Calcium (Ca²⁺) and zinc (Zn²⁺) are important cations in neuronal and glial cells and are known to play structural and signaling roles to mediate diverse cellular functions ranging from cell division and differentiation to cell death (Berridge et al., 2000; Andreini et al., 2006; Foskett et al., 2007; Granzotto et al., 2020). The concentration of intracellular Ca²⁺ ([Ca²⁺]_i) at rest is normally maintained around 100 nM and it is about 10,000-fold lower than that of extracellular environment, whereby any slight change in [Ca²⁺]_i would be perceived as specific signal coupled to a specific response (stimulus-response coupling). In addition, a multitude of molecular components are involved in Ca²⁺ signaling pathways, working in combinations to produce specific Ca²⁺ signals of different spatial and temporal profiles (Berridge et al., 2000). Similarly, the levels of labile Zn²⁺ are maintained considerably low in the cytosol with estimates corresponding to a range that extends to nanomolar ranges (Thambiayya et al., 2012; Granzotto et al., 2020). Unlike [Ca²⁺]_i, a role for intracellular Zn²⁺ ([Zn²⁺]_i) as a second messenger in certain cellular context was realized only recently (Yamasaki et al., 2007). On the other hand, the structural and catalytic functions of intracellular Zn²⁺ has long been realized (Thambiayya et al., 2012; Granzotto et al., 2020). This tight regulation of the intracellular concentrations of both ions is achieved via coordinated actions of various specialized group of proteins including channels, pumps, sensors, and buffers (McCall et al., 2000; Andreini et al., 2006; Thambiayya et al., 2012). In addition, much of the labile Ca²⁺ and Zn²⁺ is stored in the lumen of intracellular organelles such as the mitochondria, lysosomes, and endoplasmic reticulum (ER) or buffered by specialized proteins in the cytosol (Maret, 1994; Frederickson et al., 2000; Li et al., 2001; Outten and O’Halloran, 2001; Colvin et al., 2006; Hwang et al., 2008; Qin et al., 2011; Park et al., 2012; Khan et al., 2014; Lu et al., 2016). Recent studies indicate that Ca²⁺ and Zn²⁺ are intimately linked with each other, whereby Zn²⁺ contribute to shaping intracellular Ca²⁺ dynamics and vice versa specifically in excitable cells such as neurons and cardiac cells (Kiedrowski, 2012; Woodier et al., 2015; Sanford et al., 2019; Sanford and Palmer, 2020; Zhang et al., 2021).

Electric field stimulation (EFS) has been employed extensively in vitro to regulate neuronal behaviors such as excitability, migration, and regeneration (Alexander et al., 2006; Kobelt et al., 2014; Yan et al., 2014; Zhao et al., 2015; Hayashi et al., 2016; Pakhomov et al., 2017; Latchoumane et al., 2018; Tang-Schomer et al., 2018). It is well-established that EFS induces electrophysiological responses due to the invoked changes in the charged components of the membrane of the stimulated cells (Robinson, 1985; Tung and Borderies, 1992; Ye and Steiger, 2015). The electrophysiological responses may vary according to the cellular orientation and the inherent anisotropic orientation of the electric field (Tung and Borderies, 1992; Ye and Steiger, 2015). In addition, cationic currents at the plasmalemma of the stimulated cells largely depend on the activation and/or deactivation status of voltage gated ion channels (Robinson, 1985; Tung and Borderies, 1992), ultimately leading to intracellular interference with cation and/or anion homeostasis and other membrane bound receptor downstream signaling (Robinson, 1985; Taghian et al., 2015).

