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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

Cell calcium imaging is a versatile methodology to study dynamic signaling of individual cells, on mixed populations in culture or even on awakened animals, based on the expression of calcium-permeable channels/receptors that gives unique functional signatures.

Abstract

Here, we report on selective in vitro models of circuits based on glia (astrocytes, oligodendrocytes, and microglia) and/or neurons from peripheral (dorsal root ganglia) and central tissues (cortex, subventricular zone, organoid) that are dynamically studied in terms of calcium shifts. The model chosen to illustrate the results is the retina, a simple tissue with complex cellular interactions. Calcium is a universal messenger involved in most of the important cellular roles. We explain in a step-by-step protocol how retinal neuron-glial cells in culture can be prepared and evaluated, envisioning calcium shifts. In this model, we differentiate neurons from glia based on their selective response to KCl and ATP. Calcium permeable receptors and channels are selectively expressed in different compartments. To analyze calcium responses, we use ratiometric fluorescent dies such as Fura-2. This probe quantifies free Ca2+ concentration based on Ca2+-free and Ca2+-bound forms, presenting two different peaks, founded on the fluorescence intensity perceived on two wavelengths.

Introduction

Due to the universal properties of calcium as a second messenger, this ion is involved in a vast number of signaling activities: gene transcription, birth and death, proliferation, migration and differentiation, synaptic transmission, and plasticity. Hence, a method capable of tracking calcium activation dynamics with fidelity and agility would provide a way to observe unique spatial-temporal responses. Such a method is the cellular calcium imaging technique, which correlates calcium shifts functional data with specific cell phenotypes based on their distinct responses.

Ca2+ probes were first developed in the 1980's, with lat....

Protocol

All experiments involving animals were approved by and carried out following the guidelines of the Institutional Animal Care and Use Committee of the Federal University of Rio de Janeiro, following the "Principles of Laboratory Animal Care" (NIH, Bethesda, USA); permit number IBCCF-035 for fertilized White Leghorn chicken eggs.

1. Preparation of solutions

  1. Prepare Krebs solution: (132 mM NaCl, 4 mM KCl, 1.4 mM MgCl2, 2.5 mM CaCl2, 6 mM .......

Representative Results

Here, we used retina cells in culture from embryonic day 8 chicks to investigate how neurons and glia signal in terms of calcium shifts. Cultures were prepared essentially as described15,19 as mixed neuron-glial cells (at a density of ≥ 1 x 106 cell/dish) at a stage of 7 days in vitro (Figure 1A). Alternatively, enriched neuronal cells prepared in low density (5 x 105 cell/dish), seeded on treated poly-L-l.......

Discussion

We have used the retina tissue to show that calcium responses mediated by KCl or ATP are clearly compartmentalized into neuronal and glial responses, respectively (Figure 1). Although some data in the literature imply that P2X7 receptors are expressed in neurons, which regulate neuronal activity and synaptic neurotransmitter release20, other authors question the existence of neuronal P2X7 receptors. Indeed, current results sustain the idea that primary glial P2X7 rece.......

Acknowledgements

Grants, sponsors, and funding sources: MH is recipient of a PhD CNPq fellowship. HRF is recipient of a postdoc fellowship supported by CNPq (HRF grant number 152071/2020-2). RAMR is supported by CNPq and FAPERJ (grant numbers E-26/202.668/2018, E-26/010.002215/2019, 426342/2018-6 and 312157/2016-9 and INCT-INNT (National Institute for Translational Neuroscience).

....

Materials

NameCompanyCatalog NumberComments
15 mm coverslipPaul Marienfeld GmbH & Co. KG111550Cell suport
510 nm long-pass filterCarl Zeiss
ATPSigmaA1852
B-27 SupplementGibco17504044Suplement
CaCl2Sigma-AldrichC8106
CoolSNAP digital cameraRoper Scientific, Trenton, NJ
D-(+)-GlucoseNeon1466
DMEM/ F-12Gibco12400-24Cell culture medium
Excel SoftwareMicrosoft
Fetal Calf SerumSigma-AldrichF9665Suplement
Fluorescence MicroscopeAxiovert 200; Carl ZeissB 40-080
Fura-2 AMMolecular ProbesF1221Ratiometric Ca2+ indicator
Gentamicin SulfateCalbiochem1405-41-0antibiotics
HEPESSigmaH4034
KClSigmaP5405
KH2PO4SigmaP5655
Lambda DG-4 apparatusSutter Instrument, Novato, CADG-4PLUS/OF30
LamininGibco23017-015Help cell adhesion
Metafluor softwareUniversal Imaging Corp. West Chester, PA
MgCl2SigmaM4880
Na2HPO4Vetec129
NaClIsofar310
NaHCO3Vetec306
PH3 platformWarner Intruments, Hamden, CT64-0286
Pluronic F-127Molecular ProbesP6866nonionic, surfactant
Poly-L-lysineSigma-AldrichP8920Help cell adhesion
Trypsin-EDTA 0.25%Gibco25200056Dissociation enzyme

References

  1. Tsien, R. Y., Pozzan, T., Rink, T. J. Calcium homeostasis in intact lymphocytes: cytoplasmic free calcium monitored with a new, intracellularly trapped fluorescent indicator. Journal of Cell Biology. 94 (2), 325-334 (1982).
  2. Paredes, R. M., Etzler, J. C., Watts, L. T., Zheng, W., Lechleiter, J. D.

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