Étude de la structure et de la fonction de l'espace extracellulaire du cerveau à l'aide de la microscopie à super-résolution

Every cell in the brain is embedded in a fluid called the extracellular space (ECS). Its structural complexity with intercellular gaps as narrow as ten nanometers, presents a challenge of visualizing the ECS in living brain tissue. Our recently established SUSHI technique overpasses this issue enabling to image the ECS with a nanoscale resolution. During my PhD training, I unveiled new structural information about ECS, as well as characterized novel tools to study it. My PhD work was divided into three projects, all aiming at investigating the structure and function of brain ECS. (1) Using the SUSHI approach, I studied the heterogeneity of ECS structure across hippocampal layers, which are known to have a very distinct cellular organization. My results show that ECS varies in volume and width across hippocampus, raising a question whether region-based differences in ECS structure in the hippocampus could support the unique anatomical as well as functional properties of each layer.(2) Chemical fixation leads to drastic shrinkage of ECS, yet it was only investigated in a context of electron microscopy. With help of SUSHI, I performed a systematic analysis of the impact of chemical fixation on brain tissue morphology. The results revealed only minor structural alteration, meaning that chemical fixation alone is not the reason for such dramatic effects. (3) Studying calcium signals in the ECS was a challenge since all available biosensors were not designed to measure ion concentrations with a millimolar affinity. Here, I characterized a novel tool, GreenT, to capture extracellular calcium dynamics. By imaging GreenT signals during electrophysiological stimulations, I was able to measure calcium fluctuations in the ECS upon neuronal activation. This is the beginning of applying this tool for physiology studies aiming at understanding the role of extracellular calcium.Les cellules cérébrales baignent dans un fluide appelé espace extracellulaire (ECS). Sa complexité structurelle et sa géométrie, avec des espaces intercellulaires de l’ordre de quelques dizaines de nanomètres, présentent un défi pour visualiser et étudier l'ECS dans le tissu cérébral vivant. Notre technique SUSHI récemment établie permet de surmonter ce problème et d’imager cet espace avec une résolution nanométrique. Au cours de ma formation doctorale, j'ai caractérisé des outils innovant pour étudier l'ECS, ce qui m’a permis de révéler de nouvelles informations structurelles. Mon travail de doctorat a été divisé en trois projets, tous visant à étudier la structure et la fonction de l’ECS cérébral.(1) En utilisant l'approche SUSHI, j'ai étudié l'hétérogénéité de la structure de l’ECS à travers les couches de l’hippocampe, qui sont connues pour avoir une organisation cellulaire très spécifique. L'objectif de ce projet était d'aider à comprendre la nature hétérogène de la morphologie ECS. En effet, j'ai découvert que l'ECS varie en volume et en largeur à travers l'hippocampe. Cela pourrait à son tour conduire à rechercher si les différences régionales dans la structure ECS dans l'hippocampe pourraient soutenir les propriétés anatomiques et fonctionnelles uniques de chaque couche.(2) La fixation chimique conduit à un rétrécissement drastique de l'ECS, mais elle n'a été étudiée que dans le cadre de la microscopie électronique et à grande échelle. Avec l'aide de SUSHI, j'ai effectué une analyse systématique de l'impact de la fixation chimique sur la morphologie des tissus cérébraux. Les résultats ont révélé que la fixation chimique seule n'est pas la raison de ces effets dramatiques, vus par d'autres, car nous n'avons observé que des altérations structurelles mineures. (3) L'étude des signaux calciques dans l'ECS était autrefois un défi car tous les biocapteurs disponibles n'étaient pas conçus pour mesurer les concentrations d'ions avec une affinité milli-molaire. Ce projet visait à caractériser un nouvel outil, GreenT, permettant de capter la dynamique du calcium extracellulaire. J'ai testé avec succès ce capteur et en imageant ses signaux tout en mesurant l’activité électrophysiologique, j'ai pu détecter les fluctuations du calcium dans l’ECS lors de stimulation neuronale

