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Brief Report
Binge
Q1
Ethanol IntoxicationHeightens Subsequent EthanolIntake in Adolescent, But NotAdult, Rats
ABSTRACT: A question still to be answered is whether ethanol initiation has agreater effect on ethanol consumption if it occurs during adolescence than inadulthood. This study assessed the effect of ethanol initiation during adolescenceor adulthood on voluntary ethanol consumption when animals were still within
the same age range. Adolescent or adult rats were given 5, 2, or 0 ethanol expo-sures. The animals were tested for ethanol consumption through two-bottle choicetests, before undergoing a 1-week deprivation. A two-bottle assessment was con-ducted after the deprivation. Adolescents, but not adults, given two ethanoladministrations during initiation exhibited significantly higher ethanol intake dur-ing the pre-deprivation period. These adolescents also exhibited a threefold in-crease in ethanol intake after 7 days of drug withdrawal, when compared withcontrols. These findings suggest that very brief experience with binge ethanolintoxication in adolescence, but not in adulthood, impacts later predisposition todrink. 2012 Wiley Periodicals, Inc. Dev Psychobiol 9999: 110, 2012.
Keywords: adolescent; rat; ethanol initiation; ethanol intake; light-dark test
INTRODUCTION
EthanolQ4 use begins most frequently during adoles-
cence, and earlier-onset ethanol initiation is associated
with a greater probability of ethanol use disorders
(DeWit, Adlaf, Offord, & Ogborne, 2000). Whether a
causal relationship exists between these variables or
whether they are manifestations of a third phenomenon
is still unclear (Schramm-Sapyta, Kingsley, Rezvani,
Swartzwelder, & Kuhn, 2009).
The prototypical pattern of ethanol self-administration
in adolescence involves ethanol binge drinking, leading
to blood ethanol concentrations 80 mg/dl. This
binge drinking appears to facilitate the escalation of
ethanol consumption (Hargreaves, Monds, Gunasekaran,
Dawson, & McGregor, 2009).
Adolescent animal models have revealed differences
between adolescents and adults in the initial response
to ethanol (Silveri & Spear, 1998, 2001). Adolescents
are more sensitive to ethanol-induced reinforcement
and social facilitation (Pautassi, Myers, Spear, Molina,
& Spear, 2008; Spear & Varlinskaya, 2005, 2010); but
less sensitive than adults to the sedative and motor
impairing, and acute hangover effects of ethanol (Dore-
mus, Brunell, Rajendran, & Spear, 2003; Ristuccia &
Spear, 2004; Silveri & Spear, 1998, 2001). This re-
sponse pattern may represent a risk factor for the devel-
opment of problematic ethanol intake.Previous studies indicated that the adolescent con-
sumption of ethanol can be further enhanced by early
exposure to ethanol during the prenatal or early postna-
tal period (Abate, Pueta, Spear, & Molina, 2008). A
scarcity of animal studies have assessed the effects of
ethanol exposure during adolescence on later adoles-
cent ethanol self-administration. An important question
to test using animal models is whether adolescent etha-
nol initiation has a differential effect on ethanol intake
than if initiation is delayed until adulthood. Hargreaves
DEV-12-077.R2(21101)
Developmental Psychobio
Mara Carolina Fabio1,2
Michael E. Nizhnikov3
Norman E. Spear3
Ricardo Marcos Pautassi1,2
1Instituto de Investigacion Medica M. y M.Ferreyra, INIMEC-CONICET, Universidad
Nacional de Cordoba, Cordoba, C.P.5000, Argentina
E-mail: [email protected]
2Facultad de Psicologa, UniversidadNacional de Cordoba, Cordoba, C.P.
5000, Argentina
3Center for Developmental Psychobiology,Binghamton University, Binghamton, New
York 13902-6000
Manuscript Received: 4 August 2012Manuscript Accepted: 12 December 2012Correspondence to: Ricardo Marcos PautassiContract grant sponsor:NIAAAQ3
Contract grant numbers: AA018164 PIP 2010-2012 SECYT-UNCPICTPRH 246
Article first published online in Wiley Online Library(wileyonlinelibrary.com).
DOI 10.1002/dev.21101 2012 Wiley Periodicals, Inc.
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et al. (2009) found that intermittent access to ethanol-
induced binge-like drinking in adolescent but not adult
rats. Siegmund, Vengeliene, Singer, & Spanagel (2005)
provided evidence that adolescent ethanol initiation en-
hanced stress-induced ethanol consumption. A subse-
quent study found that adult female rats that initiated
ethanol self-administration during adolescence weremore sensitive to stress-induced ethanol intake than
controls that initiated ethanol intake in adulthood (Full-
grabe, Vengeliene, & Spanagel, 2007).
