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We develop an automatic method for synaptic partner identification in insect brains and use it to predict synaptic partners in a whole-brain electron microscopy dataset of the fruit fly. The predictions can be used to infer a connectivity graph with high accuracy, thus allowing fast identification of neural pathways. To facilitate circuit reconstruction using our results, we develop CIRCUITMAP, a user interface add-on for the circuit annotation tool CATMAID.A deep-learning-based approach enables automatic identification of synaptically connected neurons in electron microscopy datasets of the fly brain.

Neuronal circuit mapping using electron microscopy demands laborious proofreading or reconciliation of multiple independent reconstructions. Here, we describe new methods to apply quantitative arbor and network context to iteratively proofread and reconstruct circuits and create anatomically enriched wiring diagrams. We measured the morphological underpinnings of connectivity in new and existing reconstructions of Drosophila sensorimotor (larva) and visual (adult) systems. Synaptic inputs were preferentially located on numerous small, microtubule-free 'twigs' which branch off a single microtubule-containing 'backbone'. Omission of individual twigs accounted for 96% of errors. However, the synapses of highly connected neurons were distributed across multiple twigs. Thus, the robustness of a strong connection to detailed twig anatomy was associated with robustness to reconstruction error. By comparing iterative reconstruction to the consensus of multiple reconstructions, we show that our method overcomes the need for redundant effort through the discovery and application of relationships between cellular neuroanatomy and synaptic connectivity. The nervous system contains cells called neurons, which connect to each other to form circuits that send and process information. Each neuron receives and transmits signals to other neurons via very small junctions called synapses. Neurons are shaped a bit like trees, and most input synapses are located in the tiniest branches. Understanding the architecture of a neuron’s branches is important to understand the role that a particular neuron plays in processing information. Therefore, neuroscientists strive to reconstruct the architecture of these branches and how they connect to one another using imaging techniques. One imaging technique known as serial electron microscopy generates highly detailed images of neural circuits. However, reconstructing neural circuits from such images is notoriously time consuming and error prone. These errors could result in the reconstructed circuit being very different than the real-life circuit. For example, an error that leads to missing out a large branch could result in researchers failing to notice many important connections in the circuit. On the other hand, some errors may not matter much because the neurons share other synapses that are included in the reconstruction. To understand what effect errors have on the reconstructed circuits, neuroscientists need to have a more detailed understanding of the relationship between the shape of a neuron, its synaptic connections to other neurons, and where errors commonly occur. Here, Schneider-Mizell, Gerhard et al. study this relationship in detail and then devise a faster reconstruction method that uses the shape and other properties of neurons without sacrificing accuracy. The method includes a way to include data from the shape of neurons in the circuit wiring diagrams, revealing circuit patterns that would otherwise go unnoticed. The experiments use serial electron microscopy images of neurons from fruit flies and show that, from the tiniest larva to the adult fly, neurons form synapses with each other in a similar way. Most errors in the reconstruction only affect the tips of the smallest branches, which generally only host a single synapse. Such omissions do not have a big effect on the reconstructed circuit because strongly connected neurons make multiple synapses onto each other. Schneider-Mizell, Gerhard et al.'s approach will help researchers to reconstruct neural circuits and analyze them more effectively than was possible before. The algorithms and tools developed in this study are available in an open source software package so that they can be used by other researchers in the future.

Cells form networks in animal tissues through synaptic, chemical, and adhesive links. Invertebrate muscle cells often connect to other cells through desmosomes, adhesive junctions anchored by intermediate filaments. To study desmosomal networks, we skeletonised 853 muscle cells and their desmosomal partners in volume electron microscopy data covering an entire larva of the annelid Platynereis . Muscle cells adhere to each other, to epithelial, glial, ciliated, and bristle-producing cells and to the basal lamina, forming a desmosomal connectome of over 2000 cells. The aciculae – chitin rods that form an endoskeleton in the segmental appendages – are highly connected hubs in this network. This agrees with the many degrees of freedom of their movement, as revealed by video microscopy. Mapping motoneuron synapses to the desmosomal connectome allowed us to infer the extent of tissue influenced by motoneurons. Our work shows how cellular-level maps of synaptic and adherent force networks can elucidate body mechanics.