Investigating the structure and function of brain extracellular space using super-resolution microscopy

Les cellules cérébrales baignent dans un fluide appelé espace extracellulaire (ECS). Sa complexité structurelle et sa géométrie, avec des espaces intercellulaires de l’ordre de quelques dizaines de nanomètres, présentent un défi pour visualiser et étudier l'ECS dans le tissu cérébral vivant. Notre technique SUSHI récemment établie permet de surmonter ce problème et d’imager cet espace avec une résolution nanométrique. Au cours de ma formation doctorale, j'ai caractérisé des outils innovant pour étudier l'ECS, ce qui m’a permis de révéler de nouvelles informations structurelles. Mon travail de doctorat a été divisé en trois projets, tous visant à étudier la structure et la fonction de l’ECS cérébral.(1) En utilisant l'approche SUSHI, j'ai étudié l'hétérogénéité de la structure de l’ECS à travers les couches de l’hippocampe, qui sont connues pour avoir une organisation cellulaire très spécifique. L'objectif de ce projet était d'aider à comprendre la nature hétérogène de la morphologie ECS. En effet, j'ai découvert que l'ECS varie en volume et en largeur à travers l'hippocampe. Cela pourrait à son tour conduire à rechercher si les différences régionales dans la structure ECS dans l'hippocampe pourraient soutenir les propriétés anatomiques et fonctionnelles uniques de chaque couche.(2) La fixation chimique conduit à un rétrécissement drastique de l'ECS, mais elle n'a été étudiée que dans le cadre de la microscopie électronique et à grande échelle. Avec l'aide de SUSHI, j'ai effectué une analyse systématique de l'impact de la fixation chimique sur la morphologie des tissus cérébraux. Les résultats ont révélé que la fixation chimique seule n'est pas la raison de ces effets dramatiques, vus par d'autres, car nous n'avons observé que des altérations structurelles mineures. (3) L'étude des signaux calciques dans l'ECS était autrefois un défi car tous les biocapteurs disponibles n'étaient pas conçus pour mesurer les concentrations d'ions avec une affinité milli-molaire. Ce projet visait à caractériser un nouvel outil, GreenT, permettant de capter la dynamique du calcium extracellulaire. J'ai testé avec succès ce capteur et en imageant ses signaux tout en mesurant l’activité électrophysiologique, j'ai pu détecter les fluctuations du calcium dans l’ECS lors de stimulation neuronale.Every cell in the brain is embedded in a fluid called the extracellular space (ECS). Its structural complexity with intercellular gaps as narrow as ten nanometers, presents a challenge of visualizing the ECS in living brain tissue. Our recently established SUSHI technique overpasses this issue enabling to image the ECS with a nanoscale resolution. During my PhD training, I unveiled new structural information about ECS, as well as characterized novel tools to study it. My PhD work was divided into three projects, all aiming at investigating the structure and function of brain ECS. (1) Using the SUSHI approach, I studied the heterogeneity of ECS structure across hippocampal layers, which are known to have a very distinct cellular organization. My results show that ECS varies in volume and width across hippocampus, raising a question whether region-based differences in ECS structure in the hippocampus could support the unique anatomical as well as functional properties of each layer.(2) Chemical fixation leads to drastic shrinkage of ECS, yet it was only investigated in a context of electron microscopy. With help of SUSHI, I performed a systematic analysis of the impact of chemical fixation on brain tissue morphology. The results revealed only minor structural alteration, meaning that chemical fixation alone is not the reason for such dramatic effects. (3) Studying calcium signals in the ECS was a challenge since all available biosensors were not designed to measure ion concentrations with a millimolar affinity. Here, I characterized a novel tool, GreenT, to capture extracellular calcium dynamics. By imaging GreenT signals during electrophysiological stimulations, I was able to measure calcium fluctuations in the ECS upon neuronal activation. This is the beginning of applying this tool for physiology studies aiming at understanding the role of extracellular calcium

