Technical Report | Published:

Multiplexed peroxidase-based electron microscopy labeling enables simultaneous visualization of multiple cell types

Nature Neurosciencevolume 22pages828839 (2019) | Download Citation


Electron microscopy (EM) is a powerful tool for circuit mapping, but identifying specific cell types in EM datasets remains a major challenge. Here we describe a technique enabling simultaneous visualization of multiple genetically identified neuronal populations so that synaptic interactions between them can be unequivocally defined. We present 15 adeno-associated virus constructs and 6 mouse reporter lines for multiplexed EM labeling in the mammalian nervous system. These reporters feature dAPEX2, which exhibits dramatically improved signal compared with previously described ascorbate peroxidases. By targeting this enhanced peroxidase to different subcellular compartments, multiple orthogonal reporters can be simultaneously visualized and distinguished under EM using a protocol compatible with existing EM pipelines. Proof-of-principle double and triple EM labeling experiments demonstrated synaptic connections between primary afferents, descending cortical inputs, and inhibitory interneurons in the spinal cord dorsal horn. Our multiplexed peroxidase-based EM labeling system should therefore greatly facilitate analysis of connectivity in the nervous system.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.

Data Availability

Data supporting the findings of this study are available within the paper and its supplementary information files.

Additional information

Journal peer review information: Nature Neuroscience thanks Jennifer Bourne, Liqun Luo, and other anonymous reviewer(s) for their contribution to the peer review of this work.

Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


  1. 1.

    Daigle, T. L. et al. A suite of transgenic driver and reporter mouse lines with enhanced brain-cell-type targeting and functionality. Cell 174, 465–480.e22 (2018).

  2. 2.

    Zhu, F. et al. Architecture of the mouse brain synaptome. Neuron 99, 781–799.e10 (2018).

  3. 3.

    Feinberg, E. H. et al. GFP Reconstitution Across Synaptic Partners (GRASP) defines cell contacts and synapses in living nervous systems. Neuron 57, 353–363 (2008).

  4. 4.

    Martell, J. D. et al. A split horseradish peroxidase for the detection of intercellular protein-protein interactions and sensitive visualization of synapses. Nat. Biotechnol. 34, 774–780 (2016).

  5. 5.

    Dani, A., Huang, B., Bergan, J., Dulac, C. & Zhuang, X. Superresolution imaging of chemical synapses in the brain. Neuron 68, 843–856 (2010).

  6. 6.

    Karagiannis, E. D. & Boyden, E. S. Expansion microscopy: development and neuroscience applications. Curr. Opin. Neurobiol. 50, 56–63 (2018).

  7. 7.

    Morgan, J. L., Berger, D. R., Wetzel, A. W. & Lichtman, J. W. The fuzzy logic of network connectivity in mouse visual thalamus. Cell 165, 192–206 (2016).

  8. 8.

    Zheng, Z. et al. A complete electron microscopy volume of the brain of adult Drosophila melanogaster. Cell 174, 730–743.e22 (2018).

  9. 9.

    Hildebrand, D. G. C. et al. Whole-brain serial-section electron microscopy in larval zebrafish. Nature 545, 345–349 (2017).

  10. 10.

    Kasthuri, N. et al. Saturated reconstruction of a volume of neocortex. Cell 162, 648–661 (2015).

  11. 11.

    Atasoy, D. et al. A genetically specified connectomics approach applied to long-range feeding regulatory circuits. Nat. Neurosci. 17, 1830–1839 (2014).

  12. 12.

    Li, J., Wang, Y., Chiu, S. L. & Cline, H. T. Membrane targeted horseradish peroxidase as a marker for correlative fluorescence and electron microscopy studies. Front. Neural Circuits 4, 6 (2010).

  13. 13.

    Schikorski, T., Young, S. M. Jr. & Hu, Y. Horseradish peroxidase cDNA as a marker for electron microscopy in neurons. J. Neurosci. Methods 165, 210–215 (2007).

  14. 14.

    Joesch, M. et al. Reconstruction of genetically identified neurons imaged by serial-section electron microscopy. eLife 5, 5 (2016).

  15. 15.

    Leal-Ortiz, S. et al. Piccolo modulation of Synapsin1a dynamics regulates synaptic vesicle exocytosis. J. Cell Biol. 181, 831–846 (2008).

  16. 16.

    Shu, X. et al. A genetically encoded tag for correlated light and electron microscopy of intact cells, tissues, and organisms. PLoS Biol. 9, e1001041 (2011).

  17. 17.

    Adams, S. R. et al. Multicolor electron microscopy for simultaneous visualization of multiple molecular species. Cell Chem. Biol. 23, 1417–1427 (2016).

  18. 18.

