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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.
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.
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....
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
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.......
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.......
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).
....Name | Company | Catalog Number | Comments |
15 mm coverslip | Paul Marienfeld GmbH & Co. KG | 111550 | Cell suport |
510 nm long-pass filter | Carl Zeiss | ||
ATP | Sigma | A1852 | |
B-27 Supplement | Gibco | 17504044 | Suplement |
CaCl2 | Sigma-Aldrich | C8106 | |
CoolSNAP digital camera | Roper Scientific, Trenton, NJ | ||
D-(+)-Glucose | Neon | 1466 | |
DMEM/ F-12 | Gibco | 12400-24 | Cell culture medium |
Excel Software | Microsoft | ||
Fetal Calf Serum | Sigma-Aldrich | F9665 | Suplement |
Fluorescence Microscope | Axiovert 200; Carl Zeiss | B 40-080 | |
Fura-2 AM | Molecular Probes | F1221 | Ratiometric Ca2+ indicator |
Gentamicin Sulfate | Calbiochem | 1405-41-0 | antibiotics |
HEPES | Sigma | H4034 | |
KCl | Sigma | P5405 | |
KH2PO4 | Sigma | P5655 | |
Lambda DG-4 apparatus | Sutter Instrument, Novato, CA | DG-4PLUS/OF30 | |
Laminin | Gibco | 23017-015 | Help cell adhesion |
Metafluor software | Universal Imaging Corp. West Chester, PA | ||
MgCl2 | Sigma | M4880 | |
Na2HPO4 | Vetec | 129 | |
NaCl | Isofar | 310 | |
NaHCO3 | Vetec | 306 | |
PH3 platform | Warner Intruments, Hamden, CT | 64-0286 | |
Pluronic F-127 | Molecular Probes | P6866 | nonionic, surfactant |
Poly-L-lysine | Sigma-Aldrich | P8920 | Help cell adhesion |
Trypsin-EDTA 0.25% | Gibco | 25200056 | Dissociation enzyme |
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