Image informatics strategies for deciphering neuronal network connectivity

Brain function relies on an intricate network of highly dynamic neuronal connections that rewires dramatically under the impulse of various external cues and pathological conditions. Among the neuronal structures that show morphologi- cal plasticity are neurites, synapses, dendritic spines and even nuclei. This structural remodelling is directly connected with functional changes such as intercellular com- munication and the associated calcium-bursting behaviour. In vitro cultured neu- ronal networks are valuable models for studying these morpho-functional changes. Owing to the automation and standardisation of both image acquisition and image analysis, it has become possible to extract statistically relevant readout from such networks. Here, we focus on the current state-of-the-art in image informatics that enables quantitative microscopic interrogation of neuronal networks. We describe the major correlates of neuronal connectivity and present workflows for analysing them. Finally, we provide an outlook on the challenges that remain to be addressed, and discuss how imaging algorithms can be extended beyond in vitro imaging studies

Automated Reconstruction of Neuronal Morphology Based on Local Geometrical and Global Structural Models

Digital reconstruction of neurons from microscope images is an important and challenging problem in neuroscience. In this paper, we propose a model-based method to tackle this problem. We first formulate a model structure, then develop an algorithm for computing it by carefully taking into account morphological characteristics of neurons, as well as the image properties under typical imaging protocols. The method has been tested on the data sets used in the DIADEM competition and produced promising results for four out of the five data sets

Model and Appearance Based Analysis of Neuronal Morphology from Different Microscopy Imaging Modalities

The neuronal morphology analysis is key for understanding how a brain works. This process requires the neuron imaging system with single-cell resolution; however, there is no feasible system for the human brain. Fortunately, the knowledge can be inferred from the model organism, Drosophila melanogaster, to the human system. This dissertation explores the morphology analysis of Drosophila larvae at single-cell resolution in static images and image sequences, as well as multiple microscopy imaging modalities. Our contributions are on both computational methods for morphology quantification and analysis of the influence of the anatomical aspect. We develop novel model-and-appearance-based methods for morphology quantification and illustrate their significance in three neuroscience studies. Modeling of the structure and dynamics of neuronal circuits creates understanding about how connectivity patterns are formed within a motor circuit and determining whether the connectivity map of neurons can be deduced by estimations of neuronal morphology. To address this problem, we study both boundary-based and centerline-based approaches for neuron reconstruction in static volumes. Neuronal mechanisms are related to the morphology dynamics; so the patterns of neuronal morphology changes are analyzed along with other aspects. In this case, the relationship between neuronal activity and morphology dynamics is explored to analyze locomotion procedures. Our tracking method models the morphology dynamics in the calcium image sequence designed for detecting neuronal activity. It follows the local-to-global design to handle calcium imaging issues and neuronal movement characteristics. Lastly, modeling the link between structural and functional development depicts the correlation between neuron growth and protein interactions. This requires the morphology analysis of different imaging modalities. It can be solved using the part-wise volume segmentation with artificial templates, the standardized representation of neurons. Our method follows the global-to-local approach to solve both part-wise segmentation and registration across modalities. Our methods address common issues in automated morphology analysis from extracting morphological features to tracking neurons, as well as mapping neurons across imaging modalities. The quantitative analysis delivered by our techniques enables a number of new applications and visualizations for advancing the investigation of phenomena in the nervous system

A platform for brain-wide imaging and reconstruction of individual neurons

The structure of axonal arbors controls how signals from individual neurons are routed within the mammalian brain. However, the arbors of very few long-range projection neurons have been reconstructed in their entirety, as axons with diameters as small as 100 nm arborize in target regions dispersed over many millimeters of tissue. We introduce a platform for high-resolution, three-dimensional fluorescence imaging of complete tissue volumes that enables the visualization and reconstruction of long-range axonal arbors. This platform relies on a high-speed two-photon microscope integrated with a tissue vibratome and a suite of computational tools for large-scale image data. We demonstrate the power of this approach by reconstructing the axonal arbors of multiple neurons in the motor cortex across a single mouse brain.Howard Hughes Medical InstitutePublished versio

In situ three-dimensional reconstruction of mouse heart sympathetic innervation by two-photon excitation fluorescence imaging

