WASPSYN: A Challenge for Domain Adaptive Synapse Detection in Microwasp Brain Connectomes.

The size of image volumes in connectomics studies now reaches terabyte and often petabyte scales with a great diversity of appearance due to different sample preparation procedures. However, manual annotation of neuronal structures (e.g., synapses) in these huge image volumes is time-consuming, leading to limited labeled training data often smaller than 0.001% of the large-scale image volumes in application. Methods that can utilize in-domain labeled data and generalize to out-of-domain unlabeled data are in urgent need. Although many domain adaptation approaches are proposed to address such issues in the natural image domain, few of them have been evaluated on connectomics data due to a lack of domain adaptation benchmarks. Therefore, to enable developments of domain adaptive synapse detection methods for large-scale connectomics applications, we annotated 14 image volumes from a biologically diverse set of Megaphragma viggianii brain regions originating from three different whole-brain datasets and organized the WASPSYN challenge at ISBI 2023. The annotations include coordinates of pre-synapses and post-synapses in the 3D space, together with their one-to-many connectivity information. This paper describes the dataset, the tasks, the proposed baseline, the evaluation method, and the results of the challenge. Limitations of the challenge and the impact on neuroscience research are also discussed. The challenge is and will continue to be available at https://codalab.lisn.upsaclay.fr/competitions/9169. Successful algorithms that emerge from our challenge may potentially revolutionize real-world connectomics research and further the cause that aims to unravel the complexity of brain structure and function.

Neuronal circuits integrating visual motion information in Drosophila melanogaster.

The detection of visual motion enables sophisticated animal navigation, and studies on flies have provided profound insights into the cellular and circuit bases of this neural computation. The fly's directionally selective T4 and T5 neurons encode ON and OFF motion, respectively. Their axons terminate in one of the four retinotopic layers in the lobula plate, where each layer encodes one of the four directions of motion. Although the input circuitry of the directionally selective neurons has been studied in detail, the synaptic connectivity of circuits integrating T4/T5 motion signals is largely unknown. Here, we report a 3D electron microscopy reconstruction, wherein we comprehensively identified T4/T5's synaptic partners in the lobula plate, revealing a diverse set of new cell types and attributing new connectivity patterns to the known cell types. Our reconstruction explains how the ON- and OFF-motion pathways converge. T4 and T5 cells that project to the same layer connect to common synaptic partners and comprise a core motif together with bilayer interneurons, detailing the circuit basis for computing motion opponency. We discovered pathways that likely encode new directions of motion by integrating vertical and horizontal motion signals from upstream T4/T5 neurons. Finally, we identify substantial projections into the lobula, extending the known motion pathways and suggesting that directionally selective signals shape feature detection there. The circuits we describe enrich the anatomical basis for experimental and computations analyses of motion vision and bring us closer to understanding complete sensory-motor pathways.

En bloc preparation of Drosophila brains enables high-throughput FIB-SEM connectomics.

Deriving the detailed synaptic connections of an entire nervous system is the unrealized goal of the nascent field of connectomics. For the fruit fly Drosophila, in particular, we need to dissect the brain, connectives, and ventral nerve cord as a single continuous unit, fix and stain it, and undertake automated segmentation of neuron membranes. To achieve this, we designed a protocol using progressive lowering of temperature dehydration (PLT), a technique routinely used to preserve cellular structure and antigenicity. We combined PLT with low temperature en bloc staining (LTS) and recover fixed neurons as round profiles with darkly stained synapses, suitable for machine segmentation and automatic synapse detection. Here we report three different PLT-LTS methods designed to meet the requirements for FIB-SEM imaging of the Drosophila brain. These requirements include: good preservation of ultrastructural detail, high level of en bloc staining, artifact-free microdissection, and smooth hot-knife cutting to reduce the brain to dimensions suited to FIB-SEM. In addition to PLT-LTS, we designed a jig to microdissect and pre-fix the fly's delicate brain and central nervous system. Collectively these methods optimize morphological preservation, allow us to image the brain usually at 8 nm per voxel, and simultaneously speed the formerly slow rate of FIB-SEM imaging.

A connectome and analysis of the adult Drosophila central brain.

The neural circuits responsible for animal behavior remain largely unknown. We summarize new methods and present the circuitry of a large fraction of the brain of the fruit fly Drosophila melanogaster. Improved methods include new procedures to prepare, image, align, segment, find synapses in, and proofread such large data sets. We define cell types, refine computational compartments, and provide an exhaustive atlas of cell examples and types, many of them novel. We provide detailed circuits consisting of neurons and their chemical synapses for most of the central brain. We make the data public and simplify access, reducing the effort needed to answer circuit questions, and provide procedures linking the neurons defined by our analysis with genetic reagents. Biologically, we examine distributions of connection strengths, neural motifs on different scales, electrical consequences of compartmentalization, and evidence that maximizing packing density is an important criterion in the evolution of the fly's brain.

