Audrey Denizot

Astrocytes modulate brain processes such as neurotransmitter signaling, ion homeostasis, vascular tone, metabolism and synaptic transmission, and serve a critical role in complex brain functions. However, it remains unclear on which spatial scales these functions are organized in astrocytes and their networks: for instance, whether astrocytic interactions with neurons are organized into units at the level of individual perisynaptic processes and synapses, astrocytic domains covering synapse populations, or astrocytic networks controlling local neuronal circuits. Here we analyze astrocytic morphological features and molecular heterogeneity to define the multiple spatial scales on which astrocytes operate. We further discuss evidence regarding their intracellular and intercellular signaling, their role in neurotransmitter homeostasis and their neurotransmitter-mediated dialog with neurons, supporting the existence of astrocytic functional units with distinct spatial scales. We propose that astrocytes constitute a multilayered system of functional units that operate across multiple spatial scales, thereby increasing the degrees of freedom in brain information processing.

A growing number of studies indicate the possible involvement of astrocytes in triggering or modulating neurovascular coupling (NVC), i.e. the local dilation of blood vessels in the brain in response to neuronal activity. Astrocytes possess specialized subcellular compartments, named endfeet, that surround arterioles and capillaries, ideally positioned to mediate NVC. Various vasodilators have been shown to contribute to NVC, such as epoxyeicosatrienoic acid (EET), nitric oxide (NO), or prostaglandin E2 (PGE2), but the precise mechanisms underlying NVC and their variability remain to be fully elucidated. In particular, the involvement of astrocytes in this process is controversial. Recent translatome and proteomics data reveal that astrocytes and in particular endfeet are enriched in the proteins of the PGE2 pathway. However, how the latter could contribute to NVC remains to be characterized. Here, we develop a computational model of astrocyte-mediated NVC that recapitulates these findings and describes Ca2+ and PGE2 signaling in astrocytes, NO release by neurons, and arteriole diameter dynamics using ordinary differential equations. The model successfully reproduces the dynamics of arteriole diameter change during hyperemia from in vivo neocortical recordings in awake mice. Our simulations suggest that the astrocyte PGE2 pathway could be responsible for the late response of NVC at the arteriolar level. We further observe that PIP2-derived diacylglycerol plays a major role in driving arteriole diameter dynamics in our model, while phosphatidic acid-derived diacylglycerol, which is calcium-dependent, mainly acts as an amplifier of this response. Finally, a spatial implementation of the model using a simplified astrocyte geometry suggests that NVC is more efficient when synaptic stimulation occurs at the endfoot level rather than at other astrocytic compartments. Overall, this computational study suggests a partial role for astrocyte-mediated PGE2 release in NVC and points to astrocyte perivascular processes as sub-compartments that are ideally positioned and equipped to mediate NVC.

Simulation de la signalisation calcique dans les prolongements fins astrocytaires: Les astrocytes sont des cellules gliales du système nerveux central, essentielles à la formation des synapses, à la barrière hémato-encéphalique ainsi qu’au maintien de l'homéostasie. Récemment, les astrocytes ont été identifiés comme éléments clés du traitement de l'information dans le système nerveux central. Les astrocytes peuvent communiquer avec les neurones au niveau des synapses et moduler la communication neuronale en libérant des gliotransmetteurs et en absorbant des neurotransmetteurs. L’utilisation de nouvelles techniques comme la microscopie à super-résolution et les indicateurs calciques encodés génétiquement a permis de révéler une grande diversité spatio-temporelle des signaux calciques astrocytaires. La majorité de ces signaux sont observés au sein de leurs prolongements cellulaires, qui sont le site de communication entre neurones et astrocytes. Ces prolongements sont trop fins pour être observés en microscopie optique conventionnelle, de sorte que la microscopie à super-résolution et la modélisation informatique sont les seules méthodes adaptées à leur étude. Les travaux présentés dans cette thèse ont pour but d’étudier l'effet des propriétés spatiales (telles que la géométrie cellulaire, les distributions moléculaires et la diffusion) sur les signaux calciques dans les prolongements astrocytaires. Historiquement, les signaux calciques ont été modélisés à l'aide d'approches déterministes non spatiales. Ces modèles ont permis l'étude des signaux calciques à l’échelle de la cellule entière voire à l’échelle du réseau de cellules. Ces méthodes ne prennent cependant pas en compte la stochasticité inhérente aux interactions moléculaires ainsi que les effets de diffusion, qui jouent un rôle important dans les petits volumes. Cette thèse présente un modèle stochastique et spatial qui a été développé dans le but d’étudier les signaux calciques dans les prolongements fins astrocytaires. Ce travail a été réalisé en collaboration avec des expérimentateurs, qui nous ont fourni des données de microscopie électronique et à super-résolution. Ces données ont permis de valider le modèle. Les simulations du modèle suggèrent que (1) la diffusion moléculaire, fortement influencée par la concentration et la cinétique des buffers calciques endogènes et exogènes, (2) l'organisation spatiale intracellulaire des molécules, notamment le co-clustering des canaux calciques, (3) la géométrie du reticulum endoplasmique et sa localisation dans la cellule, (4) la géométrie cellulaire influencent fortement les signaux calciques et pourraient être responsables de leur grande diversité spatio-temporelle. Ces travaux contribuent à une meilleure compréhension du traitement de l’information par les astrocytes, un prérequis pour une meilleure compréhension de la communication entre les neurones et les astrocytes ainsi que de son influence sur le fonctionnement du cerveau.