Overall, these studies are consistent with epidemiolog-
ical data, in which the adolescent onset of ethanol expo-
sure promoted ethanol consumption (but see Tambour,
Brown, & Crabbe, 2008). One limitation of these long-
term access studies is that they relied on the self-admin-
istration of ethanol as the method of initiation. With this
method, level of intoxication is not controlled and the
influence of age of ethanol initiation is confounded by
baseline differences in drinking between adolescents andadults. Another limitation was the use of lengthy initia-
tion phases that did not allow the endpoint section of
intake tests to occur within the adolescent period.
One alternative is to expose animals to controlled
intragastric or intraperitoneal administrations of etha-
nol, which allow greater control over dose. Adolescent
rats repeatedly exposed to ethanol exhibited changes in
neurotransmitter systems (Pascual, Boix, Felipo, &
Guerri, 2009), that were associated with greater ethanol
intake in adulthood. This study, however, lacked a com-
parison with adult animals exposed to ethanol in a sim-
ilar fashion as adolescents.
The present study assessed the effects of brief etha-nol initiation during adolescence or adulthood on the
predisposition for voluntary ethanol consumption when
the animals were still within the same age range. Ani-
mals were given five or two intermittent exposures to
ethanol, or vehicle, and then were tested for ethanol
intake. The intake protocol involved an initial phase of
4 days in which subjects were tested with a two-bottle
procedure, followed by a 1-week ethanol deprivation
phase and a post-deprivation assessment of ethanol in-
take. The hypothesis was that passive exposure to etha-
nol augments ethanol consumption in adolescent but
not in adult rats. To examine the potential mechanismsthat underlie this putative effect, anxiety was measured
using the light/dark box test before self-administration
and on the last day of the ethanol-deprivation period.
MATERIALS AND METHODS
Experimental Design
AQ5 2 (age: adolescence or adulthood) 3 (ethanol initiation
treatment: 5-day exposure group, 2-day exposure group, and
control group treated with vehicle) factorial design was
employed, with 1013 animals in each group. Fourteen ani-
mals (7 adolescent and 7 adults) were included in an untreat-
ed (UT) condition.
Subjects
Seventy-four (39 adolescents and 35 adults) outbred male
Wistar rats were used. Adolescents and adults were 28 and70 postnatal days (PD) old, respectively, at the beginning
of procedures. Animals were born and reared in a tempera-
ture-controlled vivarium (21228C) at INIMEC-CONICET
(Cordoba, Argentina). Births were examined daily, and day
of parturition was considered PD0. Culling was conducted
on PD1 and pups remained with their respective dams until
PD 21. After weaning, animals were housed together until
PD24 when they were paired-housed according to their ex-
perimental condition. Half of the males of a given litter
were tested during adolescence, whereas the remaining half
was tested during adulthood. The procedures complied with
the National Institutes of Health Guide for Care and Use of
Laboratory Animals (National Research Council, 1996Q6)
and were approved by the Institutional Animal Care and
Use Committee.
Ethanol Administration (i.e., Initiation) Procedures
Ethanol initiation began on PD28 and PD70 for adolescents
and adults, respectively. The animals were given intragas-
tric administrations as described in Pautassi et al. (2008)
every other day and received five intubations of 2.5 g/kg
ethanol (5-day exposure group; ethanol on PD28, 30, 32,
34, and 36 [adolescents] or PD70, 72, 74, 76, and 78
[adults]), two intubations of 2.5 g/kg ethanol (2-day expo-
sure group; ethanol on PD28 and 32 and vehicle on PD30,
34, and 36 [adolescents] or ethanol on PD70 and 74 and
vehicle on PD72, 76, and 78 [adults]), or no intubations of
ethanol (control group treated with vehicle on PD28-36
[adolescent] or PD70-78 [adults]). The ethanol dose was
selected on the basis of studies from our laboratory
(Acevedo, Molina, Nizhnikov, Spear, & Pautassi, 2010) that
suggested its effectiveness in promoting ethanol intake in
adolescents. This was achieved by administering .015 ml/kg of
a 21% ethanol solution (96% proof ethanol, Porta Hnos.,
Cordoba, Argentina; vehicle: tap water).
Untreated animals experienced only standard housing dur-
ing PDs 28-36 or PDs 70-78. These animals were then tested
for sucrose intake on PDs 37 or 79, and for anxiety response
on PDs 38 or 80 (adolescent and adult subjects, respectively).
Untreated subjects were not tested for ethanol intake test. Thepurpose of including this group was to control for alterations
in sucrose preference or anxiety resulting from the manipula-
tions inherent to initiation procedures.
