Progress towards mammalian whole-brain cellular connectomics

Neurons are the fundamental structural units of the nervous system i.e., the Neuron Doctrine as the pioneering work of Santiago Ramon y Cajal in the 1880's clearly demonstrated through careful observation of Golgi-stained neuronal morphologies. However, at that time sample preparation, imaging methods and computational tools were either nonexistent or insufficiently developed to permit the precise mapping of an entire brain with all of its neurons and their connections. Some measure of the "mesoscopic" connectional organization of the mammalian brain has been obtained over the past decade by alignment of sparse subsets of labeled neurons onto a reference atlas or via MRI-based diffusion tensor imaging. Neither method, however, provides data on the complete connectivity of all neurons comprising an individual brain. Fortunately, whole-brain cellular connectomics now appears within reach due to recent advances in whole-brain sample preparation and high-throughput electron microscopy (EM), though substantial obstacles remain with respect to large volume electron microscopic acquisitions and automated neurite reconstructions. This perspective examines the current status and problems associated with generating a mammalian whole-brain cellular connectome and argues that the time is right to launch a concerted connectomic attack on a small mammalian whole-brain

A Pipeline for Volume Electron Microscopy of the Caenorhabditis elegans Nervous System.

The "connectome," a comprehensive wiring diagram of synaptic connectivity, is achieved through volume electron microscopy (vEM) analysis of an entire nervous system and all associated non-neuronal tissues. White et al. (1986) pioneered the fully manual reconstruction of a connectome using Caenorhabditis elegans. Recent advances in vEM allow mapping new C. elegans connectomes with increased throughput, and reduced subjectivity. Current vEM studies aim to not only fill the remaining gaps in the original connectome, but also address fundamental questions including how the connectome changes during development, the nature of individuality, sexual dimorphism, and how genetic and environmental factors regulate connectivity. Here we describe our current vEM pipeline and projected improvements for the study of the C. elegans nervous system and beyond

A serial multiplex immunogold labeling method for identifying peptidergic neurons in connectomes.

This is the final version of the article.Available from eLife Sciences Publications via the DOI in this record.Electron microscopy-based connectomics aims to comprehensively map synaptic connections in neural tissue. However, current approaches are limited in their capacity to directly assign molecular identities to neurons. Here, we use serial multiplex immunogold labeling (siGOLD) and serial-section transmission electron microscopy (ssTEM) to identify multiple peptidergic neurons in a connectome. The high immunogenicity of neuropeptides and their broad distribution along axons, allowed us to identify distinct neurons by immunolabeling small subsets of sections within larger series. We demonstrate the scalability of siGOLD by using 11 neuropeptide antibodies on a full-body larval ssTEM dataset of the annelid Platynereis. We also reconstruct a peptidergic circuitry comprising the sensory nuchal organs, found by siGOLD to express pigment-dispersing factor, a circadian neuropeptide. Our approach enables the direct overlaying of chemical neuromodulatory maps onto synaptic connectomic maps in the study of nervous systems.The research leading to these results received funding from the European Research Council under the European Union’s Seventh Framework Programme (FP7/2007-2013)/European Research Council Grant Agreement 260821. This project is supported by the Marie Curie ITN "Neptune", GA 317172, funded under the FP7, PEOPLE Work Programme of the European Commission

Large Volume Electron Microscopy and Neural Microcircuit Analysis

One recent technical innovation in neuroscience is microcircuit analysis using three-dimensional reconstructions of neural elements with a large volume Electron microscopy (EM) data set. Large-scale data sets are acquired with newly-developed electron microscope systems such as automated tape-collecting ultramicrotomy (ATUM) with scanning EM (SEM), serial block-face EM (SBEM) and focused ion beam-SEM (FIB-SEM). Currently, projects are also underway to develop computer applications for the registration and segmentation of the serially-captured electron micrographs that are suitable for analyzing large volume EM data sets thoroughly and efficiently. The analysis of large volume data sets can bring innovative research results. These recently available techniques promote our understanding of the functional architecture of the brain

Strength and dendritic organization of thalamocortical synapses onto excitatory layer 4 neurons

The thalamus is a potent driver of cortical activity, even though cortical synapses onto layer 4 (L4) neurons outnumber thalamic synapses ten to one. Previous in vitro studies have suggested that enhanced efficacy of thalamocortical (TC) relative to corticocortical (CC) synapses explains the effectiveness of the thalamus. We investigated possible key anatomical and physiological differences between these inputs onto excitatory L4 neurons in vivo. We developed a high-throughput light microscopy method, validated by electron microscopy, to completely map the locations of synapses across an entire dendritic tree. This demonstrated that TC synapses are slightly more proximal to the soma than CC synapses, but detailed compartmental modeling predicted that dendritic filtering does not appreciably favor one synaptic class over another. Measurements of synaptic strength in intact animals revealed that both TC and CC synapses are weak and approximately equivalent. We conclude that thalamic potency relies, not on enhanced TC strength, but on coincident activation of converging inputs

