A petavoxel fragment of human cerebral cortex reconstructed at nanoscale resolution.

To fully understand how the human brain works, knowledge of its structure at high resolution is needed. Presented here is a computationally intensive reconstruction of the ultrastructure of a cubic millimeter of human temporal cortex that was surgically removed to gain access to an underlying epileptic focus. It contains about 57,000 cells, about 230 millimeters of blood vessels, and about 150 million synapses and comprises 1.4 petabytes. Our analysis showed that glia outnumber neurons 2:1, oligodendrocytes were the most common cell, deep layer excitatory neurons could be classified on the basis of dendritic orientation, and among thousands of weak connections to each neuron, there exist rare powerful axonal inputs of up to 50 synapses. Further studies using this resource may bring valuable insights into the mysteries of the human brain.

A large-scale volumetric correlated light and electron microscopy study localizes Alzheimer's disease-related molecules in the hippocampus.

Connectomics is a nascent neuroscience field to map and analyze neuronal networks. It provides a new way to investigate abnormalities in brain tissue, including in models of Alzheimer's disease (AD). This age-related disease is associated with alterations in amyloid-ß (Aß) and phosphorylated tau (pTau). These alterations correlate with AD's clinical manifestations, but causal links remain unclear. Therefore, studying these molecular alterations within the context of the local neuronal and glial milieu may provide insight into disease mechanisms. Volume electron microscopy (vEM) is an ideal tool for performing connectomics studies at the ultrastructural level, but localizing specific biomolecules within large-volume vEM data has been challenging. Here we report a volumetric correlated light and electron microscopy (vCLEM) approach using fluorescent nanobodies as immuno-probes to localize Alzheimer's disease-related molecules in a large vEM volume. Three molecules (pTau, Aß, and a marker for activated microglia (CD11b)) were labeled without the need for detergents by three nanobody probes in a sample of the hippocampus of the 3xTg Alzheimer's disease model mouse. Confocal microscopy followed by vEM imaging of the same sample allowed for registration of the location of the molecules within the volume. This dataset revealed several ultrastructural abnormalities regarding the localizations of Aß and pTau in novel locations. For example, two pTau-positive post-synaptic spine-like protrusions innervated by axon terminals were found projecting from the axon initial segment of a pyramidal cell. Three pyramidal neurons with intracellular Aß or pTau were 3D reconstructed. Automatic synapse detection, which is necessary for connectomics analysis, revealed the changes in density and volume of synapses at different distances from an Aß plaque. This vCLEM approach is useful to uncover molecular alterations within large-scale volume electron microscopy data, opening a new connectomics pathway to study Alzheimer's disease and other types of dementia.

Probing Molecular Diversity and Ultrastructure of Brain Cells with Fluorescent Aptamers.

Detergent-free immunolabeling has been proven feasible for correlated light and electron microscopy, but its application is restricted by the availability of suitable affinity reagents. Here we introduce CAptVE, a method using slow off-rate modified aptamers for cell fluorescence labeling on ultrastructurally reconstructable electron micrographs. CAptVE provides labeling for a wide range of biomarkers, offering a pathway to integrate molecular analysis into recent approaches to delineate neural circuits via connectomics.

A Scalable Staining Strategy for Whole-Brain Connectomics.

Mapping the complete synaptic connectivity of a mammalian brain would be transformative, revealing the pathways underlying perception, behavior, and memory. Serial section electron microscopy, via membrane staining using osmium tetroxide, is ideal for visualizing cells and synaptic connections but, in whole brain samples, faces significant challenges related to chemical treatment and volume changes. These issues can adversely affect both the ultrastructural quality and macroscopic tissue integrity. By leveraging time-lapse X-ray imaging and brain proxies, we have developed a 12-step protocol, ODeCO, that effectively infiltrates osmium throughout an entire mouse brain while preserving ultrastructure without any cracks or fragmentation, a necessary prerequisite for constructing the first comprehensive mouse brain connectome.

Connectomics of the Octopus vulgaris vertical lobe provides insight into conserved and novel principles of a memory acquisition network.

