Neuronal wiring diagram of an adult brain

Abstract

Connections between neurons can be mapped by acquiring and analyzing electron microscopic (EM) brain images. In recent years, this approach has been applied to chunks of brains to reconstruct local connectivity maps that are highly informative, yet inadequate for understanding brain function more globally. Here, we present the first neuronal wiring diagram of a whole adult brain, containing 5×10⁷ chemical synapses between ~130,000 neurons reconstructed from a female Drosophila melanogaster. The resource also incorporates annotations of cell classes and types, nerves, hemilineages, and predictions of neurotransmitter identities. Data products are available by download, programmatic access, and interactive browsing and made interoperable with other fly data resources. We show how to derive a projectome, a map of projections between regions, from the connectome. We demonstrate the tracing of synaptic pathways and the analysis of information flow from inputs (sensory and ascending neurons) to outputs (motor, endocrine, and descending neurons), across both hemispheres, and between the central brain and the optic lobes. Tracing from a subset of photoreceptors all the way to descending motor pathways illustrates how structure can uncover putative circuit mechanisms underlying sensorimotor behaviors. The technologies and open ecosystem of the FlyWire Consortium set the stage for future large-scale connectome projects in other species.

Ext.-Figure 1-1.

Neuropils of the fly brain.

Ext. Figure 1-2.. Completeness and accuracy of FlyWire’s reconstruction.

(a) shows the result of our evaluation of proofread segments in the central brain. Experts attempted further proofreading of 826 neurons. We computed volumetric overlaps between the original and the final segment to calculate precision, recall, and F1 Scores. (b) Examples (top: before, bottom: after) of the changes made during further proofreading for a neuron scoring an F1-Score of 0.936. Arrows highlight locations that changed. (c,d) For each neuropil, we quantified what fraction of the synapses within it are pre- and postsynaptically attached to a proofread segment. (c) displays the distribution for presynaptic attachment and (d) the distribution for postsynaptic attachment. (e, f, g) Comparisons between FlyWire’s reconstruction and the hemibrain were made for overlapping neuropils. Dots represent neuropils and are colored according to Ext. Data Fig. 1-1. (e) Comparison of the number of automatically detected synapses. The axes are log-transformed. (f) Comparison of post-synaptic completion rates and (g) pre-synaptic completion rate. The axes are truncated.

Ext. Figure 1-3.

Completion rates by neuropil.

Ext. Figure 1-4.. Trachea and glia cells.

(a) Rendering of all trachea segments in the FlyWire dataset. (b) Rendering of some reconstructed glia cells in the FlyWire dataset. At the time of writing, only a subset of the glia cells, with bias towards the central brain, have been proofread and labeled. Scale bar: 100 μm; insets: 10 μm.

Extended Data Figure 3-1.. Measurements of neuron size.

Colored markers refer to neurons in Fig. 3b. (a) Neuron path lengths of intrinsic neurons, (b) afferent neurons, and (b) efferent neurons by super-class. (d) Volumes of intrinsic neurons, (e) afferent neurons, and (f) efferent neurons by super-class. (g) Comparisons of path lengths and number of incoming and outgoing synapses. (h) For intrinsic neurons, comparisons of the in- and out-degrees with the number of incoming and outgoing synapses. Every dot is a neuron. (i) Comparison of average connection strengths (synapses per connection) with the number of synapses. Every dot is a neuron. (j) In- and out-degree distributions by neurotransmitter type. (k) Neuron path lengths by neurotransmitter type.

Ext. Figure 4-1.. Neuropil-neuropil projection maps.

(a) Projection maps produced as in Fig. 4a limited to connections from cholinergic, (b) GABAergic, and (c) glutamatergic neurons. (d) The difference between the putative excitatory (acetylcholine) and the putative inhibitory (GABA, glutamate) projection maps.

Ext. Figure 4-2.. Neuropil-neuropil projections compared between hemispheres.

Each dot is a neuropil-neuropil projection in one hemisphere and the axes show the fractional weights as calculated in Fig. 4a,b. Red dots are comparisons between the same neuropils in different hemispheres (e.g. AMMC(L) -> VLP(L) vs AMMC(R) -> VLP(R). (a) Comparison of projections between neuropils in both hemispheres and between hemispheres. (b) Comparisons of projections with the center neuropils. (c) Comparisons of projections between ipsilateral and contralateral neuropil projections.

Ext. Figure 4-3.. Input side analysis.

We assigned postsynaptic locations to either the center region or the left or right hemisphere. (a-g) For each super-class, the fraction of synapses in the left vs right hemisphere is shown for those neurons receiving most of their neurons laterally (top plot). The lower plot shows the fraction of synapses in the center vs the lateral regions for all neurons. (h) Each neuron was assigned to the side where it received most of its inputs.

Ext. Figure 4-4.. Neurons on the midline with dendrites in both hemispheres.

