of brain connectivity. J. Neurosci. 24, 9228–9231 (2004).

  25. 25.

    , , & Channelrhodopsin-2-assisted circuit mapping of long-range callosal projections. Nat. Neurosci. 10, 663–668 (2007).

  26. 26.

    et al. High-speed mapping of synaptic connectivity using photostimulation in Channelrhodopsin-2 transgenic mice. Proc. Natl. Acad. Sci. USA 104, 8143–8148 (2007).

  27. 27.

    , & Wiring specificity in the direction-selectivity circuit of the retina. Nature 471, 183–188 (2011).

  28. 28.

    , , , & Single-synapse analysis of a diverse synapse population: proteomic imaging methods and markers. Neuron 68, 639–653 (2010).

  29. 29.

    , , & SignalP 4.0: discriminating signal peptides from transmembrane regions. Nat. Methods 8, 785–786 (2011).

  30. 30.

    , , , & Prediction of transmembrane alpha-helices in procaryotic membrane proteins: the Dense Alignment Surface method. Protein Eng. 10, 673–676 (1997).

  31. 31.

    et al. The Protein Data Bank. Nucleic Acids Res. 28, 235–242 (2000).

  32. 32.

    , & Production and characterization of adeno-associated viral vectors. Nat. Protoc. 1, 1412–1428 (2006).

  33. 33.

    & Cre recombinase-mediated inversion using lox66 and lox71: method to introduce conditional point mutations into the CREB-binding protein. Nucleic Acids Res. 30, e90 (2002).

  34. 34.

    , & Stereotaxic gene delivery in the rodent brain. Nat. Protoc. 1, 3166–3173 (2006).

  35. 35.

    et al. Organization of NMDA receptors at extrasynaptic locations. Neuroscience 167, 68–87 (2010).

  36. 36.

    et al. Automated reconstruction of neuronal morphology based on local geometrical and global structural models. Neuroinformatics 9, 247–261 (2011).

  37. 37.

    , , , & V3D enables real-time 3D visualization and quantitative analysis of large-scale biological image data sets. Nat. Biotechnol. 28, 348–353 (2010).

Download references

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.

Author information

Author notes

    • Jinhyun Kim
    •  & Ting Zhao

    These authors contributed equally to this work.

Affiliations

  1. Janelia Farm Research Campus, Howard Hughes Medical Institute, Ashburn, Virginia, USA.

    • Jinhyun Kim
    • , Ting Zhao
    • , Yang Yu
    • , Hanchuan Peng
    • , Eugene Myers
    •  & Jeffrey C Magee
  2. Center for Functional Connectomics, Korea Institute of Science and Technology, Seoul, Korea.

    • Jinhyun Kim
  3. Qiushi Academy for Advanced Studies, Zhejiang University, Hangzhou, China.

    • Ting Zhao
  4. National Institute on Deafness and Other Communication Disorders, National Institutes of Health, Bethesda, Maryland, USA.

    • Ronald S Petralia

Authors

  1. Search for Jinhyun Kim in:

  2. Search for Ting Zhao in:

  3. Search for Ronald S Petralia in:

  4. Search for Yang Yu in:

  5. Search for Hanchuan Peng in:

  6. Search for Eugene Myers in:

  7. Search for Jeffrey C Magee in:

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.

Competing interests

The authors declare no competing financial interests.

Corresponding authors

Correspondence to Jinhyun Kim or Jeffrey C Magee.

Supplementary information

PDF files

  1. 1.

    Supplementary Text and Figures

    Supplementary Figures 1–8, Supplementary Notes 1–3

Videos

  1. 1.

    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.

  2. 2.

    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.

Zip files

  1. 1.

    Supplementary Software

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

About this article

Publication history

Received

Accepted

Published

DOI

https://doi.org/10.1038/nmeth.1784

Further reading