Finding the right type of cell.

A new method allows researchers to automatically assign cells into different cell types and tissues, a step which is critical for understanding complex organisms.

neuPrint: An open access tool for EM connectomics.

Due to advances in electron microscopy and deep learning, it is now practical to reconstruct a connectome, a description of neurons and the chemical synapses between them, for significant volumes of neural tissue. Smaller past reconstructions were primarily used by domain experts, could be handled by downloading data, and performance was not a serious problem. But new and much larger reconstructions upend these assumptions. These networks now contain tens of thousands of neurons and tens of millions of connections, with yet larger reconstructions pending, and are of interest to a large community of non-specialists. Allowing other scientists to make use of this data needs more than publication-it requires new tools that are publicly available, easy to use, and efficiently handle large data. We introduce neuPrint to address these data analysis challenges. Neuprint contains two major components-a web interface and programmer APIs. The web interface is designed to allow any scientist worldwide, using only a browser, to quickly ask and answer typical biological queries about a connectome. The neuPrint APIs allow more computer-savvy scientists to make more complex or higher volume queries. NeuPrint also provides features for assessing reconstruction quality. Internally, neuPrint organizes connectome data as a graph stored in a neo4j database. This gives high performance for typical queries, provides access though a public and well documented query language Cypher, and will extend well to future larger connectomics databases. Our experience is also an experiment in open science. We find a significant fraction of the readers of the article proceed to examine the data directly. In our case preprints worked exactly as intended, with data inquiries and PDF downloads starting immediately after pre-print publication, and little affected by formal publication later. From this we deduce that many readers are more interested in our data than in our analysis of our data, suggesting that data-only papers can be well appreciated and that public data release can speed up the propagation of scientific results by many months. We also find that providing, and keeping, the data available for online access imposes substantial additional costs to connectomics research.

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 is not enough - what is still needed to understand the brain of Drosophila?

Understanding the structure and operation of any nervous system has been a subject of research for well over a century. A near-term opportunity in this quest is to understand the brain of a model species, the fruit fly Drosophila melanogaster. This is an enticing target given its relatively small size (roughly 200,000 neurons), coupled with the behavioral richness that this brain supports, and the wide variety of techniques now available to study both brain and behavior. It is clear that within a few years we will possess a connectome for D. melanogaster: an electron-microscopy-level description of all neurons and their chemical synaptic connections. Given what we will soon have, what we already know and the research that is currently underway, what more do we need to know to enable us to understand the fly's brain? Here, we itemize the data we will need to obtain, collate and organize in order to build an integrated model of the brain of D. melanogaster.

The connectome of the adult Drosophila mushroom body provides insights into function.

Making inferences about the computations performed by neuronal circuits from synapse-level connectivity maps is an emerging opportunity in neuroscience. The mushroom body (MB) is well positioned for developing and testing such an approach due to its conserved neuronal architecture, recently completed dense connectome, and extensive prior experimental studies of its roles in learning, memory, and activity regulation. Here, we identify new components of the MB circuit in Drosophila, including extensive visual input and MB output neurons (MBONs) with direct connections to descending neurons. We find unexpected structure in sensory inputs, in the transfer of information about different sensory modalities to MBONs, and in the modulation of that transfer by dopaminergic neurons (DANs). We provide insights into the circuitry used to integrate MB outputs, connectivity between the MB and the central complex and inputs to DANs, including feedback from MBONs. Our results provide a foundation for further theoretical and experimental work.

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 Fly Brain Atlas.

The brain's synaptic networks endow an animal with powerfully adaptive biological behavior. Maps of such synaptic circuits densely reconstructed in those model brains that can be examined and manipulated by genetic means offer the best prospect for understanding the underlying biological bases of behavior. That prospect is now technologically feasible and a scientifically enabling possibility in neurobiology, much as genomics has been in molecular biology and genetics. In Drosophila, two major advances are in electron microscopic technology, using focused ion beam-scanning electron microscopy (FIB-SEM) milling to capture and align digital images, and in computer-aided reconstruction of neuron morphologies. The last decade has witnessed enormous progress in detailed knowledge of the actual synaptic circuits formed by real neurons. Advances in various brain regions that heralded identification of the motion-sensing circuits in the optic lobe are now extending to other brain regions, with the prospect of encompassing the fly's entire nervous system, both brain and ventral nerve cord.

