Address correspondence and reprint requests to Nicholas J. Pantazis, Department of Anatomy and Cell Biology, The University of Iowa, 1-530 Bowen Science Building, 51 Newton Road, Iowa City, IA 52242-1109, USA. E-mail: email@example.com
Alcohol is a potent neuroteratogen that can trigger neuronal death in the developing brain. However, the mechanism underlying this alcohol-induced neuronal death is not fully understood. Utilizing primary cultures of cerebellar granule neurons (CGN), we tested the hypothesis that the alcohol-induced increase in intracellular calcium [Ca2+]i causes the death of CGN. Alcohol induced a dose-dependent (200–800 mg/dL) neuronal death within 24 h. Ratiometric Ca2+ imaging with Fura-2 revealed that alcohol causes a rapid (1–2 min), dose-dependent increase in [Ca2+]i, which persisted for the duration of the experiment (5 or 7 min). The alcohol-induced increase in [Ca2+]i was observed in Ca2+-free media, suggesting intracellular Ca2+ release. Pre-treatment of CGN cultures with an inhibitor (2-APB) of the inositol-triphosphate receptor (IP3R), which regulates Ca2+ release from the endoplasmic reticulum (ER), blocked both the alcohol-induced rise in [Ca2+]i and the neuronal death caused by alcohol. Similarly, pre-treatment with BAPTA/AM, a Ca2+-chelator, also inhibited the alcohol-induced surge in [Ca2+]i and prevented neuronal death. In conclusion, alcohol disrupts [Ca2+]i homeostasis in CGN by releasing Ca2+ from intracellular stores, resulting in a sustained increase in [Ca2+]i. This sustained increase in [Ca2+]i may be a key determinant in the mechanism underlying alcohol-induced neuronal death.
Alcohol abuse during pregnancy can damage multiple organ systems in the fetus, resulting in permanent functional deficits that persist into adulthood (Greenbaum and Koren 2002). As individuals exposed to alcohol during fetal life often display considerable variation in the pattern and severity of these functional deficits, the term fetal alcohol spectrum disorder (FASD) was coined to convey this widespread variability. Fetal alcohol syndrome (FAS) represents the most severe form of FASD and severe damage to the developing CNS is one of the hallmarks of FAS. Although there is now greater public awareness of the harm, which fetal alcohol exposure can induce, FASD remains a persistent public health problem. A better understanding of how fetal alcohol exposure produces such devastating injury to the CNS is necessary to develop strategies to deal with the health needs of individuals afflicted with FASD.
Studies in rodent models have identified several alcohol-induced neuropathological changes in the developing CNS, including disruption of the neuroanatomical structure of the brain (Konovalov et al. 1997; Roebuck et al. 1998), microencephaly (Pierce and West 1986; Maier et al. 1997), impaired neuronal migration (Miller 1993), changes in proliferation of neuronal precursors (Miller 1996), altered neurotransmitter receptor function (Eckardt et al. 1998) and neuronal death (Konovalov et al. 1997). As alcohol causes so many effects, it is challenging to identify the most relevant ones with regard to functional deficits. In this study, we focused on alcohol-induced neuronal death, as the loss of neurons is one of the most harmful effects of alcohol on the developing brain.
The mechanism by which alcohol induces neuronal death remains unclear. In this study, we investigated the potential role of intracellular Ca2+ [Ca2+]i in alcohol's cell death mechanism. The multifunctional role of [Ca2+]i in cell signalling is well recognized, including participation in signalling cell death (Trump and Berezesky 1995; Orrenius et al. 2003; Zhivotovsky and Orrenius 2011). Although early literature from the 1980s and 1990s indicated that alcohol disrupts [Ca2+]i homeostasis, whether this change altered cell survival was for the most part not explored. More recent studies have shown that alcohol exposure elevates [Ca2+]i in chick embryo neural crest (Debelak-Kragtorp et al. 2003; Garic-Stankovic et al. 2005) and astrocyte cultures (Holownia et al. 1997; Hirata et al. 2006), and this increase in [Ca2+]i was linked to cell death. As these prior studies focused on either avian or non-neuronal models, we felt it was important to establish the role of [Ca2+]i in the alcohol-induced death of mammalian neurons. Primary cultures of cerebellar granule neurons (CGN) derived from the cerebella of neonatal (five to seven post-natal day, PD, old) mice are well suited for this study. At PD 5 through 7, the developing mouse cerebellum is at the peak of its alcohol vulnerability (Hamre and West 1993; Maier et al. 1999), and in vivo alcohol exposure at this time induces neuronal losses of approximately 10–40% across the ten folia of the cerebellum (Bonthius and West 1991). Alcohol exposure of CGN cultures causes a similar neuronal loss, 20–30% (Pantazis et al. 1993), indicating that in terms of cell death, CGN cultures simulate the in vivo response to alcohol neurotoxicity. As a result of this strong association between the in vitro and in vivo effects of alcohol, primary cultures of CGN have become a useful model to investigate the molecular mechanisms of alcohol neurotoxicity (Luo 2012). In addition, neuroprotective agents, which can ameliorate the toxic effects of alcohol have been identified in CGN cultures. For example, several neurotrophins such as NGF (Luo et al. 1997; Heaton et al. 2000), BDNF (Heaton et al. 2000; Bonthius et al. 2003) and basic FGF (Luo et al. 1996) can reduce alcohol-induced neuronal death. Activation of the NMDA receptor can also protect CGN cultures against alcohol toxicity (Pantazis et al. 1995) by stimulating a nitric oxide (NO) signalling pathway (NO-cGMP-cGMP dependent protein kinase) (Pantazis et al. 1998; Bonthius et al. 2004).