The impact of chemical fixation on the microanatomy of mouse brain tissue

Chemical fixation using paraformaldehyde (PFA) is a standard step for preserving cells and tissues for subsequent microscopic analyses such as immunofluorescence or electron microscopy. However, chemical fixation may introduce physical alterations in the spatial arrangement of cellular proteins, organelles and membranes. With the increasing use of super-resolution microscopy to visualize cellular structures with nanometric precision, assessing potential artifacts - and knowing how to avoid them - takes on special urgency.We addressed this issue by taking advantage of live-cell super-resolution microscopy that makes it possible to directly observe the acute effects of PFA on organotypic brain slices, allowing us to compare tissue integrity in a ‘before-and-after’ experiment. We applied super-resolution shadow imaging to assess the structure of the extracellular space (ECS) and regular super-resolution microscopy of fluorescently labeled neurons and astrocytes to quantify key neuroanatomical parameters.While the ECS volume fraction and micro-anatomical organization of astrocytes remained largely unaffected by the PFA treatment, we detected subtle changes in dendritic spine morphology and observed substantial damage to cell membranes. Our experiments show that PFA application via immersion does not cause a noticeable shrinkage of the ECS in brain slices, unlike the situation in transcardially perfused animals where the ECS typically becomes nearly depleted.In addition to the super-resolved characterization of fixation artefacts in identified cellular and tissue compartments, our study outlines an experimental strategy to evaluate the quality and pitfalls of various fixation protocols for the molecular and morphological preservation of cells and tissues

The dynamic recruitment of TRBP to neuronal membranes mediates dendritogenesis during development

MicroRNAs are important regulators of local protein synthesis during neuronal development. We investigated the dynamic regulation of microRNA production and found that the majority of the microRNA‐generating complex, consisting of Dicer, TRBP, and PACT, specifically associates with intracellular membranes in developing neurons. Stimulation with brain‐derived neurotrophic factor (BDNF), which promotes dendritogenesis, caused the redistribution of TRBP from the endoplasmic reticulum into the cytoplasm, and its dissociation from Dicer, in a Ca2+‐dependent manner. As a result, the processing of a subset of neuronal precursor microRNAs, among them the dendritically localized pre‐miR16, was impaired. Decreased production of miR‐16‐5p, which targeted the BDNF mRNA itself, was rescued by expression of a membrane‐targeted TRBP. Moreover, miR‐16‐5p or membrane‐targeted TRBP expression blocked BDNF‐induced dendritogenesis, demonstrating the importance of neuronal TRBP dynamics for activity‐dependent neuronal development. We propose that neurons employ specialized mechanisms to modulate local gene expression in dendrites, via the dynamic regulation of microRNA biogenesis factors at intracellular membranes of the endoplasmic reticulum, which in turn is crucial for neuronal dendrite complexity and therefore neuronal circuit formation and function.ISSN:1469-221XISSN:1469-317

The impact of chemical fixation on the microanatomy of mouse organotypic hippocampal slices

Chemical fixation using paraformaldehyde (PFA) is a standard step for preserving cells and tissues for subsequent microscopic analyses such as immunofluorescence or electron microscopy. However, chemical fixation may introduce physical alterations in the spatial arrangement of cellular proteins, organelles and membranes. With the increasing use of super-resolution microscopy to visualize cellular structures with nanometric precision, assessing potential artifacts - and knowing how to avoid them - takes on special urgency.We addressed this issue by taking advantage of live-cell super-resolution microscopy that makes it possible to directly observe the acute effects of PFA on organotypic hippocampal brain slices, allowing us to compare tissue integrity in a 'before-and-after' experiment. We applied super-resolution shadow imaging to assess the structure of the extracellular space (ECS) and regular super-resolution microscopy of fluorescently labeled neurons and astrocytes to quantify key neuroanatomical parameters.While the ECS volume fraction and micro-anatomical organization of astrocytes remained largely unaffected by the PFA treatment, we detected subtle changes in dendritic spine morphology and observed substantial damage to cell membranes. Our experiments show that PFA application via immersion does not cause a noticeable shrinkage of the ECS in hippocampal brain slices maintained in culture, unlike the situation in transcardially perfused animals in vivo where the ECS typically becomes nearly depleted.Our study outlines an experimental strategy to evaluate the quality and pitfalls of various fixation protocols for the molecular and morphological preservation of cells and tissues.Significance StatementChemical fixation of biological samples using PFA is a standard step routinely performed in neuroscience labs. However, it is known to alter various anatomical parameters ranging from protein distribution to cell morphology, potentially affecting our interpretation of anatomical data. With the increasing use of super-resolution microscopy, understanding the extent and nature of fixation artifacts is an urgent concern.Here, we use live STED microscopy to monitor in real time the impact of PFA on the microanatomy of organotypic hippocampal brain slices. Our results demonstrate that while PFA has little impact on the extracellular space and astrocytes, it compromises cell membranes and dendritic structures. Our study provides a strategy for a direct characterization of fixation artifacts at the nanoscale, facilitating the optimization of fixation protocols.</p

