Reconstruction of neocortex: Organelles, compartments, cells, circuits, and activity.

We assembled a semi-automated reconstruction of L2/3 mouse primary visual cortex from ∼250 × 140 × 90 µm3 of electron microscopic images, including pyramidal and non-pyramidal neurons, astrocytes, microglia, oligodendrocytes and precursors, pericytes, vasculature, nuclei, mitochondria, and synapses. Visual responses of a subset of pyramidal cells are included. The data are publicly available, along with tools for programmatic and three-dimensional interactive access. Brief vignettes illustrate the breadth of potential applications relating structure to function in cortical circuits and neuronal cell biology. Mitochondria and synapse organization are characterized as a function of path length from the soma. Pyramidal connectivity motif frequencies are predicted accurately using a configuration model of random graphs. Pyramidal cells receiving more connections from nearby cells exhibit stronger and more reliable visual responses. Sample code shows data access and analysis.

FlyWire: online community for whole-brain connectomics.

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 three-dimensional 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 analyzing the connectome of mechanosensory neurons.

Oligodendrocyte precursor cells ingest axons in the mouse neocortex.

Neurons in the developing brain undergo extensive structural refinement as nascent circuits adopt their mature form. This physical transformation of neurons is facilitated by the engulfment and degradation of axonal branches and synapses by surrounding glial cells, including microglia and astrocytes. However, the small size of phagocytic organelles and the complex, highly ramified morphology of glia have made it difficult to define the contribution of these and other glial cell types to this crucial process. Here, we used large-scale, serial section transmission electron microscopy (TEM) with computational volume segmentation to reconstruct the complete 3D morphologies of distinct glial types in the mouse visual cortex, providing unprecedented resolution of their morphology and composition. Unexpectedly, we discovered that the fine processes of oligodendrocyte precursor cells (OPCs), a population of abundant, highly dynamic glial progenitors, frequently surrounded small branches of axons. Numerous phagosomes and phagolysosomes (PLs) containing fragments of axons and vesicular structures were present inside their processes, suggesting that OPCs engage in axon pruning. Single-nucleus RNA sequencing from the developing mouse cortex revealed that OPCs express key phagocytic genes at this stage, as well as neuronal transcripts, consistent with active axon engulfment. Although microglia are thought to be responsible for the majority of synaptic pruning and structural refinement, PLs were ten times more abundant in OPCs than in microglia at this stage, and these structures were markedly less abundant in newly generated oligodendrocytes, suggesting that OPCs contribute substantially to the refinement of neuronal circuits during cortical development.

Pushing photon-pair generation rate in microresonators by Q factor manipulation.

Photon pairs generated by employing spontaneous nonlinear effects in microresonators are critically essential for integrated optical quantum information technologies, such as quantum computation and quantum cryptography. Microresonators featuring high-quality (Q) factors can offer simple yet power-efficient means to generate photon pairs, thanks to the intracavity field enhancement. In microresonators, it is known that the photon-pair generation rate (PGR) is roughly proportional to the cubic power of the Q factor. However, the upper limit on PGR is also set by the Q factor: a higher Q factor brings a longer photon lifetime, which in turn leads to a lower repetition rate allowing for photon flow emitted from the microresonator, constrained by the Fourier-transform limit. Exceeding this limit will result in the overlap of photon wave packets in the time domain, thus degrading the quantum character of single-photon light beams. To push the limit of PGR in a single resonator, we propose a method by harnessing the resonance linewidth-manipulated microresonators to improve the maximum achievable photon repetition rate while keeping the power efficiency. The maximum achievable PGR and power efficiency are thus balanced by leveraging the combination of low and high-Q resonances.

Computed Tomography-Guided Microcoil Localization of Pulmonary Nodules: Effects of Multiple Punctures.