In this study, we tested two hypotheses utilizing CGN cultures. First, alcohol exposure disrupts [Ca2+]i homeostasis in these cultures, rapidly raising [Ca2+]i and sustaining [Ca2+]i at a higher level. Second, blocking the alcohol-induced increase in [Ca2+]i provides protection against alcohol neurotoxicity, preventing alcohol-induced death of CGN. The results of this study indicate that both hypotheses are true, suggesting that this alcohol-induced increase in [Ca2+]i is a key early step in the sequence of cellular events, which eventually lead to the death of vulnerable neurons hours later.
A breeding colony was established from C57BL/6; 129 mice (initially obtained from Jackson Laboratories, Bar Harbor, ME, USA) and housed in the certified animal care facility at The University of Iowa. Animal procedures were approved by the Animal Care and Use Committee.
Preparation of CGN cultures
CGN cultures were derived from five to seven post-natal day (PD) mice utilizing a protocol described previously (Pantazis et al. 1995). Briefly, cerebella were excised, pooled, minced, trypsinized (0.125%) and triturated in Dulbecco's Modified Eagle Medium (DMEM) supplemented with N2 components (Bottenstein and Sato 1979) to produce a cell suspension (plating solution). The N2 medium is a serum-free defined culture medium which contains insulin (5 μg/mL), transferrin (100 μg/mL), progesterone (20 nM), selenium (30 nM) and putrescine (100 μM). Investigators often use a high (non-physiological) concentration of K+ (25 mM) to enhance survival of cerebellar granule neurons in culture. It has been our experience that high K+ is not needed until the fourth day after establishing the primary cultures. We utilized a physiological K+ concentration (5 mM) since we used the CGN cultures within two days after plating. For alcohol neurotoxicity experiments, the cell density of the plating solution was adjusted to 1.5 × 106 cells/mL with N2-DMEM. Aliquots (0.3 mL) of this solution were added to the poly-d-lysine (PDL, 50 mg/mL per well)-coated wells of a 96-well tissue culture tray, resulting in a cell density of 4.5 × 105 cells per well. Ethanol-exposed and ethanol-free culture groups were plated into separate cell culture trays, as these trays were placed in individual sealed containers to minimize ethanol evaporation (described below). For Ca2+ imaging experiments, the cell density of the plating solution was adjusted to 1.0 × 106 cells/mL. PDL-coated (100 mg/mL) glass coverslips (25 mm diameter) were individually placed into wells of a six-well tissue culture tray, and 2.0 mL of plating solution was added (2 × 106 total cells per well). Following plating, CGN cultures were incubated overnight in humidified 5% CO2 / 95% air at 37°C and used the next day. The culture medium was not changed the next day to avoid disrupting the cells. Ethanol and all treatments were carefully added directly to the original culture medium. We (Pantazis et al. 1993) and others (Dutton 1990; Giordano and Costa 2011; Luo 2012) routinely obtain homogeneous cultures comprised of 90–95% CGN. Since the cultures were used within two days, Ara-C was not added.
Alcohol-induced cell death
Ethanol (95%), diluted in phosphate buffered saline (PBS), was added directly to the culture media to achieve final alcohol concentrations of either 200, 400 or 800 mg/dL (43, 87 or 174 mM ethanol) in dose-response experiments, or 400 mg/dL in all other experiments. For the ethanol-free culture groups, PBS replaced the alcohol. The following agents were individually tested for their effectiveness in preventing alcohol-induced neuronal death: (i) BAPTA acetoxymethyl ester (BAPTA/AM, a membrane-permeable Ca2+ chelator); (ii) 2-aminoethoxydiphenyl borate (2-APB, a membrane-permeable inositol-triphosphate receptor, IP3R, inhibitor); (iii) PBS with dimethyl sulfoxide (DMSO, vehicle control). As both BAPTA/AM and 2-APB were initially dissolved in DMSO, vehicle control groups received identical DMSO exposure (0.003% for BAPTA/AM; 0.006% for 2-APB). Solutions containing either a test agent or a vehicle control were added directly to the culture media utilizing two experimental protocols. With the pre-treatment paradigm, the test agent or the matching vehicle control was added 30-min prior to alcohol addition. With the concurrent addition paradigm, alcohol was added to the test agent solution, and an aliquot of this mixture was immediately added to the culture media. Tissue culture trays containing ethanol-exposed cultures were placed in sealed containers with 5% CO2 and an alcohol bath (ethanol concentration in the bath was equal to that in the culture media). For all experiments, matching ethanol-free culture groups were established concurrently, and these were similarly treated except the ethanol bath in the sealed containers was replaced with water.