Several studies showed that exogenous EFS induces changes to the dynamic of [Ca²⁺]_i, with varying findings regarding the kinetic and source of release (Alexander et al., 2006; Jahanshahi et al., 2014; Kobelt et al., 2014; Yan et al., 2014; Zhao et al., 2015; Hayashi et al., 2016; Pakhomov et al., 2017; Latchoumane et al., 2018; Tang-Schomer et al., 2018; Zhu et al., 2019; Meng et al., 2021). On the other hand, studies utilizing EFS to probe [Zn²⁺]_i are scarce, and only recently that pharmacological characterizations have been conducted in dissociated brain cultures in vitro to elucidate the kinetics of Zn²⁺ signaling in neurons and the consequential downstream effects (Hwang et al., 2008; Kiedrowski, 2012; Sanford et al., 2019; Sanford and Palmer, 2020). Accordingly, we investigated whether changing the excitability of the membrane by EFS can alter the dynamics of [Ca²⁺]i and [Zn²⁺]i signaling and whether an interplay between the two ions can be uncovered. Our data revealed that EFS induced hyperpolarization of the membrane which was concurrently accompanied by a rise in [Ca²⁺]_i and [Zn²⁺]_i. The EFS-induced rise in Ca²⁺ and Zn²⁺ seemed to be due mainly to release from intracellular stores, and that the dynamic profile is indicative of an interplay between the two ions. These studies are important toward building an understanding of the kinetic of Ca²⁺ and Zn²⁺ signaling in response to changing excitability of neurons by EFS in vitro.

In this study, we investigated the dynamic of [Ca²⁺]_i and [Zn²⁺]_i in response to altering excitability of the membrane using EFS in dissociated rat cortical cultures. Our results demonstrate that an EFS with an intensity of 7.69 V/cm induces membrane hyperpolarization with concomitant transient rise in [Ca²⁺]_i and in [Zn²⁺]_i. Our findings suggest that: (i) the source of this EFS-induced Ca²⁺ and Zn²⁺ rise is intracellular, and (ii) the dynamic of the release may involve an interplay between the two ions. Evidence for the latter is drawn from the finding that removal of extracellular Ca²⁺ significantly augmented the release of intracellular Ca²⁺ and Zn²⁺. Further characterizations of the source of Zn²⁺ identified membrane bound vesicular organelles with high Fluozin-1 intensity. These organelles were found to be localized in the cell body and exhibited dispersed co-localization with the ER, and lysosomes.

Electric field stimulation -induced alteration to the [Ca²⁺]_i and [Zn²⁺]_i is unlikely to be due to the effect of transmembrane potential which itself is dependent on multiple factors including cellular orientation, direction and orientation of the electric field and activation or deactivation of voltage gated ion channels (Ye and Steiger, 2015). It is also well-understood that the EFS interferes with charged components of the membrane including membrane bound receptors and ionic distribution and ultimately affects cation and/or anion homeostasis (Robinson, 1985; Taghian et al., 2015; Ye and Steiger, 2015). It has been shown that the membrane exhibits high resistance to the EFS and, as a result, induces clustering of membrane proteins and receptors, leading to subsequent activation of various transmembrane signaling pathways (Cho et al., 1999; Leppik et al., 2020). The fact that chemical hyperpolarization had no apparent effect on [Ca²⁺]_i and [Zn²⁺]_i suggests that hyperpolarization per se was not responsible for the [Ca²⁺]_i and [Zn²⁺]_I transient reported in this study. Our finding that cortical culture cells exhibited stronger and more sustained hyperpolarization in the absence of extracellular calcium may suggest a link between extracellular calcium and hyperpolarization. However, while changes in calcium can influence the activity of ion channels and contribute to changes in the membrane potential, they are not typically the primary cause of hyperpolarization. Rather, they are often involved in downstream signaling pathways that are triggered by changes in the membrane potential.

