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Studying synapses in human brain with array tomography and electron microscopy

Nature Protocols volume 8, pages 1366–1380 (2013)Cite this article

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Abstract

Postmortem studies of synapses in human brain are problematic because of the axial resolution limit of light microscopy and the difficulty in preserving and analyzing ultrastructure with electron microscopy (EM). Array tomography (AT) overcomes these problems by embedding autopsy tissue in resin and cutting ribbons of ultrathin serial sections. Ribbons are imaged with immunofluorescence, allowing high-throughput imaging of tens of thousands of synapses to assess synapse density and protein composition. The protocol takes ∼3 d per case, excluding image analysis, which is done at the end of the study. Parallel processing for transmission electron microscopy (TEM) using a protocol modified to preserve the structure in human samples allows complementary ultrastructural studies. Incorporation of AT and TEM into brain banking is a potent way of phenotyping synapses in well-characterized clinical cohorts in order to develop clinicopathological correlations at the synapse level. This will be important for research in neurodegenerative disease, developmental disease and psychiatric illness.

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Figure 1: Principles of array tomography.
Figure 2
Figure 3: Regions collected for neurodegenerative disease studies.
Figure 4: Small 1-cm3 samples from multiple brain regions are bisected, with one-half dropped into AT fixative.
Figure 5: Imaging array tomography ribbons.
Figure 6: Image analysis.
Figure 7: Examples of AT imaging.
Figure 8: Examples of EM imaging of healthy and degenerating human synapses.

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Acknowledgements

We thank the patients and their families for their generous donations. We also thank K. Micheva and S. Smith for training us in the AT technique; D. Selkoe (Harvard Medical School), V. Lee (University of Pennsylvania) and D. Walsh (Harvard Medical School) for antibodies; and K. Kuchibhotla and S. Raymond for MATLAB programming. Harvard Catalyst supplied biostatistical support. This work was supported by US National Institutes of Health (NIH) grants R00AG033670 (T.L.S.-J., K.R.K.), T32AG000277 (K.J.K., B.T.H.), P50AG05134 (B.T.H., A.S.-P., M.P.F.); Alzheimer's Research UK (A.M.P.), the Sylvia Aitken Charitable Trust (C.S., A.K.W., M.E.B., T.H.B., S.A., T.H.G.) and the Medical Research Council UK G110616 (C.S.).

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Author notes

  1. Katherine J Kopeikina

    Present address: Present address: Department of Physiology, Feinberg School of Medicine, Northwestern University, Chicago, Illinois, USA.,

Authors and Affiliations

  1. Massachusetts General Hospital and Harvard Medical School, MassGeneral Institute for Neurodegenerative Disease, Charlestown, Massachusetts, USA

    Kevin R Kay, Alberto Serrano-Pozo, Amy M Pooler, Robert Koffie, Katherine J Kopeikina, Matthew P Frosch, Bradley T Hyman & Tara L Spires-Jones

  2. University of Edinburgh, Centre for Clinical Brain Sciences, Edinburgh, UK

    Colin Smith

  3. University of Edinburgh, Euan MacDonald Centre for Motor Neurone Disease Research, Edinburgh, UK

    Ann K Wright, Mark E Bastin, Thomas H Bak, Sharon Abrahams & Thomas H Gillingwater

  4. Department of Neuroscience, King's College London, Institute of Psychiatry, London, UK

    Amy M Pooler

  5. C.S. Kubik Laboratory for Neuropathology, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts, USA

    Declan McGuone & Matthew P Frosch

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Contributions

K.R.K., T.L.S.-J., A.S.-P., A.M.P., R.K. and K.J.K. performed AT experiments. C.S. and M.P.F. consulted on neuropathology. K.R.K. C.S., D.M., A.S.-P., R.K. and T.L.S.-J. collected human brain tissue. A.K.W., M.E.B., T.H.B., S.A. and T.H.G. designed and performed electron microscopy experiments. K.R.K., A.S.-P., A.M.P., K.J.K., C.S., T.H.G., B.T.H. and T.L.S.-J. discussed the data and prepared the manuscript.

Corresponding author

Correspondence to Tara L Spires-Jones.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Figure 1

Example of stripping and re-probing array ribbons. (PDF 1719 kb)

Supplementary Table 1

Characteristics of human subjects used. (PDF 489 kb)

Supplementary Video 1

Demonstration of making AT blocks from fresh tissue. (MOV 10955 kb)

Supplementary Methods

Instructions for automated imaging of AT ribbons with AxioVision macros. (PDF 527 kb)

Supplementary Data 1

ImageJ macro examples for AT analysis. (PDF 512 kb)

Supplementary Data 2

MATLAB script for colocalization analysis of AT images. (PDF 489 kb)

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Kay, K., Smith, C., Wright, A. et al. Studying synapses in human brain with array tomography and electron microscopy. Nat Protoc 8, 1366–1380 (2013). https://doi.org/10.1038/nprot.2013.078

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