All culture groups (ethanol-treated and ethanol-free) were incubated for 24 h at 37°C prior to cell counting. Viable cell numbers were determined on a haemocytometer utilizing a dye-exclusion method (trypan blue is only taken up by dead cells). This method is straightforward and yields very reliable results. We did not use commercially available cell counting kits, as we were concerned that the alcohol-induced changes in [Ca2+]i could interfere with these kits. Following the 24-h ethanol exposure, CGN were triturated into a 0.2% trypan blue solution and viable cell numbers were determined on a haemocytometer utilizing a phase contrast microscope. Cell numbers were determined in quadruplicate. For calculation of per cent cell loss, the difference in viable cell number between the ethanol-exposed and the matching ethanol-free culture was calculated, and this difference was divided by the viable cell number of the ethanol-free culture, deriving a per cent cell loss.
Determination of intracellular calcium concentration [Ca2+]i
Alcohol-stimulated changes in [Ca2+]i were assessed by Fura-2 ratio imaging (Grynkiewicz et al. 1985) utilizing a microscopic digital imaging system (Photon Technology International, Birmingham, NJ, USA) as described previously (Sharma et al. 1995). Briefly, CGN were plated onto glass coverslips and incubated for 24 h at 37°C. Following this incubation, individual coverslips were transferred to a new well of a six-well tissue culture tray, containing 1 mL of wash solution (Hank's balanced salt solution, HBSS). The wash solution was quickly removed, and 1 mL of Fura-2/AM loading solution (1.0 μM Fura-2/AM, 0.1% DMSO in HBSS) was added to the well. Cells were loaded with Fura-2/AM for 30 min at 37°C. Following removal of the Fura-2 loading solution, 1 mL of HBSS was added to the well, and the coverslip was incubated for an additional 30 min at 37°C to complete hydrolysis of Fura-2/AM to Fura-2. The latter is membrane-impermeable and does not leach out of the cell. The coverslip was transferred to a new well containing one of the following solutions (1 mL): (i) BAPTA/AM; (ii) 2-APB; or (iii) a vehicle control containing equivalent DMSO. Coverslips were incubated for 30 min at 37°C, and then transferred to a heated (37°C) coverslip chamber, which was mounted on the stage of an inverted phase contrast microscope (Diaphot; Nikon Instruments Inc., Melville, NY, USA). Fresh test agent solution or vehicle control solution (400 μL) was added to the coverslip chamber, and data acquisition was initiated (data collected at 5-s intervals throughout the experiment). Baseline [Ca2+]i was determined for 15 s immediately prior to the addition of alcohol. Once the baseline was established, alcohol (20 μL from solutions of various dilution) was added to achieve final alcohol concentrations of 200, 400 or 800 mg/dL in the dose-response experiment, while 400 mg/dL was utilized in all other Ca2+ imaging experiments. At the end of the testing period, 10 μM ionomycin was added to the cultures producing a large Ca2+ response, which was used to verify instrumentation. To calibrate the imaging system, ratio values for maximum Ca2+-binding and minimum Ca2+-binding to Fura-2 were determined utilizing 10 μM ionomycin and 10 mM EGTA respectively. Background values were determined using empty PDL-coated coverslips with HBSS.
For each coverslip, a single view-field was randomly selected, and the microscope remained focused on this view-filed for the duration (5 or 7 min) of the experiment. Every 5 s, the [Ca2+]i was determined for each cell in this view-field. On average, approximately forty cells per coverslip (range of 15 to 50 cells) were analysed. A mean [Ca2+]i was calculated from the cell data for each 5-s time point. In most experiments, cells from several replicates of the experiment were pooled to increase the accuracy of [Ca2+]i calculations. In our experiments, cultures established from a single mouse litter constituted one experimental replicate. To verify this analysis procedure, instead of pooling cells across replicates, the cells were pooled within an experimental replicate.
For alcohol neurotoxicity studies, treatment groups within each replicate (a replicate is one litter) were established in duplicate or triplicate, and cell numbers were averaged. As differences in cell plating introduces variability in the starting cell number, data were expressed as cell numbers, as well as alcohol-induced per cent cell loss (Pantazis et al. 1995). Statistical differences in cell numbers were determined either by repeated measures or by two-way mixed-model anova with alcohol and test agent concentration as variables. Post-hoc comparisons consisted of Bonferroni-corrected pairwise comparisons or paired t-tests as directed by the anova type when a statistically significant threshold was reached (p <0.05). Statistical differences in the per cent cell loss data were determined by one-way anovas, followed by Scheffé post-hoc tests. In Ca2+ imaging experiments, comparisons were made using the maximum [Ca2+]i level (peak [Ca2+]i) that each cell attained during the experiment (5 or 7 min) (Sharma and Bhalla 1989). Statistical differences in peak [Ca2+]i data were determined by either one-way or two-way anovas with alcohol and test agent concentration as factors. Scheffé post-hoc tests followed when significance was achieved (p <0.05).