Many studies employed EFS in vitro to investigate the long-term effects on neuronal behavior and function for therapeutic purposes (Alexander et al., 2006; Jahanshahi et al., 2014; Kobelt et al., 2014; Yan et al., 2014; Zhao et al., 2015; Hayashi et al., 2016; Pakhomov et al., 2017; Latchoumane et al., 2018; Tang-Schomer et al., 2018; Zhu et al., 2019; Meng et al., 2021). Several of these studies examined the role of Ca²⁺ signaling in mediating some of the observed cellular responses induced by EFS (Alexander et al., 2006; Jahanshahi et al., 2014; Kobelt et al., 2014; Yan et al., 2014; Zhao et al., 2015; Hayashi et al., 2016; Pakhomov et al., 2017; Latchoumane et al., 2018; Tang-Schomer et al., 2018; Zhu et al., 2019; Meng et al., 2021). An EFS with an intensity of 5 V and frequency of 10 Hz for a duration of 30 min have been shown to induce a rise in [Ca²⁺]_i and consequently promote neurite outgrowth in cultured dorsal root ganglia neurons (Yan et al., 2014). Further pharmacological characterizations suggested that the source of this EFS-induced [Ca²⁺]_i rise was both intracellular release from internal stores and extracellular through voltage gated Ca²⁺ channels (Yan et al., 2014). In another study, EFS of high intensity (20–25 V/cm) and high frequency was found to induce a rise in [Ca²⁺]_i in cultured hippocampal neurons (Riquelme et al., 2011). Consequently, this rise in [Ca²⁺]_i was found to induce generation of hydrogen peroxide and stimulate activity of NF_–kB, a transcription factor known for its important role in dendritic arborization and plasticity (Riquelme et al., 2011). Further characterizations revealed that the source of this [Ca²⁺]_i rise was intracellular via activation of ryanodine receptors (Riquelme et al., 2011). These studies showed an instant increase in [Ca²⁺]_i following application of EFS. Whilst we acknowledge that pharmacological characterizations are lacking in our study, the kinetic of Ca²⁺ release reported by Riquelme et al. (2011) was different from our study. Our data indicated that only one third of the cells, under “regular” conditions, responded to EFS with a Ca²⁺ rise and that the rise was slow with a considerable lag between the EFS and the Ca²⁺ rise. This may be attributed to differences in EFS parameters applied, differences in cell types or different functional context.

Our study showed a rise in [Zn²⁺]i in response to EFS in cortical neurons and that the source of the release was intracellular stores. The cytosolic sources of labile Zn²⁺ in neurons can be either Zn²⁺ liberated from proteins such as metallothioneins or Zn²⁺ released from various intracellular organelles and compartments (Frederickson et al., 2000; Sensi et al., 2003; Colvin et al., 2006; Hwang et al., 2008; Qin et al., 2011; Park et al., 2012; Lu et al., 2016). Our studies indicate that Zn²⁺ enriched vesicles localized within the soma of cortical cells contributed to EFS-induced rise in [Zn²⁺]_i. Two possibilities exist; (i) the high fluorescence intensity of Fluozin-1 in the vesicles reflects high Zn²⁺ content and/or (ii) the high intensity reflects different intra-vesicular environment that changes the spectral characteristics of the dye independently of Zn²⁺. The latter would seem unlikely since Fluozin-1 AM intensity is highest in pH 7 and decrease at more acidic or basic pH (Martins et al., 2020). Our co-localization experiments showed that most of the zinc enriched vesicles co-localized with the ER, and lysosomes while less co-localizations have been observed in the mitochondria. This is consistent with previous studies showing that Zn²⁺ enriched vesicles in cultured neurons and astrocytes are mainly enriched in secretory vesicles, endosomes, and lysosomes (Sensi et al., 2003; Colvin et al., 2006; Hwang et al., 2008; Lu et al., 2016). Other intracellular sources may also have contributed to the liberated Zn²⁺ such as metallothioneins, but this warrants further investigations.

