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

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 Ca²⁺ 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.

Keywords: ATP; calcium imaging; fluorescent indicator; glioblastoma; neuron–glia.

FIGURE 1

Detecting intracellular calcium changes through single-cell calcium imaging. Single-cell calcium imaging evaluates intracellular Ca²⁺ dynamics through calcium probes as Fura-2 AM (A), a lipophilic acetate ester (AM) that permeates the cell membrane in an apolar environment (DMSO + pluronic F-127), allowing cleavage by intracellular esterases. These sensors show stronger fluorescence and better affinity for Ca²⁺ and selectivity against Mg²⁺ in the presence of Ca²⁺ shifts (B). Neurons and glia in bright field (C) or under fluorescence (D) when activated by KCl 50 mM (E) or ATP 1 mM (F) show selective responses. In parallel, the use of inducible genetically encoded calcium indicators and a variety of detection systems made probing neural cell behavior in live animals a possibility. Through a photoreceiver, it is possible to detect changes in cell fluorescence as the animal is awake and freely behaving. An optimized version of this technique can be obtained through multi-site photometry (G), which allows for the detection of spatiotemporal changes as the animal responds to varying environmental patterns. In this method, signals are detected by a complementary metal oxide semiconductor (CMOS) camera. Direct bidimensional fluorescence can be detected with the use of one-photon microendoscopes, capable of imaging a region of interest (ROI) inside the living brain, which in turn is detected by a CMOS camera. Two-photon endoscopes can be used to generate a high-fidelity view of a given ROI. In this case, images are generated after detection by photomultiplier tubes (H).

FIGURE 2

Matching cell phenotypes with calcium signaling activated by P2X7 receptors in retinal cells in culture. Progenitors unresponsive to ATP (A) differentiate into neurons (activated by KCl or glutamate) or glia (activated by ATP) in a cannabinoid-rich environment (B). Emerging calcium shifts induced by ATP are exclusive to Müller glia (green trace), primed by the early cannabinoid receptor activation (with WIN 55,212-2). On the other hand, neurons are activated by KCl and glutamate (red trace) (Freitas et al., 2019) (C). Instead of differentiating, some progenitors die (D) due to the activation of death-inducing pathways [bottom panel: live (calcein, green) and dead (ethidium homodimer-1, red)].

FIGURE 3

Recording dorsal root ganglia (DRG) cells from awake freely moving animals. Genetically coded calcium indicators (GECIs), which sense the concentration of Ca²⁺, allow for measurements of different cell types (A). Freely behaving mice injected with plantar formalin showed DRG neuronal activity associated with phasic pain behavior (Chen et al., 2019). This activity persisted for 5 weeks as a hallmark of neuronal hyperactivity associated with ongoing pain (B, bottom). Alternatively, GECI-expressing transgenic mice allow the study of excitatory and inhibitory synchronization properties of anesthetized and awake (B, top) animals (Knoblich et al., 2019).

FIGURE 4

Multiple Ca²⁺ pathways involved in cancer cells. P2X7 and TRPV1 receptors activate a strong calcium response in cancer cells (Strong and Daniels, 2017). Glutamate secretion upon ATP activation is excitotoxic to healthy tissue and linked to tumorigenicity into adjacent brain regions (Strong et al., 2018). The Na⁺/Ca²⁺ exchanger (NCX), which maintains cytoplasmic calcium homeostatic levels, also increases Ca²⁺ levels in glioblastoma cells (Song et al., 2014). Inhibition of the forward NCX (Ca²⁺ exit mode) induces a Ca²⁺-mediated injury in glioblastoma cells (Hu et al., 2019). Finally, G protein-coupled metabotropic receptors coupled to activation of phospholipase C result in the generation of the second messenger, inositol 1,4,5 trisphosphate, which increases intracellular Ca²⁺ and diacylglycerol, involved in cancer cell proliferation, with important participation of the large-conductance voltage- and Ca²⁺-activated K⁺ channel. Store-operated Ca²⁺ entry is mediated through Ca²⁺ release-activated Ca²⁺ current, composed by ORAI1, and stromal interaction molecule 1 (STIM-1), a luminal Ca²⁺ sensor transmembrane protein present in the endoplasmic reticulum membrane (Putney, 2009; De Bock et al., 2013). Both ORAI1 and STIM-1 are upregulated in primary human cell lines obtained from samples of glioblastoma (Motiani et al., 2013).