Homecage Ethanol Self-Administration Tests
On PD37 (adolescents) or PD79 (adults), the animals were
individually housed and exposed for 24 hr to two bottles
equipped with ball-point tubes, and filled with tap water or
1% w/v sucrose. The aim was to assess potential differences
in sucrose preference after initiation procedures.
2 Fabio et al. Developmental Psychobiology
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On PD39-42 or PD80-83 (self-administration phase for
adolescents and adults, respectively) the animals had continu-
ous access to two drinking bottles, one containing a 5.6% v/v
ethanol solution sweetened with 1% w/v sucrose and another
that contained water. Sweetened ethanol was chosen based on
the finding that adolescent Wistar rats did not voluntarily con-
sume unsweetened ethanol (5.6% v/v ethanol) but consumed
slightly sweetened (sucrose 1% w/v) ethanol (5.6% v/v) over-
night (Maldonado, Finkbeiner, & Kirstein, 2008). The avail-
able protocols to induce significant consumption of
unsweetened ethanol in rats are quite lengthy and, if used in
the present study, would not have allowed the post-depriva-
tion intake test to occur within the adolescent period. We pre-
viously employed unsweetened ethanol to test adolescent
self-administration (Ponce, Pautassi, Spear, & Molina, 2004,
2011), but these tests required extensive, 22-hr liquid depriva-
tion, which is a significant stressor.
The position of the water and ethanol bottles was varied
across sessions to avoid side-preference effects. On each test
day, the bottles were positioned at 11:00 AM to 12:00 PM,
and the animals were left undisturbed. On the following day(9:00 AM to 10:00 AM), the bottles were removed, weighed
to the nearest .1 g and refilled. During weighing and refilling
animals were allowed to interact with each other for 2 hr to
minimize stress-induced isolation. Ethanol intake is expressed
as grams of ethanol consumed per kilogram of body weight
(g/kg). Spillage was accounted for by placing water and etha-
nol bottles in an empty holding cage overnight. The differ-
ence in weight was subtracted from each animals intake
score to account for spillage that may have occurred over-
night. Food consumption was monitored by providing a pre-
measured amount of food and weighing the portion that
remained each day.
After the 4-day self-administration phase, the animals un-
derwent a 7-day ethanol recess phase, in which they had ad
libitum access to water and food. On PD50 or 92 (post-depri-
vation assessment for adolescents and adults, respectively)
animals were tested for 24 hr in ethanol self-administration,
through the two-bottle procedure described for the pre-depri-
vation self-administration phase. The rationale for the week
interval between the initial 4 days of self-administration and
the last ethanol intake assessment was to fit the treatment and
the test within the same adolescent period. We were also
guided by a recent study in which animals experienced a
short ethanol deprivation period between ethanol intake tests
(Acevedo et al., 2010).
Light/Dark Box Test
A light/dark box (LDB) test was conducted on PD38 and 48
(adolescents) or PD80 and 90 (adults). The aim was to detect
possible differences in anxiety-like behavior that could ex-
plain ethanol drinking patterns. The light/dark box exploits
the conflict between the animals tendency to explore a new
environment and its fear of bright light (Cancela, Bregonzio,
& Molina, 1995).
The apparatus (42 25 25 cm3) consisted of two com-
partments made of high impact acrylic, one dark
(17.5 25 25 cm3) illuminated with a 25 W red bulb, and
one white (24.5 25 25 cm3) illuminated by a 60 W
white bulb lamp. The compartments were separated by a di-
vider with a 6.5 6.5 cm2 opening at floor level. The test
lasted 5 min and began by gently placing the animal in the
center of the dark area, facing away from the white area. The
test was videotaped and then analyzed by a researcher blind
to the treatments. The latency to cross to the white compart-
ment, number of transfers from one compartment to the other
compartment, and time spent in the white compartment were
measured using Etholog software (Ottoni, 2000).
Data Analysis
The preliminary analysis indicated significant age-related dif-
ferences in the initial consumption of ethanol, with adolescent
subjects drinking almost twice as much ethanol as adults in
self-administration Session 1 [F1,65 5.17, p < .05]. Adults
subjects, on the other hand, exhibited greater overall locomo-
tion in the light/dark box test [F1,65 5.99, p < .01]. Base-
line differences between adolescents and adults have often
been observed across several variables, including ethanol in-take (Doremus, Brunell, Rajendran, & Spear, 2005), stress re-
sponsiveness (Varlinskaya & Spear, 2012), and preference for
tactile cues (Pautassi et al., 2008). We followed the statistical
approach of the latter studies and analyzed each dependent
variable using separate analyses of variance (ANOVAs) for
adolescents and adults. The loci of significant main effects or
significant interactions were analyzed using Tukey post hoc
tests. Planned comparisons were also conducted if justified by
previous hypotheses.
