Alcohol During Adolescence

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Alcohol. 2010 February ; 44(1): 5766. doi:10.1016/j.alcohol.2009.09.035.
Alcohol during adolescence selectively alters immediate and

long-term behavior and neurochemistry
Antoniette M. Maldonado-Devincci, M.A.1, Kimberly A. Badanich, Ph.D.3, and Cheryl L.
Kirstein, Ph.D.1,2,*
1Department
of Psychology, Cognitive and Neurosciences, University of South Florida 4202 E.

Fowler Avenue Tampa, FL 33620
2Department
of Molecular Pharmacology & Physiology, University of South Florida College of

Medicine, Tampa, FL 33612
3Center
for Drug and Alcohol Programs, Medical University of South Carolina, Charleston, SC
29425
Abstract
Alcohol use increases across adolescence and is a concern in the United States. In humans, males
and females consume different amounts of alcohol depending on the age of initiation and the longterm consequences of early ethanol consumption are not readily understood. The purpose of our
work is to better understand the immediate and long-term impact of ethanol exposure during
adolescence and the effects it can have on behavior and dopaminergic responsivity. We have
assessed sex differences in voluntary ethanol consumption during adolescence and adulthood and
the influence of binge ethanol exposure during adolescence. We have observed that males are
sensitive to passive social influences that mediate voluntary ethanol consumption and early
ethanol exposure induces long-term changes in responsivity to ethanol in adulthood. Exposure to
moderate doses of ethanol during adolescence produced alterations in dopamine (DA) in the
nucleus accumbens septi (NAcc) during adolescence and later in adulthood. Taken together, all of
these data indicate the adolescent brain is sensitive to the impact of early ethanol exposure during
this critical developmental period.
Keywords
Adolescence; alcohol; sex differences; voluntary alcohol intake; social interaction; dopamine;
neurochemistry
2009 Elsevier Inc. All rights reserved.

*
To whom correspondence should be addressed: Cheryl L. Kirstein, Ph.D., Psychology Department-PCD 4118G, University of South
Florida, 4202 East Fowler Avenue, Tampa, Florida 33620, Phone: (813) 974-9626, Fax: (813) 974-4617, [email protected].
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Maldonado-Devincci et al.
Page 2
Introduction
Initiation of alcohol use during adolescence is high in both males and females, with rates of
use starting as low as 3.9% among adolescents aged 1213 and increasing rapidly across
adolescence with rates as high as 51.6% among individuals aged 1820 (Substance Abuse
and Mental Heath Services Administration, 2007). Underage drinking is slightly higher in
males than females (Substance Abuse and Mental Heath Services Administration, 2007).
Our work focuses on the hypothesis that early initiation of alcohol use may be a predictor of
continued use into adulthood and those that begin use of alcohol at an early age may also be
at risk for developing an alcohol abuse disorder in adulthood (Grant et al., 2001; Hasin and
Glick, 1992). Early exposure to ethanol may predispose the brain to greater vulnerability to
subsequent ethanol exposure both behaviorally and neurochemically
Rodent Model of Alcohol Exposure during Adolescence