Nanoscale and functional heterogeneity of the hippocampal extracellular space

Summary: The extracellular space (ECS) and its constituents play a crucial role in brain development, plasticity, circadian rhythm, and behavior, as well as brain diseases. Yet, since this compartment has an intricate geometry and nanoscale dimensions, its detailed exploration in live tissue has remained an unmet challenge. Here, we used a combination of single-nanoparticle tracking and super-resolution microscopy approaches to map the nanoscale dimensions of the ECS across the rodent hippocampus. We report that these dimensions are heterogeneous between hippocampal areas. Notably, stratum radiatum CA1 and CA3 ECS differ in several characteristics, a difference that gets abolished after digestion of the extracellular matrix. The dynamics of extracellular immunoglobulins vary within these areas, consistent with their distinct ECS characteristics. Altogether, we demonstrate that ECS nanoscale anatomy and diffusion properties are widely heterogeneous across hippocampal areas, impacting the dynamics and distribution of extracellular molecules

Nanoscale imaging of the functional anatomy of the brain

Progress in microscopy technology has a long history of triggering major advances in neuroscience. Superresolution microscopy (SRM), famous for shattering the diffraction barrier of light microscopy, is no exception. SRM gives access to anatomical designs and dynamics of nanostructures, which are impossible to resolve using conventional light microscopy, from the elaborate anatomy of neurons and glial cells, to the organelles and molecules inside of them. In this review, we will mainly focus on a particular SRM technique (STED microscopy), and explain a series of technical developments we have made over the years to make it practical and viable in the field of neuroscience. We will also highlight several neurobiological findings on the dynamic structure-function relationship of neurons and glia cells, which illustrate the value of live-cell STED microscopy, especially when combined with other modern approaches to investigate the nanoscale behavior of brain cells

Fluorescent Sensors for Imaging Interstitial Calcium

info:eu-repo/semantics/publishe

Judaism in Contemporary Thought: Traces and Influence

The central aim of this collection is to trace the presence of Jewish tradition in contemporary philosophy. This presence is, on the one hand, undeniable, manifesting itself in manifold allusions and influences – on the other hand, difficult to define, rarely referring to openly revealed Judaic sources.Following the recent tradition of Lévinas and Derrida, this book tentatively refers to this mode of presence in terms of "traces of Judaism" and the contributors grapple with the following questions: What are these traces and how can we track them down? Is there such a thing as "Jewish difference" that truly makes a difference in philosophy? And if so, how can we define it? The additional working hypothesis, accepted by some and challenged by other contributors, is that Jewish thought draws, explicitly or implicitly, on three main concepts of Jewish theology, creation, revelation and redemption. If this is the case, then the specificity of the Jewish contribution to modern philosophy and the theoretical humanities should be found in – sometimes open, sometimes hidden – fidelity to these three categories.Offering a new understanding of the relationship between philosophy and theology, this book is an important contribution to the fields of Theology, Philosophy and Jewish Studies