BACKGROUND: The aim of the study is to analyze the effect of multiple punctures in computed tomography (CT)-guided microcoil localization of pulmonary nodules with other risk factors for common complications. METHODS: Consecutive patients who underwent CT-guided microcoil localization and subsequent video-assisted thoracoscopic surgery (VATS) between January 2020 and February 2021 were enrolled. Nodules successfully located after only one puncture were defined as the single puncture group, and nodules requiring two or more punctures were defined as the multiple puncture group. Binary logistic regression analysis was performed to assess the relationship between the number of punctures and pneumothorax and intrapulmonary hemorrhage. RESULTS: A total of 121 patients were included. There were 98 (68.1%) pulmonary nodules in the single puncture group compared with 46 (31.9%) nodules in the multiple puncture group. The frequencies of pneumothorax and intrapulmonary hemorrhage were higher in the multiple puncture group than in the single puncture group (p = 0.019 and <0.001, respectively). Binary logistic regression demonstrated that independent risk factors for developing pneumothorax included lateral positioning of the patient (p < .001) and prone positioning (p = 0.014), as well as multiple punctures (p = 0.013). Independent risk factors for intrapulmonary hemorrhage included the distance between the distal end of the coil and the surface of the pleura (p = 0.033), multiple punctures (p = 0.003), and passage through the pulmonary vasculature (p < 0.001). CONCLUSION: Multiple punctures resulted in an increased incidence of pneumothorax and intrapulmonary hemorrhage compared with single puncture during CT-guided microcoil localization of pulmonary nodules and were independently associated with both pneumothorax and intrapulmonary hemorrhage.

Outcomes of CT-Guided Deeper Localization Technique for Superficial Pulmonary Nodules.

BACKGROUND: The possibility of coil dislocation in computed tomography (CT)-guided microcoil localization of superficial pulmonary nodules is relatively high. The aim of the study is to investigate the outcomes of deeper localization technique during CT-guided microcoil localization of superficial pulmonary nodules before video-assisted thoracoscopic surgery (VATS). METHODS: Fifty-seven identified superficial pulmonary nodules (nodule-pleural distance ≤ 1 cm on CT image) from 51 consecutive patients underwent CT-guided microcoil localization, and subsequent VATSs were included. The rate of technical success, complications, and excised lung volume were compared between deeper localization technique group and conventional localization technique group. RESULTS: The technical success rate of the localization procedure was 100% (25/25) in the deeper localization group and 81.3% (26/32) in the conventional localization group (p = 0.030). Excluding one case of lobectomy, the excised lung volume in the deeper localization group and the conventional localization group was 39.3 ± 23.5 and 37.2 ± 16.2 cm3, respectively (p = 0.684). The incidence of pneumothorax was similar between the deeper localization group and the conventional localization group (24.0 vs. 21.9%, respectively, p = 0.850). The incidence of intrapulmonary hemorrhage in the deeper localization group was higher (16.0%) than that in the conventional localization group (6.3%), but the difference was not statistically significant (p = 0.388). CONCLUSION: CT-guided microcoil localization of superficial pulmonary nodules prior to VATS using a deeper localization technique is feasible. Deeper localization technique reduced the occurrence of dislocation but did not increase excised lung volume.

MORC3 restricts human cytomegalovirus infection by suppressing the major immediate-early promoter activity.

During the long coevolution of human cytomegalovirus (HCMV) and humans, the host has formed a defense system of multiple layers to eradicate the invader, and the virus has developed various strategies to evade host surveillance programs. The intrinsic immunity primarily orchestrated by promyelocytic leukemia (PML) nuclear bodies (PML-NBs) represents the first line of defense against HCMV infection. Here, we demonstrate that microrchidia family CW-type zinc finger 3 (MORC3), a PML-NBs component, is a restriction factor targeting HCMV infection. We show that depletion of MORC3 through knockdown by RNA interference or knockout by CRISPR-Cas9 augmented immediate-early protein 1 (IE1) gene expression and subsequent viral replication, and overexpressing MORC3 inhibited HCMV replication by suppressing IE1 gene expression. To relief the restriction, HCMV induces transient reduction of MORC3 protein level via the ubiquitin-proteasome pathway during the immediate-early to early stage. However, MORC3 transcription is upregulated, and the protein level recovers in the late stages. Further analyses with temporal-controlled MORC3 expression and the major immediate-early promoter (MIEP)-based reporters show that MORC3 suppresses MIEP activity and consequent IE1 expression with the assistance of PML. Taken together, our data reveal that HCMV enforces temporary loss of MORC3 to evade its repression against the initiation of immediate-early gene expression.

Percutaneous transluminal forceps biopsy with an adjustable curved sheath for obstructive jaundice.

