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Brain Res. Author manuscript; available in PMC 2007 May 24.
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PMCID: PMC1876679

Loss of Calcium and Increased Apoptosis Within the Same Neuron


Loss of neuronal calcium is associated with later apoptotic injury but observing reduced calcium and increased apoptosis in the same cell would provide more definitive proof of this apparent correlation. Thus, following exposure to vehicle or the calcium chelator BAPTA (1–20 μM), primary cortical neurons were labeled with Calcium Green-1 which was then cross-linked with EDAC, prior to immunostaining for various proteins. We found that BAPTA-induced changes in calcium were highly correlated with changes in expression of activated caspase-3 as well as the calcium binding proteins calbindin, calretinin, and parvalbumin. Additionally, in brain slices from P7 neonatal rats, BAPTA induced significant loss of calcium in a brain region we have previously shown to express only moderate levels of calcium binding proteins as well as display robust apoptosis following calcium entry blockade. In contrast, BAPTA had little influence on calcium levels in a brain region we have previously shown to express robust calcium binding proteins as well as display far less apoptosis following calcium entry blockade. These data suggest that the ability of developing neurons to buffer changes in calcium may be critical to their long-term survival.

Keywords: caspase-3, rat, brain slice, development, BAPTA, calcium binding proteins


We have shown that agents that promote loss of neuronal calcium, such as the N-methyl-D-aspartate receptor (NMDAR) antagonist MK801 (Uematsu et al., 1991), can induce neonatal brain injury by mechanisms that involve changes in expression of proteins such as the pro-apoptotic activated caspase-3 (AC3) as well as cytoskeletal and synapse associated proteins (Turner et al., 2002; Lema Tome et al., 2006a; Turner et al., 2006). Many other studies have also shown similar associations between loss of calcium and increased apoptosis (Koh and Cotman, 1992; Takadera and Ohyashiki, 1998; Gwag et al., 1999; Hwang et al., 1999; Moran et al., 1999; Takadera et al., 1999; Moulder et al., 2002). However, it is still unclear if loss of calcium is directly linked with changes in markers of injury in the same cell.

The use of fluorescent dyes such as the Fluo-series of probes, Calcium Green-1 (CG-1) or Fura-2 to estimate changes in relative calcium levels or absolute calcium concentrations have greatly advanced the field of neuroscience (Rudolf et al., 2003; Tsien, 2003). However, correlating changes in calcium with other events in the same cell requires the use of protocols that employ extensive washing and detergent solubilization. This invariably leads to leakage of the dye out of the cell, precluding comparison of calcium levels with, for example, protein expression. Recent studies suggest this problem can be overcome by using the cross-linking agent, EDAC (N-(3-dimethylaminopropyl)-N′ -ethylcarbodiimide hydrochloride) to fix the dye prior to other procedures (Tymianski et al., 1997; Dallwig and Deitmer, 2002).

Although ratiometric dyes such as Fura-2 are thought to be the superior dye of choice, a range of non-ratiometric dyes have been available for some time that have allowed important new insights into calcium signaling that would otherwise not be possible with dyes such as Fura-2 (see (Maravall et al., 2000; Rudolf et al., 2003)). Indeed, when used appropriately non-ratiometric dyes and may be superior in some circumstances (Maravall et al., 2000). Thus, to further examine this relationship between loss of calcium and cell death, neurons exposed to vehicle or the calcium chelator BAPTA were loaded with the calcium-sensitive dye Calcium Green-1 (CG-1), cross-linked using EDAC to stabilize CG-1, and immunostained for AC3 or the calcium binding proteins calbindin (CB), calretinin (CR) or parvalbumin (PV). We observed that loss of calcium was inversely correlated with AC3 expression in the presence of BAPTA. Additionally, those cells that survived BAPTA treatment had CG-1 signals that were highly correlated with expression of CB, CR or PV. Further, using brain slices from P7 neonatal rats, calcium buffering was weak in a brain region we have previously shown to display robust apoptosis following MK801 or nimodipine injection (Turner et al., 2002; Turner et al., 2006) but was strong in a brain region that displays little apoptosis following MK801 or nimodipine injection. These data support our general hypothesis that agents that lower calcium can promote apoptotic injury in developing neurons and that the inability to buffer changes in calcium is correlated with increased neuronal death.