Background Sympathetic nerve wiring in the mammalian heart has remained largely unexplored. Resolving the wiring diagram of the cardiac sympathetic network would help establish the structural underpinnings of neurocardiac coupling. New Method We used two-photon excitation fluorescence microscopy, combined with a computer-assisted 3-D tracking algorithm, to map the local sympathetic circuits in living hearts from adult transgenic mice expressing enhanced green fluorescent protein (EGFP) in peripheral adrenergic neurons. Results Quantitative co-localization analyses confirmed that the intramyocardial EGFP distribution recapitulated the anatomy of the sympathetic arbor. In the left ventricular subepicardium of the uninjured heart, the sympathetic network was composed of multiple subarbors, exhibiting variable branching and looping topology. Axonal branches did not overlap with each other within their respective parental subarbor nor with neurites of annexed subarbors. The sympathetic network in the border zone of a 2-week-old myocardial infarction was characterized by substantive rewiring, which included spatially heterogeneous loss and gain of sympathetic fibers and formation of multiple, predominately nested, axon loops of widely variable circumference and geometry. Comparison with Existing Methods In contrast to mechanical tissue sectioning methods that may involve deformation of tissue and uncertainty in registration across sections, our approach preserves continuity of structure, which allows tracing of neurites over distances, and thus enables derivation of the three-dimensional and topological morphology of cardiac sympathetic nerves. Conclusions Our assay should be of general utility to unravel the mechanisms governing sympathetic axon spacing during development and disease

Light Sheet Microscopy and Image Analysis of Neural Development and Programmed Cell Death in C. Elegans Embryos

The positioning of neuronal cell bodies and neurites is critical for intact functioning of the nervous system. Mapping the positions of the soma and neurites in the brains of developing embryos as important central nervous system structures are being created may yield novel insight into the role of distinct cell groups in creating these structures. New developments in microscopy have made this an excellent time to study neural development in the C. elegans embryo. In the past decade, implementations of highly light efficient methods such as single plane illumination microscopy have rendered it possible to follow development of embryonic structures in 3D with excellent temporal resolution (Huisken et al., 2004) and low phototoxicity. Recent work has resulted in quantitative characterization of the outgrowth of a single neurite in the late, rapidly moving three-fold stage of the C. elegans embryo for the first time (Christensen et al., 2015). In this thesis, I first describe the construction and programming of a single plane illumination microscope (SPIM) based on a design from Hari Shroff\u27s lab (Wu et al., 2011). The microscope is developed especially for use with C. elegans embryos and permits fast image acquisition without excessive photodamage, compared to other forms of microscopy. Second, I describe the use of the SPIM microscope to image the development of a subset of sublateral neurons, the earliest known entrants to the nerve ring (Rapti et al, in preparation), into which they grow in the 1.5-fold stage. I describe an algorithm for automatically aligning developing embryos onto one another until the beginning of the rapid embryonic movements known as twitching, which begin at the start of the twofold stage. I employ my algorithm to align a group of identically imaged embryos onto one another and deduce information about the positioning of the nerve ring in an approximately uniform coordinate system. I determine that nerve rings are precisely positioned in the embryo to within about a micrometer while the cell bodies that grow into the nerve ring are positioned over a much wider distance. My work suggests that the nerve ring grows out towards the ALA neuron as an anchor, and that twitching may begin when the developing nerve ring reaches the ALA. I additionally describe observation of new phenotypes related to the cam-1 mutation, which was previously identified as a regulator of anterior-posterior placement of the nerve ring (Kennerdell et al., 2009). Third, I describe an application of the SPIM microscope for imaging the death of the tail spike cell, a complex, multi-compartment differentiated cell which dies over a period of hours during the three-fold stage, when the animal is rapidly moving in its shell, and cannot be imaged otherwise than with a rapid, light efficient microscope such as the one described here. I determined the time course and confirmed the sequence of events of wild type tail spike cell death. Additionally, I report stronger phenotypes for some known tail spike cell death genes when imaged in the embryo, suggesting that eff-1 plays a stronger role than previously known in clearance of the distal part of the tail spike cell process, and additionally that ced-5 has a strong role in clearance of the same compartment (in addition to its known role in soma clearance). In an appendix I describe work beginning on an extension of the microscope, which will hopefully see the microscope used as a tool for selectively inducing fluorescence in individual cells and following the development of those cells in time. My results demonstrate the utility of single plane illumination microscopy for study of C. elegans embryogenesis and establish fundamental facts about the variability of the C. elegans central nervous system by making direct comparisons between animals. This work contributes to our understanding of the C. elegans nervous system by establishing fundamental bounds on the range of nerve ring positioning between individuals

Confocal light sheet microscopy: micron-scale neuroanatomy of the entire mouse brain.