Here, we present the first analysis of the connectome of a small volume of the Octopus vulgaris vertical lobe (VL), a brain structure mediating the acquisition of long-term memory in this behaviorally advanced mollusk. Serial section electron microscopy revealed new types of interneurons, cellular components of extensive modulatory systems, and multiple synaptic motifs. The sensory input to the VL is conveyed via~1.8 × 106 axons that sparsely innervate two parallel and interconnected feedforward networks formed by the two types of amacrine interneurons (AM), simple AMs (SAMs) and complex AMs (CAMs). SAMs make up 89.3% of the~25 × 106VL cells, each receiving a synaptic input from only a single input neuron on its non-bifurcating primary neurite, suggesting that each input neuron is represented in only~12 ± 3.4SAMs. This synaptic site is likely a 'memory site' as it is endowed with LTP. The CAMs, a newly described AM type, comprise 1.6% of the VL cells. Their bifurcating neurites integrate multiple inputs from the input axons and SAMs. While the SAM network appears to feedforward sparse 'memorizable' sensory representations to the VL output layer, the CAMs appear to monitor global activity and feedforward a balancing inhibition for 'sharpening' the stimulus-specific VL output. While sharing morphological and wiring features with circuits supporting associative learning in other animals, the VL has evolved a unique circuit that enables associative learning based on feedforward information flow.

Research on Heterogeneous Traveler Travel Mode Choices with Differences under a Mixed Traffic Environment.

Autonomous vehicles (AVs) have been made possible by advances in sensing and computing technologies. However, the high cost of AVs makes privatization take longer. Therefore, companies with autonomous vehicles can develop shared autonomous vehicle (SAV) projects. AVs with a high level of automation require high upgrade and use costs. In order to meet the needs of more customers and reduce the investment cost of the company, SAVs with different levels of automation may coexist for a long time. Faced with multiple travel modes (autonomous cars with different levels of automation, private cars, and buses), travelers' travel mode choices are worth studying. To further differentiate the types of travelers, this paper defines high-income travelers and low-income travelers. The difference between these two types of travelers is whether they have a private car. The differences in time value and willingness to pay of the two types of travelers are considered. Based on the above considerations, this paper establishes a multi-modal selection model with the goal of maximizing the total utility of all travelers and uses the imperial competition algorithm to solve it. The results show that low-income travelers are more likely to choose buses and autonomous vehicles with lower levels of automation, while high-income travelers tend to choose higher levels of automation due to their high value of travel time.

Multiplexed volumetric CLEM enabled by antibody derivatives provides new insights into the cytology of the mouse cerebellar cortex.

Mapping neuronal networks that underlie behavior has become a central focus in neuroscience. While serial section electron microscopy (ssEM) can reveal the fine structure of neuronal networks (connectomics), it does not provide the molecular information that helps identify cell types or their functional properties. Volumetric correlated light and electron microscopy (vCLEM) combines ssEM and volumetric fluorescence microscopy to incorporate molecular labeling into ssEM datasets. We developed an approach that uses small fluorescent single-chain variable fragment (scFv) immuno-probes to perform multiplexed detergent-free immuno-labeling and ssEM on the same samples. We generated eight such fluorescent scFvs that targeted useful markers for brain studies (green fluorescent protein, glial fibrillary acidic protein, calbindin, parvalbumin, voltage-gated potassium channel subfamily A member 2, vesicular glutamate transporter 1, postsynaptic density protein 95, and neuropeptide Y). To test the vCLEM approach, six different fluorescent probes were imaged in a sample of the cortex of a cerebellar lobule (Crus 1), using confocal microscopy with spectral unmixing, followed by ssEM imaging of the same sample. The results show excellent ultrastructure with superimposition of the multiple fluorescence channels. Using this approach we could document a poorly described cell type in the cerebellum, two types of mossy fiber terminals, and the subcellular localization of one type of ion channel. Because scFvs can be derived from existing monoclonal antibodies, hundreds of such probes can be generated to enable molecular overlays for connectomic studies.

Multiplexed volumetric CLEM enabled by antibody derivatives provides new insights into the cytology of the mouse cerebellar cortex.