2-Experimental Procedure

Primary Cell Cultures and Agent Exposure

All animals were used according to NIH and Wake Forest University ACUC guidelines. Unless otherwise stated all agents were from Sigma (St Louis, MO). Pregnant, embryonic day 18 (E18) rats were anesthetized (2% halothane), embryos were placed in ice-cold Hanks balanced salt solution (no Ca2+ or Mg2+ salts; Gibco-Invitrogen, Carlsbad, CA), and cerebral cortices were isolated by removing meninges and subcortical tissue. A combination of trituration and DNase/trypsin digestion was used to dissociate the tissue, followed by centrifugation and resuspension in Neurobasal medium (Brewer and Price, 1996) (supplemented with glutamine and B27) to a plating density of approximately 1x105 cells/mm2. Using a combination of NeuN-immunocytochemistry and DAPI labeling, these cultures were estimated to be about 95 % pure neurons, in general agreement with previous observations (Turner et al., 2002). Neurons were fed every 3rd day thereafter and treated with agents at 7–10 days in vitro (DIV).

Calcium Green-1 Loading, EDAC Cross-linking, Immunocytochemistry

Neurons were exposed to vehicle (0.1% DMSO) or BAPTA-AM (1,2-Bis(2-amino-5-fluorophenoxy)ethane-N,N,N',N'-tetraacetic acid-(acetoxymethyl) ester; 1–20 μM; Invitrogen, Eugene, OR) for 4 hr as previously described (Turner et al., 2002; Turner et al., 2004; Turner et al., 2006). Cells were labeled with the non-ratiometric dye, Calcium Green-1-AM (CG-1-AM; 5 μM; Invitrogen) for 45 min at 37°C, and washed in PBS three times. Internalized AM ester compounds are cleaved, thus trapping BAPTA or CG-1 inside the cell. We have performed pilot studies to determine the optimal concentration for CG-1 (which was found to be 5 μM for primary neurons) and signal intensities obtained (see below) were in close agreement with previous studies (Turner et al., 2006). To determine signal intensity prior to EDAC cross-linking, cells in some wells were immediately photomicrographed at 40X under UV light using a filter selective for 488 nm. For other wells, we next exposed cells to the cross-linking agent EDAC to fix CG-1 prior to immunocytochemistry (ICC) (Tymianski et al., 1997; Dallwig and Deitmer, 2002). We found that 60 mg/ml EDAC for 2hr at room temperature gave optimal CG-1 staining.

We have found that MAP2-ir following EDAC cross-linking was the same as that found following more conventional fixation procedures (Turner et al., 2006), suggesting immunoreactivity may be unaltered after EDAC exposure. Thus, after 5 washes with PBS (10 min each), cells were labeled with a rabbit polyclonal anti-AC3 primary antibody (1:1000; Cell Signaling, Beverly, MA) which recognizes the low molecular weight (cleaved; activated) caspase-3 (Hu et al., 2000), followed by an AlexaFluor 594 goat-anti-rabbit secondary antibody (1:200; Molecular Probes, Eugene, OR). Parallel cultures were also exposed to a mouse monoclonal, anti-CB primary antibody or mouse monoclonal, anti-PV primary antibody (1:1000; Swant, Switzerland), or mouse monoclonal, anti-CR primary antibody (1:1000; Chemicon, Temecula, CA), followed by AlexaFluor 594 donkey anti-mouse secondary antibody (1:200; Molecular Probes). All antibodies were prepared in 1% bovine serum albumin, 0.1% triton-X 100, in PBS, exposed to cells for 1 hr at room temperature, with three PBS washes between incubations. Coverslips were then inverted and mounted onto glass slides using Vectashield anti-fade mounting medium (Vector Labs, Burlingame, CA).