Elucidating the neural pathways that underlie brain function is one of the greatest challenges in neuroscience. Light sheet based microscopy is a cutting edge method to map cerebral circuitry through optical sectioning of cleared mouse brains. However, the image contrast provided by this method is not sufficient to resolve and reconstruct the entire neuronal network. Here we combined the advantages of light sheet illumination and confocal slit detection to increase the image contrast in real time, with a frame rate of 10 Hz. In fact, in confocal light sheet microscopy (CLSM), the out-of-focus and scattered light is filtered out before detection, without multiple acquisitions or any post-processing of the acquired data. The background rejection capabilities of CLSM were validated in cleared mouse brains by comparison with a structured illumination approach. We show that CLSM allows reconstructing macroscopic brain volumes with sub-cellular resolution. We obtained a comprehensive map of Purkinje cells in the cerebellum of L7-GFP transgenic mice. Further, we were able to trace neuronal projections across brain of thy1-GFP-M transgenic mice. The whole-brain high-resolution fluorescence imaging assured by CLSM may represent a powerful tool to navigate the brain through neuronal pathways. Although this work is focused on brain imaging, the macro-scale high-resolution tomographies affordable with CLSM are ideally suited to explore, at micron-scale resolution, the anatomy of different specimens like murine organs, embryos or flies. (C) 2012 Optical Society of Americ

Large-scale circuit reconstruction in medial entorhinal cortex

Es ist noch weitgehend ungeklärt, mittels welcher Mechanismen die elektrische Aktivität von Nervenzellpopulationen des Gehirns Verhalten ermöglicht. Die Orientierung im Raum ist eine Fähigkeit des Gehirns, für die im Säugetier der mediale entorhinale Teil der Großhirnrinde als entscheidende Struktur identifiziert wurde. Hier wurden Nervenzellen gefunden, die die Umgebung des Individuums in einer gitterartigen Anordnung repräsentieren. Die neuronalen Schaltkreise, welche diese geordnete Nervenzellaktivität im medialen entorhinalen Kortex (MEK) ermöglichen, sind noch wenig verstanden. Die vorliegende Dissertation hat eine Klärung der zellulären Architektur und der neuronalen Schaltkreise in der zweiten Schicht des MEK der Ratte zum Ziel. Zunächst werden die Beiträge zur Entdeckung der hexagonal angeordneten zellulären Anhäufungen in Schicht 2 des MEK sowie zur Beschreibung der Dichotomie der Haupt-Nervenzelltypen dargestellt. Im zweiten Teil wird erstmalig eine konnektomische Analyse des MEK beschrieben. Die detaillierte Untersuchung der Architektur einzelner exzitatorischer Axone ergab das überraschende Ergebnis der präzisen Sortierung von Synapsen entlang axonaler Pfade. Die neuronalen Schaltkreise, in denen diese Neurone eingebettet sind, zeigten eine starke zeitliche Bevorzugung der hemmenden Neurone. Die hier erhobenen Daten tragen zu einem detaillierteren Verständnis der neuronalen Schaltkreise im MEK bei. Sie enthalten die erste Beschreibung überraschend präziser axonaler synaptischer Ordnung im zerebralen Kortex der Säugetiere. Diese Schaltkreisarchitektur lässt einen Effekt auf die Weiterleitung synchroner elektrischer Populationsaktivität im MEK vermuten. In zukünftigen Studien muss insbesondere geklärt werden, ob es sich bei den hier berichteten Ergebnissen um eine Besonderheit des MEK oder ein generelles Verschaltungsprinzip der Hirnrinde des Säugetiers handelt.The mechanisms by which the electrical activity of ensembles of neurons in the brain give rise to an individual’s behavior are still largely unknown. Navigation in space is one important capacity of the brain, for which the medial entorhinal cortex (MEC) is a pivotal structure in mammals. At the cellular level, neurons that represent the surrounding space in a grid-like fashion have been identified in MEC. These so-called grid cells are located predominantly in layer 2 (L2) of MEC. The detailed neuronal circuits underlying this unique activity pattern are still poorly understood. This thesis comprises studies contributing to a mechanistic description of the synaptic architecture in rat MEC L2. First, this thesis describes the discovery of hexagonally arranged cell clusters and anatomical data on the dichotomy of the two principle cell types in L2 of the MEC. Then, the first connectomic study of the MEC is reported. An analysis of the axonal architecture of excitatory neurons revealed synaptic positional sorting along axons, integrated into precise microcircuits. These microcircuits were found to involve interneurons with a surprising degree of axonal specialization for effective and fast inhibition. Together, these results contribute to a detailed understanding of the circuitry in MEC. They provide the first description of highly precise synaptic arrangements along axons in the cerebral cortex of mammals. The functional implications of these anatomical features were explored using numerical simulations, suggesting effects on the propagation of synchronous activity in L2 of the MEC. These findings motivate future investigations to clarify the contribution of precise synaptic architecture to computations underlying spatial navigation. Further studies are required to understand whether the reported synaptic specializations are specific for the MEC or represent a general wiring principle in the mammalian cortex