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 later improvements allowing these molecules to be used in live cell assays1. As a chemical indicator, Fura-2 is considered to be the standard for quantitative [Ca2+]i measurements. The acetoxymethyl (AM) ester of this indicator (i.e., Fura-2 AM) easily permeates the cell membrane and can reach intracellular concentrations 20-fold greater than the incubation dilution (e.g., [5 µM]o/[100 µM]i). Another advantage of Fura-2 is that it has good photobleaching resistance; thus, imaging this indicator for longer periods of time will not greatly affect its fluorescence capabilities. Finally, Fura-2 is sensitive to a wide range of calcium levels, from ~100 nM to ~100 µM, and has a Kd of ~145 nM, which is comparable to the resting [Ca2+]i2. Later, cell calcium imaging was developed with better fluorescent microscopes and computation methods, together with ratiometric probes that are not affected by dye loading.
Every cell expresses different calcium devices (pumps, transporters, receptors, and channels) that contribute to the final response as a particular signature. The important tip is to find selective responses of different types of cells correlated with their phenotypic expression. Accordingly, there are at least two different receptors that operate through calcium shifts: ionotropic receptors that permeate Ca2+ in a fast mode and slow metabotropic receptors coupled to signaling pathways and intracellular stocks that release Ca2+ activated by second messengers, such as inositol triphosphate and cyclic ADP-ribose3.
For example, progenitor cells express nestin in the immature retina and show GABAA receptors depolarized by GABA (or muscimol)4. This happens due to the Cl– electrochemical gradient with high intracellular Cl− levels; as the tissue develops, KCC2 transporters switch from excitation on progenitors to inhibition on mature GABAergic neurons5. On the other hand, stem cells that express sox-2 at the immature subventricular zone (SVZ) of postnatal rodents also present metabotropic H1 receptors activated by histamine increasing Ca2+ in a slow manner6. A second metabotropic receptor from the protease-activated receptor-1 (PAR-1) family, activated by thrombin and downstream to G(q/11) and phospholipase C (PLC), gives slow Ca2+ shifts in oligodendrocytes (that express O4 and PLP) generated from multipotent SVZ neural stem cells7.
In general, neurons express voltage-dependent calcium channels as well as major neurotransmitter receptors permeable to Ca2+, as glutamatergic (AMPA, NMDA, kainate) and peripheral and central nicotinic receptors. Potassium chloride is usually used as a depolarizing agent to activate peripheral neurons, as the dorsal root ganglion neurons8 or central neurons, as from subventricular zone9 or retina10. On the other hand, ATP is acknowledged as the major gliotransmitter (in addition to D-serine), which activates selective Ca2+ permeable P2X members, as P2X7 and P2X4. Both receptors present equivalent Ca2+ currents, similar to the ones shown by NMDA receptors acknowledged as the largest Ca2+ currents activated by transmitters11. P2X7 receptors are highly expressed on microglia, but at a lower density on astrocytes and oligodendrocytes, having a role in the release of proinflammatory cytokines12. P2X7 receptors are also expressed on Schwann cells13 and Müller glia in the retina14,15.
The retina is known to show almost all transmitters seen in the brain. For instance, the vertical axis (photoreceptors, bipolar and retinal ganglion cells) is mainly glutamatergic, with calcium-permeable AMPA or kainate receptors expressed in OFF-bipolar cells and mgluR6 expressed in ON-bipolar cells16. Curiously, all three receptors are also found in Müller glia, which are coupled to calcium and inositol triphosphate pathways17,18. The horizontal inhibitory axis, made by horizontal and amacrine cells, secrete not only GABA, but also dopamine, acetylcholine, and other classical neurotransmitters. Amacrine cells are the main types of cells found in the avian retinal cultures, showing several types of calcium operated channels, as glutamatergic, purinergic, nicotinic and voltage dependent calcium channels. For this reason, this is an excellent model to evaluate different properties of calcium shifts among neurons and glia.
Therefore, the combination of different receptors and channels summed to selective phenotypic markers during development with distinct agonist response patterns allows unique signatures in stem, progenitor, neuron, astrocyte, oligodendrocyte, and microglia that operate through selective signaling devices.
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
2. Retina dissection and cell culture preparation
3. Loading cells with Fura-2 AM
4. Calcium imaging of cells
5. Data processing
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-lysine (10 µg/mL) coverslips at a stage of 3 days in vitro (Figure 1B). In addition, purified Müller glia were maintained for 10 days in DMEM containing 10% FCS, when neurons were removed. In order to functionally quantify the responses of neurons and glia, cells were stimulated with 50 mM KCl or 1 mM ATP. As shown (Figure 1A), out of 302 cells, 50% responded to KCl while 53% signaled to ATP. In this sense, enriched neuronal cell culture had an 89% of calcium responses to KCl compared to 17% that responded to ATP (Figure 1B). Indeed, a purified Müller glia culture, where neurons are removed after 10 days in culture, were solely activated by ATP (Figure 1C).
Figure 1. Retinal cells prepared as mixed, neuronal-enriched or glia-purified cultures show different response patterns on calcium imaging. (A) Bright and fluorescence fields of mixed embryonic retinal cells in culture. The same microscope field shown under 5 µM fura-2 AM fluorescence. 50 mM KCl activates half of the cells (neuronal phenotype), while 1 mM ATP activates the other half (glial phenotype), with high F340/380 ratios corresponding to increases in intracellular calcium ([Ca2+]i) levels. (B) The enriched neuronal cell culture had an 89% calcium response to KCl compared to 17% that responded to ATP. (C) On the other hand, a purified Müller glia culture, where neurons are removed after 10 days in culture, was solely activated by ATP. Please click here to view a larger version of this figure.
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 receptors mediate neuronal effects summed to high extracellular ATP concentrations found in unhealthy tissues21.
We have previously linked the functional differentiation of retinal cells with their phenotypic display, in a way that variations of [Ca2+]i shifts that are activated by KCl or AMPA (a glutamate agonist) express microtubule associated protein (MAP-2), marker of a mature neuron1. Alternatively, cells activated by ATP express glutamine synthetase, a typical Muller glia marker.
We have been using different types of cell cultures as shown here (mixed, neuronal enriched or purified glial cells), in addition to neurospheres derived4 to answer many questions related to different neurochemical systems as glutamatergic22, dopaminergic19, GABAergic23, cannabinoid9,10, purinergic14,24, serotoninergic25, among others to understand the neuro-glial retinal communication. Müller glia express a great number of neurotransmitter receptors26 and secrete gliotransmitters as D-serine, ATP and glutamate, which may be released in a vesicular, Ca2+ dependent manner27.
The authors have nothing to disclose.
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).
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 |