Imaging and Intensity Measurements

TIFF images of the same field were taken at 40X under UV light at 488 nm for CG-1 and 594 nm for either AC3 or each of the CaBPs, colorized in Photoshop 7.0 or imported into Image Pro Plus (MediaCybernetics, Baltimore, MD) to measure optical density (intensity) of signals using an internal gray scale of 0–255. Line scans through the cell body were performed to determine peak intensity of signal (background corrected; generally background values fell between 10–20 on this gray scale range) and the relationship between CG-1 and ICC intensities was assessed across all cells sampled (N = 20 in each treatment group) by linear regression. Data was derived from quadruplicate observations across 3 independent cultures.

Brain Slice Studies

We have previously shown that in P7 neonatal animals, agents that disrupt calcium homeostasis promote robust apoptosis in areas such as layer II of the cerebral cortex whereas the same agents have far less effect in the ventromedial caudate-putamen (Turner et al., 2002; Turner et al., 2006). Thus, P7 animals were deeply anesthetized (2% halothane; Sigma), decapitated, brains removed and placed immediately in chilled Hibernate E buffer (Brain Bits, Springfield, IL), which can maintain neuronal viability for extended periods (Brewer and Price, 1996). Brains were then chopped into approximately 400 μm slices and maintained in Neurobasal media (Jakobsen and Zimmer, 2001; Bonde et al., 2002; Noraberg et al., 2005) (media changes performed 3 times over 30–60 min) in a tissue culture incubator (37°C, 100% humidity, 5% CO2). Slices were loaded with the calcium sensitive dye, CG-1-AM (10 μM; Invitrogen, Eugene, OR) and returned to the incubator for 45–60 min. Slices were taken out of the incubator as needed and washed 3 times in fresh Hibernate E media. TIFF images were then collected at the beginning and end of exposure to vehicle (0.1% DMSO) for 3 min or BAPTA-AM (10 μM, in 0.1% DMSO; Invitrogen) for 10 min. CG-1 intensity was estimated in at least 8 cells per slice (across 3 independent slices) from TIFF images imported into ImagePro Plus 5.0 (MediaCybernetics, Baltimore, MD). After background correction, the percent change in intensity was determined for each cell according to the following equation: [(intensity at end – intensity at start)/intensity at start] x 100. Data were then expressed as mean percent change for all cells sampled across all slices (± SEM).


Differences in the means were determined by one-way ANOVA, using a Bonferroni post-test comparison (Prism 4.0, Graph Pad, San Diego, CA). Regression analysis was also performed in Prism 4.0 and Goodness of Fit (r2) and significance levels for each curve generated.


Loss of neuronal calcium leads to increased apoptosis in the same cell

Prior to the studies described later, we wished to establish if CG-1 intensity in primary cortical neurons was substantially different before and after cross-linking plus ICC. Before EDAC exposure CG-1 intensity was 195 ± 32 (grayscale units; see Methods) whereas after the cross-linking and ICC procedures was 158 ± 24 (N = 35 cells), indicating a substantial fraction of the original signal was maintained after EDAC/ICC.

Because many studies suggest an association between loss of calcium and neuronal injury (Koh and Cotman, 1992; Takadera and Ohyashiki, 1998; Gwag et al., 1999; Hwang et al., 1999; Moran et al., 1999; Han et al., 2001; Moulder et al., 2002; Turner et al., 2002; Yoon et al., 2003; Turner et al., 2004; Turner et al., 2006), we next examined if changes in both CG-1 labeling and increased AC3-immunoreactivity (ir) could be observed in the same cell following loss of intracellular calcium. Thus, primary cortical neurons were exposed to vehicle or 1–20 μM BAPTA for 4 hr, based on previous studies showing this time point is associated with robust apoptosis following loss of calcium (Turner et al., 2002; Turner et al., 2004; Turner et al., 2006). Neurons were then loaded with CG-1 for 45–60 min and cross-linked with EDAC as described (Methods).