Animal brains of all sizes, from the smallest to the largest, work in broadly similar ways. Studying the brain of any one animal in depth can thus reveal the general principles behind the workings of all brains. The fruit fly Drosophila is a popular choice for such research. With about 100,000 neurons ­ compared to some 86 billion in humans ­ the fly brain is small enough to study at the level of individual cells. But it nevertheless supports a range of complex behaviors, including navigation, courtship and learning. Thanks to decades of research, scientists now have a good understanding of which parts of the fruit fly brain support particular behaviors. But exactly how they do this is often unclear. This is because previous studies showing the connections between cells only covered small areas of the brain. This is like trying to understand a novel when all you can see is a few isolated paragraphs. To solve this problem, Scheffer, Xu, Januszewski, Lu, Takemura, Hayworth, Huang, Shinomiya et al. prepared the first complete map of the entire central region of the fruit fly brain. The central brain consists of approximately 25,000 neurons and around 20 million connections. To prepare the map ­ or connectome ­ the brain was cut into very thin 8nm slices and photographed with an electron microscope. A three-dimensional map of the neurons and connections in the brain was then reconstructed from these images using machine learning algorithms. Finally, Scheffer et al. used the new connectome to obtain further insights into the circuits that support specific fruit fly behaviors. The central brain connectome is freely available online for anyone to access. When used in combination with existing methods, the map will make it easier to understand how the fly brain works, and how and why it can fail to work correctly. Many of these findings will likely apply to larger brains, including our own. In the long run, studying the fly connectome may therefore lead to a better understanding of the human brain and its disorders. Performing a similar analysis on the brain of a small mammal, by scaling up the methods here, will be a likely next step along this path.

The Organization of the Second Optic Chiasm of the Drosophila Optic Lobe.

Visual pathways from the compound eye of an insect relay to four neuropils, successively the lamina, medulla, lobula, and lobula plate in the underlying optic lobe. Among these neuropils, the medulla, lobula, and lobula plate are interconnected by the complex second optic chiasm, through which the anteroposterior axis undergoes an inversion between the medulla and lobula. Given their complex structure, the projection patterns through the second optic chiasm have so far lacked critical analysis. By densely reconstructing axon trajectories using a volumetric scanning electron microscopy (SEM) technique, we reveal the three-dimensional structure of the second optic chiasm of Drosophila melanogaster, which comprises interleaving bundles and sheets of axons insulated from each other by glial sheaths. These axon bundles invert their horizontal sequence in passing between the medulla and lobula. Axons connecting the medulla and lobula plate are also bundled together with them but do not decussate the sequence of their horizontal positions. They interleave with sheets of projection neuron axons between the lobula and lobula plate, which also lack decussations. We estimate that approximately 19,500 cells per hemisphere, about two thirds of the optic lobe neurons, contribute to the second chiasm, most being Tm cells, with an estimated additional 2,780 T4 and T5 cells each. The chiasm mostly comprises axons and cell body fibers, but also a few synaptic elements. Based on our anatomical findings, we propose that a chiasmal structure between the neuropils is potentially advantageous for processing complex visual information in parallel. The EM reconstruction shows not only the structure of the chiasm in the adult brain, the previously unreported main topic of our study, but also suggest that the projection patterns of the neurons comprising the chiasm may be determined by the proliferation centers from which the neurons develop. Such a complex wiring pattern could, we suggest, only have arisen in several evolutionary steps.

Comparisons between the ON- and OFF-edge motion pathways in the Drosophila brain.

Understanding the circuit mechanisms behind motion detection is a long-standing question in visual neuroscience. In Drosophila melanogaster, recently discovered synapse-level connectomes in the optic lobe, particularly in ON-pathway (T4) receptive-field circuits, in concert with physiological studies, suggest a motion model that is increasingly intricate when compared with the ubiquitous Hassenstein-Reichardt model. By contrast, our knowledge of OFF-pathway (T5) has been incomplete. Here, we present a conclusive and comprehensive connectome that, for the first time, integrates detailed connectivity information for inputs to both the T4 and T5 pathways in a single EM dataset covering the entire optic lobe. With novel reconstruction methods using automated synapse prediction suited to such a large connectome, we successfully corroborate previous findings in the T4 pathway and comprehensively identify inputs and receptive fields for T5. Although the two pathways are probably evolutionarily linked and exhibit many similarities, we uncover interesting differences and interactions that may underlie their distinct functional properties.