Self-Administration During Pre-Deprivation and Post-Depri-
vation Sessions. For each age, ethanol intake (g/kg) during
the 4-day pre-deprivation phase was analyzed using two-way
mixed-factor ANOVAs. Ethanol initiation (5-day exposure, 2-day exposure, and the control group) was the between-group
factor and session was the within-group measure. Similar
ANOVAs were used to analyze water intake (ml/100 g) and
food intake (g/kg) across the procedures. One-way ANOVAs
(comparative factor: ethanol initiation condition) were per-
formed for sucrose intake on the adaptation test day (g/kg,
the UT group was also included in this ANOVA) and for eth-
anol intake (g/kg) in the post-deprivation assessment.
Light/Dark Box Tests on PD38 and 48 (Adolescents) or PD80
and 90 (Adults). The latency to cross to the white side, num-
ber of transfers from one compartment to the other compart-
ment and time spent in the white compartment were analyzedfor adolescents and adults using two-way ANOVAs (compara-
tive factor between groups: ethanol initiation condition; with-
in-group measure: day of testing). Planned comparisons were
conducted between the UT and the vehicle-exposed group, for
each of the variables measured on the first lightdark test.
We further analyzed ethanol intake measurements using a
correlational approach. Pearsons rproductmoment correlations
were separately conducted for the adolescent and adult groups,
and for each ethanol treatment at initiation. The specific question
under analysis was to what extent initial self-administration of
ethanol predicted later ethanol self-administration during the
Developmental Psychobiology BingeQ2
Ethanol Heightens Subsequent Ethanol Intake 3
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initial 4-day phase and during the post-deprivation intake ses-
sion. It has been suggested that the quality of the initial expe-
rience with ethanol significantly affects later ethanol drinking
(Schramm-Sapyta et al., 2009). Moreover, it has been ob-
served that patterns of ethanol intake are established early in
adolescent rats, with subjects that drink heavily during the
first exposure to ethanol keeping this behavior on subsequent
sessions (Schramm-Sapyta et al., 2008). It is still unknown,
however, if ethanol drinking behavior follows a similar trajec-
tory in adult subjects, and if this pattern is altered in subjects
given prior ethanol initiation.
RESULTS
Body Weight, Sucrose Consumption DuringAdaptation and Food Intake
The administration of ethanol during initiation did not
significantly alter body weight during the 4-day pre-
deprivation phase or during the post-deprivation assess-ment. As expected, the ANOVAs only indicated that
body weight in the adolescents increased as a function
of days during the pre-deprivation two-bottle intake
assessment,F3,99 361.4, p < .001. Sucrose consump-
tion (g/kg) at beginning of intake tests (PD37 or PD79)
was not affected by ethanol initiation. Sucrose intake in
animals given 5-day ethanol exposure, 2-day ethanol ex-
posure, vehicle controls and in untreated controls was
.54 .08, .52 .08, .45 .06, and .34 .10; and
2.12 .47, 2.58 .47, 2.11 .39, and 1.98 .54; for
adolescents and adults, respectively.
Food intake during the 4 days in which subjects
were tested with the two-bottle procedure increased inadolescents as a function of days [F3,99 41.83,
p < .001] but remained stable in adults. These patterns
were unaffected by ethanol initiation treatment. One-
way ANOVAs indicated that food intake during the
post-deprivation session was similar across initiated
and non-initiated adolescents or adults.
Descriptive results for body weight (g) and food in-
take (g/kg) are depicted in Table 1.