In vehicle-treated wells we found that most cells were well labeled with CG-1 (Fig. 1A1). In contrast, neurons displayed a progressively lower CG-1 signal with increasing concentration of BAPTA (compare Fig. 1A1–F1). Thus, the loss in signal we have observed in other studies following BAPTA treatment (Turner et al., 2006) is faithfully reproduced after EDAC treatment. These previous studies also show that at higher BAPTA concentrations (20 μM) most cells exhibit a profound loss in calcium signal, suggesting the buffering capacity has been overwhelmed. Following cross-linking, these same neurons were also immunostained for the pro-apoptotic enzyme AC3, using an antibody that recognizes the cleaved, lower molecular weight, protein. As expected from previous studies (Turner et al., 2002; Turner et al., 2004; Turner et al., 2006), vehicle-treated cells displayed little evidence of expression of AC3-ir (Fig. 1A2). In contrast, BAPTA-treated cells showed an increase in the number of AC3-positive cells with increasing concentration (Fig.1 B2–F2).

Fig. 1
Negative correlation between loss of calcium and AC3 expression

It may be argued that loss of CG-1 and increases in AC3 occur in separate populations of cells within the same field. However, by double labeling for CG-1 and AC3, we simultaneously monitored relative levels of both markers in the same cell. Thus, intensity of both markers was measured in 20 cells per treatment group and regression analyses of these data were performed (see Methods). We found that in vehicle-treated wells, clustering of data points favored high CG1 and low AC3 intensity, showing a modest but significant negative correlation (Fig. 1A4; for Goodness of Fit and significance levels please see panels in figure). With increasing amounts of BAPTA, there was a clear shift in the clustering of these points in favor of low CG-1 and high AC3 and the negative correlation between these markers became more robust as BAPTA concentration increased (Fig. 1B4–F4). These data clearly indicate that in the same cell, reduction in calcium is inversely related to expression of AC3. However, further demonstrating the inverse nature of this relationship, there were many examples of adjacent cells displaying high CG-1/low AC3 or low CG-1/high AC3 (Fig. 1D3; see cell groups indicated by asterisk). These data are in close agreement with previous observations showing that depletion of extracellular or intracellular calcium, disruption of store operated calcium, or blockade of calcium entry can all trigger apoptotic injury, with the latter being reversed by increasing intracellular calcium (Turner et al., 2002).

Thus, by refining the technique previously described by others (Tymianski et al., 1997; Dallwig and Deitmer, 2002), we show that cross-linking with EDAC can retain much of the CG-1 signal after extensive processing during ICC procedures. Further, the BAPTA-induced loss of calcium we have described elsewhere (Turner et al., 2002; Turner et al., 2004; Turner et al., 2006) was faithfully reproduced after EDAC cross-linking, allowing direct comparisons between current and previous studies.

Cells surviving BAPTA treatment have high CaBP expression

Calcium buffering may depend upon expression of the calcium binding proteins (CaBPs) calbindin (CB), calretinin (CR), or parvalbumin (PV) (Celio, 1990; Baimbridge et al., 1992; Rogers and Resibois, 1992; Berridge et al., 2000). We therefore exposed parallel cultures to vehicle or BAPTA (1–20 μM) and simultaneously assessed cells for CG-1 signal together with expression of one of the CaBPs, using EDAC cross-linking and ICC as described (see above).