Mapping chromatic pathways in the Drosophila visual system.

In Drosophila, color vision and wavelength-selective behaviors are mediated by the compound eye's narrow-spectrum photoreceptors R7 and R8 and their downstream medulla projection (Tm) neurons Tm5a, Tm5b, Tm5c, and Tm20 in the second optic neuropil or medulla. These chromatic Tm neurons project axons to a deeper optic neuropil, the lobula, which in insects has been implicated in processing and relaying color information to the central brain. The synaptic targets of the chromatic Tm neurons in the lobula are not known, however. Using a modified GFP reconstitution across synaptic partners (GRASP) method to probe connections between the chromatic Tm neurons and 28 known and novel types of lobula neurons, we identify anatomically the visual projection neurons LT11 and LC14 and the lobula intrinsic neurons Li3 and Li4 as synaptic targets of the chromatic Tm neurons. Single-cell GRASP analyses reveal that Li4 receives synaptic contacts from over 90% of all four types of chromatic Tm neurons, whereas LT11 is postsynaptic to the chromatic Tm neurons, with only modest selectivity and at a lower frequency and density. To visualize synaptic contacts at the ultrastructural level, we develop and apply a "two-tag" double-labeling method to label LT11's dendrites and the mitochondria in Tm5c's presynaptic terminals. Serial electron microscopic reconstruction confirms that LT11 receives direct contacts from Tm5c. This method would be generally applicable to map the connections of large complex neurons in Drosophila and other animals.

A common evolutionary origin for the ON- and OFF-edge motion detection pathways of the Drosophila visual system.

Synaptic circuits for identified behaviors in the Drosophila brain have typically been considered from either a developmental or functional perspective without reference to how the circuits might have been inherited from ancestral forms. For example, two candidate pathways for ON- and OFF-edge motion detection in the visual system act via circuits that use respectively either T4 or T5, two cell types of the fourth neuropil, or lobula plate (LOP), that exhibit narrow-field direction-selective responses and provide input to wide-field tangential neurons. T4 or T5 both have four subtypes that terminate one each in the four strata of the LOP. Representatives are reported in a wide range of Diptera, and both cell types exhibit various similarities in: (1) the morphology of their dendritic arbors; (2) their four morphological and functional subtypes; (3) their cholinergic profile in Drosophila; (4) their input from the pathways of L3 cells in the first neuropil, or lamina (LA), and by one of a pair of LA cells, L1 (to the T4 pathway) and L2 (to the T5 pathway); and (5) their innervation by a single, wide-field contralateral tangential neuron from the central brain. Progenitors of both also express the gene atonal early in their proliferation from the inner anlage of the developing optic lobe, being alone among many other cell type progeny to do so. Yet T4 receives input in the second neuropil, or medulla (ME), and T5 in the third neuropil or lobula (LO). Here we suggest that these two cell types were originally one, that their ancestral cell population duplicated and split to innervate separate ME and LO neuropils, and that a fiber crossing-the internal chiasma-arose between the two neuropils. The split most plausibly occurred, we suggest, with the formation of the LO as a new neuropil that formed when it separated from its ancestral neuropil to leave the ME, suggesting additionally that ME input neurons to T4 and T5 may also have had a common origin.

Parallel neural pathways in higher visual centers of the Drosophila brain that mediate wavelength-specific behavior.

Compared with connections between the retinae and primary visual centers, relatively less is known in both mammals and insects about the functional segregation of neural pathways connecting primary and higher centers of the visual processing cascade. Here, using the Drosophila visual system as a model, we demonstrate two levels of parallel computation in the pathways that connect primary visual centers of the optic lobe to computational circuits embedded within deeper centers in the central brain. We show that a seemingly simple achromatic behavior, namely phototaxis, is under the control of several independent pathways, each of which is responsible for navigation towards unique wavelengths. Silencing just one pathway is enough to disturb phototaxis towards one characteristic monochromatic source, whereas phototactic behavior towards white light is not affected. The response spectrum of each demonstrable pathway is different from that of individual photoreceptors, suggesting subtractive computations. A choice assay between two colors showed that these pathways are responsible for navigation towards, but not for the detection itself of, the monochromatic light. The present study provides novel insights about how visual information is separated and processed in parallel to achieve robust control of an innate behavior.

Candidate neural substrates for off-edge motion detection in Drosophila.