Astrocytes are now widely accepted as key regulators of brain function and behavior. Calcium (Ca ²⁺ ) signals in perisynaptic astrocytic processes (PAPs) enable astrocytes to fine‐tune neurotransmission at tripartite synapses. As most PAPs are below the diffraction limit, their content in Ca ²⁺ stores and the contribution of the latter to astrocytic Ca ²⁺ activity is unclear. Here, we reconstruct hippocampal tripartite synapses in 3D from a high‐resolution electron microscopy (EM) dataset and find that 75% of PAPs contain some endoplasmic reticulum (ER), a major calcium store in astrocytes. The ER in PAPs displays strikingly diverse shapes and intracellular spatial distributions. To investigate the causal relationship between each of these geometrical properties and the spatiotemporal characteristics of Ca ²⁺ signals, we implemented an algorithm that generates 3D PAP meshes by altering the distribution of the ER independently from ER and cell shape. Reaction–diffusion simulations in these meshes reveal that astrocyte activity is governed by a complex interplay between the location of Ca ²⁺ channels, ER surface–volume ratio, and spatial distribution. In particular, our results suggest that ER‐PM contact sites can act as local signal amplifiers if equipped with IP ₃ R clusters but attenuate PAP Ca ²⁺ activity in the absence of clustering. This study sheds new light on the ultrastructural basis of the diverse astrocytic Ca ²⁺ microdomain signals and on the mechanisms that regulate neuron‐astrocyte signal transmission at tripartite synapses.

Much of the Ca²⁺ activity in astrocytes is spatially restricted to microdomains and occurs in fine processes that form a complex anatomical meshwork, the so‐called spongiform domain. A growing body of literature indicates that those astrocytic Ca²⁺ signals can influence the activity of neuronal synapses and thus tune the flow of information through neuronal circuits. Because of technical difficulties in accessing the small spatial scale involved, the role of astrocyte morphology on Ca²⁺ microdomain activity remains poorly understood. Here, we use computational tools and idealized 3D geometries of fine processes based on recent super‐resolution microscopy data to investigate the mechanistic link between astrocytic nanoscale morphology and local Ca²⁺ activity. Simulations demonstrate that the nano‐morphology of astrocytic processes powerfully shapes the spatio‐temporal properties of Ca²⁺ signals and promotes local Ca²⁺ activity. The model predicts that this effect is attenuated upon astrocytic swelling, hallmark of brain diseases, which we confirm experimentally in hypo‐osmotic conditions. Upon repeated neurotransmitter release events, the model predicts that swelling hinders astrocytic signal propagation. Overall, this study highlights the influence of the complex morphology of astrocytes at the nanoscale and its remodeling in pathological conditions on neuron‐astrocyte communication at so‐called tripartite synapses, where astrocytic processes come into close contact with pre‐ and postsynaptic structures.

In this review article, we present the major insights from and challenges faced in the acquisition, analysis and modeling of astrocyte calcium activity, aiming at bridging the gap between those fields to crack the complex astrocyte “Calcium Code”. We then propose strategies to reinforce interdisciplinary collaborative projects to unravel astrocyte function in health and disease.

In this review article, we present the major insights from and challenges faced in the acquisition, analysis and modeling of astrocyte calcium activity, aiming at bridging the gap between those fields to crack the complex astrocyte \enquote{Calcium Code}. We then propose strategies to reinforce interdisciplinary collaborative projects to unravel astrocyte function in health and disease.

Astrocytes are now widely accepted as key regulators of brain function and behavior. Calcium (Ca ²⁺ ) signals in perisynaptic astrocytic processes (PAPs) enable astrocytes to fine-tune neurotransmission at tripartite synapses. As most PAPs are below the diffraction limit, their content in Ca ²⁺ stores and the contribution of the latter to astrocytic Ca ²⁺ activity is unclear. Here, we reconstruct hippocampal tripartite synapses in 3D from a high resolution electron microscopy (EM) dataset and find that 75% of PAPs contain some endoplasmic reticulum (ER), a major calcium store in astrocytes. The ER in PAPs displays strikingly diverse shapes and intracellular spatial distributions. To investigate the causal relationship between each of these geometrical properties and the spatio-temporal characteristics of Ca ²⁺ signals, we implemented an algorithm that generates 3D PAP meshes by altering the distribution of the ER independently from ER and cell shape. Reaction-diffusion simulations in these meshes reveal that astrocyte activity is governed by a complex interplay between the location of Ca ²⁺ channels, ER surface-volume ratio and spatial distribution. In particular, our results suggest that ER-PM contact sites can act as local signal amplifiers if equipped with IP ₃ R clusters but attenuate PAP Ca ²⁺ activity in the absence of clustering. This study sheds new light on the ultrastructural basis of the diverse astrocytic Ca ²⁺ microdomain signals and on the mechanisms that regulate neuron-astrocyte signal transmission at tripartite synapses. Main points: 6 nm isotropic volume EM reveals that 75% of perisynaptic astrocytic processes (PAPs) contain some ER PAPs & ER of the same cell display diverse geometric properties Simulations in EM-derived meshes hint that ER geometry governs Ca ²⁺ signals in PAPs TOC: