Evaluating signs of hippocampal neurotoxicity induced by a revisited paradigm of voluntary ethanol consumption in adult male and female Sprague-Dawley rats

Abstract

Background
Binge alcohol drinking is considered a prominent risk factor for the development of alcohol-use disorders, and could be model in rodents through the standard two-bottle preference choice test. The goal was to recreate an intermittent use of alcohol during 3 consecutive days each week to ascertain its potential impact on hippocampal neurotoxicity (neurogenesis and other neuroplasticity markers), and including sex as a biological variable, given the well-known sex differences in alcohol consumption.

Methods
Ethanol access was granted to adult Sprague–Dawley rats for 3 consecutive days per week, followed by 4 days of withdrawal, during 6 weeks, mimicking the most common pattern of intake in people, drinking over the weekends in an intensive manner. Hippocampal samples were collected to evaluate signs of neurotoxicity.

Results
Female rats consumed significantly more ethanol than males, although intake did not escalate over time. Ethanol preference levels remained below 40% over time and did not differ between sexes. Moderate signs of ethanol neurotoxicity were observed in hippocampus at the level of decreased neuronal progenitors (NeuroD + cells), and these effects were independent of sex. No other signs of neurotoxicity were induced by ethanol voluntary consumption when measured through several key cell fate markers (i.e., FADD, Cyt c, Cdk5, NF-L) by western blot analysis.

Conclusions
Overall, the present results suggest that even though we modeled a situation where no escalation in ethanol intake occurred across time, mild signs of neurotoxicity emerged, suggesting that even the use of ethanol during adulthood in a recreational way could lead to certain brain harm.

Full text

Vol.:(0123456789)
1 3
Pharmacological Reports
https://doi.org/10.1007/s43440-023-00464-6
ARTICLE
Evaluating signs ofhippocampal neurotoxicity induced byarevisited
paradigm ofvoluntary ethanol consumption inadult male andfemale
Sprague‑Dawley rats
CarlesColom‑Rocha1,2· CristianBis‑Humbert1,2,3· M.JuliaGarcía‑Fuster1,2
Received: 15 November 2022 / Revised: 7 February 2023 / Accepted: 8 February 2023
© The Author(s) 2023
Abstract
Background Binge alcohol drinking is considered a prominent risk factor for the development of alcohol-use disorders, and
could be model in rodents through the standard two-bottle preference choice test. The goal was to recreate an intermittent
use of alcohol during 3 consecutive days each week to ascertain its potential impact on hippocampal neurotoxicity (neuro-
genesis and other neuroplasticity markers), and including sex as a biological variable, given the well-known sex differences
in alcohol consumption.
Methods Ethanol access was granted to adult Sprague–Dawley rats for 3 consecutive days per week, followed by 4days
of withdrawal, during 6weeks, mimicking the most common pattern of intake in people, drinking over the weekends in an
intensive manner. Hippocampal samples were collected to evaluate signs of neurotoxicity.
Results Female rats consumed significantly more ethanol than males, although intake did not escalate over time. Ethanol
preference levels remained below 40% over time and did not differ between sexes. Moderate signs of ethanol neurotoxicity
were observed in hippocampus at the level of decreased neuronal progenitors (NeuroD + cells), and these effects were inde-
pendent of sex. No other signs of neurotoxicity were induced by ethanol voluntary consumption when measured through
several key cell fate markers (i.e., FADD, Cyt c, Cdk5, NF-L) by western blot analysis.
Conclusions Overall, the present results suggest that even though we modeled a situation where no escalation in ethanol
intake occurred across time, mild signs of neurotoxicity emerged, suggesting that even the use of ethanol during adulthood
in a recreational way could lead to certain brain harm.
Keywords Alcohol voluntary drinking· Sex differences· Hippocampus· Neurogenesis· Rat
Introduction
Alcohol is the legal drug most consumed at world level; in
2020, the last National Survey on Drug Use and Health [1]
reported that 50% of people in the USA, ages 12 or older,
used alcohol in the past month, and from those, around 44%
were classified as binge drinkers and 13% as heavy drinkers.
Binge drinking, defined by the National Institute on Alcohol
Abuse and Alcoholism (NIAAA) as an enduring period of
observable behavioral intoxication that brings blood alcohol
concentration to 0.08g percent (or 80mg%) or above (e.g., 5
or more drinks for males or 4 or more drinks for females in
about 2h), is a prominent risk factor for later development
of alcohol-use disorders (e.g., [2]).
At the preclinical level, binge modeling of ethanol drink-
ing across laboratories has been proven challenging in terms
of achieving comparable intoxication levels (as measured
by high blood ethanol concentrations), since when provided
in a 24-h span, rodents distribute their intake throughout
the day. Distinctive paradigms have been characterized for
adult rodents utilizing variations of the standard two-bottle
preference choice test, which initially allowed continuous
unlimited access to one bottle containing an ethanol solution
(i.e., [3]; and hundreds of publications afterwards), to then
* M. Julia García-Fuster
j.garcia@uib.es
1 IUNICS, University oftheBalearic Islands, Cra. de
Valldemossa Km 7.5, 07122Palma, Spain
2 Health Research Institute oftheBalearic Islands (IdISBa),
Palma, Spain
3 Present Address: Psychobiology ofDrug Addiction,
Neurocentre Magendie, INSERM U1215, Bordeaux, France