PURPOSE: To evaluate the feasibility and safety of percutaneous transluminal forceps biopsy (PTFB) with an adjustable curved sheath in patients with obstructive jaundice. MATERIAL AND METHODS: Forty-two patients who underwent PTFB with an adjustable curved sheath were analyzed retrospectively. Sensitivity, specificity, negative predictive value (NPV), positive predictive value (PPV) and accuracy were calculated for all populations and in different situations. Technical success and safety were evaluated. RESULTS: The technical success rate was 100%. Thirty-five of 42 cases were diagnosed malignant diseases, the sensitivity of PTFB with an adjustable curved sheath was 74.29% (26/35), the specificity was 100%, the positive predictive value was 100%, the negative predictive value was 43.75% (7/16), and the accuracy rate was 78.57% (33/42). There was a better sensitivity for bile duct malignancies when compared with non-bile duct malignancies (p = 0.012). No statistical difference was found in the sensitivity of the upper part of the biliary tree and the lower part of the biliary tree, and none in the sensitivity of different approaches (left vs. right). The complication rate was 11.90%, and no serious complications were observed. CONCLUSIONS: PTFB with an adjustable curved sheath is an effective and safe technique, without being limited by approaches and obstruction sites.

Direct 3D Analyses Reveal Barrel-Specific Vascular Distribution and Cross-Barrel Branching in the Mouse Barrel Cortex.

Whether vascular distribution is spatially specific among cortical columns is a fundamental yet controversial question. Here, we have obtained 1-µm resolution 3D datasets that cover the whole mouse barrel cortex by combining Nissl staining with micro-optical sectioning tomography to simultaneously visualize individual cells and blood vessels, including capillaries. Pinpointing layer IV of the posteromedial barrel subfield, direct 3D reconstruction and quantitative analysis showed that (1) penetrating vessels preferentially locate in the interbarrel septa/barrel wall (75.1%) rather than the barrel hollows, (2) the branches of 70% penetrating vessels only reach the neighboring but not always all the neighboring barrels and the other 30% extend beyond the neighboring barrels and may provide cross-barrel blood supply or drainage, (3) the branches of 59.6% penetrating vessels reach all the neighboring barrels, while the rest only reach part of them, and (4) the length density of microvessels in the interbarrel septa/barrel wall is lower than that in the barrel hollows with a ratio of 0.92. These results reveal that the penetrating vessels and microvessels exhibit a barrel-specific organization, whereas the branches of penetrating vessels do not, which suggests a much more complex vascular distribution pattern among cortical columns than previously thought.

3D BrainCV: simultaneous visualization and analysis of cells and capillaries in a whole mouse brain with one-micron voxel resolution.

Systematic cellular and vascular configurations are essential for understanding fundamental brain anatomy and metabolism. We demonstrated a 3D brainwide cellular and vascular (called 3D BrainCV) visualization and quantitative protocol for a whole mouse brain. We developed a modified Nissl staining method that quickly labeled the cells and blood vessels simultaneously in an entire mouse brain. Terabytes 3D datasets of the whole mouse brains, with unprecedented details of both individual cells and blood vessels, including capillaries, were simultaneously imaged at 1-µm voxel resolution using micro-optical sectioning tomography (MOST). For quantitative analysis, we proposed an automatic image-processing pipeline to perform brainwide vectorization and analysis of cells and blood vessels. Six representative brain regions from the cortex to the deep, including FrA, M1, PMBSF, V1, striatum, and amygdala, and six parameters, including cell number density, vascular length density, fractional vascular volume, distance from the cells to the nearest microvessel, microvascular length density, and fractional microvascular volume, had been quantitatively analyzed. The results showed that the proximity of cells to blood vessels was linearly correlated with vascular length density, rather than the cell number density. The 3D BrainCV made overall snapshots of the detailed picture of the whole brain architecture, which could be beneficial for the state comparison of the developing and diseased brain.

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.

Cell-type-specific inhibitory circuitry from a connectomic census of mouse visual cortex.