All statistical analyses were performed using SPSS Statistics (IBM, New York, NY, USA).
Alcohol exposure induces neuronal death in CGN cultures
Exposure of CGN cultures to increasing concentrations of alcohol causes a significant (repeated measures anova, p <0.001) decrease in cell number (Fig. 1). This cell loss is because of an induction of cell death by alcohol. Our previous studies (Pantazis et al. 1993) established that CGN cultures do not proliferate, thus ruling out the alternative possibility that alcohol inhibits CGN proliferation. The alcohol-induced cell death is a dose-dependent effect ranging from 13% to 32%. As noted in the introduction, in vivo studies revealed a similar level of alcohol-induced neuronal death, indicating that alcohol causes a significant, but far from total loss of CGN in both animals and cell cultures.
Alcohol exposure induces a rapid and sustained rise in [Ca2+]i in CGN cultures
As shown in the top row of photomicrographs (Basal) in Fig. 2a, prior to alcohol exposure, most of the cells are blue (indicating low [Ca2+]i) with a few green cells (higher [Ca2+]i), but no yellow cells are seen. The second row of photomicrographs shows the same field after 1 min of alcohol exposure, and there are greater numbers of green–yellow cells, indicating a higher [Ca2+]i. The photomicrographs in Fig. 2a also show that this alcohol effect on [Ca2+]i is dose-dependent with the greatest increase in [Ca2+]i occurring at the highest alcohol dose.
In our study, an experimental replicate is defined as data derived from a single litter of mouse pups with each replicate performed at different times. To increase the numbers of cells for analysis, data sets from multiple experimental replicates were combined, generating cellular [Ca2+]i values for hundreds of cells. A mean [Ca2+]i was calculated from the cellular [Ca2+]i values at each 5-s interval and these means are shown in Fig. 2b. Baseline [Ca2+]i was determined for 15 s prior to initiating alcohol exposure (Fig. 2b). The addition of alcohol at time 0 rapidly (within a minute or two) increased the [Ca2+]i. Over time the [Ca2+]i stopped increasing, but it did not return to baseline, instead remaining at a constant level that was higher than baseline [Ca2+]i. Every culture group which received alcohol had a higher [Ca2+]i compared with the ethanol-free group. The rate of increase in [Ca2+]i was dependent on alcohol dose and was greatest at 800 mg/dL, while the 400 and 200 mg/dL cultures displayed more moderate rates of increase. Both the 800 and 400 mg/dL cultures levelled off at a higher [Ca2+]i level (~120 nM) compared with the 200 mg/dL cultures (~90 nM). Note, the [Ca2+]i level never returned to baseline, and remained elevated throughout the course of the experiment (7 min).
Instead of deriving means for [Ca2+]i from hundreds of cells and displaying data as [Ca2+]i traces as in Fig. 2b, we used an alternative method of data analysis, called peak [Ca2+]i, in which data can be more readily quantified and statistically analysed (Sharma and Bhalla 1989). With peak [Ca2+]i analysis, the objective was to select the highest [Ca2+]i level (peak [Ca2+]i) that an individual cell achieved during the 7 min immediately following the addition of alcohol. This procedure generated peak [Ca2+]i values for hundreds of cells in each treatment group, and a mean peak [Ca2+]i value was derived (Fig. 2c). Alcohol induced a significant (one-way anova, p <0.001) dose-dependent increase in peak [Ca2+]i.
The results shown in Fig. 2b and c were generated by pooling cells from the replicates of the experiment. To be assured that pooling cells across experimental replicates did not skew our results, we used an alternative method of analysis in which cells were grouped within individual replicates, and peak [Ca2+]i values were derived (Fig. 2d). Grouping the data within replicates did not change the overall result; alcohol still induced a significant (p <0.05) increase in [Ca2+]i. Because of the greater statistical power derived by combining cells across replicates, this method was used in subsequent analyses.
