Whole-brain annotation and multi-connectome cell typing of Drosophila.

The fruit fly Drosophila melanogaster has 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). Of 8,453 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.

Automatic detection of synaptic partners in a whole-brain Drosophila electron microscopy data set.

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.

The connectome of an insect brain.

Brains contain networks of interconnected neurons and so knowing the network architecture is essential for understanding brain function. We therefore mapped the synaptic-resolution connectome of an entire insect brain (Drosophila larva) with rich behavior, including learning, value computation, and action selection, comprising 3016 neurons and 548,000 synapses. We characterized neuron types, hubs, feedforward and feedback pathways, as well as 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 state-of-the-art deep learning architectures. The identified brain architecture provides a basis for future experimental and theoretical studies of neural circuits.

Whole-brain annotation and multi-connectome cell typing quantifies circuit stereotypy in Drosophila.

The fruit fly Drosophila melanogaster 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 1 , 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 2 . Crucially, this resource includes 4,552 cell types. 3,094 are rigorous consensus validations of cell types previously proposed in the hemibrain connectome 3 . 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.

Desmosomal connectomics of all somatic muscles in an annelid larva.

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.

Structured sampling of olfactory input by the fly mushroom body.

Associative memory formation and recall in the fruit fly Drosophila melanogaster is subserved by the mushroom body (MB). Upon arrival in the MB, sensory information undergoes a profound transformation from broadly tuned and stereotyped odorant responses in the olfactory projection neuron (PN) layer to narrowly tuned and nonstereotyped responses in the Kenyon cells (KCs). Theory and experiment suggest 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 brain-spanning neurons. Here, we used a recent whole-brain electron microscopy volume of the adult fruit fly to map PN-to-KC connectivity at synaptic resolution. The PN-KC connectome revealed unexpected structure, with preponderantly food-responsive PN types converging at above-chance levels on downstream KCs. Axons of the overconvergent PN types tended to arborize near one another in the MB main calyx, making local KC dendrites more likely to receive input from those types. Overconvergent PN types preferentially co-arborize and connect with dendrites of αß and α'ß' KC subtypes. Computational simulation of the observed network showed degraded discrimination performance compared with a random network, except when all signal flowed through the overconvergent, primarily food-responsive PN types. Additional theory and experiment will be needed to fully characterize the impact of the observed non-random network structure on associative memory formation and recall.

Quantitative neuroanatomy for connectomics in Drosophila.

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.

Endogenously tagged rab proteins: a resource to study membrane trafficking in Drosophila.

Membrane trafficking is key to the cell biological mechanisms underlying development. Rab GTPases control specific membrane compartments, from core secretory and endocytic machinery to less-well-understood compartments. We tagged all 27 Drosophila Rabs with YFP(MYC) at their endogenous chromosomal loci, determined their expression and subcellular localization in six tissues comprising 23 cell types, and provide this data in an annotated, searchable image database. We demonstrate the utility of these lines for controlled knockdown and show that similar subcellular localization can predict redundant functions. We exploit this comprehensive resource to ask whether a common Rab compartment architecture underlies epithelial polarity. Strikingly, no single arrangement of Rabs characterizes the five epithelia we examine. Rather, epithelia flexibly polarize Rab distribution, producing membrane trafficking architectures that are tissue- and stage-specific. Thus, the core machinery responsible for epithelial polarization is unlikely to rely on polarized positioning of specific Rab compartments.