Tiny Sea Anemone from the Lower Cambrian of China

Background Abundant fossils from the Ediacaran and Cambrian showing cnidarian grade grossly suggest that cnidarian diversification occurred earlier than that of other eumetazoans. However, fossils of possible soft-bodied polyps are scanty and modern corals are dated back only to the Middle Triassic, although molecular phylogenetic results support the idea that anthozoans represent the first major branch of the Cnidaria. Because of difficulties in taxonomic assignments owing to imperfect preservation of fossil cnidarian candidates, little is known about forms ancestral to those of living groups. Methods and Findings We have analyzed the soft-bodied polypoid microfossils Eolympia pediculata gen. et sp. nov. from the lowest Cambrian Kuanchuanpu Formation in southern China by scanning electron microscopy and computer-aided microtomography after isolating fossils from sedimentary rocks by acetic acid maceration. The fossils, about a half mm in body size, are preserved with 18 mesenteries including directives bilaterally arranged, 18 tentacles and a stalk-like pedicle. The pedicle suggests a sexual life cycle, while asexual reproduction by transverse fission also is inferred by circumferential grooves on the body column. Conclusions The features found in the present fossils fall within the morphological spectrum of modern Hexacorallia excluding Ceriantharia, and thus Eolympia pediculata could be a stem member for this group. The fossils also demonstrate that basic features characterizing modern hexacorallians such as bilateral symmetry and the reproductive system have deep roots in the Early Cambrian.Funding was provided by the National Science Foundation of China (http://www.nsfc.gov.cn/) grants 40830208, 40602003, 50702005 to J. Han and D. G. Shu, and by MOST Special Fund from the State Key Laboratory of Continental Dynamics, Northwest University, China (http://sklcd.nwu.edu.cn/) to J. Han and D. G. Shu. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Peer reviewe

Automatic Mitochondria Segmentation for EM Data Using a 3D Supervised Convolutional Network

Recent studies have supported the relation between mitochondrial functions and degenerative disorders related to ageing, such as Alzheimer's and Parkinson's diseases. Since these studies have exposed the need for detailed and high-resolution analysis of physical alterations in mitochondria, it is necessary to be able to perform segmentation and 3D reconstruction of mitochondria. However, due to the variety of mitochondrial structures, automated mitochondria segmentation and reconstruction in electron microscopy (EM) images have proven to be a difficult and challenging task. This paper puts forward an effective and automated pipeline based on deep learning to realize mitochondria segmentation in different EM images. The proposed pipeline consists of three parts: (1) utilizing image registration and histogram equalization as image pre-processing steps to maintain the consistency of the dataset; (2) proposing an effective approach for 3D mitochondria segmentation based on a volumetric, residual convolutional and deeply supervised network; and (3) employing a 3D connection method to obtain the relationship of mitochondria and displaying the 3D reconstruction results. To our knowledge, we are the first researchers to utilize a 3D fully residual convolutional network with a deeply supervised strategy to improve the accuracy of mitochondria segmentation. The experimental results on anisotropic and isotropic EM volumes demonstrate the effectiveness of our method, and the Jaccard index of our segmentation (91.8% in anisotropy, 90.0% in isotropy) and F1 score of detection (92.2% in anisotropy, 90.9% in isotropy) suggest that our approach achieved state-of-the-art results. Our fully automated pipeline contributes to the development of neuroscience by providing neurologists with a rapid approach for obtaining rich mitochondria statistics and helping them elucidate the mechanism and function of mitochondria

Cellular mechanics and intracellular organization

Mechanical signals affect and regulate many aspects of the cell behaviour, including growth, differentiation, gene expression and cell death. This thesis investigates the manner by which mechanical stress perturbs the intracellular structures of the cell and induces mechanical responses. In order to correlate mechanical perturbations to cellular responses, a combined fluorescence-atomic force microscope (AFM) was used to produce well defined nanomechanical perturbations while simultaneously tracking the real-time motion of fluorescently labelled intracellular organelles in live cells. By tracking instantaneous displacements of mitochondria far from the point of indentation, insights can be gained into the long-distance propagation of forces and the role of the cytoskeleton in force transmission. Quantitative analysis and tracking of mitochondria, using several image registration and tracking techniques, revealed an increase of approximately 40% in the mean mitochondrial displacement following AFM perturbation. Furthermore, when either the actin cytoskeleton or microtubules were disrupted using anti-cytoskeletal drugs, no significant change in mitochondrial displacement was observed following indentation, revealing the crucial role of both cytoskeletal networks in the long-distance transmission of forces through the cell. In addition, the effect of retinol and conjugated linoleic acid (CLA), compounds that have diverse effects on various cellular processes, on the mechanical behaviour of the cell was examined: both compounds were found to have a significant detrimental effect on the formation of focal adhesions, which was directly correlated to the measured cell elasticity (Young’s modulus) of the cell. Furthermore, quantification of mitochondrial displacements in response to applied AFM perturbations showed force propagation through the cytoskeleton to be blunted. Treatment of the two compounds in combination showed an additive effect. These results may broaden our understanding of the interplay between cell mechanics and cellular contact with the external microenvironment, and help to shed light on the important role of retinoids and CLA in health and disease

