Biology subjects

Arizono, M.

Publications and source records attributed to Arizono, M..

5 recordsLinked to original sources

bioRxiv2023

Imaging 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.

neuroscience↗

bioRxiv2022

Shadow imaging for panoptical visualisation of living brain tissue

Progress in neuroscience research hinges on technical advances in visualizing living brain tissue with high fidelity and facility. Current neuroanatomical imaging approaches either require tissue fixation, do not have cellular resolution or only give a fragmented view. Here, we show how regular light microscopy together with fluorescence labeling of the interstitial fluid in the extracellular space provide comprehensive optical access in real-time to the anatomical complexity and dynamics of living brain tissue.

neuroscience↗

bioRxiv2021

Astrocyte nanoscale morphology controls Ca2+ signals 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. Table of ContentsO_ST_ABSMain PointsC_ST_ABSO_LIAstrocyte nano-morphology favors the compartmentalization of biochemical signals C_LIO_LIThis compartmentalization promotes local Ca2+ activity and signal propagation robustness C_LIO_LIIn contrast, its pathological remodeling upon swelling attenuates Ca2+ activity C_LI Table of Contents Image O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=102 SRC="FIGDIR/small/432635v4_fig1.gif" ALT="Figure 1"> View larger version (27K): org.highwire.dtl.DTLVardef@22b80forg.highwire.dtl.DTLVardef@1621218org.highwire.dtl.DTLVardef@191e971org.highwire.dtl.DTLVardef@751637_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOFigure 1:C_FLOATNO Proposed mechanisms that regulate astrocytic Ca2+ activity in perisynaptic astrocytic processes. Our simulation results demonstrate that the nano-morphology of astrocytic processes, consisting in the alternation of nodes and shafts, favors the compartmentalization of biochemical signals. This compartmentalization promotes local Ca2+ activity and signal propagation robustness. Astrocyte swelling, observed in pathological conditions such as brain injury, stroke and epilepsy, results in an increased shaft width without altering node size. Our results suggest that such pathological alterations of the nanoscale morphology of astrocytes result in a decreased local Ca2+ activity, which we confirm experimentally in hypo-osmotic conditions. Upon repeated neuronal stimuli, we predict that swelling hinders astrocytic signal propagation. Overall, this study highlights the impact of astrocyte nano-morphology on astrocyte activity at tripartite synapses, in health and disease. C_FIG

neuroscience↗

bioRxiv2021

Super-resolution shadow imaging reveals local remodeling of astrocytic microstructures and brain extracellular space after osmotic challenge

The extracellular space (ECS) plays a central role for brain physiology, shaping the time course and spread of neurochemicals, ions and nutrients that ensure proper brain homeostasis and neuronal communication. Astrocytes are the most abundant type of glia cell in the brain, whose processes densely infiltrate the brains parenchyma. As astrocytes are highly sensitive to changes in osmotic pressure, they are capable of exerting a potent physiological influence on the ECS. However, little is known about the spatial distribution and temporal dynamics of the ECS that surrounds astrocytes, owing mostly to a lack of appropriate techniques to visualize the ECS in live brain tissue. Mitigating this technical limitation, we applied the recent SUper-resolution SHadow Imaging technique (SUSHI) to astrocyte-labeled organotypic hippocampal brain slices, which allowed us to concurrently image the complex morphology of astrocytes and the ECS with nanoscale resolution in a live experimental setting. Focusing on ring-like astrocytic microstructures in the spongiform domain, we found them to enclose sizable pools of interstitial fluid and cellular structures like dendritic spines. Upon an experimental osmotic challenge, these microstructures remodeled and swelled up at the expense of the pools, effectively increasing the physical contact between astrocytic and cellular structures. Our study reveals novel facets of the dynamic microanatomical relationships between astrocytes, neuropil and the ECS in living brain tissue, which could be of functional relevance for neuronglia communication in a variety of (patho)physiological settings, e.g. LTP induction, epileptic seizures or acute ischemic stroke, where osmotic disturbances are known to occur.

neuroscience↗

bioRxiv2019

Simulation of calcium signaling in fine astrocytic processes: effect of spatial properties on spontaneous activity

Astrocytes, a glial cell type of the central nervous system, have emerged as detectors and regulators of neuronal information processing. Astrocyte excitability resides in transient variations of free cytosolic calcium concentration over a range of temporal and spatial scales, from sub-microdomains to waves propagating throughout the cell. Despite extensive experimental approaches, it is not clear how these signals are transmitted to and integrated within an astrocyte. The localization of the main molecular actors and the geometry of the system, including calcium channels IP3R spatial organization, are deemed essential. However, as most calcium signals occur in astrocytic ramifications that are too fine to be resolved by conventional light microscopy, most of those spatial data are unknown and computational modeling remains the only methodology to study this issue. Here, we propose an IP3R-mediated calcium signaling model for dynamics in such small sub-cellular volumes. To account for the expected stochasticity and low copy numbers, our model is both spatially explicit and particle-based. Extensive simulations show that spontaneous calcium signals arise in the model via the interplay between excitability and stochasticity. The model reproduces the main forms of calcium signals and indicates that their frequency crucially depends on the spatial organization of the IP3R channels. Importantly, we show that two processes expressing exactly the same calcium channels can display different types of calcium signals depending on channels spatial organization. Our model with realistic process volume and calcium concentrations successfully reproduces spontaneous calcium signals that we measured in calcium micro-domains with confocal microscopy. To our knowledge, this model is the first model suited to investigate calcium dynamics in fine astrocytic processes and to propose plausible mechanisms responsible for their variability.

neuroscience↗