    Drawitsch, F., Karimi, A., Boergens, K. M. & Helmstaedter, M. FluoEM, virtual labeling of axons in three-dimensional electron microscopy data for long-range connectomics. eLife 7, 7 (2018).

  19. 19.

    Fang, T. et al. Nanobody immunostaining for correlated light and electron microscopy with preservation of ultrastructure. Nat. Methods 15, 1029–1032 (2018).

  20. 20.

    Lin, T. Y. et al. Mapping chromatic pathways in the Drosophila visual system. J. Comp. Neurol. 524, 213–227 (2016).

  21. 21.

    Machida, A. et al. Intraperitoneal administration of AAV9-shRNA inhibits target gene expression in the dorsal root ganglia of neonatal mice. Mol. Pain 9, 36 (2013).

  22. 22.

    Rhee, H. W. et al. Proteomic mapping of mitochondria in living cells via spatially restricted enzymatic tagging. Science 339, 1328–1331 (2013).

  23. 23.

    Han, C. et al. Integrins regulate repulsion-mediated dendritic patterning of Drosophila sensory neurons by restricting dendrites in a 2D space. Neuron 73, 64–78 (2012).

  24. 24.

    Lam, S. S. et al. Directed evolution of APEX2 for electron microscopy and proximity labeling. Nat. Methods 12, 51–54 (2015).

  25. 25.

    Hung, V. et al. Spatially resolved proteomic mapping in living cells with the engineered peroxidase APEX2. Nat. Protoc. 11, 456–475 (2016).

  26. 26.

    Martell, J. D. et al. Engineered ascorbate peroxidase as a genetically encoded reporter for electron microscopy. Nat. Biotechnol. 30, 1143–1148 (2012).

  27. 27.

    Gerfen, C. R., Paletzki, R. & Heintz, N. GENSAT BAC cre-recombinase driver lines to study the functional organization of cerebral cortical and basal ganglia circuits. Neuron 80, 1368–1383 (2013).

  28. 28.

    Abraira, V. E. & Ginty, D. D. The sensory neurons of touch. Neuron 79, 618–639 (2013).

  29. 29.

    Sugiura, Y., Lee, C. L. & Perl, E. R. Central projections of identified, unmyelinated (C) afferent fibers innervating mammalian skin. Science 234, 358–361 (1986).

  30. 30.

    Alvarez, F. J., Kavookjian, A. M. & Light, A. R. Ultrastructural morphology, synaptic relationships, and CGRP immunoreactivity of physiologically identified C-fiber terminals in the monkey spinal cord. J. Comp. Neurol. 329, 472–490 (1993).

  31. 31.

    Abraira, V. E. et al. The cellular and synaptic architecture of the mechanosensory dorsal horn. Cell 168, 295–310.e219 (2017).

  32. 32.

    Rau, K. K. et al. Mrgprd enhances excitability in specific populations of cutaneous murine polymodal nociceptors. J. Neurosci. 29, 8612–8619 (2009).

  33. 33.

    Vrontou, S., Wong, A. M., Rau, K. K., Koerber, H. R. & Anderson, D. J. Genetic identification of C fibres that detect massage-like stroking of hairy skin in vivo. Nature 493, 669–673 (2013).

  34. 34.

    Ribeiro-da-Silva, A. & Coimbra, A. Two types of synaptic glomeruli and their distribution in laminae I-III of the rat spinal cord. J. Comp. Neurol. 209, 176–186 (1982).

  35. 35.

    Castle, M. J., Turunen, H. T., Vandenberghe, L. H. & Wolfe, J. H. Controlling AAV tropism in the nervous system with natural and engineered capsids. Methods Mol. Biol. 1382, 133–149 (2016).

  36. 36.

    Madisen, L. et al. Transgenic mice for intersectional targeting of neural sensors and effectors with high specificity and performance. Neuron 85, 942–958 (2015).

  37. 37.

    Hu, H., Gan, J. & Jonas, P. Interneurons. Fast-spiking, parvalbumin+ GABAergic interneurons: from cellular design to microcircuit function. Science 345, 1255263 (2014).

  38. 38.

    Vong, L. et al. Leptin action on GABAergic neurons prevents obesity and reduces inhibitory tone to POMC neurons. Neuron 71, 142–154 (2011).

  39. 39.

    Rudomin, P. & Schmidt, R. F. Presynaptic inhibition in the vertebrate spinal cord revisited. Exp. Brain Res. 129, 1–37 (1999).

  40. 40.

    Valtschanoff, J. G., Weinberg, R. J. & Rustioni, A. Amino acid immunoreactivity in corticospinal terminals. Exp. Brain Res. 93, 95–103 (1993).

  41. 41.

