Corrado Calì

Three-dimensional immersive virtual reality for studying cellular compartments in 3D models from EM preparations of neural tissues

Advances in the application of electron microscopy (EM) to serial imaging are opening doors to new ways of analyzing cellular structure. New and improved algorithms and workflows for manual and semiautomated segmentation allow us to observe the spatial arrangement of the smallest cellular features with unprecedented detail in full three‐dimensions. From larger samples, higher complexity models can be generated; however, they pose new challenges to data management and analysis. Here we review some currently available solutions and present our approach in detail. We use the fully immersive virtual reality (VR) environment CAVE (cave automatic virtual environment), a room in which we are able to project a cellular reconstruction and visualize in 3D, to step into a world created with Blender, a free, fully customizable 3D modeling software with NeuroMorph plug‐ins for visualization and analysis of EM preparations of brain tissue. Our workflow allows for full and fast reconstructions of volumes of brain neuropil using ilastik, a software tool for semiautomated segmentation of EM stacks. With this visualization environment, we can walk into the model containing neuronal and astrocytic processes to study the spatial distribution of glycogen granules, a major energy source that is selectively stored in astrocytes. The use of CAVE was key to the observation of a nonrandom distribution of glycogen, and led us to develop tools to quantitatively analyze glycogen clustering and proximity to other subcellular features. J. Comp. Neurol. 524:23–38, 2016.

Abstractocyte: A Visual Tool for Exploring Nanoscale Astroglial Cells

This paper presents Abstractocyte, a system for the visual analysis of astrocytes and their relation to neurons, in nanoscale volumes of brain tissue. Astrocytes are glial cells, i.e., non-neuronal cells that support neurons and the nervous system. The study of astrocytes has immense potential for understanding brain function. However, their complex and widely-branching structure requires high-resolution electron microscopy imaging and makes visualization and analysis challenging. Furthermore, the structure and function of astrocytes is very different from neurons, and therefore requires the development of new visualization and analysis tools. With Abstractocyte, biologists can explore the morphology of astrocytes using various visual abstraction levels, while simultaneously analyzing neighboring neurons and their connectivity. We define a novel, conceptual 2D abstraction space for jointly visualizing astrocytes and neurons. Neuroscientists can choose a specific joint visualization as a point in this space. Interactively moving this point allows them to smoothly transition between different abstraction levels in an intuitive manner. In contrast to simply switching between different visualizations, this preserves the visual context and correlations throughout the transition. Users can smoothly navigate from concrete, highly-detailed 3D views to simplified and abstracted 2D views. In addition to investigating astrocytes, neurons, and their relationships, we enable the interactive analysis of the distribution of glycogen, which is of high importance to neuroscientists. We describe the design of Abstractocyte, and present three case studies in which neuroscientists have successfully used our system to assess astrocytic coverage of synapses, glycogen distribution in relation to synapses, and astrocytic-mitochondria coverage.

The effects of aging on neuropil structure in mouse somatosensory cortex—A 3D electron microscopy analysis of layer 1

This study has used dense reconstructions from serial EM images to compare the neuropil ultrastructure and connectivity of aged and adult mice. The analysis used models of axons, dendrites, and their synaptic connections, reconstructed from volumes of neuropil imaged in layer 1 of the somatosensory cortex. This shows the changes to neuropil structure that accompany a general loss of synapses in a well-defined brain region. The loss of excitatory synapses was balanced by an increase in their size such that the total amount of synaptic surface, per unit length of axon, and per unit volume of neuropil, stayed the same. There was also a greater reduction of inhibitory synapses than excitatory, particularly those found on dendritic spines, resulting in an increase in the excitatory/inhibitory balance. The close correlations, that exist in young and adult neurons, between spine volume, bouton volume, synaptic size, and docked vesicle numbers are all preserved during aging. These comparisons display features that indicate a reduced plasticity of cortical circuits, with fewer, more transient, connections, but nevertheless an enhancement of the remaining connectivity that compensates for a generalized synapse loss.

Norepinephrine stimulates glycogenolysis in astrocytes to fuel neurons with lactate

The mechanism of rapid energy supply to the brain, especially to accommodate the heightened metabolic activity of excited states, is not well-understood. We explored the role of glycogen as a fuel source for neuromodulation using the noradrenergic stimulation of glia in a computational model of the neural-glial-vasculature ensemble (NGV). The detection of norepinephrine (NE) by the astrocyte and the coupled cAMP signal are rapid and largely insensitive to the distance of the locus coeruleus projection release sites from the glia, implying a diminished impact for volume transmission in high affinity receptor transduction systems. Glucosyl-conjugated units liberated from glial glycogen by NE-elicited cAMP second messenger transduction winds sequentially through the glycolytic cascade, generating robust increases in NADH and ATP before pyruvate is finally transformed into lactate. This astrocytic lactate is rapidly exported by monocarboxylate transporters to the associated neuron, demonstrating that the astrocyte-to-neuron lactate shuttle activated by glycogenolysis is a likely fuel source for neuromodulation and enhanced neural activity. Altogether, the energy supply for both astrocytes and neurons can be supplied rapidly by glycogenolysis upon neuromodulatory stimulus.

