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      Cell Calcium Imaging as a Reliable Method to Study Neuron–Glial Circuits

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          Abstract

          Complex dynamic cellular networks have been studied in physiological and pathological processes under the light of single-cell calcium imaging (SCCI), a method that correlates functional data based on calcium shifts operated by different intracellular and extracellular mechanisms integrated with their cell phenotypes. From the classic synaptic structure to tripartite astrocytic model or the recent quadripartite microglia added ensemble, as well as other physiological tissues, it is possible to follow how cells signal spatiotemporally to cellular patterns. This methodology has been used broadly due to the universal properties of calcium as a second messenger. In general, at least two types of receptor operate through calcium permeation: a fast-acting ionotropic receptor channel and a slow-activating metabotropic receptor, added to exchangers/transporters/pumps and intracellular Ca 2+ release activated by messengers. These prototypes have gained an enormous amount of information in dynamic signaling circuits. SCCI has also been used as a method to associate phenotypic markers during development and stage transitions in progenitors, stem, vascular cells, neuro- and glioblasts, neurons, astrocytes, oligodendrocytes, and microglia that operate through ion channels, transporters, and receptors. Also, cancer cells or inducible cell lines from human organoids characterized by transition stages are currently being used to model diseases or reconfigure healthy cells in terms of the expression of calcium-binding/permeable molecules and shed light on therapy.

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          Most cited references134

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          The capsaicin receptor: a heat-activated ion channel in the pain pathway.

          Capsaicin, the main pungent ingredient in 'hot' chilli peppers, elicits a sensation of burning pain by selectively activating sensory neurons that convey information about noxious stimuli to the central nervous system. We have used an expression cloning strategy based on calcium influx to isolate a functional cDNA encoding a capsaicin receptor from sensory neurons. This receptor is a non-selective cation channel that is structurally related to members of the TRP family of ion channels. The cloned capsaicin receptor is also activated by increases in temperature in the noxious range, suggesting that it functions as a transducer of painful thermal stimuli in vivo.
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            Fluorescent indicators for Ca2+ based on green fluorescent proteins and calmodulin.

            Important Ca2+ signals in the cytosol and organelles are often extremely localized and hard to measure. To overcome this problem we have constructed new fluorescent indicators for Ca2+ that are genetically encoded without cofactors and are targetable to specific intracellular locations. We have dubbed these fluorescent indicators 'cameleons'. They consist of tandem fusions of a blue- or cyan-emitting mutant of the green fluorescent protein (GFP), calmodulin, the calmodulin-binding peptide M13, and an enhanced green- or yellow-emitting GFP. Binding of Ca2+ makes calmodulin wrap around the M13 domain, increasing the fluorescence resonance energy transfer (FRET) between the flanking GFPs. Calmodulin mutations can tune the Ca2+ affinities to measure free Ca2+ concentrations in the range 10(-8) to 10(-2) M. We have visualized free Ca2+ dynamics in the cytosol, nucleus and endoplasmic reticulum of single HeLa cells transfected with complementary DNAs encoding chimaeras bearing appropriate localization signals. Ca2+ concentration in the endoplasmic reticulum of individual cells ranged from 60 to 400 microM at rest, and 1 to 50 microM after Ca2+ mobilization. FRET is also an indicator of the reversible intermolecular association of cyan-GFP-labelled calmodulin with yellow-GFP-labelled M13. Thus FRET between GFP mutants can monitor localized Ca2+ signals and protein heterodimerization in individual live cells.
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              Neurophysiological investigation of the basis of the fMRI signal.

              Functional magnetic resonance imaging (fMRI) is widely used to study the operational organization of the human brain, but the exact relationship between the measured fMRI signal and the underlying neural activity is unclear. Here we present simultaneous intracortical recordings of neural signals and fMRI responses. We compared local field potentials (LFPs), single- and multi-unit spiking activity with highly spatio-temporally resolved blood-oxygen-level-dependent (BOLD) fMRI responses from the visual cortex of monkeys. The largest magnitude changes were observed in LFPs, which at recording sites characterized by transient responses were the only signal that significantly correlated with the haemodynamic response. Linear systems analysis on a trial-by-trial basis showed that the impulse response of the neurovascular system is both animal- and site-specific, and that LFPs yield a better estimate of BOLD responses than the multi-unit responses. These findings suggest that the BOLD contrast mechanism reflects the input and intracortical processing of a given area rather than its spiking output.
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                Author and article information

                Contributors
                URI : http://loop.frontiersin.org/people/108791/overview
                URI : http://loop.frontiersin.org/people/506501/overview
                URI : http://loop.frontiersin.org/people/1087580/overview
                Journal
                Front Neurosci
                Front Neurosci
                Front. Neurosci.
                Frontiers in Neuroscience
                Frontiers Media S.A.
                1662-4548
                1662-453X
                02 October 2020
                2020
                : 14
                : 569361
                Affiliations
                [1] 1Laboratório de Neuroquímica, Instituto de Biofísica Carlos Chagas Filho, CCS, Universidade Federal do Rio de Janeiro , Rio de Janeiro, Brazil
                [2] 2Department of Pathology and Laboratory Medicine, MIND Institute, University of California, Davis , Sacramento, CA, United States
                Author notes

                Edited by: João O. Malva, University of Coimbra, Portugal

                Reviewed by: Xin Yu, Max Planck Institute for Biological Cybernetics, Germany; Fahmeed Hyder, Yale University, United States

                *Correspondence: Ricardo Augusto de Melo Reis, ramreis@ 123456biof.ufrj.br

                This article was submitted to Brain Imaging Methods, a section of the journal Frontiers in Neuroscience

                Article
                10.3389/fnins.2020.569361
                7566175
                33122991
                3d5e8c4b-e762-407c-9a4b-bc3e198ba7a4
                Copyright © 2020 de Melo Reis, Freitas and de Mello.

                This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

                History
                : 03 June 2020
                : 24 August 2020
                Page count
                Figures: 4, Tables: 0, Equations: 0, References: 134, Pages: 14, Words: 0
                Funding
                Funded by: Fundação Carlos Chagas Filho de Amparo à Pesquisa do Estado do Rio de Janeiro 10.13039/501100004586
                Funded by: Conselho Nacional de Desenvolvimento Científico e Tecnológico 10.13039/501100003593
                Funded by: Instituto Nacional de Ciência e Tecnologia de Neurociência Translacional 10.13039/501100007384
                Award ID: 465346/2014-6
                Categories
                Neuroscience
                Review

                Neurosciences
                calcium imaging,neuron–glia,atp,glioblastoma,fluorescent indicator
                Neurosciences
                calcium imaging, neuron–glia, atp, glioblastoma, fluorescent indicator

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