(a) All symmetric neurons with a cell body on the midline (N=106). (b-e) examples of individual neurons. Scale bar: 100 μm, inset: 50 μm

Ext. Figure 4-5.

Renderings of neurons for each cross-hemisphere category (up to 3,000 neurons rendered per group). Scale bar: 100 μm

Extended Data Figure 6-1.. Percentile ranks for every modality.

(a) For each sensory modality we used the traversal distances to establish a neuron ranking. Each panel shows the distributions of neurons of each super-class within the sensory modality specific rankings. (b) Same as in (a) for the fast neurotransmitters. (c) Neurons in the UMAP plot are colored by the rank order in which they are reached from a given seed neuron set. Red neurons are reached earlier than blue neurons.

Extended Data Figure 6-2.. Rank-based UMAP projection and neuropils.

(a) Every neuron in the central brain was assigned to the neuropil where it received the most synapses. Every dot is then colored by the assigned neuropil (Ext. Data Fig. 1-1). (b) Same as in a but limited to the central complex neurons. Neurons in the central complex with an assigned neuropil other than the ones shown are colored black.

Extended Data Figure 7-1.. Ocellar circuit.

(a) Renderings of all neurons (excluding the photoreceptors) with arbors in the ocellar ganglion. “Information flow” from pre- and postsynapses is indicated by arrows along the arbors. (b) Overview of the three ocelli (left, medial, right) which are positioned on the top of the head. Photoreceptors from each ocellus project to a specific subregion of the ocellar ganglion which are separated by glia (marked with black lines on the EM). (c) Top view of the dendritic arbors within the ocellar ganglion of each DNp28 (brown) and OCG01 (blue: cholinergic, green: glutamatergic). The render on the lower shows all 12 OCG01s and 2 DNp28s. Each other render shows one neuron in color and all others in the background in gray for reference. (d) Comparison of number of synapses from OCG01 neurons and visual projection neurons onto descending neurons. (e) Connectivity matrix for connections between ocellar centrifugal neurons and ocellar projection neurons. (f) Inputs to ocellar centrifugal neurons by neuropil. Scale bars: 100 μm (a), 20 μm (c)

Figure 1.. A connectomic reconstruction of a whole fly brain.

(a) All neuron morphologies reconstructed with FlyWire. All neurons in the central brain and both optic lobes were segmented and proofread. Note: image and dataset are mirror inverted relative to the native fly brain. (b) An overview of many of the FlyWire resources which are being made available. FlyWire leverages existing resources for EM imagery by Zheng et al., synapse predictions by Buhmann et al.^, and neurotransmitter predictions by Eckstein et al.. Annotations of the FlyWire dataset such as hemilineages, nerves, and hierarchical classes are established in our companion paper by Schlegel et al. (c) FlyWire uses CAVE (in prep) for proofreading, data management, and analysis backend. The data can be accessed programmatically through the CAVEclient, navis and natverse, and through the browser in Codex, Catmaid Spaces and braincircuits.io. Static exports of the data are also available. (d) The Drosophila brain can be divided into spatially defined regions based on neuropils (Ext. Data Fig. 1-1). Neuropils for the lamina are not shown. (e) Synaptic boutons in the fly brain are often polyadic such that there are multiple postsynaptic partners per presynaptic bouton. Each link between a pre- and a postsynaptic location is a synapse. (f) Neuron tracts, trachea, neuropil, cell bodies can be readily identified from the EM data which was acquired by Zheng et al.. Scale bar: 10 μm

Figure 2.. Neuron categories.

(a) We grouped neurons in the fly brain by “flow”: intrinsic, afferent, efferent. Each flow class is further divided into “super-classes” based on location and function. Neuron annotations are described in more detail in our companion paper by . The first public release is missing ~8,000 retinula cells in the compound eyes and four eyelets in one hemisphere which are indicated by hatched bars. (b) Using these neuron annotations, we created an aggregated synapse graph between the super-classes in the fly brain. (c) Renderings of all neurons in each super-class. (d) There are eight nerves into each hemisphere in addition to the ocellar nerve and the cervical connective (CV). All neurons traversing the nerves have been reconstructed and accounted for. (e) Sensory neurons can be subdivided by the sensory modality they respond to. In FlyWire, almost all sensory neurons have been typed by modality. The counts for the medial ocelli were omitted and are shown in Fig. 7b. (f) Renderings of all non-visual sensory neurons. Scale bar: 100 μm

Figure 3.. Neuron and connection sizes.