The peak [Ca2+]i values shown in Fig. 2c are means derived from populations of several hundred cells. It is not clear from this data whether alcohol is increasing [Ca2+]i by a small amount in a large number of cells or alternatively increasing [Ca2+]i by a large amount in a small number of cells. Or, possibly both mechanisms are involved. To address this question, the peak [Ca2+]i values for individual cells, which were used to derive the means in Fig. 2c, were grouped into bins of 20 nM increments in peak [Ca2+]i. The number of cells in each bin was determined and expressed as a per cent of the total cell population to derive the histograms (Hegarty et al. 1997) shown in Fig. 2e. To facilitate interpretation of these histograms, we selected a reference peak [Ca2+]i value, which was greater than the peak [Ca2+]i for the vast majority of cells in the ethanol-free (0 mg/dL) group. A reference line (shown as a vertical line in Fig. 2e) of 200 nM peak [Ca2+]i was ideal as essentially all the cells in the ethanol-free group have peak [Ca2+]i below 200 nM with very few cells (4%) having [Ca2+]i above 200 nM. With alcohol exposure (200, 400, or 800 mg/dL), the majority of cells remained below 200 nM [Ca2+]i, suggesting that alcohol has little effect on peak [Ca2+]i in most cells. However, as the alcohol concentration is increased, a greater number of cells display peak [Ca2+]i values above 200 nM. For example, with alcohol concentrations of 0, 200, 400 and 800 mg/dL, the per cent of cells above the 200 nM reference line are 4%, 14%, 23%, and 29%, respectively, which is a significant increase [Pearson's chi-squared test, χ2 (3, n = 1389) = 71.18, p <0.001]. In summary, although alcohol has little effect on peak [Ca2+]i in most cells, there is a substantial increase in [Ca2+]i in a small number of cells. The number of cells experiencing this large rise in peak [Ca2+]i increases with increasing ethanol. Whether the cells displaying this enhanced [Ca2+]i increase are the cells which die 24 h later awaits further investigation. For comparison, Fig. 1 shows that alcohol exposures of 200, 400 and 800 mg/dL alcohol cause cell losses of 13%, 22% and 32%, respectively, whereas these same alcohol concentrations increase [Ca2+]i above 200 nM in 14%, 23% and 29% of the cells. The magnitude of the alcohol-induced cell loss approximates the percentage of cells that have experienced a marked increase in [Ca2+]i.
Alcohol exposure elevates [Ca2+]i in Ca2+-free medium, suggesting intracellular release of Ca2+
CGN cultures were exposed to alcohol in Ca2+-free media, thus eliminating the extracellular source of Ca2+. Alcohol induced a [Ca2+]i surge in Ca2+-free media, which was similar to that observed in Ca2+-containing media (Fig. 3a). Furthermore, peak [Ca2+]i values were similar in Ca2+-containing and Ca2+-free media (Fig. 3b). These results suggest that intracellular release of Ca2+ is responsible for the increase in [Ca2+]i caused by alcohol.
Chelation of Ca2+ with cell-permeant BAPTA/AM eliminates both the increase in [Ca2+]i and the neuronal death caused by alcohol in CGN cultures
We next tested the hypothesis that preventing the alcohol-induced increase in [Ca2+]i ameliorates the cell death caused by alcohol. A membrane-permeable chelator, BAPTA/AM, was used to prevent the alcohol-induced increase in [Ca2+]i. In our experimental protocol, Fura-2-loaded CGN were pre-treated with either 1.0 μM BAPTA/AM or a vehicle control solution containing DMSO, thirty min before initiating alcohol exposure. In the absence of BAPTA/AM, alcohol exposure rapidly increased [Ca2+]i, while there was little change in the alcohol-free culture group (Fig. 4a). In contrast, alcohol did not raise [Ca2+]i in CGN cultures pre-treated with BAPTA/AM, and the [Ca2+]i levels in this alcohol-exposed group were similar to the BAPTA/AM pre-treated cultures, which never received alcohol. A peak [Ca2+]i analysis was performed (Fig. 4b) and statistical analysis (two-way anova) of this data revealed the main effects of alcohol (p <0.001), BAPTA/AM pre-treatment (p <0.001) and an interaction (p <0.001). These results indicate that BAPTA/AM effectively blocks the alcohol-induced increase in [Ca2+]i.
On the basis of these observations, we tested the hypothesis that BAPTA/AM is a neuroprotective agent capable of reducing the alcohol-induced cell death in CGN cultures. Analysis (two-way repeated measures anova) of the number of surviving CGNs following a 24-h alcohol exposure revealed that without BAPTA/AM (Fig. 5a), alcohol caused a significant loss of viable cells compared with the matching alcohol-free control group (p <0.001). Pre-treatment with BAPTA/AM produced a dose-dependent neuroprotective effect. A significant (but reduced) alcohol-induced cell loss continued to be observed following pre-treatment with 0.1 μM BAPTA/AM (Fig. 5a). However, there was no significant alcohol-induced cell loss when CGN cultures were pre-treated with the higher dose of BAPTA/AM (1.0 μM). To demonstrate better the magnitude of the alcohol-induced cell death and the effectiveness of BAPTA/AM to prevent it, the cell number data in Fig. 5a were expressed as a per cent cell loss (Fig. 5b). In the absence of BAPTA/AM pre-treatment, alcohol exposure reduced viable cell numbers by 18 ± 2%. Pre-treatment of the CGN cultures with either 0.1 μM or 1.0 μM BAPTA/AM significantly (p <0.001) reduced the alcohol-induced cell losses to 7 + 2% and 4 ± 2% respectively. As a further test of the neuroprotective effect of chelators, membrane-permeable EGTA/AM (1.0 μM) also significantly (p <0.001) reduced alcohol-induced cell loss, much like BAPTA/AM (data not shown).
