Teravoxel volume electron microscopy data sets from neural tissue can now be acquired in weeks, but data analysis requires years of manual labor. We developed the SyConn framework, which uses deep convolutional neural networks and random forest classifiers to infer a richly annotated synaptic connectivity matrix from manual neurite skeleton reconstructions by automatically identifying mitochondria, synapses and their types, axons, dendrites, spines, myelin, somata and cell types. We tested our approach on serial block-face electron microscopy data sets from zebrafish, mouse and zebra finch, and computed the synaptic wiring of songbird basal ganglia. We found that, for example, basal-ganglia cell types with high firing rates in vivo had higher densities of mitochondria and vesicles and that synapse sizes and quantities scaled systematically, depending on the innervated postsynaptic cell types.
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We thank W. Denk for generous support and many helpful discussions. We thank M.S. Fee, K. Doroschak, S. Mikula, L. Heinrich and W. Denk for comments on the manuscript. We thank R. Illichmann, D. Dimitrov, O. Fedorashko, K. Kiesl and G. Patzer for ground truth annotations and many others for the skeleton annotations, and the KNOSSOS development team for continuous software support. We thank J. Buhmann for neurite segmentation training data, L. Heinrich for optimizing the watershed parameters, L. Schott for myelin training data, L. Burchartz for spine ground truth, C. Roome and M. Hilpert for IT support, and J. Kuhl for support with the figures. J.K. and F.S. were supported by the Boehringer Ingelheim Fonds.
The authors declare no competing financial interests.
Integrated supplementary information
Supplementary Figure 1 Failure modes of ray casting and watershed for an erroneous barrier prediction probability map
(a) Raw data and boundary prediction map (b) with a clear hole (arrow). (c) The watershed segmentation floods the adjacent process while (d) rays cast through the hole were removed during the filtering stage. Scale bar: 1 μm.
(a) Raw image data of mouse dataset. (b) Corresponding barrier prediction of a recursive 3D CNN, trained only on the bird dataset training data. (c) Corresponding barrier prediction after (d) an iterative post-training procedure with focused generation of minimal additional ground truth. Scale bars: 1 μm.
(a) Crosshair indicates probability map threshold and size filter parameters after connected-component analysis with maximum F1 score on training set. (b-d) Representative synapse examples of different sizes. Many found synaptic junction objects below the size filter threshold were false positives. Scale bars: 1 μm.
(a) Classification performance of an RFC spatially restricted to a local feature context, illustrating that neurites can be classified with good performance locally. (b) Left: MSN reconstruction with soma, dendrite and axon, identified by typical spiny dendritic morphology. Right: MSN axon fragment, identified by typical ultrastructural appearance of MSN synapses. (c) Separation of neurite reconstructions in principal component 1 vs. 2 space. Data points colored by ground truth annotations, which shows that cell types do not form easily separable clusters. Markers indicate MSN neurites from (b), that fall into different clusters. (d) and (e) components 1 vs. 3 and 2 vs 3. (f) Explained data variance by principal component.
(a)-(c) Two neurite reconstructions (red and blue shaded) with a tip contact that was missed during automatic contact site detection. Scale bar: 500 nm.
(a) Comparison of synapse sizes, labeled by 3 annotators vs SyConn, at the same synapse locations. Per synapse mean with s.d. of annotators on y-axis. (b) Comparison of synapse size distributions for SyConn and the 3 annotators. (c) Correlation-matrix of synapse sizes, reported are pairwise Pearson’s r-values. Note that some annotators correlate particularly well (annotator 1 and 3), while there is overall variability in the annotation of synapse sizes on the level of individual synapses.
(a) Connectivity matrix of Area X dataset; rows describe outgoing connections, columns incoming connections. Each matrix entry (enlarged for better visualization) is colored by synaptic type and shaded according to the number of synapses between the two neurons. (b) Average number of synapses of all connections between cell type pairs divided by the total number of cell type pair matrix elements.
Supplementary Figures 1–7 and Supplementary Tables 1, 2 and 5 (PDF 1426 kb)
Parameters of the CNNs used in this study. (XLSX 57 kb)
Boundaries used for the volume annotations in this study. (XLSX 15 kb)
Source code and documentation. (ZIP 14646 kb)
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Dorkenwald, S., Schubert, P., Killinger, M. et al. Automated synaptic connectivity inference for volume electron microscopy. Nat Methods 14, 435–442 (2017). https://doi.org/10.1038/nmeth.4206
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