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 4D analysis of dendritic spine morphology: applications to stimulus-induced spine remodeling and pharmacological rescue in a disease model

Uncovering the mechanisms that regulate dendritic spine morphology has been limited, in part, by the lack of efficient and unbiased methods for analyzing spines. Here, we describe an automated 3D spine morphometry method and its application to spine remodeling in live neurons and spine abnormalities in a disease model. We anticipate that this approach will advance studies of synapse structure and function in brain development, plasticity, and disease

Dense 4D nanoscale reconstruction of living brain tissue

Three-dimensional (3D) reconstruction of living brain tissue down to an individual synapse level would create opportunities for decoding the dynamics and structure–function relationships of the brain’s complex and dense information processing network; however, this has been hindered by insufficient 3D resolution, inadequate signal-to-noise ratio and prohibitive light burden in optical imaging, whereas electron microscopy is inherently static. Here we solved these challenges by developing an integrated optical/machine-learning technology, LIONESS (live information-optimized nanoscopy enabling saturated segmentation). This leverages optical modifications to stimulated emission depletion microscopy in comprehensively, extracellularly labeled tissue and previous information on sample structure via machine learning to simultaneously achieve isotropic super-resolution, high signal-to-noise ratio and compatibility with living tissue. This allows dense deep-learning-based instance segmentation and 3D reconstruction at a synapse level, incorporating molecular, activity and morphodynamic information. LIONESS opens up avenues for studying the dynamic functional (nano-)architecture of living brain tissue

Three-dimensional reengineering of neuronal microcircuits : The cortical column in silico

The presented thesis will describe a pipeline to reengineer three-dimensional, anatomically realistic, functional neuronal networks with subcellular resolution. The pipeline consists of five methods: 1. "NeuroCount" provides the number and three-dimensional distribution of all neuron somata in large brain regions. 2. "NeuroMorph" provides authentic neuron tracings, comprising dendrite and axon morphology. 3. "daVinci" registers the neuron morphologies to a standardized reference framework. 4. "NeuroCluster" objectively groups the standardized tracings into anatomical neuron types. 5. "NeuroNet" combines the number and distribution of neurons and neuron-types with the standardized tracings and determines the neuron-type- and position-specific number of synaptic connections for any two types of neuron. The developed methods are demonstrated by reengineering the thalamocortical lemniscal microcircuit in the somatosensory system of rats. There exists an one-to-one correspondence between the sensory information obtained by a single facial whisker and segregated areas in the thalamus and cortex. The reengineering of this pathway results in a column-shaped network model of ~15200 excitatory full-compartmental cortical neurons. This network is synaptically connected to ~285 pre-synaptic thalamic neurons. Animation of this "cortical column in silico" with measured physiological input will help to gain a mechanistic understanding of neuronal sensory information processing in the mammalian brain

Automated dendritic spine tracking on 2-photon microscopic images (2-Foton mikroskopi görüntülerinde otomatik dendritik diken takibi)

The rapid and spontaneous morphological changes of dendritic spines have been an important observation to understand how information is stored in brain. Manual assessment of spine structure has been a useful tool to understand the differences between wild type (normal) and diseased cases. In order to perform a more through analysis, automatic tools need to be developed due to the immense amount of image data collected throughout the experiments. Additionally, dendritic spines are very dynamic structures and florescence microscopy contains high level of noise, blur and shift due to the optical properties. In this study, we track locations of dendritic spines in a full series of a time-lapse two photon microscopic images. To achieve this we propose a combined detection and tracking framework. For the detection we use a SIFT based algorithm, while the tracking requires a combination of registration and distance based spine matching. Experimental results show that this technique helps to track detected spines in time series even though the noise or blur deformed the image and complicated the detection

Technologies for imaging neural activity in large volumes

Neural circuitry has evolved to form distributed networks that act dynamically across large volumes. Collecting data from individual planes, conventional microscopy cannot sample circuitry across large volumes at the temporal resolution relevant to neural circuit function and behaviors. Here, we review emerging technologies for rapid volume imaging of neural circuitry. We focus on two critical challenges: the inertia of optical systems, which limits image speed, and aberrations, which restrict the image volume. Optical sampling time must be long enough to ensure high-fidelity measurements, but optimized sampling strategies and point spread function engineering can facilitate rapid volume imaging of neural activity within this constraint. We also discuss new computational strategies for the processing and analysis of volume imaging data of increasing size and complexity. Together, optical and computational advances are providing a broader view of neural circuit dynamics, and help elucidate how brain regions work in concert to support behavior

Enabling Scalable Neurocartography: Images to Graphs for Discovery

In recent years, advances in technology have enabled researchers to ask new questions predicated on the collection and analysis of big datasets that were previously too large to study. More specifically, many fundamental questions in neuroscience require studying brain tissue at a large scale to discover emergent properties of neural computation, consciousness, and etiologies of brain disorders. A major challenge is to construct larger, more detailed maps (e.g., structural wiring diagrams) of the brain, known as connectomes. Although raw data exist, obstacles remain in both algorithm development and scalable image analysis to enable access to the knowledge within these data volumes. This dissertation develops, combines and tests state-of-the-art algorithms to estimate graphs and glean other knowledge across six orders of magnitude, from millimeter-scale magnetic resonance imaging to nanometer-scale electron microscopy. This work enables scientific discovery across the community and contributes to the tools and services offered by NeuroData and the Open Connectome Project. Contributions include creating, optimizing and evaluating the first known fully-automated brain graphs in electron microscopy data and magnetic resonance imaging data; pioneering approaches to generate knowledge from X-Ray tomography imaging; and identifying and solving a variety of image analysis challenges associated with building graphs suitable for discovery. These methods were applied across diverse datasets to answer questions at scales not previously explored

Partial connectomes of labeled dopaminergic circuits reveal non-synaptic communication and axonal remodeling after exposure to cocaine

Dopaminergic (DA) neurons exert profound influences on behavior including addiction. However, how DA axons communicate with target neurons and how those communications change with drug exposure remains poorly understood. We leverage cell type-specific labeling with large volume serial electron microscopy to detail DA connections in the nucleus accumbens (NAc) of the mouse (Mus musculus) before and after exposure to cocaine. We find that individual DA axons contain different varicosity types based on their vesicle contents. Spatially ordering along individual axons further suggests that varicosity types are non-randomly organized. DA axon varicosities rarely make specific synapses (<2%, 6/410), but instead are more likely to form spinule-like structures (15%, 61/410) with neighboring neurons. Days after a brief exposure to cocaine, DA axons were extensively branched relative to controls, formed blind-ended ‘bulbs’ filled with mitochondria, and were surrounded by elaborated glia. Finally, mitochondrial lengths increased by ~2.2 times relative to control only in DA axons and NAc spiny dendrites after cocaine exposure. We conclude that DA axonal transmission is unlikely to be mediated via classical synapses in the NAc and that the major locus of anatomical plasticity of DA circuits after exposure to cocaine are large-scale axonal re-arrangements with correlated changes in mitochondria