C.Colom-Rocha et al.
1 3
limit its scheduling to better mimic human alcohol intake
(i.e., reviewed by [4, 5]). Providing an intermittent access to
a bottle with ethanol (i.e., 10–20% solution range) models a
progressive increase in intake across time following cycles
of exposure and drug removal, with increased preference
ratios when ethanol is reintroduced (reviewed by [4, 5]). The
most commonly used schedule allows unlimited access to
rats for periods of 24h on alternate days (Mondays, Wednes-
days and Fridays), creating multiple 24-h short-withdrawal
periods in between ethanol drinking sessions, which have
proven to lead to severe consequences (i.e., kindling effect
[6, 7] and/or negative affect [8]). Over a period comprising
several weeks, rats show a progressive increase in voluntary
ethanol intake, modeling the development of an addictive-
like phenotype (e.g., [9]; reviewed by [4, 5]). Other inter-
mittent models are based on given access to ethanol only
for a limited time each day (e.g., 4h per day during 4 con-
secutive days a week [10]). Moreover, additional schedules
mimicking the development of addictive-like features, either
allowing continuous access to ethanol during several weeks
or giving access to ethanol at night when rats are active
“drinking in the dark” model (reviewed by [4]), have been
extensively utilized leading to multiple publications.
In the present study, however, we decided to revisit the
most used paradigm that allows access to ethanol for 3 non-
consecutive days a week (e.g., Monday, Wednesday and Fri-
day) with alternate 24-h periods of access and/or withdrawal,
to explore the response of a continuous 3-day access (i.e.,
72h of unlimited access) followed by a period of 4days of
withdrawal, mimicking the most common pattern of intake
in people, drinking over the weekends in a voluntary man-
ner. This pattern of consumption, although it might not nec-
essarily lead to intoxication and/or alcohol-use disorder, it
could still be used as a model in which to study sex-specific
neuronal adaptations and/or complications. This is particu-
larly relevant given the well-known differential influence of
sex when modeling addiction in animals (e.g., [11]); female
rats of many strains and ages (adolescent and/or adult) drink
more alcohol than their male counterparts (e.g., [12–14]).
Also, ethanol is predominantly harmful in hippocampus
(both structural and function-dependent; e.g., [15–18]), and
there are well-known sex differences in hippocampal dam-
age following alcohol use (e.g., [19, 20]). Therefore, our
goal was to revisit and recreate an intermittent use of alcohol
in rats of both sexes, with greater behavioral translation to
the pattern observed in humans, in which to ascertain the
potential impact on hippocampal neurotoxicity.
In this context, signs of hippocampal neurotoxicity were
evaluated at different levels. The negative effects of ethanol
on impairing the novel generation of adult neurons in hip-
pocampus are well known (i.e., for its consequences on the
different stages of adult neurogenesis see [21]; reviewed by
[22]). Prior studies reported sex differences in hippocampal
damage (e.g., [19]), including changes in the regulation of
adult neurogenesis following ethanol use (e.g., [19, 20]),
and other sex-specific differential neurotoxic events (e.g.,
miRNAs expression [23]; microglia number and reactiv-
ity [24]). However, there is still scarce knowledge on the
magnitude and/or direction of the events taking place in the
hippocampus of female rodents following ethanol exposure.
In this context, we compared the potential sex differences in
the regulation of the initial stages of adult neurogenesis (i.e.,
cell proliferation and early neuronal survival). Moreover,
to deepen our understanding on the sex-related neurotoxic
events that might be taking place in this brain region, we
selected some key molecular markers, with certain links to
the regulation of adult hippocampal neurogenesis that were
previously characterized for adult male rodents in the con-
text of other drugs of abuse. For example, the dysregulation
of Fas-Associated protein with Death Domain (FADD), a
key cell fate player that could balance cell death vs. plas-
ticity events (reviewed by [25, 26]), paralleled decreased
levels of cell proliferation in hippocampus following cocaine
exposure [27], suggestive of neurotoxic events in this brain
region in male rats. We also studied another marker of the
apoptotic pathway (Cytochrome c, Cty c)whose expres-
sion was altered by drugs of abuse (i.e., cocaine, MDMA)
in hippocampus in conjunction with FADD [28, 29]. Also,
Cyclin-dependent kinase-5 (Cdk5)was evaluated since is
key in the regulation of neurogenesis [30] and was shown
to be modulated in parallel to FADD in hippocampus (see
[31]). Finally, the potential structural damage of ethanol was
evaluated at the level of neurofilament proteins (e.g., NF-L)
as certain drugs of abuse induced neurotoxicity in male rats
by decreasing its hippocampal content (e.g., [29]); it was
found hyperphosphorylated in hippocampus in response to
ethanol toxicity [32], and its circulating levels were altered
in heavy drinking in association with lower gray matter
thickness [33]. Exploring these molecular events will give
us an idea of which pathways and/or events should be further
explored in the context of ethanol toxicity in hippocampus
for each sex.
Materials andmethods
Animals
A total of 42 adult Sprague-Dawley rats (21 males and 21
females) bred in the animal facility at the University of
the Balearic Islands were used in this study. During the
experimental procedures, rats were housed individually in
standard cages following a 12-h light/dark schedule (lights
on at 8:00AM) in a climate-controlled room (22°C, 70%
humidity) and with unlimited access to a standard diet and
water. Rats were given at least 1week to acclimatize to