Inhibition of the inositol-triphosphate receptor (IP3R) with 2-APB eliminates both the increase in [Ca2+]i and the neuronal death caused by alcohol in CGN cultures
To verify that inhibition of the alcohol-induced rise in [Ca2+]i prevents the subsequent neuronal death caused by alcohol, we blocked the rise in [Ca2+]i by an alternative mechanism. As our experiment in Ca2+-free media indicated that alcohol raised [Ca2+]i from an intracellular source, we inhibited the IP3R, which regulates Ca2+ release from the endoplasmic reticulum (ER) (Vermassen et al. 2004). Fura-2-loaded CGN were pre-treated (30 min prior to alcohol exposure) with either 2-APB (5 μM), a specific inhibitor of IP3R, or vehicle control (DMSO). As shown in Fig. 6a, alcohol exposure in the absence of 2-APB pre-treatment increased the [Ca2+]i, consistent with our previous results. In contrast, pre-treatment of CGN cultures with 2-APB prevented this alcohol-induced rise in [Ca2+]i, and this culture group had [Ca2+]i levels which were essentially identical to 2-APB pre-treated cultures that were never exposed to alcohol (Fig. 6a).
Peak [Ca2+]i analysis verified that 2-APB pre-treatment reduced the alcohol-induced [Ca2+]i surge to a level that was identical to cultures which never were exposed to alcohol (Fig. 6b). Statistical analysis (two-way anova) of the peak [Ca2+]i data revealed a main effect of alcohol (p <0.001), and an interaction between 2-APB pre-treatment and alcohol exposure (p <0.001). No main effect of 2-APB treatment on peak [Ca2+]i levels was observed.
As 2-APB blocks the alcohol-induced increase in [Ca2+]i, much like BAPTA/AM, we tested the hypothesis that 2-APB is a neuroprotective agent and prevents the cell death caused by alcohol. In the absence of 2-APB pre-treatment, alcohol caused significant cell death (Fig. 7a), resulting in a 23% cell loss (Fig. 7b). A reduced alcohol-induced celI loss (14%), which did not reach significance, continued to be observed when cultures were pre-treated with 0.3 μM 2-APB (Fig. 7b). The higher doses of 2-APB (1.3 μM and 5.0 μM) significantly reduced alcohol-induced cell loss to 5% and 0% respectively. Two-way anova revealed a significant interaction of 2-APB and alcohol (p <0.005). Post-hoc tests revealed that the two highest concentrations of 2-APB significantly reduced this cell loss (Scheffé, p <0.05). As further verification that inhibition of the IP3R rescues CGN cultures from alcohol neurotoxicity, the IP3R was inhibited with Xestospongin C, which effectively reduced (p <0.001) alcohol-induced cell death from 24% to 1.0% (data not shown).
Alcohol neurotoxicity returns when the mitigating effect of either BAPTA/AM or 2-APB on [Ca2+]i homeostasis is aborted
To examine further the neuroprotective effects of BAPTA/AM and 2-APB, the 30-min pre-treatment protocol was modified, and these agents were added to the cultures at the same time as alcohol (concurrent addition). Concurrent addition of either BAPTA/AM (Fig. 8a) or 2-APB (Fig. 8b) with alcohol rendered both agents ineffective in preventing the alcohol-induced increase in [Ca2+]i. In contrast, a 30-min pre-treatment with either agent replicated our previous results, and the alcohol-induced increase in [Ca2+]i, was reduced (Fig. 8a and b). Not only did the concurrent addition eliminate the effectiveness of BAPTA/AM and 2-APB to block the rise in [Ca2+]i, but these agents no longer ameliorated the alcohol-induced neuronal loss (Fig. 8c and d) when concurrent addition was utilized. In contrast, the 30-min pre-treatment with either BAPTA/AM (Fig. 8c) or 2-APB (Fig. 8d) continued to effectively mitigate the cell loss caused by alcohol. These results provide additional evidence that the rise in [Ca2+]i and the cell death caused by alcohol are closely linked; blocking the [Ca2+]i surge ameliorates the cell death.
Previous studies from our laboratory have shown that alcohol exposure of CGN cultures causes neuronal loss (Pantazis et al. 1993). Herein, we show that alcohol treatment of CGN disrupts [Ca2+]i levels in part by release of Ca2+ from intracellular stores. When the increase in [Ca2+]i is prevented by cell-permeable chelators or specific inhibitors of the IP3R, the alcohol-induced neuronal death is ameliorated, suggesting a strong association between these two effects. These are the first data to identify alcohol-mediated release of Ca2+ from intracellular sources as a mechanism by which alcohol diminishes CGN survival.