    Nassar, M. A. et al. Nociceptor-specific gene deletion reveals a major role for Nav1.7 (PN1) in acute and inflammatory pain. Proc. Natl Acad. Sci. USA 101, 12706–12711 (2004).

  42. 42.

    Shields, S. D. et al. Nav1.8 expression is not restricted to nociceptors in mouse peripheral nervous system. Pain 153, 2017–2030 (2012).

  43. 43.

    Ng, J. et al. Genetically targeted 3D visualisation of Drosophila neurons under electron microscopy and X-ray microscopy using miniSOG. Sci. Rep. 6, 38863 (2016).

  44. 44.

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

  45. 45.

    Lee, W. C. et al. Anatomy and function of an excitatory network in the visual cortex. Nature 532, 370–374 (2016).

  46. 46.

    Briggman, K. L., Helmstaedter, M. & Denk, W. Wiring specificity in the direction-selectivity circuit of the retina. Nature 471, 183–188 (2011).

  47. 47.

    Deverman, B. E. et al. Cre-dependent selection yields AAV variants for widespread gene transfer to the adult brain. Nat. Biotechnol. 34, 204–209 (2016).

  48. 48.

    Lakso, M. et al. Efficient in vivo manipulation of mouse genomic sequences at the zygote stage. Proc. Natl Acad. Sci. USA 93, 5860–5865 (1996).

  49. 49.

    Rodríguez, C. I. et al. High-efficiency deleter mice show that FLPe is an alternative to Cre-loxP. Nat. Genet. 25, 139–140 (2000).

  50. 50.

    Martell, J. D., Deerinck, T. J., Lam, S. S., Ellisman, M. H. & Ting, A. Y. Electron microscopy using the genetically encoded APEX2 tag in cultured mammalian cells. Nat. Protoc. 12, 1792–1816 (2017).

  51. 51.

    Hua, Y., Laserstein, P. & Helmstaedter, M. Large-volume en-bloc staining for electron microscopy-based connectomics. Nat. Commun. 6, 7923 (2015).

  52. 52.

    Cardona, A. et al. TrakEM2 software for neural circuit reconstruction. PLoS One 7, e38011 (2012).

  53. 53.

    Saalfeld, S., Cardona, A., Hartenstein, V. & Tomančák, P. As-rigid-as-possible mosaicking and serial section registration of large ssTEM datasets. Bioinformatics 26, i57–i63 (2010).

  54. 54.

    Saalfeld, S., Fetter, R., Cardona, A. & Tomancak, P. Elastic volume reconstruction from series of ultra-thin microscopy sections. Nat. Methods 9, 717–720 (2012).

Download references


We thank P. Kaeser, G. Fishell and members of the Ginty laboratory for discussions and comments on this manuscript, the Boston Children’s Hospital Viral Core Facility for sharing AAV production reagents, E. Raviola, C. Bolger and the Harvard Medical School Electron Microscopy Facility for EM assistance, and the Boston Children’s Hospital Mouse Gene Manipulation Core Facility for assistance in generation of mouse lines. We thank K. Deisseroth for providing the AAV expression vector and H. Zeng for providing the Ai65 targeting vector. This work was supported by NIH grants R35NS097344 (D.D.G.), RF1MH114047 (W.-C.A.L.), and U54HD090255 (Boston Children’s Hospital Mouse Gene Manipulation Core Facility, Intellectual and Developmental Disabilities Research Center), and the Edward R. and Anne G. Lefler Center for Neurodegenerative Disorders (D.L.P. and D.D.G.). Q.Z. is supported by the Stuart H.Q. & Victoria Quan fellowship at Harvard Medical School. D.D.G. is supported by the Howard Hughes Medical Institute.

Author information


  1. Department of Neurobiology, Harvard Medical School, Boston, MA, USA

    • Qiyu Zhang
    • , David L. Paul
    •  & David D. Ginty
  2. Howard Hughes Medical Institute, Harvard Medical School, Boston, MA, USA

    • Qiyu Zhang
    •  & David D. Ginty
  3. F.M. Kirby Neurobiology Center, Boston Children’s Hospital, Boston, MA, USA

    • Wei-Chung A. Lee


  1. Search for Qiyu Zhang in:

  2. Search for Wei-Chung A. Lee in:

  3. Search for David L. Paul in:

  4. Search for David D. Ginty in:


Q.Z., D.L.P. and D.D.G. conceived the study. Q.Z. and D.L.P. generated the AAV constructs and mouse lines. Q.Z. conducted the LM and EM experiments. W.-C.A.L. assisted with EM experiments and analysis. Q.Z. and D.L.P. quantified the EM volume. Q.Z., D.L.P. and D.D.G. wrote the paper, with input from W.-C.A.L.

Competing interests

The authors declare no competing interests.

Corresponding authors

Correspondence to David L. Paul or David D. Ginty.