Mapping neuronal networks that underlie behavior has become a central focus in neuroscience. While serial section electron microscopy (ssEM) can reveal the fine structure of neuronal networks (connectomics), it does not provide the molecular information that helps identify cell types or their functional properties. Volumetric correlated light and electron microscopy (vCLEM) combines ssEM and volumetric fluorescence microscopy to incorporate molecular labeling into ssEM datasets. We developed an approach that uses small fluorescent single-chain variable fragment (scFv) immuno-probes to perform multiplexed detergent-free immuno-labeling and ssEM on the same samples. We generated eight such fluorescent scFvs that targeted useful markers for brain studies (green fluorescent protein, glial fibrillary acidic protein, calbindin, parvalbumin, voltage-gated potassium channel subfamily A member 2, vesicular glutamate transporter 1, postsynaptic density protein 95, and neuropeptide Y). To test the vCLEM approach, six different fluorescent probes were imaged in a sample of the cortex of a cerebellar lobule (Crus 1), using confocal microscopy with spectral unmixing, followed by ssEM imaging of the same sample. The results show excellent ultrastructure with superimposition of the multiple fluorescence channels. Using this approach we could document a poorly described cell type in the cerebellum, two types of mossy fiber terminals, and the subcellular localization of one type of ion channel. Because scFvs can be derived from existing monoclonal antibodies, hundreds of such probes can be generated to enable molecular overlays for connectomic studies.

Evidences of Topological Surface States in the Nodal-Line Semimetal SnTaS2 Nanoflakes.

Exploring the topological surface state of a topological semimetal by the transport technique has always been a big challenge because of the overwhelming contribution of the bulk state. In this work, we perform systematic angular-dependent magnetotransport measurements and electronic band calculations on SnTaS2 crystals, a layered topological nodal-line semimetal. Distinct Shubnikov-de Haas quantum oscillations were observed only in SnTaS2 nanoflakes when the thickness was below about 110 nm, and the oscillation amplitudes increased significantly with decreasing thickness. By analysis of the oscillation spectra, together with the theoretical calculation, a two-dimensional and topological nontrivial nature of the surface band is unambiguously identified, providing direct transport evidence of drumhead surface state for SnTaS2. Our comprehensive understanding of the Fermi surface topology of the centrosymmetric superconductor SnTaS2 is crucial for further research on the interplay of superconductivity and nontrivial topology.

Immersion Fixation and Staining of Multicubic Millimeter Volumes for Electron Microscopy-Based Connectomics of Human Brain Biopsies.

Connectomics allows mapping of cells and their circuits at the nanometer scale in volumes of approximately 1 mm3. Given that the human cerebral cortex can be 3 mm in thickness, larger volumes are required. Larger-volume circuit reconstructions of human brain are limited by 1) the availability of fresh biopsies; 2) the need for excellent preservation of ultrastructure, including extracellular space; and 3) the requirement of uniform staining throughout the sample, among other technical challenges. Cerebral cortical samples from neurosurgical patients are available owing to lead placement for deep brain stimulation. Described here is an immersion fixation, heavy metal staining, and tissue processing method that consistently provides excellent ultrastructure throughout human and rodent surgical brain samples of volumes 2 × 2 × 2 mm3 and up to 37 mm3 with one dimension ≤2 mm. This method should allow synapse-level circuit analysis in samples from patients with psychiatric and neurologic disorders.

Post-embryonic remodeling of the C. elegans motor circuit.

During development, animals can maintain behavioral output even as underlying circuitry structurally remodels. After hatching, C. elegans undergoes substantial motor neuron expansion and synapse rewiring while the animal continuously moves with an undulatory pattern. To understand how the circuit transitions from its juvenile to mature configuration without interrupting functional output, we reconstructed the C. elegans motor circuit by electron microscopy across larval development. We observed the following: First, embryonic motor neurons transiently interact with the developing post-embryonic motor neurons prior to remodeling of their juvenile wiring. Second, post-embryonic neurons initiate synapse development with their future partners as their neurites navigate through the juvenile nerve cords. Third, embryonic and post-embryonic neurons sequentially build structural machinery needed for the adult circuit before the embryonic neurons relinquish their roles to post-embryonic neurons. Fourth, this transition is repeated region by region along the body in an anterior-to-posterior sequence, following the birth order of neurons. Through this orchestrated and programmed rewiring, the motor circuit gradually transforms from asymmetric to symmetric wiring. These maturation strategies support the continuous maintenance of motor patterns as the juvenile circuit develops into the adult configuration.

Connectomes across development reveal principles of brain maturation.