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.

A resource for the Drosophila antennal lobe provided by the connectome of glomerulus VA1v.

Using FIB-SEM we report the entire synaptic connectome of glomerulus VA1v of the right antennal lobe in Drosophila melanogaster. Within the glomerulus we densely reconstructed all neurons, including hitherto elusive local interneurons. The fruitless-positive, sexually dimorphic VA1v included >11,140 presynaptic sites with ~38,050 postsynaptic dendrites. These connected input olfactory receptor neurons (ORNs, 51 ipsilateral, 56 contralateral), output projection neurons (18 PNs), and local interneurons (56 of >150 previously reported LNs). ORNs are predominantly presynaptic and PNs predominantly postsynaptic; newly reported LN circuits are largely an equal mixture and confer extensive synaptic reciprocity, except the newly reported LN2V with input from ORNs and outputs mostly to monoglomerular PNs, however. PNs were more numerous than previously reported from genetic screens, suggesting that the latter failed to reach saturation. We report a matrix of 192 bodies each having >50 connections; these form 88% of the glomerulus' pre/postsynaptic sites.

Fully-Automatic Synapse Prediction and Validation on a Large Data Set.

Extracting a connectome from an electron microscopy (EM) data set requires identification of neurons and determination of connections (synapses) between neurons. As manual extraction of this information is very time-consuming, there has been extensive research efforts to automatically segment the neurons to help guide and eventually replace manual tracing. Until recently, there has been comparatively little research on automatic detection of the actual synapses between neurons. This discrepancy can, in part, be attributed to several factors: obtaining neuronal shapes is a prerequisite for the first step in extracting a connectome, manual tracing is much more time-consuming than annotating synapses, and neuronal contact area can be used as a proxy for synapses in determining connections. However, recent research has demonstrated that contact area alone is not a sufficient predictor of a synaptic connection. Moreover, as segmentation improved, we observed that synapse annotation consumes a more significant fraction of overall reconstruction time (upwards of 50% of total effort). This ratio will only get worse as segmentation improves, gating the overall possible speed-up. Therefore, we address this problem by developing algorithms that automatically detect presynaptic neurons and their postsynaptic partners. In particular, presynaptic structures are detected using a U-Net convolutional neural network (CNN), and postsynaptic partners are detected using a multilayer perceptron (MLP) with features conditioned on the local segmentation. This work is novel because it requires minimal amount of training, leverages advances in image segmentation directly, and provides a complete solution for polyadic synapse detection. We further introduce novel metrics to evaluate our algorithm on connectomes of meaningful size. When applied to the output of our method on EM data from Drosphila, these metrics demonstrate that a completely automatic prediction can be used to effectively characterize most of the connectivity correctly.

Analysis Tools for Large Connectomes.