Accumulating evidence highlights the important roles for Zn²⁺ and Ca²⁺ interactive signaling in several functional contexts that extend from modulating cellular excitability to mediating cellular injury and cell death (Claiborne et al., 1989; Frederickson and Danscher, 1990; Li et al., 2001; Quinta-Ferreira et al., 2004; Huang et al., 2008; Ketterman and Li, 2008; Liu et al., 2008; Hershfinkel et al., 2009; Sindreu et al., 2011; Khan et al., 2014; Vergnano et al., 2014; Woodier et al., 2015; Ha et al., 2018; Levaot and Hershfinkel, 2018; Sanford et al., 2019; Morabito et al., 2022). Ca²⁺ dependent Zn²⁺ release is well-documented in several studies. It has been shown that labile Zn²⁺ is abundant in a subset of glutamatergic neurons, specifically localized in synaptic vesicles and, like conventional neurotransmitters, Zn²⁺ is released in a depolarization and Ca²⁺ dependent manner (Frederickson et al., 2000; Li et al., 2001; Khan et al., 2014). Additionally, it has been shown that stimulation with KCl or glutamate in dissociated hippocampal neurons induces a rise in [Zn²⁺]i that is downstream from Ca²⁺ signaling and that the source of Zn²⁺ release is from intracellular stores (Kiedrowski, 2012; Sanford et al., 2019; Sanford and Palmer, 2020). The other way of interaction also exists, whereby Zn²⁺ modulates Ca²⁺ dynamic and signaling in excitable cells. It has been shown that intracellular Zn²⁺ has a higher affinity to bind to the cardiac ryanodine receptor (RYR) than Ca²⁺ and that a millimolar rise in intracellular levels of free Zn²⁺ inhibits the channel opening, thereby regulating Ca²⁺ release (Woodier et al., 2015). In addition, Zn²⁺ dependent Ca²⁺ influx in cortical neurons has been shown to contribute to neuronal cell death during oxidative stress (Park et al., 2019).

Our study revealed a possible interaction between intracellular Ca²⁺ and Zn²⁺ signaling in response to EFS. EFS in the absence of extracellular Ca²⁺ evoked significantly higher maximum release of intracellular Zn²⁺, indicating that extracellular Ca²⁺ may interfere with intracellular release of Zn²⁺. It is therefore tempting to speculate that, similar to the Zn²⁺ modulation of the cardiac RYR, Ca²⁺ entry through voltage sensitive Ca²⁺ channels may be also modulated and/or inhibited by binding of intracellular Zn²⁺ and that in the absence of extracellular Ca²⁺, Zn²⁺ will not bind to these channels leading to higher levels of free Zn²⁺ (Sun et al., 2007; Woodier et al., 2015). This may also explain the reduced release of intracellular Ca²⁺ in response to EFS in the presence of extracellular Ca²⁺. Further investigations are required to decipher details of this interdependence relationship between Ca²⁺ and Zn²⁺ and to elucidate the downstream signaling and determine its consequence.

In this study, we normalized fluorescence intensity to baseline in order to quantify the relative changes in intracellular Ca²⁺ and Zn²⁺. Normalization to baseline assumes that the treatment does not affect baseline levels, which might not be always true. We therefore provided evidence of the effect of the various treatments on baseline values (Supplementary Figure 5). We show that cellular Fluo-4 AM fluorescence intensity of cells bathed in calcium-free Krebs medium exhibited no apparent difference from that of cells bathed in normal calcium medium indicating no effect on baseline level of Fluo-4 AM fluorescence (Supplementary Figure 5A). Manipulation of extracellular calcium would lead to changes in intracellular calcium set-point (al-Mohanna et al., 1992) however, the relatively short time of exposure to calcium free-medium might explain the lack of effect we have noticed. In addition, the possibility that these cultured cortical neurons might be more resistant to changes in extracellular calcium than others (within the time scale of the experiments), could not be excluded. Future research would clear this point. On the other hand, we have found a significant difference in the baseline Fluozin-1 AM intensity levels under the different extracellular calcium levels (Supplementary Figure 5B). As for TPEN treatment, we found no statistical difference in the Fluozin-1 AM intensity levels in cells treated with TPEN compared to untreated cells (Supplementary Figure 5C).

In summary, we showed that modifying membrane potential of cortical neurons by EFS resulted in hyperpolarization that is concurrently accompanied by release of Ca²⁺ and Zn²⁺ from intracellular sources. Our study highlights an association between EFS-induced modification of membrane excitability and [Ca²⁺]_i and [Zn²⁺]_i signaling and supports the use of EFS as a tool to interrogate the dynamics of intracellular ions homeostasis.

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

The animal study was reviewed and approved by the Animal Care and Use Committee of the King Faisal Specialist Hospital and Research Centre.

AA and FAM: conception and design of the study, data analysis and interpretation, and manuscript writing. AA, SA, and FAM: collection and/or assembly of data. FAM: provision of study material and administrative and financial support. All authors contributed to final approval of the manuscript.