The fruit fly Drosophila melanogasterhas emerged as a key model organism in neuroscience, in large part due to the concentration of collaboratively generated molecular, genetic and digital resources available for it. Here we complement the approximately 140,000 neuron FlyWire whole-brain connectome1 with a systematic and hierarchical annotation of neuronal classes, cell types and developmental units (hemilineages). Of8,453 annotated cell types, 3,643 were previously proposed in the partial hemibrain connectome2, and 4,581 are new types, mostly from brain regions outside the hemibrain subvolume. Although nearly all hemibrain neurons could be matched morphologically in FlyWire, about one-third of cell types proposed for the hemibrain could not be reliably reidentified. We therefore propose a new definition of cell type as groups of cells that are each quantitatively more similar to cells in a different brain than to any other cell in the same brain, and we validate this definition through joint analysis of FlyWire and hemibrain connectomes. Further analysis defined simple heuristics for the reliability of connections between brains, revealed broad stereotypy and occasional variability in neuron count and connectivity, and provided evidence for functional homeostasis in the mushroom body through adjustments of the absolute amount of excitatory input while maintaining the excitation/inhibition ratio. Our work defines a consensus cell type atlas for the fly brain and provides both an intellectual framework and open-source toolchain for brain-scale comparative connectomics.

The fruit fly combines surprisingly sophisticated behaviour with a highly tractable nervous system. A large part of the fly's success as a model organism in modern neuroscience stems from the concentration of collaboratively generated molecular genetic and digital resources. As presented in our FlyWire companion paper , this now includes the first full brain connectome of an adult animal. Here we report the systematic and hierarchical annotation of this ~130,000-neuron connectome including neuronal classes, cell types and developmental units (hemilineages). This enables any researcher to navigate this huge dataset and find systems and neurons of interest, linked to the literature through the Virtual Fly Brain database . Crucially, this resource includes 4,552 cell types. 3,094 are rigorous consensus validations of cell types previously proposed in the hemibrain connectome . In addition, we propose 1,458 new cell types, arising mostly from the fact that the FlyWire connectome spans the whole brain, whereas the hemibrain derives from a subvolume. Comparison of FlyWire and the hemibrain showed that cell type counts and strong connections were largely stable, but connection weights were surprisingly variable within and across animals. Further analysis defined simple heuristics for connectome interpretation: connections stronger than 10 unitary synapses or providing >1% of the input to a target cell are highly conserved. Some cell types showed increased variability across connectomes: the most common cell type in the mushroom body, required for learning and memory, is almost twice as numerous in FlyWire as the hemibrain. We find evidence for functional homeostasis through adjustments of the absolute amount of excitatory input while maintaining the excitation-inhibition ratio. Finally, and surprisingly, about one third of the cell types proposed in the hemibrain connectome could not yet be reliably identified in the FlyWire connectome. We therefore suggest that cell types should be defined to be robust to inter-individual variation, namely as groups of cells that are quantitatively more similar to cells in a different brain than to any other cell in the same brain. Joint analysis of the FlyWire and hemibrain connectomes demonstrates the viability and utility of this new definition. Our work defines a consensus cell type atlas for the fly brain and provides both an intellectual framework and open source toolchain for brain-scale comparative connectomics.

Cells form networks in animal tissues through synaptic, chemical and adhesive links. Invertebrate muscle cells often connect to other cells through desmosomes, adhesive junctions anchored by intermediate filaments. To study desmosomal networks, we skeletonised 853 muscle cells and their desmosomal partners in volume electron microscopy data covering an entire larva of the annelid Platynereis. Muscle cells adhere to each other, to epithelial, glial, ciliated, and bristle-producing cells and to the basal lamina, forming a desmosomal connectome of over 2,000 cells. The aciculae, chitin rods that form an endoskeleton in the segmental appendages, are highly connected hubs in this network. This agrees with the many degrees of freedom of their movement, as revealed by video microscopy. Mapping motoneuron synapses to the desmosomal connectome allowed us to infer the extent of tissue influenced by motoneurons. Our work shows how cellular-level maps of synaptic and adherent force networks can elucidate body mechanics. Competing Interest Statement The authors have declared no competing interest. Footnotes * Several figures and the text has been updated. The raw data and code to analyse the data is available on https://github.com/JekelyLab/Jasek_et_al * https://catmaid.jekelylab.ex.ac.uk/