Evaluating signs ofhippocampal neurotoxicity induced byarevisited paradigm ofvoluntary…
1 3
the housing conditions and the handling prior to the actual
experiments, which were performed during the light period
(between 10:00 and 12:00h). All procedures complied with
the ARRIVE Guidelines [34], the EU Directive 2010/63/
EU and the Spanish Royal Decree 53/2013 for animal
experiments, and were approved both by the Local Bioethi-
cal Committee (CEEA 100/10/18) and the Regional Gov-
ernment (Exp.: 2018/14/AEXP). All efforts were made to
minimize the number of rats used and their suffering. In this
context, and to prevent the induction of unnecessary stress in
female rats during the experimental procedure, the specific
stages of the estrous cycle were not examined. This decision
was based on the fact that prior studies suggested that the
estrous cycle stage might not be a significant player in the
amount of ethanol consumption for female rats (e.g., [35]),
but mainly because the cyclicity of females was not part of
our research question (see [36]).
Intermittent access to20% ethanol inatwo‑bottle
choice test
Rats of each sex were randomly allocated into two exper-
imental groups (Control, n = 10 and Ethanol, n = 11 per
sex; see Fig.1a) and were exposed to two bottles with
fluids during 6 consecutive weeks. Each week, rats from
the ethanol groups were given unlimited voluntary access
to ethanol (20% ethanol vs. water) during 3 consecutive
days (i.e., every Tuesdays, Wednesdays and Thursdays)
followed by a period of 4days of withdrawal (all rats
had access to 2 bottles of water). Rats from the control
groups were always exposed to two bottles of water. Each
rat was exposed to a total of 18 ethanol (or control) ses-
sions during the 6-week procedure. The placement of the
ethanol bottle was alternated daily to account for side
preferences and bottles were weighed every morning dur-
ing the 3days of ethanol access. Total fluid intake (sum
of both bottles in ml) and the amount of water and/or
ethanol consumed (in ml) was recorded on each session
day. Ethanol preference was calculated as the amount of
ethanol consumed divided by total fluid intake and multi-
plied by 100 (% values). Weights were monitored weekly
(D1 of each week) throughout the course of 6weeks as
detailed in Fig.1a, and were used to calculate the amount
of ethanol consumed (g/kg) daily and cumulative (i.e.,
ethanol load throughout the whole experimental proce-
dure). Results are expressed as daily consumption, aver-
age consumption per week and average consumption dur-
ing the 18 sessions.
Fig. 1 Experimental design. a Sprague-Dawley rats were exposed
during 6 consecutive weeks to a weekly schedule consisting of a
3-day continued voluntary ethanol access (two-bottle choice: 20%
ethanol vs. water; D1–D3) followed by a 4-day withdrawal period
(two bottles of water). Rats were killed on the last day of ethanol
exposure on week 6 (D38). b Changes in body weight across weeks
(g). Groups of treatment: Control-male (n = 10); Ethanol-male
(n = 11); Control-female (n = 10); Ethanol-female (n = 11). Columns
represent mean ± SEM of change in body weight (g). Individual sym-
bols are shown for each rat. #p < 0.05 when comparing the effect of
sex (female vs. male rats; three-way RM ANOVA)