In vehicle-treated wells, we frequently observed that cells with a high CG-1 intensity were often well labeled with CB (Fig. 2A1–A3). Regression analysis revealed a modest correlation was apparent between these two markers (Fig. 2C1). However, with higher BAPTA concentrations, the smaller fraction of cells that still had a measurable CG-1 signal were more frequently associated with expression of CB (Fig. 2B1–B3). Regression analyses of all experimental groups showed that the correlation between CG-1 and CB increased as the concentration of BAPTA also increased (Fig. 2C2–C6). We found the same outcome when we analyzed cultures stained for CG-1 and CR (Fig. 3) or CG-1 and PV (Fig. 4). For example at 20 μM, CG-1 was highly correlated with CB or PV expression (r2 = 0.85 for CB and 0.79 for PV) and moderately correlated with CR expression (r2 = 0.54). It should be noted, however, that most cells are already overwhelmed by BAPTA treatment (particularly at 10–20 μM) and the increase in correlation between CG-1 and CaBPs represents data from surviving cells only. Collectively, these data suggest that those cells that have high enough calcium levels to yield a still detectable CG-1 signal do so because they express a CaBP. Stated in terms of cell viability, survival of developing neurons, when challenged by calcium chelation, may be dependent on whether they possess the molecular machinery to cope with a significant drop in free calcium.

Fig. 2
Positive correlation between CG-1 intensity and CB expression
Fig. 3
Positive correlation between CG-1 intensity and CR expression
Fig. 4
Positive correlation between CG-1 intensity and PV expression

P7 brain slices: region-specific lack of buffering and injury

We have previously shown that, following disruption of calcium homeostasis in P7 neonates, brain regions, such as layer II of the cerebral cortex, rapidly display robust AC3 expression (Turner et al., 2002; Turner et al., 2006). However, other brain regions, such as the ventromedial caudate putamen, display far less evidence of apoptosis. Further, this injury has been shown to peak at P7 (Ikonomidou et al., 1999) and is inversely related to expression of CaBPs (Lema Tome et al., 2006b). Thus, using CG-1-loaded brain slices from P7 animals (that included either cortical layer II or the ventromedial caudate-putamen), we examined relative changes in intracellular calcium following exposure to vehicle (3 min) or the calcium chelator BAPTA (20 μM; 10 min). The percent change in CG-1 intensity was then estimated per cell and averaged across all cells sampled (see Methods).

We found that in cortical layer II, whereas CG-1 intensity was relatively stable in the presence of vehicle, a highly significant loss of CG-1 signal was observed at the end of BAPTA exposure (Fig. 5A, B & E). In contrast, within the ventromedial caudate-putamen, whereas vehicle exposure caused little change in CG-1 intensity, BAPTA caused a slight but significant elevation in this signal at the end of the 10 min exposure period (Fig. 5C, D & E). Collectively, these and other studies suggest that injury induced in P7 slices following reduction in intracellular calcium may depend on whether cells have developed the capacity to buffer such a change, as suggested in earlier studies (Turner et al., 2002; Turner et al., 2006) and consistent with data from primary neuronal cultures (Fig. 14).

Fig. 5
Regional variation in calcium buffering in brain slices


Whereas we have previously shown that loss of calcium is highly correlated with increased apoptosis (Turner et al., 2002; Turner et al., 2004; Turner et al., 2006) in close agreement with many other studies (Koh and Cotman, 1992; Takadera and Ohyashiki, 1998; Gwag et al., 1999; Hwang et al., 1999; Moran et al., 1999; Takadera et al., 1999; Han et al., 2001; Moulder et al., 2002), it was not presently clear if both events can be observed in the same cell. However, we now show that, in the same neuron, decreased CG-1 intensity is correlated with increased AC3-ir, providing new and important evidence that loss of calcium is directly linked to increased apoptosis. This direct correlation further supports a growing body of evidence from our lab that developing neurons are especially vulnerable to loss of calcium (Turner et al., 2002; Turner et al., 2004; Turner et al., 2006). Indeed, we have recently shown that nimodipine can promote similar apoptotic injury to that already shown for MK801 (Turner et al., 2006), thus providing the first in vivo evidence that calcium channel blockade promotes the same injury as NMDAR blockade.

