Studying Axon-Astrocyte Functional Interactions by 3D Two-Photon Ca2+ Imaging: A Practical Guide to Experiments and “Big Data” Analysis

Savtchouk, Iaroslav; Carriero, Giovanni; Volterra, Andrea

doi:10.3389/fncel.2018.00098

Cited by 16 publications

(10 citation statements)

References 47 publications

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“…Conventional 2D imaging of cells using fluorescent microscopes presents several problems including background noise arising from scattering, photobleaching, and an inability to capture cells from various planes (Gualda et al, 2014; Jonkman & Brown, 2015; Savtchouk, Carriero, & Volterra, 2018), thus leading to a reduction in the volume and total number of cells imaged. In contrast, confocal imaging provides control in choosing focal depth, while allowing higher spatial resolution through sampling larger number of z‐stacks (Jonkman & Brown, 2015).…”

Section: Discussionmentioning

confidence: 99%

“…To overcome these limitations with existing imaging modalities, we performed 3D imaging using resonance scanning, which permitted imaging of thick samples (Figure S5) and the visualization of cells at various depths. Resonance scanning also enabled imaging at higher speeds than possible with normal scanning (Figure S3), thus possibly reducing photobleaching and phototoxicity (Savtchouk et al, 2018). Though traditional laser scanning is typically limited in acquisition speed, the use of resonance mode enabled faster scanning because the galvanometer mirrors scanned back and forth (Jonkman & Brown, 2015).…”

Section: Discussionmentioning

confidence: 99%

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Three‐dimensional imaging and quantification of real‐time cytosolic calcium oscillations in microglial cells cultured on electrospun matrices using laser scanning confocal microscopy

Venkateswarlu

Gare

Dhyani

et al. 2020

Biotech & Bioengineering

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The development of a minimally invasive, robust, and inexpensive technique that permits real‐time monitoring of cell responses on biomaterial scaffolds can improve the eventual outcomes of scaffold‐based tissue engineering strategies. Towards establishing correlations between in situ biological activity and cell fate, we have developed a comprehensive workflow for real‐time volumetric imaging of spatiotemporally varying cytosolic calcium oscillations in pure microglial cells cultured on electrospun meshes. Live HMC3 cells on randomly oriented electrospun fibers were stained with a fluorescent dye and imaged using a laser scanning confocal microscope. Resonance scanning provided high‐resolution in obtaining the time‐course of intracellular calcium levels without compromising spatial and temporal resolution. Three‐dimensional reconstruction and depth‐coding enabled the visualization of cell location and intracellular calcium levels as a function of sample thickness. Importantly, changes in cell morphology and in situ calcium spiking were quantified in response to a soluble biochemical cue and varying matrix architectures (i.e., randomly oriented and aligned fibers). Importantly, raster plots generated from spiking data revealed calcium signatures specific to culture conditions. In the future, our approach can be used to elucidate correlations between calcium signatures and cell phenotype/activation, and facilitate the rational design of scaffolds for biomedical applications.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Three‐dimensional imaging and quantification of real‐time cytosolic calcium oscillations in microglial cells cultured on electrospun matrices using laser scanning confocal microscopy

Venkateswarlu

Gare

Dhyani

et al. 2020

Biotech & Bioengineering

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“…Another potential caveat of Ca 2+ imaging in slices was that the initiation points of Ca 2+ events may be located outside of the focal plane. This issue can be addressed in the future experiments with emerging technologies such as three-dimensional Ca 2+ imaging (Savtchouk, Carriero, & Volterra, 2018).…”

Section: Discussionmentioning

confidence: 99%

Morphological profile determines the frequency of spontaneous calcium events in astrocytic processes

Gordleeva²,

Tang

et al. 2018

Glia

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Astrocytes express a complex repertoire of intracellular Ca2+ transients (events) that represent a major form of signaling within individual cells and in astrocytic syncytium. These events have different spatiotemporal profiles, which are modulated by neuronal activity. Spontaneous Ca2+ events appear more frequently in distal astrocytic processes and independently from each other. However, little is known about the mechanisms underlying such subcellular distribution of the Ca2+ events. Here, we identify the initiation points of the Ca2+ events within the territory of single astrocytes expressing genetically encoded Ca2+ indicator GCaMP2 in culture or in hippocampal slices. We found that most of the Ca2+ events start in an optimal range of thin distal processes. Our mathematical model demonstrated that a high surface‐to‐volume of the thin processes leads to increased amplitude of baseline Ca2+ fluctuations caused by a stochastic opening of Ca2+ channels in the plasma membrane. Suprathreshold fluctuations trigger Ca2+‐induced Ca2+ release from the Ca2+ stores by activating inositol 1,4,5‐trisphosphate (IP3) receptors. In agreement with the model prediction, the spontaneous Ca2+ events frequency depended on the extracellular Ca2+ concentration. Astrocytic depolarization by high extracellular K+ increased the frequency of the Ca2+ events through activation of voltage‐gated Ca2+ channels in cultured astrocytes. Our results suggest that the morphological profile of the astrocytic processes is responsible for tuning of the Ca2+ events frequency. Therefore, structural plasticity of astrocytic processes can be directly translated into changes in astrocytic Ca2+ signaling. This may be important for both physiological and pathological astrocyte remodeling.

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“…Beyond characterizing the functionality of neurons, the ability to analyze interactions of neurons to glia is paramount to faithfully Kim et al 2018) and ischemic stroke (Hersh and Yang 2018). Powerful tools such as genetically encoded calcium reporters have allowed for the rapid analysis of signaling within astrocyte populations in response to neuronal activity (Savtchouk et al 2018). Apart from rapid signaling, characterizing the metabolic outputs resulting from neuron:astrocyte interaction has been studied in 3D substrates, specifically the monitoring of glucose and acetate metabolism (Simao et al 2016).…”

Section: Cell-cell Interactionsmentioning

confidence: 99%

Advances in 3D neuronal microphysiological systems: towards a functional nervous system on a chip

Anderson

Bosak

Högberg

et al. 2021

In Vitro Cell.Dev.Biol.-Animal

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Microphysiological systems (MPS) designed to study the complexities of the peripheral and central nervous systems have made marked improvements over the years and have allowed researchers to assess in two and three dimensions the functional interconnectivity of neuronal tissues. The recent generation of brain organoids has further propelled the field into the nascent recapitulation of structural, functional, and effective connectivities which are found within the native human nervous system. Herein, we will review advances in culture methodologies, focused especially on those of human tissues, which seek to bridge the gap from 2D cultures to hierarchical and defined 3D MPS with the end goal of developing a robust nervous system-on-a-chip platform. These advances have far-reaching implications within basic science, pharmaceutical development, and translational medicine disciplines.

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Studying Axon-Astrocyte Functional Interactions by 3D Two-Photon Ca2+ Imaging: A Practical Guide to Experiments and “Big Data” Analysis

Cited by 16 publications

References 47 publications

Three‐dimensional imaging and quantification of real‐time cytosolic calcium oscillations in microglial cells cultured on electrospun matrices using laser scanning confocal microscopy

Three‐dimensional imaging and quantification of real‐time cytosolic calcium oscillations in microglial cells cultured on electrospun matrices using laser scanning confocal microscopy

Morphological profile determines the frequency of spontaneous calcium events in astrocytic processes

Advances in 3D neuronal microphysiological systems: towards a functional nervous system on a chip

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