Mammalian cortex features a vast diversity of neuronal cell types, each with characteristic anatomical, molecular and functional properties. Synaptic connectivity powerfully shapes how each cell type participates in the cortical circuit, but mapping connectivity rules at the resolution of distinct cell types remains difficult. Here, we used millimeter-scale volumetric electron microscopy1 to investigate the connectivity of all inhibitory neurons across a densely-segmented neuronal population of 1352 cells spanning all layers of mouse visual cortex, producing a wiring diagram of inhibitory connections with more than 70,000 synapses. Taking a data-driven approach inspired by classical neuroanatomy, we classified inhibitory neurons based on the relative targeting of dendritic compartments and other inhibitory cells and developed a novel classification of excitatory neurons based on the morphological and synaptic input properties. The synaptic connectivity between inhibitory cells revealed a novel class of disinhibitory specialist targeting basket cells, in addition to familiar subclasses. Analysis of the inhibitory connectivity onto excitatory neurons found widespread specificity, with many interneurons exhibiting differential targeting of certain subpopulations spatially intermingled with other potential targets. Inhibitory targeting was organized into "motif groups," diverse sets of cells that collectively target both perisomatic and dendritic compartments of the same excitatory targets. Collectively, our analysis identified new organizing principles for cortical inhibition and will serve as a foundation for linking modern multimodal neuronal atlases with the cortical wiring diagram.

Continuously tracing brain-wide long-distance axonal projections in mice at a one-micron voxel resolution.

Revealing neural circuit mechanisms is critical for understanding brain functions. Significant progress in dissecting neural connections has been made using optical imaging with fluorescence labels, especially in dissecting local connections. However, acquiring and tracing brain-wide, long-distance neural circuits at the neurite level remains a substantial challenge. Here, we describe a whole-brain approach to systematically obtaining continuous neuronal pathways in a fluorescent protein transgenic mouse at a one-micron voxel resolution. This goal is achieved by combining a novel resin-embedding method for maintaining fluorescence, an automated fluorescence micro-optical sectioning tomography system for long-term stable imaging, and a digital reconstruction-registration-annotation pipeline for tracing the axonal pathways in the mouse brain. With the unprecedented ability to image a whole mouse brain at a one-micron voxel resolution, the long-distance pathways were traced minutely and without interruption for the first time. With advancing labeling techniques, our method is believed to open an avenue to exploring both local and long-distance neural circuits that are related to brain functions and brain diseases down to the neurite level.

CT-guided microcoil localization for scapula-blocked pulmonary nodules using penetrating lung puncture before video-assisted thoracic surgery.

PURPOSE: To retrospectively analyze the effectiveness and safety of computed tomography (CT)-guided microcoil localization for scapula-blocked pulmonary nodules using penetrating lung puncture prior to video-assisted thoracic surgery (VATS). METHODS: One hundred thirty-eight patients with 138 pulmonary nodules were included in this single-center retrospective study. Among them, 110 patients who underwent CT-guided microcoil localization using the routine puncture technique formed the routine group; the other 28 patients who underwent the CT-guided microcoil localization using the penetrating lung puncture technique formed the penetrating lung group. The main outcomes were the success rate and complication rate of the two groups. RESULTS: The localization success rate was 95.5% (105/110) in the routine group and 89.3% (25/28) in the penetrating lung group (P = 0.205). There was no statistical difference in any of the complications (pneumothorax, intrapulmonary hemorrhage, or moderate and severe chest pain) in both groups (P = 0.178, P = 0.204, P = 0.709, respectively). Localization procedure time was significantly increased in the penetrating lung group compared with the routine group (31.0 ± 3.0 min vs. 21.2 ± 2.8 min, P < 0.001). CONCLUSION: CT-guided microcoil localization for scapula-blocked pulmonary nodules using penetrating lung puncture prior to VATS resection is effective and safe. However, the deployment of the microcoil using penetrating lung puncture required more time than the routine puncture method.

A complete reconstruction of the early visual system of an adult insect.

For most model organisms in neuroscience, research into visual processing in the brain is difficult because of a lack of high-resolution maps that capture complex neuronal circuitry. The microinsect Megaphragma viggianii, because of its small size and non-trivial behavior, provides a unique opportunity for tractable whole-organism connectomics. We image its whole head using serial electron microscopy. We reconstruct its compound eye and analyze the optical properties of the ommatidia as well as the connectome of the first visual neuropil-the lamina. Compared with the fruit fly and the honeybee, Megaphragma visual system is highly simplified: it has 29 ommatidia per eye and 6 lamina neuron types. We report features that are both stereotypical among most ommatidia and specialized to some. By identifying the "barebones" circuits critical for flying insects, our results will facilitate constructing computational models of visual processing in insects.

