Visualizing Shifts on Neuron-Glia Circuit with the Calcium Imaging Technique

• Published in: Journal of visualized experiments no. 182
• Main Authors: Tempone, Matheus H, Freitas, Hercules R, Schitine, Clarissa S, de Melo Reis, Ricardo A
• Format: Journal Article
• Language: English
• Published: United States 08-04-2022
• Subjects: Astrocytes; Calcium; Cell Communication - physiology; Neuroglia; Neurons
• ISSN: 1940-087X
• DOI: 10.3791/63338

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 Ca
concentration based on Ca
-free and Ca
-bound forms, presenting two different peaks, founded on the fluorescence intensity perceived on two wavelengths.