In rodent models, the time frame encompassing adolescence has yet to reach consensus.
Adolescence in the rodent has been defined using various factors indicative of
developmental transitions in human adolescents. These factors include changes in behavioral
patterns, hormonal patterns, and/or primary sexual characteristics. In a comprehensive
review, Spear (2000) argues that adolescence cannot be defined based solely on the
characteristics associated with puberty and sexual maturation, but rather, that adolescence is
a period of soft events that should be viewed as a time of transitions that cannot be clearly
delineated. Using the appearance of growth spurt, pruning of excitatory synapses as well as
unique behavioral transitions in the rat (e.g., increased peer interaction, play and exploratory
behavior in the wild), Spear (2000) defined adolescence broadly from postnatal day (PND)
28 to PND 42, with the acknowledgement that in males some traits may appear as late as
PND 55. Other reviews have suggested similar broad classifications defined by the
appearance of mature hormonal cycling (PND 2830) and the subsequent appearance of
reproductive maturity which can occur as late as PND 60 in males (Ojeda and Urbanski,
1994; Smith, 2003). Within this broad range, some researchers have identified the categories
of early- (PND 2134), mid- (PND 3446) and late- (PND 4659) adolescence centered
around the appearance of puberty (between PND 3344) and encompassed by weaning on
PND 21 and reproductive maturity on PND 59 (Tirelli et al., 2003). The beginning of
preadolescence in the rat, in terms of sexual maturation begins at approximately PND 20 for
females and PND 30 for males (Odell, 1990) with significant changes continuing between
PND 2855 (Ojeda & Urbanski, 1994).
Ethanol has been shown to affect multiple neurotransmitter systems during early
development as well as adulthood. Most studies to date have not focused on adolescent
animals but have been conducted during the early postnatal period (Druse, 1992; Light,
Serbus & Santiago, 1989; Martin, Savage & Swartzwelder, 1992; Savage et al., 1991) or in
older, mature animals (see Hunt, 1985, for review). Therefore it is our aim to investigate the
behavioral effects of alcohol during adolescence and to determine how alcohols effects on
mesolimbic DA within adolescence is critical in addressing the long-term effects of early
exposure to alcohol. For these reasons our work has examined the effects of alcohol
exposure during the adolescent transition period from PND 3050.
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Social Interaction
One of the behavioral aspects that guides some of the recent work we have conducted
includes a preclinical approach to understanding environmental factors, specifically the
influence of social interaction and its impact on passive social modeling behavior, which can
influence early alcohol-seeking behavior in adolescent male and female rats. Social
influences, including passive social influences, such as social modeling, contribute to future
alcohol use in adolescent humans (Graham, Marks and Hansen, 1991). Previous studies have
shown the ability of alcohol to facilitate social interactions as an important factor in
adolescent drinking behaviors (Beck, Thombs and Summons, 1993; Beck and Treiman,
1996; Smith, Goldman, Greenbaum and Christiansen, 1995; Thombs, Beck, Mahoney,
Bromley and Bezon, 1994).
In recent work, we have employed the demonstrator-observer paradigm developed by Strupp

and Levitsky (1984). The paradigm is based on the interaction between two animals; one
serving as a demonstrator, which serves as part of the experimental paradigm and the other
as an observer, which serves as the experimental animal in which changes in behavior are
assessed. Social behaviors peak during adolescence, and adolescent rats engage in
distinctive forms of social behavior, including play behavior, social investigation and social
contact (Panksepp, 1981; Pellis, Field, Smith and Pellis, 1997; Pellis and Pellis, 1997;
Varlinskaya, Spear and Spear, 2001, Varlinskaya and Spear, 2002). It has been suggested
that social play represents a separate category of behavior that is different from adult typical
social, sexual and aggressive types of behaviors (for review see Vanderschuren et al., 1997).
While play behavior peaks during adolescence, as adults mature their behavior becomes
more aggressive (Meaney and Stewart, 1981). These data demonstrate that the peak in social
behavior is present during the adolescent period of development, and not in younger or older
animals. Therefore, assessing the influence of social behaviors on passive social learning
specifically during adolescence is appropriate.
Acute ethanol increases social interaction in adolescent rats (Varlinskaya et al., 2001), and
produces social inhibition in adult rats (Varlinskaya and Spear, 2006). Given the increased
social interaction observed in adolescent rats administered ethanol, the demonstratorobserver model has been applied to assess preference for alcohol in adolescent rodents after
social interaction with a familiar alcohol-intoxicated peer (e.g. Fernandez-Vidal and Molina,
2004; Hunt, Holloway and Scordalakes, 2001). One experiment was designed to extend on
the findings of Hunt and colleagues, in which they observed increased ethanol intake in
adolescent rats following social interaction with a familiar alcohol-intoxicated peer (Hunt et
al., 2001). The present work assessed the effects of social familiarity during social
interaction on voluntary ethanol intake using a two-bottle choice test with a choice between
a sweetened ethanol solution and water in male and female adolescent rats (Maldonado,
Finkbeiner and Kirstein, 2008). Consistent with previous work in rodents (Hunt et al., 2001),
we found adolescent males and females that socially interacted with an alcohol-intoxicated
familiar peer voluntarily consumed significantly more sweetened ethanol relative to those
that socially interacted with an alcohol-free peer (Maldonado et al., 2008a). In contrast,
adolescent males that socially interacted with an unfamiliar alcohol-intoxicated peer
consumed significantly less sweetened ethanol relative to adolescent males that socially
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interacted with an unfamiliar alcohol-free peer (Maldonado et al., 2008a). Adolescent

females that socially interacted with an unfamiliar alcohol-intoxicated peer showed similar
increases in voluntary sweetened ethanol intake relative to those that socially interacted with
a familiar alcohol-intoxicated peer (Maldonado et al., 2008a). It is interesting to note the
striking sex differences observed between males and females when the paradigm was altered
by familiarity of the peer.