Ethanol and Water Self-Administration DuringEthanol Intake Tests
Adolescent Rats. The ANOVA for the 4-day phase in
which subjects were tested with the two-bottle proce-
dure revealed a significant main effect of session,
F3,99 5.19, p < .005. The post hoc tests indicated
greater ethanol consumption during Session 1 than in
the subsequent sessions, which in turn did not differ
Table 1. Body Weight (g), Water Intake (ml/100 g of Body Weight), Food Intake (g/kg of Body Weight) and Ethanol
Intake (ml/100 g of Body Weight); in Adolescent and Adult Rats During the 4 Days in Which Subjects Were Tested With a
Two-Bottle Procedure (Pre-Deprivation Intake Sessions) and During the Post-Deprivation Intake Session
Adolescent Rats Adult Rats
5-Day Group 2-Day Group Control 5-Day Group 2-Day Group Control
Body weight (g)
Pre-deprivation intake session 1 185.92 5.91 17 6.83 4.10 182.09 5.93 443.82 13.10 433.75 10.99 456.58 9.00
Pre-deprivation intake session 2 196.62 6.31 18 7.83 4.70 195.64 5.32 449.36 13.12 439.25 10.77 462.75 9.27
Pre-deprivation intake session 3 205.08 6.51 19 9.25 5.10 206.72 5.60 454.45 13.54 440.33 12.34 466.67 9.55
Pre-deprivation intake session 4 214.54 7.08 20 7.75 5.15 214 5.56 458.27 13.56 419.42 26.16 468.92 9.39
Post-deprivation intake session 285.54 10.98 278.83 7.99 16.65 4.59 482.64 16.79 470.67 11.45 496.33 11.05
Food intake (g/kg)
Pre-deprivation intake session 1 4.24 .26 3.83 .17 4.37 .20 13.19 .65 12.59 .68 13.41 .86
Pre-deprivation intake session 2 4.72 .37 4.20 .39 4.80 .41 12.86 .84 13.63 .81 14.42 .57
Pre-deprivation intake session 3 5.24 .33 5.02 .22 5.46 .25 13.36 1.05 13.29 .78 14.15 .71
Pre-deprivation intake session 4 8.61 3.55 5.08 .24 5.68 .40 12.57 1.01 12.17 .95 13.91 .69
Post-deprivation intake session 7.86 .67 7.38 .53 8.29 .56 13.45 .70 13.74 .75 13.30 1.39
Water intake (ml/100 g)
Pre-deprivation intake session 1 9.70
1.62 8.94
1.82 12.99
1.84 11.11
1.31 9.10
1.48 7.67
1.02Pre-deprivation intake session 2 9.28 1.70 10.19 1.86 12.20 2.00 11.18 1.82 10.23 1.61 8.64 1.32
Pre-deprivation intake session 3 11.91 1.51 12.99 1.90 14.39 2.15 9.21 2.53 9.25 1.75 8.60 1.39
Pre-deprivation intake session 4 11.8 1.39 12.34 1.55 12.65 1.65 10.52 1.73 12.19 3.88 7.21 1.55
Post-deprivation intake session 6.76 1.36 6.68 1.38 7.98 1.58 10.63 2.66 10.28 2.24 7.63 1.66
Ethanol intake (ml/100 g)
Pre-deprivation intake session 1 7.12 1.94 10.55 2.42 5.43 1.94 2.56 1.02 5.38 1.56 3.20 .88
Pre-deprivation intake session 2 7.44 2.09 9.37 2.39 4.93 1.96 3.63 1.31 4.02 1.70 3.92 1.61
Pre-deprivation intake session 3 5.15 1.58 5.49 2.00 3.04 1.57 6.07 1.85 3.54 1.13 3.32 .78
Pre-deprivation intake session 4 8.69 1.57 10.07 1.94 5.78 1.63 8.80 .24 10.47 1.40 8.57 .18
Post-deprivation intake session 18.89 .62 19.40 .52 18.83 .52 7.59 1.89 6.51 1.83 7.88 1.25
Results are presented as mean standard error of the means.
4 Fabio et al. Developmental Psychobiology
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between each other. Guided by a priori hypothesis,
planned comparisons were conducted between the
5-day or the 2-day ethanol treatment group and the
control group, for each intake session. These analyses
revealed significantly greater ethanol intake scores
(g/kg) in adolescents given 2-day exposure than in con-
trol counterparts, during pre-deprivation intake sessions
1 and 2 (F1,33 4.61, F1,33 5.24, both ps < .05; for
Sessions 1 and 2, respectively). These results are
depicted in Figure 1.
FIGURE 1 Ethanol intake (g/kg) in adolescent (upper panels) and adult (lower panels) male
rats during the 4-day self-administration phase and during the post-deprivation assessment.
Ethanol intake during the initial 4 days of self-administration is depicted as a function of day
of assessment (Sessions 1, 2, 3, and 4) and ethanol treatment during initiation. Initiation oc-
curred on postnatal days 28-36 (PD28-36) or PD70-78 for adolescents and adults, respectively.
During initiation, the animals were given intragastric administrations every other day and re-
ceived five 2.5 g/kg ethanol intubations (5-day exposure group; ethanol on PD28, 30, 32, 34,
and 36 [adolescents] or PD70, 72, 74, 76, and 78 [adults]), two 2.5 g/kg ethanol intubations
(2-day exposure group; ethanol on PD28 and 32 and vehicle on PD30, 24, and 36 (adoles-cents) or ethanol on PD70 and 74 and vehicle on PD72, 76, and 78 [adults]), or no ethanol
intubations (control group treated with vehicle on PD28-36 [adolescents] or PD70-78 [adults]).
The initial self-administration phase (i.e., pre-deprivation assessment of ethanol intake) lasted
for 4 days, in which the animals were given continuous, 22 hr two-bottle choice between
ethanol (5.6% v/v) and water in the homecage. After the last acquisition session, the animals
had ad libitum access to only water and food for 7 days, and then a single 22-hr test of ethanol
intake was conducted (two-bottle homecage choice test between 5.6% v/v ethanol and water;
post-deprivation assessment). Adolescents but not adults given two ethanol exposures during
initiation exhibited significantly greater ethanol intake than controls during pre-deprivation
intake Sessions 1 and 2, and during the post-deprivation assessment. These significant effects
of ethanol initiation are indicated by the asterisk. The vertical bars indicate SEM.