NANOCHANNEL-ASSISTED ACTIVE CONTROL OF MASS TRANSPORT IN POLYDIMETHYLSILOXANE-BASED MICRO- /NANOFLUIDIC SYSTEMS

Department of Mechanical EngineeringNanofluidic devices have been extensively studied due to a fascinating nature of their small size which facilitates biosensing, bio-chemical separations, seawater desalination, nanofluidic transistors, protein, and preconcentration for a Lab-on-a-Chip (LOC). Such applications could be achieved by a control of electrokinetic transport in a nanochannel produced by sophisticated nanofabrication technique. However, it has been a challenge from a fabrication to the control of electrokinetic phenomena in nanochannel because of the cost, time, incompatibility, and addressability issues. Therefore, an innovative method is required to achieve simple fabrication and versatile operations of micro/nanofluidic device with limited resources. This dissertation proposes a new method for nanochannel-assisted active manipulation of mass transport by switching physicochemical environment. In the early chapters of this dissertation, unconventional fabrication methods for hybrid-scale micro-/nanofluidic devices is described by using both crack-photolithography and polydimethylsiloxane (PDMS) based soft lithography. The late chapters introduce the mechanism of the mass transport in micro/nanofluidic device using solutes gradient and humidity for manipulation of colloidal motion and molecule valves, respectively. These studies can be introduced as follows. First, crack-photolithography is employed to facilitate large-scale reproducible channel fabrication through a single molding process and thus enable the fabrication of hybrid-scale micro-/nanofluidic devices at a wafer level with advantages seen in the throughput, cost-effectiveness, reliability, and reproducibility. In addition, modified soft lithography process is developed to fabricate stable nanochannel which is free from the collapse and the crumbling. Second, crack-assisted nanochannel is introduced to manipulate physicochemical environment of neighboring microchamber. Diffusion-controlled ion transport produces solutes gradient inducing spontaneous electric field which affects the motion of colloidal particles. Since the single nanochannel allows the production of concentration gradient in a long-term and stable manner, least source is required to maintain the spontaneous electric field without any external power source, which is appropriate for a portable and self-containable LOC. As a practical application, integrated micro/nanofluidic device facilitates concentration, on-demand extraction, and separation of the colloidal particles. Third, gas permeable PDMS nanochannel with high hydraulic resistance is employed to develop humidity-based gating nanochannel. The rate of mass transport can be manipulated by humidity due to the evaporation of water and the adsorption of solutes to the wall of channel. To demonstrate functionality of humidity for liquid gating or capacitor of molecules, the effect of humidity on mass transport was investigated. This new concept of manipulation of nanofluidic transport made it possible to successfully perform individual mass transport control in a nanochannel array, which is difficult with conventional technique using electricity. It further facilitated on-demand addressable bio/chemical assay using humidity-based molecule valves and pumps. The role of nanochannel as a passage for mass transfer is essential to allow stable and precise control of transport of ions and molecules in the microchannel. It provides wide range of applications using a diffusion-based control of microfluidic environment to induce not only solute gradient for production of electric field but also liquid gating for a valve at molecular level. Thus, achievements of this dissertation contribute to raise the insight about nanochannel-assisted system for simple and precise control of mass transport in hybrid-scale micro-/nanofluidic devices, which is facilitated by the help of the cracking-assisted micro-/nanofabrication technologies.clos

An electron microscopic method to identify peptidergic neurons in connectomes

Animal nervous systems are complex networks of connections between diverse types of neurons and effector tissues. Mapping the connections of molecularly identified neuron types at the synaptic level would greatly enhance our understanding of the structure and function of the nervous system. In this thesis, I created a large serial EM dataset for neuronal network analysis, circuit reconstruction and stereotypy studies in the nervous system of the larvae of the marine annelid Platynereis dumerilii. I also developed an innovative method to assign molecular identities to peptidergic neurons directly in the EM dataset. I used serial-section transmission electron microscopy (ssTEM) combined with serial multiplex immunogold labeling (siGOLD) to molecularly identify multiple different neuron types. siGOLD is the method of labeling subsets of sections with various antibodies to reveal the molecular identities of specific neurons. I used neuropeptide antibodies to establish this method, taking advantage of the high immunogenicity of neuropeptides and their broad distribution throughout the axons. I demonstrate the effectiveness of siGOLD by using 11 neuropeptide antibodies to specifically label several distinct types of peptidergic neurons on a full-body larval ssTEM dataset of a Platynereis larva. siGOLD was also applied in the reconstruction of a peptidergic circuit comprising the sensory nuchal organs that express the circadian neuropeptide pigment-dispersing factor (PDF). Overall, this approach enables the direct overlaying of chemical neuromodulatory maps onto synaptic connectomic maps in the study of nervous systems. The full-body Platynereis EM dataset provided evidence for stereotypy between neuronal circuit anatomy and function in individuals of the same species. It also provided a database that can be reconstructed into a full-body connectome in the near future