(a) The synapse-rich (synapses in blue) neuropil is surrounded by a layer of nuclei (random colors) located at the outside of the brain as well as between the optic lobes (purple) and the central brain (blue). (b) An LPsP neuron can be divided into morphologically distinct regions. Synapses (purple and blue) are found on the neuronal twigs and only rarely on the backbone. (c) We selected seven diverse neurons as a reference for the following panels. (d) The morphology of a neuron can be reduced to a skeleton from which the path length can be measured. The histograms show the distribution of path length and volume (the sum of all internal voxels) for all neurons. The triangles on top of the distributions indicate the measurements of the neurons in (b). (e) Connections in the fly brain are usually multisynaptic as in this example of neurons connecting with 71 synapses. (f) The number of connections with a given number of synapses and a fitted truncated power law distribution. (g) In degree and out degree of intrinsic neurons in the fly brain are linearly correlated (R=0.76). (h) The number of synapses per neuron varies between neurons by over a magnitude and the number of incoming and outgoing synapses is linearly correlated (R=0.80). Only intrinsic neurons were included in this plot. Scale bars: 50 μm (b, c), 10 μm (b-insets)

Figure 4.. Neuropil projections and analysis of crossing neurons.

(a) Whole brain neuropil-neuropil connectivity matrix. The main matrix was generated from intrinsic neurons, and afferent and efferent neuron classes are shown on the side. Incoming synapses onto afferent neurons and outgoing synapses from efferent neurons were not considered for this matrix. See Ext. Data Fig. 4-1 for neurotransmitter specific matrices. (b) Cartoon describing the generation of the matrix in (a). Each neuron’s connectivity is mapped onto synaptic projections between different neuropils. (c) shows examples from the matrix with each render corresponding to one row or column in the matrix and (d) shows examples from the matrix with each render corresponding to one square in the matrix. (e) Most neurons have pre- and postsynaptic locations in less than four neuropils. (f) Renderings (subset of 3,000 each) and input and output fractions of neurons projecting to (N=11916) and from (N=7528) the SEZ. The SEZ is roughly composed of five neuropils (the AMMC has a left and right homologue). Average input and output fractions were computed by summing the row and column values of the SEZ neuropils in the super-class specific projection matrices. (g) Fraction of contralateral synapses for each central brain neuron. (h) Fraction of ipsilateral, bilateral, contralateral, neurons projecting to and from the center neuropils per super-class. Scale bars: 100 μm

Figure 5:. Optic lobes.

(a) Rendering of a subset of the neurons in the fly brain. A cut through the optic lobe is highlighted. (b) All 779 Mi1 neurons in the right optic lobe. (c) A single Mi1 neuron, (d) all neurons crossing through the column in c as defined by a cylinder in the medulla with 1 μm radius through it, and (e) all neurons sharing a connection with the single Mi1 neuron shown in (c) (≥ 5 synapses) - 3 large neurons (CT1, OA-AL2b2, Dm17) were excluded for the visualization. (f) The two LPi1-2 neurons in the right lobula plate (neuropil shown in background). Scale bars: 50 μm (b,c,d,e,f), 10 μm (b-inset)

Figure 6.. Information flowthrough the Drosophila central brain

(a) We applied the information flow model for connectomes by Schlegel et al. to the connectome of the central brain neurons. Neurons are traversed probabilistically according to the ratio of incoming synapses from neurons that are in the traversed set. The information flow calculations were seeded with the afferent classes of neurons (including the sensory categories). (b) We rounded the traversal distances to assign neurons to layers. For gustatory neurons, we show a subset of the neurons (up to 1,000) that are reached in each layer. (c) For each sensory modality we used the traversal distances to establish a neuron ranking. Each panel shows the distributions of neurons of each super-class within the sensory modality specific rankings (see Ext. Data Fig. 6-1a for the complete set). (d) We assign neurons to neurotransmitter types and show their distribution within the traversal rankings similar to (c). The arrows highlight the sequence of GABA - glutamate peaks found for almost all sensory modalities (see Ext. Data Fig. 6-1b for the complete set). (e) We UMAP projected the matrix of traversal distances to obtain a 2d representation of each neuron in the central brain. Neurons from the same class co-locate (see also Ext. Data Fig. 6-2) (f) Neurons in the UMAP plot are colored by the rank order in which they are reached from a given seed neuron set. Red neurons are reached earlier than blue neurons (see Ext. Data Fig. 6-1c for the complete set).

Figure 7.. Ocellar circuits and their integration with visual projection neurons.

(a) Overview of the three ocelli (left, medial, right) which are positioned on the top of the head. Photoreceptors from each ocellus project to a specific subregion of the ocellar ganglion which are separated by glia (marked with black lines on the EM). (b) Renderings of the axons of the photoreceptors and their counts, and (c) OCG01, OCG02 and DNp28 neurons with arbors. “Information flow” from pre- and postsynapses is indicated by arrows along the arbors. (d) Connectivity matrix of connections between photoreceptors and ocellar projection neurons, including two descending neurons (DNp28). (e) Comparison of number of glutamatergic and cholinergic synapses from ocellar projection neurons onto downstream neurons colored by super-class (R=.65, p<1e-21). (f) Summary of the observed connectivity between ocellar projection neurons, visual projection neurons and descending neurons. Scale bar: 100 μm