Developmental Psychobiology Binge Ethanol Heightens Subsequent Ethanol Intake 5
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Ethanol initiation also appeared to heighten ethanol
intake during the post-deprivation assessment (see
Fig. 1). The one-way ANOVA revealed a significant
main effect of treatment at initiation (F2,33 3.73,
p < .005). Subsequent post hoc tests indicated signifi-
cantly greater consumption of ethanol in adolescents
given 2-day exposure than in the control group. Ani-mals that received five intubations also exhibited great-
er ethanol intake than controls, but the difference did
not achieve statistical significance.
The ANOVA for water intake during pre-deprivation
intake sessions indicated only a significant main effect
of session (F3,99 4.41, p < .005), and the post hoc
tests revealed greater water intake on Days 3 and 4 of
the pre-deprivation intake phase than on Days 1 and 2.
Treatment at initiation did not modify water intake dur-
ing pre-deprivation sessions or during the post-depriva-
tion session. The average mean (SEM) water intake
and ethanol intake (ml/100 g) in adolescents given 5-day exposure, and 2-day exposure and controls is
depicted in Table 1.
Adult Rats. Ethanol drinking in adult rats remained
stable during the 4 days in which subjects were tested
with a two-bottle procedure and was not affected by
the ethanol initiation procedures. The ANOVAs indicat-
ed a lack of significant main effects or significant inter-
actions. The planned comparisons between the 5-day or
the 2-day ethanol treatment group and the control
group did not achieved significance. Unlike adolescent
subjects, post-deprivation ethanol intake in adult rats
was insensitive to previous passive exposure to ethanol(see Fig. 1). The ANOVA revealed that ethanol-initiat-
ed and control (vehicle-treated) subjects exhibited simi-
lar levels of ethanol consumption.
The ANOVAs for water intake during the four pre-
deprivation sessions and during the post-deprivation
session indicated no significant main effects or signifi-
cant interactions. Average mean and SEM water intake
and ethanol intake (ml/100 g) across sessions is pre-
sented in Table 1.
Light/Dark Box Test
Adolescent Rats. Adolescents given 0, 2, or 5 ethanol
exposures at initiation exhibited similar latency to cross
to the white side, number of transfers from one com-
partment to the other, and similar time spent in the
bright side of the light/dark box. This pattern was fairly
similar during the first and second light/dark box tests.
The ANOVA revealed a significant main effect of day
of testing on the number of transfers, [F1,33 4.27,
p > .05], indicating a reduction of overall locomotor
activity in the second test session.
Adult Rats. Ethanol initiation did not exert significant
main effects or significantly interact with the remaining
variables in any of the measures. The number of trans-
fers tended to decrease in the second test, as indicated
by a borderline main effect of day of testing (p .056).
Planned comparisons indicated that anxiety during
the first lightdark test did not significantly differ be-tween the untreated and vehicle controls, neither in
adolescents nor in adults.
Correlation Between Measures of Ethanol Intake
Adolescent Rats. Ethanol intake was highly correlated
during the four pre-deprivation intake sessions in all of
the ethanol initiation conditions. Post-deprivation etha-
nol intake was predicted in the control groupbut not
in the 5- or 2-day exposure groupby ethanol intake
during acquisition sessions 14 (r .75, .89, .80, and
.77; respectively) (Tab. 2).
Adult Rats. In control adults ethanol intake on pre-dep-
rivation day 2 was predictive of ethanol intake on pre-
deprivation day 3 and the latter predicted ethanol intake
at pre-deprivation day 4. The animals given five ethanol
exposures exhibited a significant association between
ethanol intake on pre-deprivation day 1 and ethanol
self-administration on pre-deprivation days 2 and 4. In
this group, post-deprivation ethanol consumption was
predicted by pre-deprivation day 3. In the 2-day ethanol
initiation group ethanol intake on pre-deprivation day 1
predicted intake on pre-deprivation days 2 and 3, and
post-deprivation ethanol intake was predicted by etha-
nol intake on pre-deprivation days 1, 2, and 3 (r .81,.73, and .93, respectively).
