nihms419319

INTRACELLULAR CALCIUM PLAYS A CRITICAL ROLE IN THE
ALCOHOL-MEDIATED DEATH OF CEREBELLAR GRANULE
NEURONS
Dimitrios E. Kouzoukas, PhD*, Guiying Li, MD*, Maysaam Takapoo*, Thomas Moninger!,
Ramesh C. Bhalla, PhD, DVM*, and Nicholas J. Pantazis, PhD*
*Department of Anatomy and Cell Biology, Carver College of Medicine, The University of Iowa
!University of Iowa Central Microscopy Research Facilities
Abstract
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 hours. Ratiometric Ca2+
imaging with Fura-2 revealed that alcohol causes a rapid (one-two minutes), dose-dependent
increase in [Ca2+]i, which persisted for the duration of the experiment (5 or 7 minutes). 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.
Keywords
alcohol neurotoxicity; fetal alcohol syndrome; fetal alcohol spectrum disorder; intracellular
calcium; inositol triphosphate receptor; neurodevelopment
Introduction
Alcohol abuse during pregnancy can damage multiple organ systems in the fetus, resulting in
permanent functional deficits that persist into adulthood (Greenbaum & Koren 2002). Since
Corresponding author: Nicholas J. Pantazis, PhD, Department of Anatomy and Cell Biology, The University of Iowa, 1-530 Bowen
Science Building, 51 Newton Road, Iowa City, IA 52242-1109, 319-335-7732 (phone), 319-335-7198 (fax), nicholas-
pantazis@uiowa.edu.
The authors have no conflict of interest to declare.
HHS Public Access
Author manuscript
J Neurochem. Author manuscript; available in PMC 2016 November 07.
Published in final edited form as:
J Neurochem. 2013 February ; 124(3): 323–335. doi:10.1111/jnc.12076.
AuthorManuscriptAuthorManuscriptAuthorManuscriptAuthorManuscript

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 in order 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 (Maier et al. 1997, Pierce &
West 1986), 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). Since 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, since 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 signaling is well recognized,
including participation in signaling cell death (Orrenius et al. 2003, Trump & Berezesky
1995, Zhivotovsky & 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 (Hirata et al. 2006, Holownia et al. 1997), and
this increase in [Ca2+]i was linked to cell death. Since 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 (5 – 7 postnatal day, PD) mice are
well suited for this work. At PD 5 through 7, the developing mouse cerebellum is at the peak
of its alcohol vulnerability (Hamre & 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 & 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
Kouzoukas et al. Page 2