We show here that CG-1 intensity in primary neurons is positively correlated with expression of CB, CR or PV. Further, cells with still detectable CG-1 signal following exposure to BAPTA were more likely to express one of these CaBPs, suggesting cell survival may be linked to expression of these proteins, in general agreement with other studies (Hof et al., 1991; Mattson et al., 1991; Freund et al., 1992; Iacopino et al., 1992; Burke and Baimbridge, 1993; Lukas and Jones, 1994; Prendergast et al., 2001; Mulholland et al., 2003) (though see (Mockel and Fischer, 1994; Airaksinen et al., 1997; Klapstein et al., 1998; Bouilleret et al., 2000; Isaacs et al., 2000)). The data we describe here are consistent with our hypothesis that loss of calcium triggers apoptotic injury in the developing brain and is inversely related to expression of CB, CR, and PV (Lema Tome et al., 2006b). Thus, neurons may be more sensitive to loss of calcium if they do not have the molecular machinery to buffer potentially pathological changes in this divalent cation.

Calcium Buffering and Neurotoxicity

We have previously shown that agents such as MK801 and nimodipine, which block calcium entry into neurons, can promote caspase-3-dependent injury in an age-dependent manner (Turner et al., 2002; Turner et al., 2006). We also have shown that such injury may involve loss of calcium (Turner et al., 2002; Turner et al., 2006) and that MK801-induced injury occurs in neuronal populations that have low expression of the CaBPs CB, CR, and PV (Lema Tome et al., 2006b). In contrast, cells that do not display such injury robustly express one of these CaBPs. Further, recent evidence suggests that CaBPs play a role in buffering ionotropic glutamate receptor-induced changes in calcium (Ikenaga et al., 2006) and age-related changes in calcium buffering in the hippocampus are thought to be coupled to age-dependent changes in CB expression (Sleeper et al., 2005). Thus, developmentally regulated apoptosis appears to be associated with an inability to buffer calcium. Consistent with this hypothesis, we found that cells in brain slices were unable to buffer changes in calcium in a brain region (cortical layer II) we have previously shown to be exquisitely sensitive to calcium-reducing agents such as MK801 (Turner et al., 2002; Turner et al., 2006) but lack significant CaBP expression (Lema Tome et al., 2006b). In contrast, calcium buffering was evident in a brain region (ventromedial caudate putamen) we previously have shown to display far less sensitivity to MK801 but robustly expresses CaBPs. These and other studies from our lab, as well as studies elsewhere, strongly suggest that, in general, any agent (or event) that changes intracellular calcium may promote injury in developing neurons if they lack a CaBP. Conversely, if cells express a CaBP, then buffering agent-induced changes in calcium is possible, thereby protecting cells from injury- or event-promoting changes in this cation.

The collective observations made in this lab suggest that developmental regulation of CaBP expression significantly influences neuronal responses to changes in calcium, such that cells that express CaBPs will be equipped with the molecular machinery to not only meet the changing physiological demands of neuronal maturation but also buffer any potentially pathological swings in calcium concentrations. Thus, the use of agents to lower intracellular calcium following neonatal brain trauma, control obstetric complications, or maintain anesthesia during neonatal surgery, may induce unintended injury that could have lasting effects on child development.


This work was supported by a Wake Forest University School of Medicine Faculty Development fund and NIH RO1 NS051632. Cell culture – slr; CG-1, EDAC, immunocytochemistry – cpt; data acquisition and analysis – jc, kb, cpt; manuscript preparation – jc, cpt.


activated caspase-3
1,2-Bis(2-amino-5-fluorophenoxy)ethane-N,N,N',N'-tetraacetic acid-(acetoxymethyl) ester
days in vitro
N-(3-dimethylaminopropyl)-N′ -ethylcarbodiimide hydrochloride
calcium binding protein
Calcium Green-1
N-methyl-D-aspartate receptor


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