An animal's nervous system changes as its body grows from birth to adulthood and its behaviours mature1-8. The form and extent of circuit remodelling across the connectome is unknown3,9-15. Here we used serial-section electron microscopy to reconstruct the full brain of eight isogenic Caenorhabditis elegans individuals across postnatal stages to investigate how it changes with age. The overall geometry of the brain is preserved from birth to adulthood, but substantial changes in chemical synaptic connectivity emerge on this consistent scaffold. Comparing connectomes between individuals reveals substantial differences in connectivity that make each brain partly unique. Comparing connectomes across maturation reveals consistent wiring changes between different neurons. These changes alter the strength of existing connections and create new connections. Collective changes in the network alter information processing. During development, the central decision-making circuitry is maintained, whereas sensory and motor pathways substantially remodel. With age, the brain becomes progressively more feedforward and discernibly modular. Thus developmental connectomics reveals principles that underlie brain maturation.

[Advanced Treatment of Tail Water Using Pilot-scale Horizontal and Vertical Subsurface Flow Constructed Wetlands in Low-temperature Seasons].

There were significant differences in the working efficiency and mechanism of constructed wetlands between low temperature and suitable temperature conditions. This study designed a horizontal subsurface flow constructed wetland (HFCW) and a vertical subsurface flow constructed wetland (VFCW) to explore their performance differences in advanced treatment of sewage based on contaminant degradation analysis including the removal of organic matters, total nitrogen (TN), and total phosphorus (TP), as well as the analysis of microbial community structure. The results showed that when the COD concentration of influent was between 37.50 to 80.00 mg·L-1, the concentration of total nitrogen and total phosphorus were within the first level A criteria specified in the discharge standard of pollutants for municipal wastewater treatment plant at the continuous flow of 2 m3·d-1:①Both HFCW and VFCW showed stable degradation ability of organic matter in influent and good resistance to high organic load. ②Supplementation of the carbon source significantly improved the nitrogen removal efficiency of two subsurface flow constructed wetlands. HFCW achieved the average removal rate of TN at 76.01%, and the average removal rate of TN by VFCW reached 71.69% after the carbon addition. In contrast, dosage of an external carbon source showed limited effect on phosphorus removal. Furthermore, it worked more effectively for performance improvement of HFCW than that of VFCW. ③The analysis of microbial community structure in wetland substrate and plant rhizosphere samples revealed that Proteobacteria, Firmicutes, and Verrucomicrobia were the dominant phylum in two series of wetland samples. For the dominant microbiota at the genus level, there were more significant differences in microbial community structure in wetland substrate samples than that in plant rhizosphere samples. Hydrogenophaga, Erysipelothrix, and Devosia contributed the most to the differences between the microbial communities of HFCW and VFCW. Overall, the species diversity and abundance of microbial samples from VFCW was higher than those from HFCW.

Absolute Frequency Measurement of

We report the precision measurement of the absolute frequencies, hyperfine splitting, and 2P fine structure splitting in cold atoms of ^{6}Li. Using the stabilized optical frequency comb and developed heterodyne detection technique, the photon shot-noise limited optical spectroscopy is achieved. The measurement of absolute frequencies of D_{1} lines is reached with an uncertainty of about 1 kHz, which is 1 order of magnitude more accurate than previous measurements. The hyperfine splitting of the D_{1} line and 2P fine structure splitting of ^{6}Li are 26.103 1 (14) and 10 052.780 4 (18) MHz, respectively, in agreement with recent theoretical calculations. Our results could provide a benchmark to test the theory at the higher precision and help to resolve large discrepancies among previous experiments.

Single crystal growth and ferromagnetism of Cr-doped Sb4Te3.

Here we report the single crystal growth, magnetic and transport properties of Cr-doped Sb4Te3, (Sb1-x Cr x )4Te3, with doping concentrations x = 0.25%, 0.5%, 0.75%, and 1%. The samples with lower doping concentrations are paramagnetic, while ferromagnetism appears in higher doped samples with the highest Curie temperature of 7 K when x = 1%. Anomalous Hall effect with clear hysteresis loop is observed in the samples with x = 1%, indicating the intrinsic ferromagnetism in the system. Hall resistivity measurements show the dominant charge carriers are holes and the density of holes increases with the doping concentration. This work provides a possible single-crystalline platform for further experimental researches on the nontrivial band topology in Sb4Te3, and enriches the ferromagnetic members in the transition metal doped (Sb2) m -Sb2Te3 topological material series.

Engineering brain activity patterns by neuromodulator polytherapy for treatment of disorders.