toxicity (Pantazis et al. 1995) by stimulating a nitric oxide (NO) signaling 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.
METHODS
Animals
A breeding colony was established from C57BL/6;129 mice (initially obtained from Jackson
Laboratories, Bar Harbor, ME) 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 5 to 7 postnatal 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 & 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 X 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 X 105 cells per well.
Ethanol-exposed and ethanol-free culture groups were plated into separate cell culture trays,
since these trays were placed in individual sealed containers in order to minimize ethanol
evaporation (described below). For Ca2+ imaging experiments, the cell density of the plating
solution was adjusted to 1.0 X 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 in order 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 & 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: 1)
BAPTA acetoxymethyl ester (BAPTA/AM, a membrane-permeable Ca2+ chelator); 2) 2-
aminoethoxydiphenyl borate (2-APB, a membrane-permeable inositol-triphosphate receptor,
IP3R, inhibitor); 3) PBS with dimethyl sulfoxide (DMSO, vehicle control). Since 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 pretreatment paradigm, the test agent or the
matching vehicle control was added 30-minutes 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 hours at 37°C
prior to cell counting. Viable cell numbers were determined on a hemocytometer utilizing a
dye-exclusion method (trypan-blue is only taken up by dead cells). This method is straight-
forward and yields very reliable results. We did not use commercially available cell counting
kits, since we were concerned that the alcohol-induced changes in [Ca2+]i could interefere
with these kits. Following the 24-hour ethanol exposure, CGN were triturated into a 0.2%
trypan blue solution and viable cell numbers were determined on a hemocytometer utilizing
a phase-contrast microscope. Cell numbers were determined in quadruplicate. For
calculation of percent 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 percent 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) as described previously (Sharma et al. 1995). Briefly, CGN were plated onto
glass coverslips and incubated for 24-hours 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 minutes 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 minutes at 37°C in order 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): 1) BAPTA/AM; 2) 2-APB; or 3) a vehicle control containing equivalent
DMSO. Coverslips were incubated for 30 minutes 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 (Nikon Diaphot). 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-
second intervals throughout the experiment). Baseline [Ca2+]i was determined for 15
seconds 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. In order 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 (five or seven minutes) of the experiment. Every
five seconds, the [Ca2+]i was determined for each cell in this view-field. On average,
approximately forty cells per coverslip (range of fifteen to fifty cells) were analyzed. A
mean [Ca2+]i was calculated from the cell data for each five-second time point. In most
experiments, cells from several replicates of the experiment were pooled in order to increase
the accuracy of [Ca2+]i calculations. In our experiments, cultures established from a single
mouse litter constituted one experimental replicate. In order to verify this analysis
procedure, instead of pooling cells across replicates, the cells were pooled within an
experimental replicate.
Statistical analysis
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. Since
differences in cell plating introduces variability in the starting cell number, data were
expressed as cell numbers, as well as alcohol-induced percent 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 percent 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 minutes) (Sharma & 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).
RESULTS
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 due
to 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
one minute 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 work, an experimental replicate is defined as data derived from a single litter of
mouse pups with each replicate performed at different times. In order 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 five second interval and these means are
shown in Fig 2B. Baseline [Ca2+]i was determined for 15-seconds 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 to 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 leveled off at a higher [Ca2+]i level (~120 nM)
compared to 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 minutes).
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 analyzed (Sharma & 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 seven minutes 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 Figs 2B and 2C were generated by pooling cells from the replicates of
the experiment. In order 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. In order 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 percent of the total cell population in order to
derive the histograms (Hegarty et al. 1997) shown in Figure 2E. In order 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 since 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 percent 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 hours 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 xtracellular
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 pretreated with either 1.0 µM BAPTA/AM or a vehicle control
solution containing DMSO, thirty minutes 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 pretreated with BAPTA/AM, and the [Ca2+]i levels in this alcohol-exposed group
were similar to the BAPTA/AM-pretreated 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 pretreatment (p < 0.001)
and an interaction (p < 0.001). These results indicate that BAPTA/AM effectively blocks the
alcohol-induced increase in [Ca2+]i.
Based on 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-hour
alcohol exposure revealed that without BAPTA/AM (Fig 5A), alcohol caused a significant
loss of viable cells compared to the matching alcohol-free control group (p < 0.001).
Pretreatment with BAPTA/AM produced a dose-dependent neuroprotective effect. A
significant (but reduced) alcohol-induced cell loss continued to be observed following
pretreatment with 0.1 µM BAPTA/AM (Fig 5A). However, there was no significant alcohol-
induced cell loss when CGN cultures were pretreated with the higher dose of BAPTA/AM
(1.0 µM). In order 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 was expressed
as a percent cell loss (Fig 5B). In the absence of BAPTA/AM pretreatment, alcohol exposure
reduced viable cell numbers by 18 ± 2%. Pretreatment 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. Since 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 pretreated
(30 minutes 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 pretreatment increased the [Ca2+]i, consistent with our previous results. In contrast,
pretreatment 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-
pretreated cultures that were never exposed to alcohol (Fig 6A).
Peak [Ca2+]i analysis verified that 2-APB pretreatment 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 pretreatment and alcohol exposure (p
< 0.001). No main effect of 2-APB treatment on peak [Ca2+]i levels was observed.
Since 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 pretreatment, 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
pretreated 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-minute
pretreatment 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-minute pretreatment with either agent
replicated our previous results, and the alcohol-induced increase in [Ca2+]i, was reduced
(Figs 8A and 8B). 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 (Figs 8C and 8D) when concurrent addition was utilized.
In contrast, the 30-minute pretreatment 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.
Discussion
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 minutes). 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 hours to become
evident (Ramachandran et al. 2003, Pantazis et al. 1993, Pantazis et al. 1995). 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 & 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 (Gerstin et al. 1998, Mullikin-
Kilpatrick & Treistman 1994, Walter & 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 & Weight 1988) and the glutamate
(NMDA)-gated Ca2+ channel (Gerstin et al. 1998, Mullikin-Kilpatrick & Treistman 1994,
Walter & Messing 1999). However, long-term (days) alcohol exposure can upregulate 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, Hoek & Higashi 1991, Higashi &
Hoek 1991, Hoek et al. 1992, Hoek & Kholodenko 1998), murine embryos (Stachecki &
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 hydrolyze
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 & 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.
Since 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-minute pretreatment, 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 signaling. 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). Since β-
catenin enhances cell survival (Kohn & 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 & 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 & 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 minutes. 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 one
minute (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 minutes) increase in [Ca2+]i did not result in cell death,
whereas a prolonged (~10 minutes) increase effectively killed the cells, supporting the
hypothesis that the duration of [Ca2+]i increase is a key determinant in [Ca2+]i-mediated
death signaling. One additional point regarding the Norberg study cited above (Norberg et
al. 2008). This work 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, the present 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, since
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.

