When 3D electron microscopy and calcium imaging are used to investigate the structure and function of neural circuits, the resulting datasets pose new challenges of visualization and interpretation. Here, we present a new kind of digital resource that encompasses almost 400 ganglion cells from a single patch of mouse retina. An online "museum" provides a 3D interactive view of each cell's anatomy, as well as graphs of its visual responses. The resource reveals two aspects of the retina's inner plexiform layer: an arbor segregation principle governing structure along the light axis and a density conservation principle governing structure in the tangential plane. Structure is related to visual function; ganglion cells with arbors near the layer of ganglion cell somas are more sustained in their visual responses on average. Our methods are potentially applicable to dense maps of neuronal anatomy and physiology in other parts of the nervous system.

Due to advances in automated image acquisition and analysis, whole-brain connectomes with 100,000 or more neurons are on the horizon. Proofreading of whole-brain automated reconstructions will require many person-years of effort, due to the huge volumes of data involved. Here we present FlyWire, an online community for proofreading neural circuits in a Drosophila melanogaster brain, and explain how its computational and social structures are organized to scale up to whole-brain connectomics. Browser-based 3D interactive segmentation by collaborative editing of a spatially chunked supervoxel graph makes it possible to distribute proofreading to individuals located virtually anywhere in the world. Information in the edit history is programmatically accessible for a variety of uses such as estimating proofreading accuracy or building incentive systems. An open community accelerates proofreading by recruiting more participants and accelerates scientific discovery by requiring information sharing. We demonstrate how FlyWire enables circuit analysis by reconstructing and analysing the connectome of mechanosensory neurons.

The value of an integrated approach for understanding the neocortex by combining functional characterization of single neuron activity with the underlying circuit architecture has been understood since the dawn of modern neuroscience. However, in practice, anatomical connectivity and physiology have been studied mostly separately. Following in the footsteps of previous studies that have combined physiology and anatomy in the same tissue, here we present a unique functional connectomics dataset that contains calcium imaging of an estimated 75,000 neurons from primary visual cortex (VISp) and three higher visual areas (VISrl, VISal and VISlm), that were recorded while a mouse viewed natural movies and parametric stimuli. The functional data were co-registered with electron microscopy (EM) data of the same volume which were automatically segmented, reconstructing more than 200,000 cells (neuronal and non-neuronal) and 524 million synapses. Subsequent proofreading of some neurons in this volume yielded reconstructions that include complete dendritic trees as well the local and inter-areal axonal projections. The largest proofread excitatory axon reached a length of 19 mm and formed 1,893 synapses, while the largest inhibitory axon formed 10,081 synapses. Here we release this dataset as an open access resource to the scientific community including a set of analysis tools that allows easy data access, both programmatically and through a web user interface.

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 5x10^7 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.