C.Colom-Rocha et al.
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Immunohistochemistry
Rats were killed by rapid decapitation on the last session/day
of week 6 (D38; see Fig.1a) and the left half-brain was snap-
frozen with – 30°C isopentane (Panreac Química, Barcelona,
Spain, cat #143501) and stored at – 80°C to latter quantify
hippocampal neural progenitors by immunohistochemistry
at specific periods of regulation (i.e., cell proliferation with
Ki-67 and early neuronal survival with NeuroD). Tissue was
cryostat-cut (30µm sections) and slide-mounted throughout
the whole hippocampal extent ( – 1.72 to – 6.80mm from
Bregma) in 3 series containing the most anterior, the middle
part and the most posterior part of this region as described
in detail before [37–39]. For each marker, experiments were
performed in 1 slide per rat containing 8 tissue-sections from
the middle portion of the hippocampus that were post-fixed
in 4% paraformaldehyde (Merck, Darmstadt, Germany, cat
#76240). Then, sections were exposed to several steps, such
as epitope retrieval in 10% sodium citrate dihydrate (Thermo
Fisher Scientific, Waltham, MA, USA, cat #BP327-1) pH
6.0 at 90°C for 1h, 0.3% peroxidase solution (Thermo
Fisher Scientific, cat #426000010) and BSA blocking
(Merck, cat #A7906) before an overnight incubation with
rabbit anti-Ki-67 (1:20000) (provided by Drs. Huda Akil
and Stanley J. Watson, University of Michigan, MI, USA,
cat #B7) or goat anti-NeuroD (1:25000; Santa Cruz Biotech-
nology, CA, USA, cat #sc-1084). The next steps included
a series of sequential incubations, first with the second-
ary antibody (biotinylated anti-rabbit or anti-goat, 1:1000
respectively, Vector Laboratories, CA, USA, cat #BA-1000
and BA-5000 respectively), followed by the Avidin/Biotin
complex (Vectastain Elite ABC kit; Vector Laboratories,
cat #PK-6100), and the chromogen 3,3’-diaminobenzidine
(DAB; Merck, cat #D8001) for signal detection(for NeuroD
with nickel chloride; Merck, cat #339350). Tissue for Ki-67
quantification was counterstained with cresyl violet (Thermo
Fisher Scientific, cat #405760100). Finally, sections were
dehydrated in graded alcohols, immersed in xylene (Shar-
lab, Barcelona, Spain, cat #XI0052) and cover-slipped with
Permount® (Thermo Fisher Scientific, cat #SP15-500). Pos-
itive cells were quantified in the dentate gryus of 8 sections/
rat by an experimenter blind to the treatment groups with a
Leica DMR light microscope (63 × objective lens). The total
number of positive cells is represented in relation to the %
number of cells present in control-male rats.
Western blot
Hippocampal cell fate markers were evaluated by Western
blot analysis. To do so, total homogenates were prepared
from the right hippocampus as previously described in detail
before (see [28, 29]). Brain proteins (40μg; protein amount
assessed by BCA, Thermo Fisher Scientific, cat #23225)
were separated by electrophoresis on 10–15% SDS-PAGE
minigels (Bio-Rad Laboratories, Hercules, CA, USA) and
transferred (110V, during 2h 30min) to nitrocellulose
membranes that were incubated with appropriate primary
antibody whose vendors and dilution conditions were the
following: (1) Santa Cruz Biotechnology (CA, USA): anti-
FADD (H-181) (1:2500; cat #sc-5559); (2) BD Biosciences
(CA, USA): anti-Cyt c(1:5000; cat #556433); (3) Lab
Vision Corporation (CA, USA): anti-Cdk5 (DC17) (1:1000;
cat #DC17); and (4) Sigma-Aldrich (MO, USA): anti-NF-
L (N5139) (1:1000; cat #5139NR4), anti-β-actin (clone
AC-15) (1:10000; cat #A1978). Following incubation with
the appropriate secondary antibody (anti-rabbit or -mouse
IgG linked to horseradish peroxidase; 1:5000; Cell Signal-
ing; cat #7074 and 7076, respectively), the immunoreactivity
of selected proteins was detected by ECL reagents (Amer-
sham, Buckinghamshire, UK) and signal of bound antibody
was visualized with autoradiographic films (Amersham ECL
Hyperfilm). Each band of interest was quantified by densito-
metric scanning (GS-800 Imaging Calibrated Densitometer,
Bio-Rad), and percent changes in immunoreactivity for each
marker were calculated for each rat with respect to control-
male samples (100%) in various gels (each sample was run
at least 2–3 times in different gels), and the mean value was
used as a final estimate. Data were not normalized to any
protein, since β-actin analysis served as a loading control
(i.e., its content was not altered by any treatment conditions).
Data statistical analyses
GraphPad Prism, Version 9.5 (GraphPad Software, USA)
was used to analyze and plot all graphs following the guide-
lines in experimental pharmacology for displaying data
and statistical methods [40]. Results are reported as mean
values ± standard error of the mean (SEM), and individual
symbols for each rat are shown within bar graphs. Paramet-
ric tests were used for statistical comparisons, since assump-
tions for normality of data distribution and homogeneity of
variance (in case of analysis of variance) were met (i.e.,
D’Agostino-Pearson normality test). Three-way repeated-
measure (RM) ANOVAs were performed when analyzing
potential changes in body weight (g), total fluid (sum of
liquid consumed from both bottles independently of its
contents and expressed in ml) and water (volume consumed
from a single water bottle through the course of the experi-
mental procedures and expressed in ml). The independent
variables of study were Sex, Bottle Choice (Ethanol vs.
Water) and Time of Analysis (Day or Week). When meas-
uring ethanol preference (%) and ethanol dose consumed (g/
kg) data were analyzed with two-way RM ANOVAs (inde-
pendent variables: Sex and Time of Analysis), since all rats
were exposed to the choice of one ethanol bottle. Cumulative
ethanol load was compared between male and female rats