Acknowledgements
This work 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.
DEK, GL, RCB and NJP were involved in experimental design. DEK, GL, MT and TM were responsible for
performing the work. DEK, RCB and NJP wrote the manuscript. All authors approved this version of the
manuscript.
Abbreviations
2-APB 2-aminoethoxydiphenyl borate
AIF apoptosis-inducing factor
BAPTA/AM 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid tetrakis
(acetoxymethyl ester)
[Ca2+]i intracellular Ca2+ concentration
CaMKII Ca2+/calmodulin-dependent protein kinase II
CGN cerebellar granule neuron
DMEM Dulbecco's Modified Eagle Medium
DMSO dimethyl sulfoxide
FAS fetal alcohol syndrome
FASD fetal alcohol spectrum disorders
HBSS Hank’s balanced salt solution
IP3R inositol triphosphate receptor
LGCC ligand-gated Ca2+ channels
PBS phosphate buffed saline
PDL poly-D-lysine
PD postnatal day
VGCC voltage-gated Ca2+ channels
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Figure 1. Alcohol reduces cell number in CGN primary cultures
CGN cultures derived from 13 litters (n = 13) were exposed to ethanol for 24 hours. Cell
numbers were determined on a hemocytometer utilizing trypan blue exclusion to verify cell
death. Values represent the mean number of cells/well ± SEM. Percent cell loss is shown at
the top of each bar. Statistical differences were determined by a repeated measures ANOVA.
* Means differ significantly (p < 0.05) from all other groups as determined by Bonferroni-
adjusted pair-wise comparisons.

Figure 2. Alcohol rapidly increases intracellular Ca2+ [Ca2+]i
Ratiometric analyses were performed on Fura-2-loaded CGN. (A) Representative
pseudocolor 340 / 380 ratio images of CGN cultures derived from a single mouse litter. The
scale bar to the right reflects [Ca2+]i with blue and red depicting low and high [Ca2+]i,
respectively. (a–d) Representative view-fields of CGN cultures before addition of alcohol.
(e–f) The identical view-fields after one-minute of alcohol exposure. Comparisons of the
panels in the top row with the corresponding bottom-row panels indicate that a one-minute
alcohol exposure increases [Ca2+]i. Panels e-h reveal that raising the alcohol concentration
increases the numbers of cells with higher [Ca2+]i. (B) Traces chart the mean [Ca2+]i ± SEM
in alcohol-exposed CGN cultures over time (plotted in five second intervals). In order to
acquire values from hundreds of cells, each trace combines data from four mouse litters, i.e.
four experimental replicates. (C) Peak [Ca2+]i levels were derived from the data in Figure 2B
by determining the highest [Ca2+]i that each cell attains over the seven minute duration of
the experiment. A mean ± SEM was derived from this data and is shown here. The solid line
indicates baseline [Ca2+]i, which is the mean [Ca2+]i level of the cells as measured for 15
seconds prior to the addition of alcohol. (D) In Figure 2C, the cell data from four
experimental replicates (four litters) were combined and used to derive peak [Ca2+]i levels.
In Figure 2D, the cell data within each replicate were kept separate, not pooled, and mean