Conventional drug screens and treatments often ignore the underlying complexity of brain network dysfunctions, resulting in suboptimal outcomes. Here we ask whether we can correct abnormal functional connectivity of the entire brain by identifying and combining multiple neuromodulators that perturb connectivity in complementary ways. Our approach avoids the combinatorial complexity of screening all drug combinations. We develop a high-speed platform capable of imaging more than 15000 neurons in 50ms to map the entire brain functional connectivity in large numbers of vertebrates under many conditions. Screening a panel of drugs in a zebrafish model of human Dravet syndrome, we show that even drugs with related mechanisms of action can modulate functional connectivity in significantly different ways. By clustering connectivity fingerprints, we algorithmically select small subsets of complementary drugs and rapidly identify combinations that are significantly more effective at correcting abnormal networks and reducing spontaneous seizures than monotherapies, while minimizing behavioral side effects. Even at low concentrations, our polytherapy performs superior to individual drugs even at highest tolerated concentrations.

Brain activity patterns in high-throughput electrophysiology screen predict both drug efficacies and side effects.

Neurological drugs are often associated with serious side effects, yet drug screens typically focus only on efficacy. We demonstrate a novel paradigm utilizing high-throughput in vivo electrophysiology and brain activity patterns (BAPs). A platform with high sensitivity records local field potentials (LFPs) simultaneously from many zebrafish larvae over extended periods. We show that BAPs from larvae experiencing epileptic seizures or drug-induced side effects have substantially reduced complexity (entropy), similar to reduced LFP complexity observed in Parkinson's disease. To determine whether drugs that enhance BAP complexity produces positive outcomes, we used light pulses to trigger seizures in a model of Dravet syndrome, an intractable genetic epilepsy. The highest-ranked compounds identified by BAP analysis exhibit far greater anti-seizure efficacy and fewer side effects during subsequent in-depth behavioral assessment. This high correlation with behavioral outcomes illustrates the power of brain activity pattern-based screens and identifies novel therapeutic candidates with minimal side effects.

Automated deep-phenotyping of the vertebrate brain.

Here, we describe an automated platform suitable for large-scale deep-phenotyping of zebrafish mutant lines, which uses optical projection tomography to rapidly image brain-specific gene expression patterns in 3D at cellular resolution. Registration algorithms and correlation analysis are then used to compare 3D expression patterns, to automatically detect all statistically significant alterations in mutants, and to map them onto a brain atlas. Automated deep-phenotyping of a mutation in the master transcriptional regulator fezf2 not only detects all known phenotypes but also uncovers important novel neural deficits that were overlooked in previous studies. In the telencephalon, we show for the first time that fezf2 mutant zebrafish have significant patterning deficits, particularly in glutamatergic populations. Our findings reveal unexpected parallels between fezf2 function in zebrafish and mice, where mutations cause deficits in glutamatergic neurons of the telencephalon-derived neocortex.

Guided Homing of Cells in Multi-Photon Microfabricated Bioscaffolds.

Tissues contain exquisite vascular microstructures, and patterns of chemical cues for directing cell migration, homing, and differentiation for organ development and function. 3D microfabrication by multi-photon photolithography is a flexible, high-resolution tool for generating 3D bioscaffolds. However, the combined fabrication of scaffold microstructure simultaneously with patterning of cues to create both geometrically and chemically defined microenvironments remains to be demonstrated. This study presents a high-speed method for micron-resolution fabrication of scaffold microstructure and patterning of protein cues simultaneously using native scaffold materials. By the simultaneous microfabrication of arbitrary microvasculature geometries, and patterning selected regions of the microvasculature with the homing ligand P-selectin, this study demonstrates adhesion, rolling, and selective homing of cells in defined 3D regions. This novel ability to generate high-resolution geometries replete with patterned cues at high speed enables the construction of biomimetic microenvironments for complex 3D assays of cellular behavior.

Protecting a quantum memory for a photonic polarization qubit in a cold atomic ensemble by dynamical decoupling.

We report an experimental demonstration of storage of photonic polarization qubit (PPQ) protected by dynamical decoupling (DD). PPQ's states are stored as a superposition of two spin waves by electromagnetically-induced-transparency (EIT). Carr-Purcell-Meiboom-Gill (CPMG) DD sequences are applied to the spin-wave superposition to suppress its decoherence. Thus, the quantum process fidelity remains better than 0.8 for up to 800 µs storage time, which is 3.4-times longer than the corresponding storage time of ~180 µs without the CPMG sequences. This work is a key step towards the storage of single-photon polarization qubit protected by the CPMG sequences.