BACKGROUND: In the fly's visual motion pathways, two cell types-T4 and T5-are the first known relay neurons to signal small-field direction-selective motion responses [1]. These cells then feed into large tangential cells that signal wide-field motion. Recent studies have identified two types of columnar neurons in the second neuropil, or medulla, that relay input to T4 from L1, the ON-channel neuron in the first neuropil, or lamina, thus providing a candidate substrate for the elementary motion detector (EMD) [2]. Interneurons relaying the OFF channel from L1's partner, L2, to T5 are so far not known, however. RESULTS: Here we report that multiple types of transmedulla (Tm) neurons provide unexpectedly complex inputs to T5 at their terminals in the third neuropil, or lobula. From the L2 pathway, single-column input comes from Tm1 and Tm2 and multiple-column input from Tm4 cells. Additional input to T5 comes from Tm9, the medulla target of a third lamina interneuron, L3, providing a candidate substrate for L3's combinatorial action with L2 [3]. Most numerous, Tm2 and Tm9's input synapses are spatially segregated on T5's dendritic arbor, providing candidate anatomical substrates for the two arms of a T5 EMD circuit; Tm1 and Tm2 provide a second. Transcript profiling indicates that T5 expresses both nicotinic and muscarinic cholinoceptors, qualifying T5 to receive cholinergic inputs from Tm9 and Tm2, which both express choline acetyltransferase (ChAT). CONCLUSIONS: We hypothesize that T5 computes small-field motion signals by integrating multiple cholinergic Tm inputs using nicotinic and muscarinic cholinoceptors.

A systematic nomenclature for the insect brain.

Despite the importance of the insect nervous system for functional and developmental neuroscience, descriptions of insect brains have suffered from a lack of uniform nomenclature. Ambiguous definitions of brain regions and fiber bundles have contributed to the variation of names used to describe the same structure. The lack of clearly determined neuropil boundaries has made it difficult to document precise locations of neuronal projections for connectomics study. To address such issues, a consortium of neurobiologists studying arthropod brains, the Insect Brain Name Working Group, has established the present hierarchical nomenclature system, using the brain of Drosophila melanogaster as the reference framework, while taking the brains of other taxa into careful consideration for maximum consistency and expandability. The following summarizes the consortium's nomenclature system and highlights examples of existing ambiguities and remedies for them. This nomenclature is intended to serve as a standard of reference for the study of the brain of Drosophila and other insects.

Systematic analysis of neural projections reveals clonal composition of the Drosophila brain.

BACKGROUND: During development neurons are generated by sequential divisions of neural stem cells, or neuroblasts. In the insect brain progeny of certain stem cells form lineage-specific sets of projections that arborize in distinct brain regions, called clonal units. Though this raises the possibility that the entire neural network in the brain might be organized in a clone-dependent fashion, only a small portion of clones has been identified. RESULTS: Using Drosophila melanogaster, we randomly labeled one of about 100 stem cells at the beginning of the larval stage, analyzed the projection patterns of their progeny in the adult, and identified 96 clonal units in the central part of the fly brain, the cerebrum. Neurons of all the clones arborize in distinct regions of the brain, though many clones feature heterogeneous groups of neurons in terms of their projection patterns and neurotransmitters. Arborizations of clones overlap preferentially to form several groups of closely associated clones. Fascicles and commissures were all made by unique sets of clones. Whereas well-investigated brain regions such as the mushroom body and central complex consist of relatively small numbers of clones and are specifically connected with a limited number of neuropils, seemingly disorganized neuropils surrounding them are composed by a much larger number of clones and have extensive specific connections with many other neuropils. CONCLUSIONS: Our study showed that the insect brain is formed by a composition of cell-lineage-dependent modules. Clonal analysis reveals organized architecture even in those neuropils without obvious structural landmarks.

Flybrain neuron database: a comprehensive database system of the Drosophila brain neurons.

The long history of neuroscience has accumulated information about numerous types of neurons in the brain of various organisms. Because such neurons have been reported in diverse publications without controlled format, it is not easy to keep track of all the known neurons in a particular nervous system. To address this issue we constructed an online database called Flybrain Neuron Database (Flybrain NDB), which serves as a platform to collect and provide information about all the types of neurons published so far in the brain of Drosophila melanogaster. Projection patterns of the identified neurons in diverse areas of the brain were recorded in a unified format, with text-based descriptions as well as images and movies wherever possible. In some cases projection sites and the distribution of the post- and presynaptic sites were determined with greater detail than described in the original publication. Information about the labeling patterns of various antibodies and expression driver strains to visualize identified neurons are provided as a separate sub-database. We also implemented a novel visualization tool with which users can interactively examine three-dimensional reconstruction of the confocal serial section images with desired viewing angles and cross sections. Comprehensive collection and versatile search function of the anatomical information reported in diverse publications make it possible to analyze possible connectivity between different brain regions. We analyzed the preferential connectivity among optic lobe layers and the plausible olfactory sensory map in the lateral horn to show the usefulness of such a database.