peak [Ca2+]i levels were derived from the four experimental replicates. Since the conclusion
from Figures 2C and 2D is similar that alcohol raises Peak [Ca2+]i, this indicates that
analyzing the data as a pool of all the replicates or as individual replicates does not alter the
final outcome. In Figure 2E, the peak neuronal [Ca2+]i levels for each alcohol treatment
condition were grouped into 20 nM bins and plotted in the histograms. The vertical line is
drawn at a [Ca2+]i concentration of 200 nM. As can be seen, alcohol increased the range of
the peak Ca2+ response. * Statistically different from ethanol-free (0 mg/dl) cultures as
determined by one-way ANOVA with Scheffé post-hoc comparisons and p < 0.05.

Figure 3. The ethanol-induced rise in [Ca2+]i occurs in Ca2+ - free media
Immediately before imaging, CGN-containing coverslips were transferred to HBSS media
containing Ca2+ (+Ca2+) or Ca2+-free (−Ca2+) media. (A) Alcohol was added to both the
+Ca2+ and −Ca2+ culture groups at 0 seconds. As in Figure 2B, cell data from five
experimental replicates were combined in order to obtain mean [Ca2+]i ± SEM values for
several hundred cells. (B) Mean Peak [Ca2+]i values were calculated from the data in Figure
3A. Alcohol continues to induce a rapid rise in [Ca2+]i in Ca2+-free media, suggesting that

the alcohol-induced increase in [Ca2+]i originates from an intracellular, rather than an
extracellular source.

Figure 4. Pretreatment with BAPTA/AM, a membrane-permeable Ca2+ chelator, eliminates the
alcohol-induced rise in [Ca2+]i
CGN cultures were pre-treated with either 1.0 µM BAPTA/AM (filled circles in figure) or
vehicle control, 0.003% DMSO (hollow circles) for 30 minutes prior to receiving 400 mg/dl
ethanol (red lines) or PBS for ethanol-free cultures (blue lines). The duration of the
experiment was reduced from seven to five minutes since the results of Figs 2B and 3A
indicate that there is little change in [Ca2+]i after five minutes. (A) The data from five
replicates of the experiment were combined and mean [Ca2+]i ± SEM were calculated. (B)
Mean Peak [Ca2+]i ± SEM for the four treatment groups are shown. * indicates that the

Mean Peak [Ca2+]i differs significantly from that of all other groups (Scheffé, p < 0.001).
Pretreatment with BAPTA/AM eliminates the alcohol-induced rise in [Ca2+]i. Although
BAPTA/AM (with and without alcohol) appeared to lower [Ca2+]i with respect to the control
group (no alcohol, no BAPTA/AM), this effect is primarily due to the difference in start
point for the basal [Ca2+]i level (observed fifteen seconds prior to alcohol exposure).