New reconstruction techniques are generating connectomes of unprecedented size. These must be analyzed to generate human comprehensible results. The analyses being used fall into three general categories. The first is interactive tools used during reconstruction, to help guide the effort, look for possible errors, identify potential cell classes, and answer other preliminary questions. The second type of analysis is support for formal documents such as papers and theses. Scientific norms here require that the data be archived and accessible, and the analysis reproducible. In contrast to some other "omic" fields such as genomics, where a few specific analyses dominate usage, connectomics is rapidly evolving and the analyses used are often specific to the connectome being analyzed. These analyses are typically performed in a variety of conventional programming language, such as Matlab, R, Python, or C++, and read the connectomic data either from a file or through database queries, neither of which are standardized. In the short term we see no alternative to the use of specific analyses, so the best that can be done is to publish the analysis code, and the interface by which it reads connectomic data. A similar situation exists for archiving connectome data. Each group independently makes their data available, but there is no standardized format and long-term accessibility is neither enforced nor funded. In the long term, as connectomics becomes more common, a natural evolution would be a central facility for storing and querying connectomic data, playing a role similar to the National Center for Biotechnology Information for genomes. The final form of analysis is the import of connectome data into downstream tools such as neural simulation or machine learning. In this process, there are two main problems that need to be addressed. First, the reconstructed circuits contain huge amounts of detail, which must be intelligently reduced to a form the downstream tools can use. Second, much of the data needed for these downstream operations must be obtained by other methods (such as genetic or optical) and must be merged with the extracted connectome.

A connectome of a learning and memory center in the adult Drosophila brain.

Understanding memory formation, storage and retrieval requires knowledge of the underlying neuronal circuits. In Drosophila, the mushroom body (MB) is the major site of associative learning. We reconstructed the morphologies and synaptic connections of all 983 neurons within the three functional units, or compartments, that compose the adult MB's α lobe, using a dataset of isotropic 8 nm voxels collected by focused ion-beam milling scanning electron microscopy. We found that Kenyon cells (KCs), whose sparse activity encodes sensory information, each make multiple en passant synapses to MB output neurons (MBONs) in each compartment. Some MBONs have inputs from all KCs, while others differentially sample sensory modalities. Only 6% of KC>MBON synapses receive a direct synapse from a dopaminergic neuron (DAN). We identified two unanticipated classes of synapses, KC>DAN and DAN>MBON. DAN activation produces a slow depolarization of the MBON in these DAN>MBON synapses and can weaken memory recall.

The comprehensive connectome of a neural substrate for 'ON' motion detection in Drosophila.

Analysing computations in neural circuits often uses simplified models because the actual neuronal implementation is not known. For example, a problem in vision, how the eye detects image motion, has long been analysed using Hassenstein-Reichardt (HR) detector or Barlow-Levick (BL) models. These both simulate motion detection well, but the exact neuronal circuits undertaking these tasks remain elusive. We reconstructed a comprehensive connectome of the circuits of Drosophila's motion-sensing T4 cells using a novel EM technique. We uncover complex T4 inputs and reveal that putative excitatory inputs cluster at T4's dendrite shafts, while inhibitory inputs localize to the bases. Consistent with our previous study, we reveal that Mi1 and Tm3 cells provide most synaptic contacts onto T4. We are, however, unable to reproduce the spatial offset between these cells reported previously. Our comprehensive connectome reveals complex circuits that include candidate anatomical substrates for both HR and BL types of motion detectors.

Synaptic circuits and their variations within different columns in the visual system of Drosophila.

We reconstructed the synaptic circuits of seven columns in the second neuropil or medulla behind the fly's compound eye. These neurons embody some of the most stereotyped circuits in one of the most miniaturized of animal brains. The reconstructions allow us, for the first time to our knowledge, to study variations between circuits in the medulla's neighboring columns. This variation in the number of synapses and the types of their synaptic partners has previously been little addressed because methods that visualize multiple circuits have not resolved detailed connections, and existing connectomic studies, which can see such connections, have not so far examined multiple reconstructions of the same circuit. Here, we address the omission by comparing the circuits common to all seven columns to assess variation in their connection strengths and the resultant rates of several different and distinct types of connection error. Error rates reveal that, overall, <1% of contacts are not part of a consensus circuit, and we classify those contacts that supplement (E+) or are missing from it (E-). Autapses, in which the same cell is both presynaptic and postsynaptic at the same synapse, are occasionally seen; two cells in particular, Dm9 and Mi1, form ≥ 20-fold more autapses than do other neurons. These results delimit the accuracy of developmental events that establish and normally maintain synaptic circuits with such precision, and thereby address the operation of such circuits. They also establish a precedent for error rates that will be required in the new science of connectomics.