DISCUSSION
The present study analyzed whether brief and intermit-
tent exposure to ethanol heightens later ethanol intake,
particularly after a period of ethanol deprivation, and
tested the age-specificity of this effect. Consistent with
previous studies (e.g., Doremus et al., 2005), the initial
self-administration of ethanol was greater in adoles-
cents than in adults. The most important new finding
was that adolescent rats exposed to binge ethanol intox-ication subsequently exhibited greater ethanol intake
than control subjects. Specifically, binge ethanol in-
duced a significant increase in ethanol intake in adoles-
cents during intake Sessions 1 and 2 of the initial self-
administration phase. Moreover, when tested after
7 days of drug withdrawal, adolescents given two pas-
sive ethanol intubations exhibited a significant threefold
increase in ethanol intake compared with age-matched
controls. These effects were not found in adults and no
changes in water intake were observed.
6 Fabio et al. Developmental Psychobiology
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Rodd-Henricks et al. (2002a, 2002b) gave adolescent
or adult ethanol-preferring rats free choice access to
ethanol or standard housing. During adulthood, ethanol
operant self-administration was greater in animals that
had been initiated to ethanol during adolescence. This
and others (e.g., Fullgrabe et al., 2007; Siegmund et al.,
2005) are similar to the present study in that they ex-
amined the predisposition for ethanol intake after ado-
lescent exposure. The previous studies, however,
employed extensive initiation phases (e.g., >7 months,
Tambour et al., 2008) that lasted past adolescence, not
allowing the intake test to occur within the adolescent
Table 2. Pearson Correlations Between Ethanol Intake (g/kg) During Acquisition Days 14 and During the
Post-Deprivation Assessment, for Adolescent and Adult Rats Given 5 [5-Day Exposure Group], 2 [2-Day Exposure Group]
or 0 [Control Group Treated With Vehicle] Administrations of 2.5 g/kg Ethanol During Ethanol Initiation Procedures
Ethanol Intake
at Acquisition
Day 1
Ethanol Intake
at Acquisition
Day 2
Ethanol Intake
at Acquisition
Day 3
Ethanol Intake
at Acquisition
Day 4
Post-Deprivation
Assessment of
Ethanol Intake
Adolescent rats
Ethanol intake at Acquisition day 1
5-Day group
2-Day group
Control
Ethanol intake at Acquisition day 2
5-Day group .65
2-Day group .87
Control .82
Ethanol intake at Acquisition day 3
5-Day group .69 .68
2-Day group .78 .95
Control .65 .93
Ethanol intake at Acquisition day 45-Day group .64 .76 .90
2-Day group .79 .94 .97
Control .64 .91 .99
Post-deprivation assessment of ethanol intake
5-Day group .27 .47 .27 .46
2-Day group .29 .27 .11 .21
Control .75 .89 .80 .77
Adult rats
Ethanol intake at Acquisition day 1
5-Day group
2-Day group
Control
Ethanol intake at Acquisition day 2
5-Day group .76
2-Day group .88
Control .12
Ethanol intake at Acquisition day 3
5-Day group .53 .41
2-Day group .75 .67
Control .25 .71
Ethanol intake at Acquisition day 4
5-Day group .79 .97 .44
2-Day group .06 .12 .33
Control .33 .10 .50
Post-deprivation assessment of ethanol intake
5-Day group .45 .57 .82 .57
2-Day group .81 .73 .93 .10 Control .06 .29 .08 .10
Correlations significant at p
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period. Moreover, in these studies the initiation phase
consisted of self-administration of ethanol, which
implies habituation of neophobia, familiarization with
the flavor of ethanol and usually results in baseline dif-
ferences in drinking across sessions. The latter factor is
particularly important when comparing adults and ado-
lescents, given the propensity of adolescents to drinksignificantly more ethanol than adults (Doremus et al.,
2005). In the present study, the choice of intragastric
intubation as the method for ethanol initiation equated
level of ethanol exposure across ages. The present
study is also significant in that it specifically investigat-
ed whether early-onset drinking in an early stage of
adolescence facilitates the escalation of ethanol con-
sumption during adolescence.
The alcohol exposure procedure employed in the
present study involved 5 or 2 binge exposures, followed
by 4 days of self-administration to a moderate concen-
tration of alcohol. Caution should be taken, therefore,when comparing the results of this study with others
that employed protracted exposure to ethanol or repeat-
ed cycles of access to and withdrawal from ethanol
(e.g., Bell et al., 2008; Spanagel, 2000). Those proce-
dures are specifically aimed to model alcoholism and
proven to induce tolerance, physical dependence, and
withdrawal symptoms (Spanagel, 2000).
Another caveat of the present study is the use of
sweetened ethanol. The enhanced predisposition for
ethanol drinking in ethanol-initiated adolescents may
have been attributable to early ethanol exposure alter-
ing the palatability of sucrose or inducing sucrose seek-
ing due to its nutritional value. There were, however,no differences in sucrose intake across ethanol initia-
tion conditions for adolescents or adults, when the
sweet tastant was tested alone against water. Likewise,
no differences were found in food intake or body
weight that could have indicated altered nutritional sta-
tus due to early ethanol exposure.
