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Dense EM-based reconstruction of the interglomerular projectome in the zebrafish olfactory bulb

Nature Neuroscience volume 19, pages 816825 (2016) | Download Citation

Abstract

The dense reconstruction of neuronal circuits from volumetric electron microscopy (EM) data has the potential to uncover fundamental structure–function relationships in the brain. To address bottlenecks in the workflow of this emerging methodology, we developed a procedure for conductive sample embedding and a pipeline for neuron reconstruction. We reconstructed 98% of all neurons (>1,000) in the olfactory bulb of a zebrafish larva with high accuracy and annotated all synapses on subsets of neurons representing different types. The organization of the larval olfactory bulb showed marked differences from that of the adult but similarities to that of the insect antennal lobe. Interneurons comprised multiple types but granule cells were rare. Interglomerular projections of interneurons were complex and bidirectional. Projections were not random but biased toward glomerular groups receiving input from common types of sensory neurons. Hence, the interneuron network in the olfactory bulb exhibits a specific topological organization that is governed by glomerular identity.

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Acknowledgements

We thank C. Bleck, B. Anderson and B. Titze for inputs and comments on the manuscript, and we thank W. Ong, L. Ong and R. Ong for help with tracer training and management. This work was supported by the Novartis Research Foundation, the Human Frontiers Science Program (HFSP) and the Swiss National Science Foundation (SNF).

Author information

Affiliations

  1. Friedrich Miescher Institute for Biomedical Research, Basel, Switzerland.

    • Adrian A Wanner
    • , Christel Genoud
    • , Tafheem Masudi
    • , Léa Siksou
    •  & Rainer W Friedrich
  2. University of Basel, Basel, Switzerland.

    • Adrian A Wanner
    • , Tafheem Masudi
    •  & Rainer W Friedrich

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Contributions

A.A.W. participated in all tasks. He developed EE embedding, CORE, PyKNOSSOS and analysis procedures. He acquired images from the larval OB, founded ariadne-service, supervised tracers, reconstructed all neurons, annotated synapses, analyzed the data, and participated in writing the manuscript. C.G. participated in sample preparation, methods development, and image acquisition. T.M. participated in sample preparation and image acquisition of the stack from the adult OB. L.S. participated in sample preparation. R.W.F. participated in data analysis and wrote the manuscript.

Competing interests

Part of the results disclosed herein have been included in European patent application EP14736451 and US patent application US14897514. A.A.W. is the founder and owner of ariadne-service.

Corresponding author

Correspondence to Rainer W Friedrich.

Integrated supplementary information

Supplementary information

PDF files

  1. 1.

    Supplementary Text and Figures

    Supplementary Figures 1–10 and Supplementary Software Information

  2. 2.

    Supplementary Methods Checklist

Videos

  1. 1.

    SBEM stack of an EE-embedded sample (telencephalon of adult zebrafish).

    Pixel size 12 × 12 nm2; section thickness 25 nm; acquisition rate 1 MHz; EL = 1.5 keV; De = 8.7 e nm–2; high vacuum).

  2. 2.

    SBEM stack from the adult zebrafish OB (Fig. 1f).

    Each image consisted of 40 tiles, each with 4,096 × 4,096 pixels. EE embedding; pixel size 9 × 9 nm2; section thickness 25 nm; acquisition rate 2 MHz; EL = 1.5 keV; De = 15.9 – 17.3 e nm–2; high vacuum; lvSEM. Image resolution has been severely reduced and only a subset of sections is shown to minimize movie size.

  3. 3.

    Zoom into an image from large SBEM stack (Fig. 1f and Supplementary Movie 2).

    Image consisted of 40 tiles, each with 4,096 × 4,096 pixels. Enlarged region contains overlap between adjacent tiles. EE embedding; pixel size 9 × 9 nm2; section thickness 25 nm; acquisition rate 2 MHz; EL = 1.5 keV; De = 15.9 – 17.3 e nm–2; high vacuum; lvSEM. Image quality has been slightly reduced to reduce movie size.

  4. 4.

    Zoom into an SBEM stack from the larval zebrafish OB (Supplementary Fig. 4).

    EE embedding; pixel size 9.25 × 9.25 nm2; section thickness 25 nm; acquisition rate 200 kHz; EL = 2 keV; De = 17.5 e nm−2; high vacuum; vpSEM.

  5. 5.

    Subvolume of SBEM stack from the larval zebrafish OB (Supplementary Fig. 4).

    EE embedding; pixel size 9.25 × 9.25 nm2; section thickness 25 nm; acquisition rate 200 kHz; EL = 2 keV; De = 17.5 e nm−2; high vacuum; vpSEM. The four quadrants show the four viewports of PyKNOSSOS (red: xy; blue: yz; green: xz; yellow: arbitrary). Arbitrary viewport (yellow) was set to be dorsal up and ventral down, lateral to the right and medial to the left.

  6. 6.

    Distribution of somata in the OB and adjacent areas.

    Somata are color-coded as in Supplementary Figure 6 (blue, reconstructed OB neurons; pale blue spheres: putative OB neurons; red, reconstructed non-OB cells; pale red, putative non-OB cells). Gray, glomeruli. Scale bar, 10 μm.

  7. 7.

    1,022 neurons reconstructed in the OB of a zebrafish larva.

    Color encodes soma position along dorsoventral axis. Scale bar, 10 μm.

  8. 8.

    Large olfactory bulb cells (LOC).

    Shaded volumes are outlines of glomeruli and color-coded according to the relative innervation by the two LOCs (Fig. 5g). Scale bars, 10 μm.

  9. 9.

    Soma of a LOC.

    Scale bars (black edges), 2 μm.

  10. 10.

    Glomeruli of the larval OB.

    Shaded volumes show glomeruli, color-coded according to OSN-defined glomerular groups (Fig. 6a). Scale bars (red edges), 20 μm.

  11. 11.

    Associations between mitral cells and glomeruli.

    Glomeruli are represented by large shaded volumes. Superimposed are individual mitral cells with neurites innervating three glomeruli. Mitral cell somata are represented by small shaded volumes. Long processes are axons projecting out of the olfactory bulb. Dendrites of individual mitral cells are closely associated with single glomeruli. Scale bars, 10 μm.

  12. 12.

    All mitral cells and their associations with glomeruli.

    Mitral cells are color-coded according to the OSN-defined group of their parent glomerulus (color code as in Fig. 6a). The parent glomerulus is the glomerulus containing the majority of intraglomerular neurites. Note that somata of mitral cells are superficial and spatially clustered by OSN-defined groups. Scale bar, 10 μm.

  13. 13.

    Associations between interneurons and glomeruli.

    Glomeruli are represented by large shaded volumes. Superimposed are individual interneurons with neurites innervating three glomeruli. Somata are represented by small shaded volumes. Individual interneurons associated with the same glomerulus show different morphologies. Scale bars, 10 μm.

  14. 14.

    All INs and their associations with glomeruli.

    INs are color-coded according to the OSN-defined group of their parent glomerulus (color code as in Fig. 6a). The parent glomerulus is the glomerulus containing the majority of intraglomerular neurites. Note that most somata of INs are located in deep layers and that spatial clustering is less pronounced than for mitral cells. Scale bar, 10 μm.

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DOI

https://doi.org/10.1038/nn.4290

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