The alcohol-induced increase in [Ca2+]i is a rapid, dose-dependent effect, which is sustained for the duration of the experiments (5 or 7 min). Similar to the increase in [Ca2+]i, the neuronal death caused by alcohol in CGN cultures is also a dose-dependent effect, but unlike alcohol's rapid effect on increasing [Ca2+]i, the neuronal death requires h to become evident (Pantazis et al. 1993, 1995; Ramachandran et al. 2003). Although alcohol concentrations of 200, 400 and 800 mg/dL induce significant increases in [Ca2+]i and neuronal death, we most often utilized 400 mg/mL (0.4%). A blood alcohol concentration of 400 mg/dL is a high and potentially lethal dose for individuals who do not abuse alcohol. In contrast, alcohol abusers can reach this blood alcohol level and not only survive, but may appear to be only slightly inebriated (Chesher and Greeley 1992). Pregnant women, who abuse alcohol and reach these high blood alcohol levels, are most at risk for having a child with FASD.
Research by several laboratories has demonstrated effects of alcohol on stimulated Ca2+ responses, such as voltage-gated Ca2+ channels (VGCC) and ligand-gated Ca2+ channels (LGCC), which are found on excitable cells, such as neurons. Acute (minutes) alcohol exposure inhibits several types of VGCC including the L-type (Mullikin-Kilpatrick and Treistman 1994; Gerstin et al. 1998; Walter and Messing 1999), N-type (McMahon et al. 2000) and P/Q-type (Solem et al. 1997). Acute alcohol exposure also inhibits LGCC such as the acetylcholine (muscarinic)-gated Ca2+ channel (Rabe and Weight 1988) and the glutamate (NMDA)-gated Ca2+ channel (Mullikin-Kilpatrick and Treistman 1994; Gerstin et al. 1998; Walter and Messing 1999). However, long-term (days) alcohol exposure can up-regulate the expression of VGCC and LGCC, possibly as part of a compensatory response (Katsura et al. 2006; Mah et al. 2011), and thereby enhance Ca2+ uptake.
Two observations in our study indicate that alcohol can induce intracellular Ca2+ release in resting neurons. First, the alcohol-induced increase in [Ca2+]i occurs in Ca2+-free medium, suggesting an intracellular source. Second, 2-APB, a specific inhibitor of the IP3R found in the ER, inhibits the alcohol-induced increase in [Ca2+]i. Previous studies in other model systems such as hepatocyte cultures (Hoek et al. 1987, 1992; Higashi and Hoek 1991; Hoek and Higashi 1991; Hoek and Kholodenko 1998), murine embryos (Stachecki and Armant 1996) and chick neural crest (Debelak-Kragtorp et al. 2003; Garic-Stankovic et al. 2005) have suggested that alcohol increases [Ca2+]i from intracellular sources by enhancing the activity of phosphoinositide-specific phospholipase C (PI-PLC), which can hydrolyse phosphatidylinositol 4,5-bisphosphate (PIP2) to generate inositol 1,4,5-trisphosphate. Inositol 1,4,5-trisphosphate activates the IP3R, resulting in Ca2+ release from the ER (Furuichi and Mikoshiba 1995; Ma et al. 2000). In agreement with these studies, our preliminary evidence (data not shown) suggests that inhibition of PLC prevents the alcohol-induced increase in [Ca2+]i in CGN cultures. Our studies have extended these observations by providing direct evidence that alcohol increases [Ca2+]i in part by Ca2+ release from intracellular sources.
As a key goal for this study was to gain a better understanding of the mechanism by which alcohol causes neuronal death, we explored the possibility that the disruption in [Ca2+]i homeostasis by alcohol is linked to its neurotoxicity. Our experimental approach was to block the alcohol-induced rise in [Ca2+]i and determine what effect this had on the neuronal death caused by alcohol in CGN cultures. We blocked the alcohol-induced rise in [Ca2+]i by two independent mechanisms, either chelation of Ca2+ with BAPTA/AM or inhibition of the IP3R with 2-APB. Both mechanisms prevented alcohol-induced neuronal death. Furthermore, when the effectiveness of the chelator or the IP3R inhibitor to modulate [Ca2+]i was circumvented by adding these agents concurrently with alcohol, rather than as a 30-min pre-treatment, their protective effect to prevent cell death was lost. Therefore, Ca2+ release from intracellular stores may be a primary cause of alcohol-induced neuronal death. Other studies (Camandola et al. 2005) utilizing alternative culture models (fibroblasts, primary cortical neurons and TNFα or glutamate as toxins) have shown that blocking the function of the IP3R, thereby reducing release of intracellular Ca2+, enhances cell survival in toxic conditions.