Evaluating signs ofhippocampal neurotoxicity induced byarevisited paradigm ofvoluntary…
1 3
with an unpaired two-tailed t-test. Finally, the regulation of
hippocampal markers of induced neurotoxicity (i.e., neural
progenitors and/or cell fate markers) was performed with a
two-way ANOVA (independent variables: Sex and Experi-
mental Group). Individual values were normalized to control
male rats, to estimate the % magnitude of change. Tukey's
or Sidak's multiple comparisons tests were performed for
post hoc pair-wise statistical comparisons when appropriate.
The level of significance was set at p ≤ 0.05. Interactions
among variables were only reported when relevant and/or
significant.
Results
All datasets generated during and/or analyzed during the
current study are available from the corresponding author
on reasonable request. Moreover, a table with full statistical
analysis is included as Supplementary Materials.
No changes inbody weight byethanol voluntary
consumption
The impact of voluntary ethanol consumption on weekly
body weight was analyzed (see Supplementary TableS1).
While there was no effect of Bottle of Choice (F1,38 = 0.16,
p = 0.692), the expected significant effects of Sex
(F1,38 = 281.8, ###p < 0.001; Fig.1b) and Time of Analy-
sis (F6,228 = 323.6, p < 0.001) were observed (i.e., increased
weight in male vs. female rats, and progressive body weight
gain over time during adulthood).
Intermittent access to20% ethanol inatwo‑bottle
choice test for3 consecutive days
When evaluating how the procedure might have affected
daily total fluid intake (ml) (a total of 18days; 3days/
week), the results showed that there were no significant
effects of Bottle Choice (F1,38 = 1.48, p = 0.231) or Sex
(F1,38 = 0.14, p = 0.709), but a significant effect of Time of
Analysis (F17,646 = 25.63, p < 0.001) (see Fig.2a and Sup-
plementary TableS1); results that could also be represented
as the average 3-day consumption in a given week (see
Fig.2b). When calculating the average daily intake across
all 18days, male rats consumed an average of 45.9ml of
daily fluid, while female rats consumed 44.8ml, and these
total volumes were not affected by the fact that rats had or
not access to ethanol (Fig.2c and Supplementary TableS1).
However, for water intake, the results showed a significant
effect of Bottle Choice (F1,38 = 19.84, p < 0.001) and Time
of Analysis (F17,646 = 20.49, p < 0.001), but no effect of Sex
(F1,38 = 0.35, p = 0.555) (see Fig.2d); results that could also
be represented as the average 3-day consumption in a given
week (see Fig.2e). Rats that had access to an ethanol bot-
tle drank more water than those with no access (i.e., water
intake was calculated as the average of both water bottles),
as represented by the daily intake averaged across all 18days
(see statistics analysis in Supplementary TableS1). While
male and female control rats consumed similar amounts of
water (an average of 26.8 vs. 25.8ml per day, respectively),
rats with access to one bottle of ethanol drank, in aver-
age, more water every day (males: + 9.7ml, ***p < 0.001;
females: + 7.2ml, *p = 0.023 vs. controls; Fig.2f) as rep-
resented by the significant effect of Bottle of Choice (Sup-
plementary TableS1).
When calculating ethanol preference (%) for rats exposed
to both liquids, the results showed that there was no sig-
nificant effect of Sex (F1,20 = 0.74, p = 0.400), but a signifi-
cant effect of Time of Analysis (F17,340 = 3.54, p < 0.001)
(see Fig.2g and Supplementary TableS1). The fluctua-
tions observed across days dissipated when results were
represented as the average 3-days consumption in a given
week (F5,100 = 2.02, p = 0.083; see Fig.2h, Supplementary
TableS1). In general, no sex differences were observed
in ethanol preference, with male rats showing an average
preference of 37.0% while females showed a preference of
39.2% (Fig.2i). Although the preference for ethanol was
the same for both sexes, when evaluating the dose of etha-
nol consumed (g/kg), a significant effect of Sex displayed
in the statistical analysis (F1,20 = 7.82 #p = 0.011; Fig.2j),
together with an effect of Time of Analysis (F17,340 = 13.07,
p < 0.001); results also detected when the average 3-day con-
sumption in a given week was represented (see Fig.2k, Sup-
plementary TableS1). Female rats showed a higher dose of
ethanol consumed (12.2g/kg) when compared to male rats
(8.0g/kg), with a mean increase of 4.2g/kg (*p = 0.011 vs.
male rats; Fig.2l, Supplementary TableS1). Interestingly,
when calculating the cumulative ethanol load across all
experimental days, female rats consumed a total of 219.2g/
kg as compared to males that consumed only 143.8g/kg
(t = 2.80, df = 20, p = 0.011; data not shown in figures).
Decreased survival ofneural progenitors byethanol
voluntary consumption asasign ofethanol‑induced
neurotoxicity
The number of Ki-67 + cells was used to measure poten-
tial changes in cell proliferation for all groups as compared
to control-male rats and expressed as % of its mean value
(700 ± 102 Ki-67 + cells; expected number of cells for male
rats of this age [37, 39]). The results showed no significant
effects of Bottle Choice (F1,36 = 0.01, p = 0.925) or Sex
(F1,36 = 0.24, p = 0.627) (see Fig. 3a, and Supplementary
TableS1). As for NeuroD, the mean number of + cells in
control-male rats (used to calculate the % changes for the
rest of the groups) were 6616 ± 246 NeuroD + cells, also