Cyclic structure with cellular precision in a vertebrate sensorimotor neural circuit.

Neuronal wiring diagrams reconstructed by electron microscopy1,2,3,4,5 pose new questions about the organization of nervous systems following the time-honored tradition of cross-species comparisons.6,7 The C. elegans connectome has been conceptualized as a sensorimotor circuit that is approximately feedforward,8,9,10,11 starting from sensory neurons proceeding to interneurons and ending with motor neurons. Overrepresentation of a 3-cell motif often known as the "feedforward loop" has provided further evidence for feedforwardness.10,12 Here, we contrast with another sensorimotor wiring diagram that was recently reconstructed from a larval zebrafish brainstem.13 We show that the 3-cycle, another 3-cell motif, is highly overrepresented in the oculomotor module of this wiring diagram. This is a first for any neuronal wiring diagram reconstructed by electron microscopy, whether invertebrate12,14 or mammalian.15,16,17 The 3-cycle of cells is "aligned" with a 3-cycle of neuronal groups in a stochastic block model (SBM)18 of the oculomotor module. However, the cellular cycles exhibit more specificity than can be explained by the group cycles-recurrence to the same neuron is surprisingly common. Cyclic structure could be relevant for theories of oculomotor function that depend on recurrent connectivity. The cyclic structure coexists with the classic vestibulo-ocular reflex arc for horizontal eye movements,19 and could be relevant for recurrent network models of temporal integration by the oculomotor system.20,21.

Neuronal wiring diagram of an adult brain.

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 5×107 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.

NEURD: automated proofreading and feature extraction for connectomics.

We are now in the era of millimeter-scale electron microscopy (EM) volumes collected at nanometer resolution (Shapson-Coe et al., 2021; Consortium et al., 2021). Dense reconstruction of cellular compartments in these EM volumes has been enabled by recent advances in Machine Learning (ML) (Lee et al., 2017; Wu et al., 2021; Lu et al., 2021; Macrina et al., 2021). Automated segmentation methods can now yield exceptionally accurate reconstructions of cells, but despite this accuracy, laborious post-hoc proofreading is still required to generate large connectomes free of merge and split errors. The elaborate 3-D meshes of neurons produced by these segmentations contain detailed morphological information, from the diameter, shape, and branching patterns of axons and dendrites, down to the fine-scale structure of dendritic spines. However, extracting information about these features can require substantial effort to piece together existing tools into custom workflows. Building on existing open-source software for mesh manipulation, here we present "NEURD", a software package that decomposes each meshed neuron into a compact and extensively-annotated graph representation. With these feature-rich graphs, we implement workflows for state of the art automated post-hoc proofreading of merge errors, cell classification, spine detection, axon-dendritic proximities, and other features that can enable many downstream analyses of neural morphology and connectivity. NEURD can make these new massive and complex datasets more accessible to neuroscience researchers focused on a variety of scientific questions.

Functional connectomics reveals general wiring rule in mouse visual cortex.

To understand how the brain computes, it is important to unravel the relationship between circuit connectivity and function. Previous research has shown that excitatory neurons in layer 2/3 of the primary visual cortex of mice with similar response properties are more likely to form connections. However, technical challenges of combining synaptic connectivity and functional measurements have limited these studies to few, highly local connections. Utilizing the millimeter scale and nanometer resolution of the MICrONS dataset, we studied the connectivity-function relationship in excitatory neurons of the mouse visual cortex across interlaminar and interarea projections, assessing connection selectivity at the coarse axon trajectory and fine synaptic formation levels. A digital twin model of this mouse, that accurately predicted responses to arbitrary video stimuli, enabled a comprehensive characterization of the function of neurons. We found that neurons with highly correlated responses to natural videos tended to be connected with each other, not only within the same cortical area but also across multiple layers and visual areas, including feedforward and feedback connections, whereas we did not find that orientation preference predicted connectivity. The digital twin model separated each neuron's tuning into a feature component (what the neuron responds to) and a spatial component (where the neuron's receptive field is located). We show that the feature, but not the spatial component, predicted which neurons were connected at the fine synaptic scale. Together, our results demonstrate the "like-to-like" connectivity rule generalizes to multiple connection types, and the rich MICrONS dataset is suitable to further refine a mechanistic understanding of circuit structure and function.