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.

A genetically specified connectomics approach applied to long-range feeding regulatory circuits.

Synaptic connectivity and molecular composition provide a blueprint for information processing in neural circuits. Detailed structural analysis of neural circuits requires nanometer resolution, which can be obtained with serial-section electron microscopy. However, this technique remains challenging for reconstructing molecularly defined synapses. We used a genetically encoded synaptic marker for electron microscopy (GESEM) based on intra-vesicular generation of electron-dense labeling in axonal boutons. This approach allowed the identification of synapses from Cre recombinase-expressing or GAL4-expressing neurons in the mouse and fly with excellent preservation of ultrastructure. We applied this tool to visualize long-range connectivity of AGRP and POMC neurons in the mouse, two molecularly defined hypothalamic populations that are important for feeding behavior. Combining selective ultrastructural reconstruction of neuropil with functional and viral circuit mapping, we characterized some basic features of circuit organization for axon projections of these cell types. Our findings demonstrate that GESEM labeling enables long-range connectomics with molecularly defined cell types.

Toward large-scale connectome reconstructions.

Recent results have shown the possibility of both reconstructing connectomes of small but biologically interesting circuits and extracting from these connectomes insights into their function. However, these reconstructions were heroic proof-of-concept experiments, requiring person-months of effort per neuron reconstructed, and will not scale to larger circuits, much less the brains of entire animals. In this paper we examine what will be required to generate and use substantially larger connectomes, finding five areas that need increased attention: firstly, imaging better suited to automatic reconstruction, with excellent z-resolution; secondly, automatic detection, validation, and measurement of synapses; thirdly, reconstruction methods that keep and use uncertainty metrics for every object, from initial images, through segmentation, reconstruction, and connectome queries; fourthly, processes that are fully incremental, so that the connectome may be used before it is fully complete; and finally, better tools for analysis of connectomes, once they are obtained.

A visual motion detection circuit suggested by Drosophila connectomics.

Animal behaviour arises from computations in neuronal circuits, but our understanding of these computations has been frustrated by the lack of detailed synaptic connection maps, or connectomes. For example, despite intensive investigations over half a century, the neuronal implementation of local motion detection in the insect visual system remains elusive. Here we develop a semi-automated pipeline using electron microscopy to reconstruct a connectome, containing 379 neurons and 8,637 chemical synaptic contacts, within the Drosophila optic medulla. By matching reconstructed neurons to examples from light microscopy, we assigned neurons to cell types and assembled a connectome of the repeating module of the medulla. Within this module, we identified cell types constituting a motion detection circuit, and showed that the connections onto individual motion-sensitive neurons in this circuit were consistent with their direction selectivity. Our results identify cellular targets for future functional investigations, and demonstrate that connectomes can provide key insights into neuronal computations.

Minimizing manual image segmentation turn-around time for neuronal reconstruction by embracing uncertainty.

The ability to automatically segment an image into distinct regions is a critical aspect in many visual processing applications. Because inaccuracies often exist in automatic segmentation, manual segmentation is necessary in some application domains to correct mistakes, such as required in the reconstruction of neuronal processes from microscopic images. The goal of the automated segmentation tool is traditionally to produce the highest-quality segmentation, where quality is measured by the similarity to actual ground truth, so as to minimize the volume of manual correction necessary. Manual correction is generally orders-of-magnitude more time consuming than automated segmentation, often making handling large images intractable. Therefore, we propose a more relevant goal: minimizing the turn-around time of automated/manual segmentation while attaining a level of similarity with ground truth. It is not always necessary to inspect every aspect of an image to generate a useful segmentation. As such, we propose a strategy to guide manual segmentation to the most uncertain parts of segmentation. Our contributions include 1) a probabilistic measure that evaluates segmentation without ground truth and 2) a methodology that leverages these probabilistic measures to significantly reduce manual correction while maintaining segmentation quality.