C.Colom-Rocha et al.
1 3
in line with prior data in male rats of this age [37, 39]. In
this case, ethanol access decreased the survival of neu-
ral progenitors, as measured through the number of Neu-
roD + cells, and as observed by the significant effect of
Bottle Choice (F1,37 = 8.98, **p = 0.005); rats with access
to ethanol displayed an average of 22.7% lower Neu-
roD + cells when compared to control rats, independently
of sex (Fig.3b, Supplementary TableS1). This effect was

Evaluating signs ofhippocampal neurotoxicity induced byarevisited paradigm ofvoluntary…
1 3
observed for each sex when analyzed separately (male rats:
-26%, *p = 0.042; female rats: -19%, *p = 0.05; see Fig.3b,
Supplementary TableS1). Moreover, female rats showed
lower NeuroD + cells (a general drop of 15.5%) than male
rats as observed by the significant effect of Sex (F1,37 = 4.19,
#p = 0.048) (Fig.3b). Correlation analysis were performed
between the different parameters of ethanol consumption
(i.e., average intake, preference and even with cumulative
intake across all days) and the number of hippocampal Neu-
roD + cells; however, none of these variables were predictive
of the later changes observed (data not shown).
Besides, no other signs of neurotoxicity were induced
by ethanol voluntary consumption during adulthood when
measured through several key cell fate markers (i.e., FADD,
Cyt c, Cdk5, NF-L) by western blot analysis at the specific
time-point of consideration (see Fig.3c-f, Supplementary
TableS1). The only significant change observed through the
two-way ANOVAs performedwas the significant effect of
sex for FADD (F1,38 = 5.84, #p = 0.021; + 16.7% more FADD
content for female adult rats independently of Bottle Choice;
see Fig.3c). β-actin was quantitated and used as a loading
control as it was not altered by any treatment conditions
(see Fig.3g).
Discussion
The main results showed moderate signs of ethanol neuro-
toxicity in hippocampus at the level of decreased neuronal
progenitors (NeuroD + cells), effects that were independent
of sex. Overall, the present results suggested that even in
a situation where no escalation of ethanol intake occurred
across time (a pattern mimicking a potential recreational use
of ethanol in people), certain signs of neurotoxicity emerged,
suggesting that sporadic ethanol use could still induce cer-
tain consequences in adult rats.
Our results aligned with several prior studies report-
ing that adult female rats, when given access to ethanol,
consumed more (in g/kg), than their male counterparts (e.g.,
[12–14, 35],). In fact, the absolute volume of ethanol con-
sumption did not different between sexes, however, given the
smaller body weight of adult females, their total consump-
tion in a g/kg basis was higher (around 4.2g/kg higher per
day in average). However, in comparison with other intermit-
tent paradigms of ethanol accessibility (i.e., 24-h periods of
access on alternate days of the week), the schedule followed
in the present study (ethanol access continuously for 3days
a week followed by 4days of withdrawal during 6weeks)
did not lead to a temporal increase in intake, neither for
male nor forfemale rats. In our particular conditions, the
range of intake (lowest and highest value) among individual
rats from each group fluctuated between 5.5 and 12.8g/kg
(mean value of 8.0g/kg) for adult males, and between 6.6
and 20.1g/kg (mean value of 12.2g/kg) for adult female
rats, which are considered moderate to large doses of ethanol
(reviewed by [4]). This differs from prior data describing
that Sprague-Dawley rats consume low to moderate levels
of ethanol and achieve lower ethanol preference rates than
other strains (reviewed by [5]). That seems to be one of the
reasons why the majority of studies reported in the literature
were done in Long-Evan or Wistar rats, although escalation
in ethanol intake has been reported for all strains, including
Sprague-Dawley (reviewed by [5]). Moreover, many studies
have revealed that not only strain, but also many other fac-
tors could affect the behavioral results, such as age, sex and
particular environmental conditions (e.g., number of avail-
able bottles, ethanol concentration and temporal accessibil-
ity, etc.; reviewed by [41]), and could justify the lack of
escalation in ethanol intake (g/kg) and/or in ethanol prefer-
ence observed with the current paradigm.
In terms of ethanol preference across time, our results
reported no significant differences by sex, with male rats
showing an average preference of 37.0% while females
showed a preference of 39.2%. The preference was below
50% since rats with access to ethanol drank more water
than ethanol, although the total amount of fluid intake
did not change when compared to their respective control
group (i.e., comparing rats exposed to two-water bottles
vs. rats given unlimited access to ethanol). Still, these
levels of preference seemed comparably higher to others
previously reported in the literature showing ethanol pref-
erences starting around 15% on day 1, and then escalating
up to 35–40% following several weeks of exposure (e.g.,
[9]). Our results reported similar amounts of ethanol con-
sumption every week (no drop in consumption observed
with time), but no escalation in preference over time, sug-
gesting no progression into an addictive-like phenotype.
Contrary, we could argue that in our particular experimen-
tal conditions, and right from the start of the procedure
(week 1 and onward), rats showed goodpreference levels
for ethanol, in line with the high values of intake described
Fig. 2 Intermittent access to 20% ethanol in a two-bottle choice
test for 3 consecutive days. a–c Total fluid (ml) and d–f Water (ml)
intake daily (a–d), weekly (b–e) and expressed as the 18days over-
all average (c–f). Groups of treatment: Control-male (n = 10); Etha-
nol-male (n = 11); Control-female (n = 10); Ethanol-female (n = 11).
Columns represent mean ± SEM of the amount of liquid consumed
in ml. Individual symbols are shown for each rat. Three-way RM
ANOVAs showed no significant effects of Bottle Choice or Sex. g–i
Ethanol preference (%) and j–l Ethanol (g/kg) consumption daily
(g–j), weekly (h–k) and expressed as the 18 days overall average
(i–l). Groups of treatment: Ethanol-male (n = 11); Ethanol-female
(n = 11). Columns represent mean ± SEM of the preference for etha-
nol (expressed as a % value) or ethanol dose consumed (g/kg). Indi-
vidual symbols are shown for each rat. #p < 0.05 when comparing the
general effect of sex (two-way repeated-measures ANOVA); *p < 0.05
when comparing the dose consumed by female rats vs. male rats (Stu-
dent’s t-test)
◂