The experiments described above indicate exposure to an alcohol-intoxicated peer altered

ethanol consumption after just one pairing of social interaction with an alcohol-intoxicated
peer. In the experiment described below (Figure 1), preliminary data are presented to further
investigate the impact of repeated pairings of environmental cues paired with social
interaction on preference for the associated environment in alcohol-nave adolescent
observer rats. In this experiment (Figure 1), we aimed to expand on the findings of
Fernandez-Vidal and Molina (2004), in which they found that adolescent rats that repeated
socially interacted with an alcohol-intoxicated peer showed a change in preference for an
alcohol odor as compared to an appetitive banana odor. It is important to note in this social
interaction experiment, the observer was never orally administered water or ethanol,
therefore the observer rats were in a drug-free state and we assessed the passive social
influence of social interaction with an alcohol-intoxicated peer on changes in behavior in the
ethanol-nave adolescent observer. Specifically, we assessed the impact of repeated pairings
of social interaction with an alcohol-intoxicated peer paired with environmental cues on
changes in preference for the chamber paired with social interaction with an alcoholintoxicated peer in adolescent male and female rats. Our paradigm followed the same age
and number of pairings of social interaction with an alcohol-intoxicated peer as that used in
the Fernandez-Vidal and Molina (2004) study. In the present work, rats were given a 15minute baseline conditioning assessment on PND 30 in which rats had free access to both
chambers to assess baseline preferences for each chamber. The chamber the adolescent
observer rat spent the least amount of time in was paired with social interaction with an
alcohol-intoxicated peer. Control rats socially interacted with the same alcohol-free peer in
both chambers. Conditioning occurred over a period of four days from PND 3134.
Specifically, in the morning, all rats underwent social isolation in a holding cage and water
administration to the demonstrator after the initial 30 minutes of social isolation followed by
an additional 30 minutes of social isolation. Rats were then given the opportunity to socially
interact with an alcohol-free peer in the initially most-preferred chamber for 30 minutes.
Four hours later, the same paradigm was repeated, however the same demonstrator was
orally administered ethanol (0.5 or 1.5 g/kg) or water in control rats and the dyad were
confined to the alternate chamber for 30 minutes. Each adolescent observer socially
interacted with the same adolescent demonstrator for all social interaction sessions.
However, the demonstrator was sober in the morning sessions of social interaction and
intoxicated in the afternoon sessions of social interaction. Control rats socially interacted
with the same sober peer for all social interaction sessions. For each dose tested in which the
observer socially interacted with an alcohol-intoxicated or non-intoxicated demonstrator,
separate rats were used, for a total of three groups of observer-demonstrator dyads (control,
0.5 and 1.5 g/kg). This cycle was repeated three additional times for a total of four pairings
with an alcohol-free peer and four pairings with an alcohol-intoxicated peer (or eight
Page 5
pairings with an alcohol-free peer for control rats). Each observer socially interacted with
the same demonstrator for all conditioning sessions. On the final day, PND 35, all rats
underwent a post-conditioning test to assess changes in time spent in the designated paired
chamber (Maldonado and Kirstein, unpublished data).
Note that adolescent observers were never administered water or ethanol in this experiment,
but were given the opportunity to socially interact with an alcohol-intoxicated demonstrator
to assess changes in behavior. As depicted in Figure 2, adolescent ethanol nave observer
male rats that socially interacted with an alcohol-intoxicated peer administered the highest
dose (1.5 g/kg) showed a significant increase in time spent in the chamber paired with the
alcohol-intoxicated peer at the post-conditioning test relative to the preconditioning baseline
assessment as compared to all other groups. There were no other significant differences
observed in adolescent ethanol nave male observers that socially interacted with an alcoholintoxicated peer administered the lower ethanol dose (0.5 g/kg) relative to their controls. Nor
were there changes in preference for the environment paired with social interaction with an
alcohol-intoxicated demonstrator at either dose in ethanol-nave female observers relative to
their controls (Maldonado and Kirstein, unpublished data). Together, these data indicate
adolescent male rats are especially sensitive to environmental conditions surrounding the
influence of the peer on changes in behavior following social interaction with an alcoholintoxicated peer.
Social play in adolescent animals has been suggested to be rewarding using the conditioned
place preference (CPP) paradigm (Calcagnetti & Schechter, 1992). Consistent with
Calcagnetti and Schechter (1992), there were no significant sex differences in time spent in
the initially least preferred side after conditioning in control animals in the social interaction
conditioning experiment discussed above (Figures 1 and 2). Extending upon the work of
Calcagnetti and Schechter (1992), using the conditioned place preference paradigm in
adolescent animals, in conjunction with the effects of ethanol on facilitating social
interaction, adolescent male rats appear to be the most sensitive to the effects of cues paired
with social interaction with an alcohol-intoxicated peer. Overall, the present results suggest
adolescent males are particularly sensitive to environmental conditions when socially
interacting with an alcohol-intoxicated peer, which may alter subsequent responding for
alcohol in social settings.
The ability of alcohol to facilitate social interactions plays a key role in adolescent drinking
behaviors (Beck et al., 1993; Beck and Treiman, 1996; Smith et al., 1995). Additionally, one
of the most important factors implicated in alcohol use in high school students is its effects
on social facilitation (Beck & Treiman, 1996; Thombs et al., 1994). Passive social
influences have been shown to affect future alcohol use in adolescent humans (Graham et
al., 1991) and the results obtained from the present experiments and previous preclinical
work (Fernandez-Vidal & Molina, 2004; Hunt et al., 2001) confirms this notion. More work
should be conducted to identify the specific mechanisms that may mediate these results and
how they may alter adolescent alcohol-seeking behavior.
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Voluntary Ethanol Consumption
The method of initial exposure to ethanol can alter future drinking patterns in rats (Files et
al., 1997). Ethanol consumption during adolescence appears to have no effect on subsequent
ethanol intake in adulthood (Slawecki and Betancourt, 2002; Tolliver and Samson, 1991;
Vetter et al., 2007). Adolescent rats voluntarily consume more ethanol relative to adults
(Truxell et al., 2007) when animals were allowed voluntary access to ethanol under
continuous access conditions (Brunell and Spear, 2005; Doremus et al., 2005; Vetter et al.,
2007). The same effect was observed using a modified sucrose-substitution paradigm under
limited access conditions (Maldonado, Finkbeiner, Alipour and Kirstein, 2008).