Figure 5. Pretreatment with BAPTA/AM rescues neurons from alcohol-induced cell loss
Thirty minutes before the addition of alcohol, CGN cultures received either BAPTA/AM or
0.003% DMSO (vehicle control). Viable cell numbers were determined following a twenty-
four hour alcohol exposure. (A) Cell number values are the means ± SEM derived from 12
experimental replicates. * indicates significant difference from the matching ethanol-free (0
mg/dl) cultures, as determined by follow-up paired t-tests. The higher dose (1.0 µM) of
BAPTA/AM protected the cells against alcohol neurotoxicity. (B) Percent cell loss was
calculated from the cell number data in Panel A and is shown above each bar. Error bars

represent ± SEM. ** indicates a significant difference from cultures, which did not receive
BAPTA/AM pretreatment (determined by one-way ANOVA and Scheffé post-hoc tests, p <
0.05). When data was expressed as a percent cell loss, both the low and high concentrations
of BAPTA/AM significantly reduced the cell loss caused by alcohol.

Figure 6. Pretreatment with 2-APB, an IP3R inhibitor that blocks Ca2+ release from internal
stores, prevents the alcohol-induced rise in [Ca2+]i
CGN cultures received either 5 µM 2-APB (filled circles) or vehicle control, 0.006% DMSO
(hollow circles) for 30 minutes prior to receiving 400 mg/dl ethanol (red lines) or PBS for
ethanol-free cultures (blue lines). (A) The data from five replicates were combined and mean
[Ca2+]i ± SEM were calculated. (B) Mean Peak [Ca2+]i ± SEM for the four treatment groups
are shown. * indicates treatment group differs significantly from all other groups (Scheffé, p
< 0.005). The alcohol-induced rise in [Ca2+]i is prevented by 2-APB. Although 2-APB (with
or without alcohol) appeared to raise [Ca2+]i with respect to the control group (no alcohol,

no 2-APB), this effect is primarily due to the difference in starting point for the basal [Ca2+]i
level (observed fifteen seconds prior to alcohol exposure).

Figure 7. 2-APB eliminates alcohol-induced cell loss
Thirty minutes before the addition of alcohol, CGN cultures received either the IP3R
inhibitor, 2-APB or 0.006% DMSO (vehicle control). Viable cell numbers were determined
following a 24-hour alcohol exposure. (A) The cell number values are the means ± SEM
derived from six experimental replicates. * indicates significant difference from the
matching ethanol-free group (determined by follow-up paired t-tests, p < 0.05). There is no
significant ethanol-induced cell loss with the higher 2-APB concentrations (1.3 and 5.0 µM).
(B) The percent cell loss caused by alcohol was calculated from the cell number data in

Figure 7A and is shown above each bar. ** indicates significant difference from cultures
which were not pretreated with 2-APB (Scheffé, p < 0.01). Blocking intracellular Ca2+
release from internal stores with 2-APB eliminates alcohol-induced cell loss.

Figure 8. BAPTA/AM and 2-APB must precede alcohol exposure in order to prevent the rapid
alcohol-induced rise in [Ca2+]i and cell loss in CGN cultures
CGN cultures received 1.0 µM BAPTA/AM, 5.0 µM 2-APB, or an equivalent concentration
of DMSO (vehicle control) either 30 minutes before (pretreatment) or at the same time as
400 mg/dl ethanol (concurrent). (A) Mean Peak [Ca2+]i ± SEM pooled from five
experimental replicates treated with BAPTA/AM. (B) Mean Peak [Ca2+]i ± SEM derived
from three experimental replicates treated with 2-APB. * and ** indicate that the Mean Peak
[Ca2+]i differs significantly from all other treatment groups (Scheffé, p < 0.01). BAPTA/AM
and 2-APB are only effective in preventing the alcohol-induced rise in [Ca2+]i when they are

added 30 minutes before, but not concurrent with, the addition of alcohol. (C and D) The
ethanol-induced percent cell loss was calculated from the cell number data (not shown).
Values are expressed as means ± SEM. * and ** indicate that the percent cell loss differs
significantly from all other treatment groups (Scheffé, p < 0.001). BAPTA/AM and 2-APB
are only effective in preventing alcohol-induced cell loss when they are added 30 minutes
before, but not concurrently with, alcohol.

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