C.Colom-Rocha et al.
1 3
above, and as compared to prior studies (reviewed by [4,
5]), and maybe representing steady celling values prevent-
ing further escalation. In fact, we propose that this pattern
of consumption might be mimicking the recreational use
of ethanol during weekends in people. Overall, the present
paradigm did not show escalation of ethanol intake, but
it reproduced high levels of consumption, mimicking a
common paradigm of intake for most adult individuals, in
which to evaluate potential signs of neurotoxicity induced
by ethanol in hippocampus.
Fig. 3 Evaluating signs of ethanol-induced neurotoxicity in hip-
pocampus. Quantitative analysis of a Ki-67 and b NeuroD + cells in
the left dentate gyrus of the hippocampus by immunohistochemis-
try or of c FADD, d Cytochrome c (Cyt c) e Cdk5, f NF-L and g
β-actin protein content by western blot analysis in the hippocampus
of male and female rats exposed to the two-bottle choice test (Con-
trol vs. Ethanol) on D38. Groups of treatment: Control-male (n = 10);
Ethanol-male (n = 11); Control-female (n = 10); Ethanol-female
(n = 11). Columns represent mean ± SEM of the number of + cells
quantified in 8 sections from the middle part of the hippocampus
and expressed as % change vs. control-male rats or of n experiments
per group and expressed as a percentage of Control-male-treated
rats. Individual symbols are shown for each rat. Two-way ANOVAs
evaluating the potential effects of Bottle Choice (ethanol vs. water)
and Sex. #p < 0.05 when comparing the general effect of sex and
**p < 0.01 when comparing the effects of having access to a bottle
with ethanol vs. control (general effect independently of sex). Bottom
panels: representative images showing individual Ki-67 (brown labe-
ling in the blue granular layer) and NeuroD (dark blue labeling in the
blue granular layer) cells taken with a light microscope (40 × objec-
tive lens) or representative immunoblots depicting labeling of FADD,
Cyt c, Cdk5, NF-L and β-actin are shown for each set of experiments.
Other representative images for Ki-67 and NeuroD labeling and/or
full immunoblots from which images were taken could be found in
Supplementary Materials