GLAM: Glycogen-derived Lactate Absorption Map for visual analysis of dense and sparse surface reconstructions of rodent brain structures on desktop systems and virtual environments

Abstract Human brain accounts for about one hundred billion neurons, but they cannot work properly without ultrastructural and metabolic support. For this reason, mammalian brains host another type of cells called “glial cells”, whose role is to maintain proper conditions for efficient neuronal function. One type of glial cell, astrocytes, are involved in particular in the metabolic support of neurons, by feeding them with lactate, one byproduct of glucose metabolism that they can take up from blood vessels, and store it under another form, glycogen granules. These energy-storage molecules, whose morphology resembles to spheres with a diameter ranging 10–80 nanometers roughly, can be easily recognized using electron microscopy, the only technique whose resolution is high enough to resolve them. Understanding and quantifying their distribution is of particular relevance for neuroscientists, in order to understand where and when neurons use energy under this form. To answer this question, we developed a visualization technique, dubbed GLAM (Glycogen-derived Lactate Absorption Map), and customized for the analysis of the interaction of astrocytic glycogen on surrounding neurites in order to formulate hypotheses on the energy absorption mechanisms. The method integrates high-resolution surface reconstruction of neurites, astrocytes, and the energy sources in form of glycogen granules from different automated serial electron microscopy methods, like focused ion beam scanning electron microscopy (FIB-SEM) or serial block face electron microscopy (SBEM), together with an absorption map computed as a radiance transfer mechanism. The resulting visual representation provides an immediate and comprehensible illustration of the areas in which the probability of lactate shuttling is higher. The computed dataset can be then explored and quantified in a 3D space, either using 3D modeling software or virtual reality environments. Domain scientists have evaluated the technique by either using the computed maps for formulating functional hypotheses or for planning sparse reconstructions to avoid excessive occlusion. Furthermore, we conducted a pioneering user study showing that immersive VR setups can ease the investigation of the areas of interest and the analysis of the absorption patterns in the cellular structures.

Adding large EM stack support

Serial section electron microscopy (SSEM) image stacks generated using high throughput microscopy techniques are an integral tool for investigating brain connectivity and cell morphology. FIB or 3View scanning electron microscopes easily generate gigabytes of data. In order to produce analyzable 3D dataset from the imaged volumes, efficient and reliable image segmentation is crucial. Classical manual approaches to segmentation are time consuming and labour intensive. Semiautomatic seeded watershed segmentation algorithms, such as those implemented by ilastik image processing software, are a very powerful alternative, substantially speeding up segmentation times. We have used ilastik effectively for small EM stacks - on a laptop, no less; however, ilastik was unable to carve the large EM stacks we needed to segment because its memory requirements grew too large - even for the biggest workstations we had available. For this reason, we refactored the carving module of ilastik to scale it up to large EM stacks on large workstations, and tested its efficiency. We modified the carving module, building on existing blockwise processing functionality to process data in manageable chunks that can fit within RAM (main memory). We review this refactoring work, highlighting the software architecture, design choices, modifications, and issues encountered.

Dysfunction of homeostatic control of dopamine by astrocytes in the developing prefrontal cortex leads to cognitive impairments

Astrocytes orchestrate neural development by powerfully coordinating synapse formation and function and, as such, may be critically involved in the pathogenesis of neurodevelopmental abnormalities and cognitive deficits commonly observed in psychiatric disorders. Here, we report the identification of a subset of cortical astrocytes that are competent for regulating dopamine (DA) homeostasis during postnatal development of the prefrontal cortex (PFC), allowing for optimal DA-mediated maturation of excitatory circuits. Such control of DA homeostasis occurs through the coordinated activity of astroglial vesicular monoamine transporter 2 (VMAT2) together with organic cation transporter 3 and monoamine oxidase type B, two key proteins for DA uptake and metabolism. Conditional deletion of VMAT2 in astrocytes postnatally produces loss of PFC DA homeostasis, leading to defective synaptic transmission and plasticity as well as impaired executive functions. Our findings show a novel role for PFC astrocytes in the DA modulation of cognitive performances with relevance to psychiatric disorders.

A Process for Digitizing and Simulating Biologically Realistic Oligocellular Networks Demonstrated for the Neuro-Glio-Vascular Ensemble

One will not understand the brain without an integrated exploration of structure and function, these attributes being two sides of the same coin: together they form the currency of biological computation. Accordingly, biologically realistic models require the re-creation of the architecture of the cellular components in which biochemical reactions are contained. We describe here a process of reconstructing a functional oligocellular assembly that is responsible for energy supply management in the brain and creating a computational model of the associated biochemical and biophysical processes. The reactions that underwrite thought are both constrained by and take advantage of brain morphologies pertaining to neurons, astrocytes and the blood vessels that deliver oxygen, glucose and other nutrients. Each component of this neuro-glio-vasculature ensemble (NGV) carries-out delegated tasks, as the dynamics of this system provide for each cell-type its own energy requirements while including mechanisms that allow cooperative energy transfers. Our process for recreating the ultrastructure of cellular components and modeling the reactions that describe energy flow uses an amalgam of state-of the-art techniques, including digital reconstructions of electron micrographs, advanced data analysis tools, computational simulations and in silico visualization software. While we demonstrate this process with the NGV, it is equally well adapted to any cellular system for integrating multimodal cellular data in a coherent framework.