The most intriguing result of the study was the
significant increase in ethanol self-administration ob-
served in adolescents after just 2 days of ethanol ex-
posure. These animals drank more ethanol than non-
initiated counterparts during the first 2 days of self-
administration as well as during the post-deprivationassessment. What is the potential mechanism underly-
ing higher ethanol consumption in ethanol-initiated
adolescents? Ethanol exposure during passive initia-
tion or during the initial self-administration phase
could conceivably alter anxiety-like behavior. The
results of the light/dark box test, however, indicated
similar patterns of exploration in ethanol-initiated
and non-initiated animals.
Another possibility is that adolescent ethanol initia-
tion facilitates subsequent ethanol intake by altering the
normal pattern of development of specific transmitter
systems. A previous study found that chronic intermit-
tent ethanol exposure during adolescence altered basal
level of dopamine in nucleus accumbens and induced a
down-regulation of dopaminergic and glutamatergic
receptors in prefrontral cortex (PFC; Pascual et al.,
2009). Fabio, Nizhnikov, Spear, & Pautassi (2012)revealed that earlier ethanol initiation in the rat (during
the last 4 days of gestation) also altered basal neural
activity at PFC, when measured at adolescence. These
effects were associated with heightened ethanol
consumption.
Intriguingly, ethanol exposure on PD28 and PD32
increased later ethanol intake, but the 5-day exposure
treatment apparently did not. It could be that ethanol
initiation enhances subsequent ethanol intake when ini-
tiation occurs during restricted developmental time-
frames, such as during early adolescence.
Dopaminergic receptors, which modulate ethanol-in-duced appetitive learning, reach their peak at PD28,
and then decline significantly (Tarazi & Baldessarini,
2000). The present results are also in agreement with
Acevedo et al. (2010), in which ethanol exposure at
PD28 but not at PD31, enhanced subsequent ethanol
consumption. It is also possible that the 5-day exposure
treatment lacked an effect on subsequent intake due to
this treatment inducing tolerance to the appetitive
effects of ethanol.
Yet another possibility is that animals exposed twice
to ethanol on PD28 and PD32 acquired a conditioned
preference for ethanols orosensory cues due to percep-
tion of non-metabolic ethanol excretion (by urine orperspiration) in close contiguity with pharmacological
properties of the drug (Molina, Chotro, & Spear, 1989).
On the other hand, animals exposed to the 5-day expo-
sure treatment may have developed a conditioned aver-
sion, which competed with the preference acquired on
the first days of exposure. In other words, the more
protracted ethanol experience may have induced taste
aversion to ethanol chemosensory properties and this
memory affected subsequent ethanol intake.
The possibility that repeated administration of etha-
nol induced metabolic tolerance in adolescents, but not
in adults, cannot be dismissed. A recent study (VanSkike, Novier Diaz-Granados, & Matthews, 2012)
found metabolic tolerance in adolescent rats that had
been exposed to ethanol vapor 16 hr a day for 4 days.
In adolescent animals ethanol intake across the initial
4 days of self-administration was highly correlated; and
in control adolescents there was a significant associa-
tion between ethanol drinking before and after drug
deprivation. It seems that patterns of ethanol intake
were established early in adolescence and, for those
given vehicle at initiation, maintained through the post-
8 Fabio et al. Developmental Psychobiology
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8/12/2019 35 Dp Init - Brief
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deprivation test. Ethanol-initiated animals, on the other
hand, did not exhibit a significant correlation between
pre- and post-deprivation scores. It seems that, similar
to the results found by Schramm-Sapyta et al. (2008),
those control adolescents that consumed the most dur-
ing the first exposure to ethanol kept drinking heavily
during subsequent sessions and exhibited the highestdrinking scores at the post-deprivation assessment. Pas-
sive exposure to ethanol disrupted this normal trajecto-
ry of ethanol intake otherwise seen in adolescents.
The present study is consistent with findings sug-
gesting that onset of ethanol consumption exerts greater
impact on predisposition to drink when it occurs at ad-
olescence than when it is delayed until adulthood. The
study shows that even brief binge ethanol intoxication
during adolescence can increase drinking. Specifically,
significantly greater ethanol drinking was observed in
adolescents after just 2 days of alcohol exposure. In
agreement with recent work (Schramm-Sapyta et al.,2008), adolescents also exhibited greater susceptibly to
ethanol drinking relapse than adults.
NOTES
This work, a collaborative project between the Research
Foundation of SUNY Binghamton and Instituto Ferreyra, was
supported by NIAAA grants AA018164 to MN and grants
PIP 2010-2012, SECYT-UNC and PICTPRH 246 to RMP.
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