Evaluating signs ofhippocampal neurotoxicity induced byarevisited paradigm ofvoluntary…
1 3
Overall, the neurochemical results showed a moderate
impact of ethanol on the markers evaluated in hippocampus.
In particular, both male and female rats exposed to ethanol
intermittently during 6weeks presented lower hippocampal
NeuroD + cells, a sign of decreased survival rate of neural
progenitors (immature neurons). These results align with
prior studies reporting damaging effects of ethanol on sev-
eral stages of adult neurogenesis, including neuronal pro-
genitors (e.g., [21]; reviewed by [22]), to describe a similar
impact but following a distinct scheduling and including
both sexes. Contrarily to other studies that reported sex dif-
ferences in the regulation of hippocampal neurogenesis fol-
lowing ethanol use (e.g., [19, 20]), the present data showed
similar changes on NeuroD for bothsexes, even though
females consumed more ethanol (in g/kg). This was inter-
esting given the basal differences observed in Neuro + cells,
having female rats lower neuronal progenitors as compared
to male rats. However, independent of these basal differ-
ences, ethanol exposure affected both sexes similarly. This
goes along with the sex effects priory reported in the differ-
ent stages of hippocampal neurogenesis in the literature [42,
43], and in particular with the reported slower hippocampal
neuronal maturation in females compared to males [43, 44].
In line with these baseline sex disparities, adult female
rats also showed higher hippocampal FADD protein content
than males, suggesting differences in baseline hippocampal
neurotoxicity rates in line with their, just reported, lower
NeuroD levels. Interestingly, FADD and/or the rest of the
potential markers evaluated (Cyt c, Cdk5, NF-L) showed
no signs of induced toxicity by ethanol exposure that could
accompany the observed moderate decreased number of
neuronal progenitors, even though prior studies have paired
decreases in NeuroD with increases in FADD content but
following cocaine exposure [27]. Although no prior stud-
ies have evaluated hippocampal FADD regulation following
ethanol consumption invivo, indications of ethanol-induced
apoptosis through Fas-mediated pathways were described
in several invitro lines of human liver adenocarcinoma
cells [45, 46]. Considering the present results, probably
the amount of ethanol exposure was not sufficient to induce
changes in FADD protein. Alternatively, the expected ini-
tial acute change might have adapted following a 6-week
repeated ethanol exposure. These explanations might also
justify the lack of effects on the other markers evaluated in
hippocampus by ethanol exposure. For example, no changes
were observed in Cdk5 regulation, although prior studies
suggested that ethanol exposure resulted in Cdk5 overactiva-
tion in cortex and cerebellum [47], as well as in increased
levels of Cdk5 and its activator p25 in hippocampus. These
prior experiments suggested that interfering with this path-
way might serve as a potential therapeutic approach to pre-
vent ethanol-induced neurotoxicity in the brain [48], and
although our results did not find Cdk5 altered at the end of
the procedure, maybe it still might be regulated during etha-
nol consumption. Similarly, we found no signs of neurotox-
icity as measured by NF-L protein content in hippocampus,
though it was found hyperphosphorylated in hippocampus
in response to ethanol toxicity [32], and other drugs of abuse
(i.e., opiates/opioids and MDMA) decreased its content, pro-
posing NF-L as a marker of structural damage to particular
brain regions [29, 49].
In conjunction, the present results demonstrated a mod-
erate neurotoxic effect of ethanol exposure as observed by
decreased NeuroD + cells in the dentate gyrus of male and
female rats, without any other ethanol-evoked changes (i.e.,
number of Ki-67 + cells or protein contents of FADD, Cyt
c, Cdk5, NF-L). The discrepancies with prior reports that
suggested signs of neurotoxicity through the dysregulation
of these markers by ethanol exposure could be due to dif-
ferent experimental conditions, including the paradigm of
ethanol used, the specific time of evaluation, and/or whether
experiments were performed invitro or invivo, among other
factors. Therefore, our pool of data is limited by how the
experimental procedures were designed and could only be
discussed in that context, yet presented relevant results while
including sex as a biological variable. Other complementary
experiments, both behavioral and neurochemical, would be
required to demonstrate other signs of neurotoxicity, besides
our moderate changes, and thus our conclusions are limited
to the current design which particularly studied the regu-
lation of the proposed markers on a single time-point of
analysis at the end of the experiment.
In conclusion, this study sheds light on some of the poten-
tial negative consequences induced by a common pattern of
ethanol consumption during adulthood, with no apparent
signs of increased intake and/or changes in ethanol prefer-
ence, but with certain signs of moderate neurotoxicity. Over-
all, our data suggest that the consumption of ethanol during
adulthood, although at a recreational level, could also lead
to certain brain harm.
Supplementary Information The online version contains supplemen-
tary material available at https:// doi. org/ 10. 1007/ s43440- 023- 00464-6.
Acknowledgements The authors would like to thank Drs. Huda Akil
and Stanley J. Watson (University of Michigan, Ann Arbor, MI, USA)
for generously providing the primary antibody for Ki-67 labeling.
Author contributions MJG-F was responsible for the study concept
and design with the contribution of CC-R and CB-H. CC-R and CB-H
conducted the experiments. CC-R analyzed the data. MJG-F revised
the data, plotted the figures and drafted the manuscript. All authors
have critically contributed and have approved the final version of the
manuscript for publication.
Funding Open Access funding provided thanks to the CRUE-CSIC
agreement with Springer Nature. This work was supported by 'Del-
egación del Gobierno para el Plan Nacional sobre Drogas' (2020/001,
Ministerio de Sanidad, Spain) to MJG-F; a pre-doctoral scholarship
(FPU2022-012-A; Conselleria de Fons Europeus, Universitat i Cultura

C.Colom-Rocha et al.
1 3
del Govern de les Illes Balears) to CC-R; and “TECH” from project
“TALENT PLUS Construint Salut, Generant Valor" (IdISBa, GOIB) to
CB-H. MJG-F and CC-R are members of RIAPAd (RD21/0009/0008;
ISCIII, Plan de Recuperación, Transformación y Resilencia,
NextGenerationEU).
Declarations
Conflict of interest None.
Open Access This article is licensed under a Creative Commons Attri-
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tion, distribution and reproduction in any medium or format, as long
as you give appropriate credit to the original author(s) and the source,
provide a link to the Creative Commons licence, and indicate if changes
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otherwise in a credit line to the material. If material is not included in
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