Increase, intracellular calcium

Calcium is arguably the most versatile and important intracellular messenger in neurons (Berridge et al., 2000). Interestingly, although calcium may often promote neuronal death, it can also activate pathways that promote survival. For example, calcium can promote survival through a pathway involving activation of protein kinase B (PKB/Akt) by calcium/calmodulin-dependent protein kinase (Yano et al., 1998). Calcium is a prominent regulator of cellular responses to stress, activating transcription through the cyclic-AMP response element-binding protein (CREB), which can promote neuron survival in experimental models of developmental cell death (Hu et al., 1999). Calcium can also activate a rapid neuroprotective signalling pathway in which the calcium-activated actin-severing protein gelsolin induces actin depolymerization, resulting in suppression of calcium influx through membrane NMDA (N-methyl-d-aspartate) receptors and voltage-dependent calcium channels (Furukawa et al., 1997). This may occur through intermediary actin-binding proteins that interact with NMDA receptor and calcium channel proteins. Finally, signals such as calcium and secreted amyloid precursor protein-α (sAPP-α), which increase cyclic GMP production, can induce activation of potassium channels and the transcription factor NF-κB, and thereby increase resistance of neurons to excitotoxic apoptosis (Furukawa et al., 1996).

An increase in [Ca^2+]i was measured using Fluo3 AM as an indicator dye after the addition of metals (single or in mixture) to the culture wells following an optimized protocol (Arey et al., 2022). The fluorescent signals were read by fluorescence imaging plate reader Synergy HT (BioTek, Winooski, VT) (Rai and others 2010).

Briefly, Ca2+ levels in human astrocytes were monitored by fluorescence microscopy using the Ca2+ indicator fluo-4. Slices were incubated with fluo-4-AM (2–5 µL of 2 mM dye were dropped over the tissue, attaining a final concentration of 2–10 µM and 0.01% of pluronic) and Sulforhodamine 101 (100 µM) for 30–60 min at room temperature (Navarrete and others 2013). In these conditions, most of the Fluo-4-loaded cells were astrocytes as indicated by their SR101 staining (Nimmerjahn et al., 2004; Dombeck et al., 2007; Kafitz et al., 2008; Takata and Hirase 2008), and confirmed in some cases by their electrophysiological properties. Astrocytes were imaged with an Olympus FV300 laser-scanning confocal microscope or a CCD camera (Retiga EX) attached to the Olympus BX50WI microscope (Navarrete and others 2013).

Diversity of endogenous Ca2+ activity in a mature hippocampal astrocyte in situ: Ca2+ signals in cell body and processes are different. (A) Cumulative Ca2+ activity recorded in an astrocyte over a 165 s period revealed by the calcium indicator Fluo4-AM. The visible boundaries of the astrocyte are shown in white. Note the different intensities of spatially- confined local activity in the astrocyte cell body (s), primary process (p1) stemming from the soma and secondary processes (p2) branching from a primary process. Intensity of the normalized cumulative activity is expressed in arbitrary units (a.u.) and shown in pseudocolour, from dark (lowest) to white (highest). (B) Frequency map of the Ca2+ activity in the astrocyte during the 165 s period as in A. Activity is measured in individual pixels, expressed in mHz and color-coded from black (never active) to dark red (frequently active). Most of the activity is within the white boundaries and the most frequently active pixels are in defined small regions (arrowheads) of the primary and secondary processes (30 mHz), whereas pixels of the soma are less active (~10 mHz) (Volterra et al., 2014). 

Free intracellular calcium ions were measured using the fluorescent calcium indicator FLUO-3/AM (Molecular probes, Eugene, OR, USA). Cells (4 × 10⁴ cells/cm²) were seeded in 24-well plates for 24 h to reach 60%–70%, and then treated for 24 h with As(III) (0.5 and 1 mg/l), or coexposed to As(III) (1 mg/l) and F (2.5, 5, and 10 mg/l). After treatment, supernatant was collected and combined with trypsinized cells. Pelleted samples were resuspended in 500 μl of FLUO-3/AM (4 μmol/l) and incubated at 37 °C for 30 min. After centrifugation, cells were washed with HBSS (Hank's Buffered Salt Solution, Sigma), made up to 400 μl with HBSS and analyzed by flow cytometry. The signal from FLUO-3/AM bound to Ca²⁺ was recorded using the Fl-1 channel (Rocha et al., 2011).

Fluo-4/AM was used as an intracellular free Ca²⁺ fluorescent probe to analyze [Ca²⁺]_i in Cd-exposed cerebral cortical neurons. In short, the harvested cells were incubated with Fluo-4/AM (5 µmol/L final concentration) for 30 min at 37°C in the dark, washed with PBS, and analyzed on a BD-FACS Aria flow cytometry. Intracellular [Ca²⁺]_i levels were represented by fluorescent intensity. Fluorescent intensity was recorded by excitation at 494 nm and emission at 516 nm. The data were analyzed by Cell Quest program (Becton Dickinson), and the mean fluorescence intensity was obtained by histogram statistics (Yuan et al., 2013).

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