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Most digital brain atlases have macroscopic resolution and are confined to a single imaging modality. Here we present a new kind of resource that combines dense maps of anatomy and physiology at cellular resolution. The resource encompasses 5 almost 400 ganglion cells from a single patch of mouse retina, and a digital "museum" provides a 3D interactive view of each cell's anatomy as well as graphs of its visual responses. To demonstrate the utility of the resource, we use it to divide the inner plexiform layer of the retina into four sublaminae defined 10 by a purely anatomical principle of arbor segregation. We also test the hypothesis that the aggregate neurite density of a ganglion cell type should be approximately uniform ("density conservation"). Finally, we find that ganglion cells arborizing in the inner marginal sublamina of the inner plexiform layer ex- 15hibit significantly more sustained visual responses on average.Calcium imaging followed by 3D electron microscopy (EM) has become established as a powerful approach for obtaining anatomical and physiological information about the same neurons. The approach has been used to study visual neurons in mouse cortex 20 [Bock et al., 2011, Lee et al., 2016 and retina [Briggman et al., 2011], and oculomotor neurons in larval zebrafish hindbrain [Vishwanathan et al., 2017]. These prior studies have been limited to tens of neurons sparsely sampled from a single individual. Here we present as a resource the anatomy and physiology of almost 25 400 ganglion cells (GCs) densely sampled from a single patch of mouse retina.To facilitate exploratory data analysis using the resource, we have constructed the Eyewire Museum (http://museum. eyewire.org), where every reconstructed ganglion cell can be in-30 teractively viewed along with its visual response properties. Due to its cellular and subcellular resolution, the Museum is novel relative to traditional brain atlases, which typically divide the brain into macroscopic regions [Lein et al., 2007, Amunts et al., 2013, Zingg et al., 2014. Because it fuses anatomy with physiology, 35 the Museum is also novel relative to traditional atlases of neuronal morphologies such as neuromorpho.org [Ascoli et al., 2007] and wormatlas.org [Hall et al., 2007]. Previous dense EM reconstructions in the mouse retina [Helmstaedter et al., 2013] and larval zebrafish olfactory bulb [Wanner et al., 2016] were limited to 40 anatomy only. A recent large-scale calcium imaging study contains the visual responses of more than 11,000 neurons from 50 retinas, but less than 1% of the cells had their dendritic arbors reconstructed [Baden et al., 2016].To illustrate the utility of our resource, we use it to reveal new 45 principles of retinal organization. First, we show how to optimally subdivide the inner plexiform layer (IPL) of the retina using the purely anatomical principle that arbors should segregate into distinct sublamina. For GC dendritic arbors, segregation is maximized by subdividing the IPL into two marginal sublamina flanking a...

Mammalian cortex features a large diversity of neuronal cell types, each with characteristic anatomical, molecular and functional properties. Synaptic connectivity rules powerfully shape how each cell type participates in the cortical circuit, but comprehensively mapping connectivity at the resolution of distinct cell types remains difficult. Here, we used millimeter-scale volumetric electron microscopy to investigate the connectivity of inhibitory neurons across a dense neuronal population spanning all layers of mouse visual cortex with synaptic resolution. We classified all 1183 excitatory neurons within a 100 micron column into anatomical subclasses using quantitative morphological and synapse features based on full dendritic reconstructions, finding both familiar subclasses corresponding to axonal projections and novel intralaminar distinctions based on synaptic properties. To relate these subclasses to single-cell connectivity, we reconstructed all 164 inhibitory interneurons in the same column, producing a wiring diagram of inhibition with more than 70,000 synapses. We found widespread cell-type-specific inhibition, including interneurons selectively targeting certain excitatory subpopulations among spatially intermingled neurons in layer 2/3, layer 5, and layer 6. Globally, inhibitory connectivity was organized into "motif groups," heterogeneous collections of cells that collectively target both perisomatic and dendritic compartments of the same combinations of excitatory subtypes. We also discovered a novel category of disinhibitory-specialist interneurons that preferentially targets basket cells. Collectively, our analysis revealed new organizing principles for cortical inhibition and will serve as a powerful foundation for linking modern multimodal neuronal atlases with the cortical wiring diagram.

Due to advances in automated image acquisition and analysis, new whole-brain connectomes beyond C. elegans are finally on the horizon. Proofreading of whole-brain automated reconstructions will require many person-years of effort, due to the huge volumes of data involved. Here we present FlyWire, an online community for proofreading neural circuits in a fly brain, and explain how its computational and social structures are organized to scale up to whole-brain connectomics. Browser-based 3D interactive segmentation by collaborative editing of a spatially chunked supervoxel graph makes it possible to distribute proofreading to individuals located virtually anywhere in the world. Information in the edit history is programmatically accessible for a variety of uses such as estimating proofreading accuracy or building incentive systems. An open community accelerates proofreading by recruiting more participants, and accelerates scientific discovery by requiring information sharing. We demonstrate how FlyWire enables circuit analysis by reconstructing and analysing the connectome of mechanosensory neurons.