Integrated supplementary information

  1. Supplementary Figure 1 AAV9 IP injection efficiently transduces DRG neurons.

    Confocal images showing the transduction efficiency of DRG neurons by neonatal AAV9 i.p. injections. AAV9-mCherry was used in this experiment. Note the efficient labeling but highly variable expression levels, with small-diameter neurons generally expressing the transgene at higher levels than large-diameter neurons. NeuN was used to label neurons. Solid arrowhead: example small-diameter neuron with high transgene expression level. Open arrowhead: example large-diameter neuron with low transgene expression level. n = 2 animals and experiments. Scale bar: 50 μm.

  2. Supplementary Figure 2 Insufficient staining using previously reported constructs.

    (a) LM image of HEK293T cells after transfection with HRP-TM. n = 3 experiments. (b) EM image showing plasma membrane labeling in a HEK293T cell transfected with HRP-TM (asterisk). n = 2 experiments. (c) LM image of the cortex after parenchymal injection of AAV1-HRP-TM. Staining could be observed with LM. n = 4 animals and experiments. (d) EM image of the cortex after parenchymal injection of AAV1-HRP-TM. Little if any discernible DAB staining was observed. n = 2 animals and experiments. Scale bars: a: 100 μm, b: 2 μm, c: 500 μm, d: 0.5 μm.

  3. Supplementary Figure 3 Comparison of peroxidase staining conditions.

    LM images of the spinal cord dorsal horn of the same animal after systemic transduction of AAV9-dAPEX2 stained with different conditions. 0.003% hydrogen peroxide gave the highest staining intensity regardless of the DAB concentration. Staining intensity observed under LM was positively correlated with DAB concentration. n = 2 animals and experiments. Scale bar: 200 μm.

  4. Supplementary Figure 4 Excessively high concentrations of DAB could cause staining artifacts.

    (a, b) EM images of the spinal cord dorsal horn of the same animal after systemic transduction of AAV9-Matrix-dAPEX2 stained with 0.003% hydrogen peroxide and 1 mg/mL DAB (a) or 0.3 mg/mL DAB (b). The ultrastructure of the IMS was better preserved with 0.3 mg/mL DAB staining. n = 2 animals and experiments. Scale bar: 0.2 μm.

  5. Supplementary Figure 5 Excessive osmication could cause staining artifacts.

    (a, b) EM images of the spinal cord dorsal horn of the same ThT2A-CreER animal transduced with AAV9-DIO-Matrix-dAPEX2 and treated with tamoxifen at P14–21 to label C-LTMRs prepared with the rOTO protocol (a) or the reduced osmium protocol (b). Strong spurious staining was seen in the IMS in the samples prepared with the rOTO protocol. This spurious staining was not seen in unlabeled mitochondria. Reduced osmium staining preserved the DAB staining while providing sufficient counterstaining contrast. n = 2 animals and experiments. (c) EM images showing a sample prepared with the rOTO protocol with double labeling of cortical layer 5 pyramidal neurons (ER) using Tg(Rbp4-Cre)KL100 and AAV1-DIO-ER-dAPEX2 (red asterisks), and fast-spiking GABAergic interneurons (mitochondrial matrix) using PvalbT2A-FlpO and AAV1-FDIO-Matrix-dAPEX2 (green asterisks), equivalent to the experiment in Fig. 3a. Arrowhead: symmetric perisomatic synapse made by fast-spiking interneurons onto layer 5 pyramidal neurons. Note while spurious staining was present in the IMS, the mitochondrial matrix staining could still be easily distinguished from the ER staining. n = 2 animals and experiments. Scale bars: a, b: 0.2 μm, c: 0.5 μm.

Supplementary information

  1. Supplementary Figures 1–5.

  2. Reporting Summary

  3. Supplementary Table 1

    List of all constructs tested

  4. Supplementary Video 1

    Image volume of the reconstruction. Full image volume of the reconstruction shown in Fig. 4a. A primary afferent (green), an axon of an inhibitory interneuron (light red), a dendrite of an inhibitory interneuron (dark red), an axoaxonic synapse between an inhibitory interneuron and the primary afferent (magenta), and an axodendritic synapse between the primary afferent and an inhibitory interneuron (blue) are shown. Scale bar: 1 μm.

  5. Supplementary Video 2

    3D reconstruction. 3D reconstruction shown in Fig. 4b. A primary afferent (green), an axon of an inhibitory interneuron (light red), a dendrite of an inhibitory interneuron (dark red), an axoaxonic synapse between an inhibitory interneuron and the primary afferent (magenta), an axodendritic synapse between the primary afferent and an inhibitory interneuron (blue), and labeled mitochondria (grey) are shown.

About this article

Publication history




Issue Date