It is now widely recognized that disruption of [Ca2+]i homeostasis can lead to cell death (Berridge et al. 1998; Hajnoczky et al. 2003; Orrenius et al. 2003). Alcohol toxicity has also been linked to [Ca2+]i signalling. For example, a modest alcohol-induced rise in [Ca2+]i causes cell death in chick neural crest (Debelak-Kragtorp et al. 2003; Garic-Stankovic et al. 2005) and astrocytes (Hirata et al. 2006). At first glance, it would appear that the modest increase in [Ca2+]i by alcohol is not sufficient to induce cell death. However, it is important to note that in our study, this modest increase in [Ca2+]i is sustained. Possibly, the rise in [Ca2+]i in combination with the persistence of this disruption is most damaging to the cell (more discussion below).
Utilizing a neural crest model of ethanol-induced cell death, Susan Smith and colleagues have shown that alcohol exposure induces a rapid rise in [Ca2+]i, which in turn activates Ca2+/calmodulin-dependent protein kinase II (CaMKII) (Garic et al. 2011). CaMKII has been linked to apoptotic pathways (Timmins et al. 2009). A subsequent study suggested that CaMKII phosphorylates the transcription effector β-catenin, eventually leading to the ubiquitination and degradation of this transcription factor (Flentke et al. 2011). As β-catenin enhances cell survival (Kohn and Moon 2005; Holowacz et al. 2011), the loss of this important transcription factor could be responsible for the death of neural crest cells.
The link between the dysregulation of [Ca2+]i and cell death is now well recognized (Zhivotovsky and Orrenius 2011). In our study, the alcohol-induced increase in [Ca2+]i is not a large effect, but it is sufficient to cause cell death in CGN cultures. Then again, the in vitro (Pantazis et al. 1995) and in vivo (Bonthius and West 1991) loss of cerebellar neurons by alcohol is not total and ranges from 10 to 40% of the neuronal population. The magnitude of alcohol's effect on [Ca2+]i in our experiments is consistent with the magnitude of cerebellar neuron loss caused by alcohol. Another consideration is the persistence of the increase in [Ca2+]i. In our studies, the alcohol-induced rise in [Ca2+]i never returns to baseline, remaining elevated for up to 7 min. The combination of an increase in [Ca2+]i coupled with the persistence of this effect may be most disruptive for the neuron.
With regard to the duration of the [Ca2+]i response, our result is in contrast to that seen in the neural crest model. Although alcohol exposure of the neural crest rapidly increases [Ca2+]i, much like our observation in CGN cultures, the [Ca2+]i returns to baseline in 1 min (Garic et al. 2011). Astrocyte cultures have also been used as a model to investigate alcohol-induced cell death, and in this case, alcohol induced a rapid and persistent rise in [Ca2+]i, which was linked to cell death (Hirata et al. 2006), similar to results presented here. In another culture model, a prolonged elevation of [Ca2+]i was linked to staurosporin-induced cell death in pheochromocytoma PC12, a neuronal-like cell line (Kruman et al. 1998). The cell death in PC12 cultures was prevented by chelating Ca2+ with BAPTA/AM, similar to results presented here. Finally a study, utilizing a human cancer cell line and staurosporin-induced cell death as a model, directly investigated whether the duration of elevated [Ca2+]i altered the eventual death of the cell (Norberg et al. 2008). The results indicated that a transient (less than 3 min) increase in [Ca2+]i did not result in cell death, whereas a prolonged (~10 min) increase effectively killed the cells, supporting the hypothesis that the duration of [Ca2+]i increase is a key determinant in [Ca2+]i-mediated death signalling. One additional point regarding the Norberg study cited above (Norberg et al. 2008). This study also showed that the staurosporin-induced rise in [Ca2+]i promoted the release of apoptosis-inducing factor (AIF) from the inner mitochondrial membrane into the cytosol. The release of AIF was triggered by the Ca2+-induced activation of a Ca2+-dependent protease, calpain (Kar et al. 2010), which cleaves AIF from the mitochondrial membrane. Interestingly, AIF is linked to alcohol-induced toxicity, as alcohol exposure of fetal cortical neurons releases AIF from the mitochondria and eventually cell death (Cherian et al. 2008). Thus, AIF may be a downstream effector for the [Ca2+]i-mediated cell death induced by alcohol.
In summary, this study provides the first evidence that a persistent alcohol-induced increase in [Ca2+]i is a key determinant for neuronal death in CGN cultures. There is a strong link between the sustained rise in [Ca2+]i and alcohol-induced neuronal death, as chelation of Ca2+ with BAPTA/AM or inhibition of the IP3R with 2-APB prevents this cell death. Therapeutic intervention aimed at alleviating the alcohol-induced disruption of [Ca2+]i homeostasis may potentially lessen neuronal loss, one of the most damaging effects of alcohol on the developing brain.
This study was supported by NIAAA grant AA011577 and support from the University of Iowa Graduate College. The technical assistance of the University of Iowa Central Microscopy Research Facilities is acknowledged. The authors have no conflict of interest to declare. DEK, GL, RCB and NJP were involved in experimental design. DEK, GL, MT and TM were responsible for performing the study. DEK, RCB and NJP wrote the manuscript. All authors approved this version of the manuscript.