Brains contain networks of interconnected neurons, so knowing the network architecture is essential for understanding brain function. We therefore mapped the synaptic-resolution connectome of an insect brain (Drosophila larva) with rich behavior, including learning, value-computation, and action-selection, comprising 3,013 neurons and 544,000 synapses. We characterized neuron-types, hubs, feedforward and feedback pathways, and cross-hemisphere and brain-nerve cord interactions. We found pervasive multisensory and interhemispheric integration, highly recurrent architecture, abundant feedback from descending neurons, and multiple novel circuit motifs. The brain's most recurrent circuits comprised the input and output neurons of the learning center. Some structural features, including multilayer shortcuts and nested recurrent loops, resembled powerful machine learning architectures. The identified brain architecture provides a basis for future experimental and theoretical studies of neural circuits.Competing Interest StatementThe authors have declared no competing interest.

Associative memory formation and recall in the adult fruit fly Drosophila melanogaster is subserved by the mushroom body (MB). Upon arrival in the MB, sensory information undergoes a profound transformation. Olfactory projection neurons (PNs), the main MB input, exhibit broadly tuned, sustained, and stereotyped responses to odorants; in contrast, their postsynaptic targets in the MB, the Kenyon cells (KCs), are nonstereotyped, narrowly tuned, and only briefly responsive to odorants. Theory and experiment have suggested that this transformation is implemented by random connectivity between KCs and PNs. However, this hypothesis has been challenging to test, given the difficulty of mapping synaptic connections between large numbers of neurons to achieve a unified view of neuronal network structure. Here we used a recent whole-brain electron microscopy (EM) volume of the adult fruit fly to map large numbers of PN-to-KC connections at synaptic resolution. Comparison of the observed connectome to precisely defined null models revealed unexpected network structure, in which a subset of food-responsive PN types converge on individual downstream KCs more frequently than expected. The connectivity bias is consistent with the neurogeometry: axons of the overconvergent PNs tend to arborize near one another in the MB main calyx, making local KC dendrites more likely to receive input from those types. Computational modeling of the observed PN-to-KC network showed that input from the overconvergent PN types is better discriminated than input from other types. These results suggest an 'associative fovea' for olfaction, in that the MB is wired to better discriminate more frequently occurring and ethologically relevant combinations of food-related odors. Competing Interest Statement The authors have declared no competing interest. Footnotes * corrected author affiliation & added orcid IDs

Large-scale neuronal circuit mapping using electron microscopy demands laborious proofreading by humans who resolve local ambiguities with larger contextual cues or by reconciling multiple indepen- dent reconstructions. We developed a new method that empowers expert neuroanatomists to apply quantitative arbor and network context to proofread and reconstruct neurons and circuits. We implemented our method in the web application CATMAID, supporting a group of collaborators to concurrently reconstruct neurons in the same circuit. We measured the neuroanatomical underpinnings of circuit connectivity in Drosophila neurons. We found that across life stages and cell types, synaptic inputs were preferentially located on spine-like microtubule-free branches, \"twigs\", while synaptic outputs were typically on microtubule-containing \"backbone\". The differential size and tortuosity of small twigs and rigid backbones was reflected in reconstruction errors, with nearly all errors being omission or truncation of twigs. The combination of redundant twig connectivity and low backbone error rates al- lows robust mapping of Drosophila circuits without time-consuming independent reconstructions. As a demonstration, we mapped a large sensorimotor circuit in the larva. We found anatomical pathways for proprioceptive feedback into motor circuits and applied novel methods of representing neuroanatomical compartments to describe their detailed structure. Our work suggests avenues for incorporating neuroanatomy into machine-learning approaches to connectomics and reveals the largely unknown circuitry of larval locomotion.