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

Abstract

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.

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Fig. 1: dAPEX2 is a sensitive reporter for visualizing axons of long-projection neurons with EM.
Fig. 2: Peroxidase constructs targeted to different subcellular compartments for multiplexed EM labeling.
Fig. 3: Double and triple EM labeling using orthogonal peroxidase reporter constructs.
Fig. 4: Multiplexed peroxidase labeling in volume EM.
Fig. 5: Generation of recombinase-dependent mouse dAPEX2 reporter lines.
Fig. 6: Mouse dAPEX2 reporter lines exhibit robust EM staining.

Data Availability

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

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Acknowledgements

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

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Authors

Contributions

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.

Corresponding authors

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

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Competing interests

The authors declare no competing interests.

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.

Integrated supplementary information

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.

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.

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.

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.

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

Supplementary Figures 1–5.

Reporting Summary

Supplementary Table 1

List of all constructs tested

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.

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.

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Zhang, Q., Lee, WC.A., Paul, D.L. et al. Multiplexed peroxidase-based electron microscopy labeling enables simultaneous visualization of multiple cell types. Nat Neurosci 22, 828–839 (2019). https://doi.org/10.1038/s41593-019-0358-7

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