Importantly, some of our most recent work has examined some of the sex-related differences
that may accompany the age-related differences in voluntary ethanol consumption under
limited access conditions. In the most common paradigm we employ in our experiments, rats
are given daily 30 minutes access to sweetened ethanol solutions using sucrose or saccharin.
Rats are initially placed in a holding cage for 3060 minutes to allow them to acclimate to
the behavioral testing room and subsequently given access to ethanol and water for 30
minutes in rats that are neither food, nor water deprived. We have observed interesting sex
and age-related differences in voluntary ethanol consumption under these limited access
conditions. As described recently, adolescent male rats consume significantly more ethanol
than adult male rats at higher sweetened ethanol concentrations (10% and 20% v/v ethanol)
(Maldonado et al., 2008b). In contrast, adult female rats consume significantly more ethanol
than adolescent female rats at lower sweetened ethanol concentrations (5% and 10%).
Consistent with other work, we have observed increased voluntary sweetened ethanol intake
in females as compared to males, when voluntary sweetened ethanol intake was equated for
body weight. These data highlight the importance of examining sex differences when
conducting developmental experiments. It appears early during adolescence there are no
differences in voluntary sweetened ethanol consumption (Maldonado and Kirstein,
unpublished data), however sex differences in voluntary ethanol consumption are observed
in adulthood (Almeida et al., 1998; Blanchard et al., 1993; Juarez and Barrios de Tomasi,
1999; Lancaster et al., 1996). Additionally, across development from adolescence into
young adulthood, there appears to be a natural decrease in voluntary ethanol consumption
(Vetter et al., 2007). Given that sex differences emerge in adulthood, early exposure to
ethanol during adolescence could alter the normal pattern of ethanol consumption in
adulthood.
Interestingly, when using a binge model of adolescent ethanol exposure and assessing
subsequent sweetened ethanol intake under limited access conditions in young adulthood in
male and female rats, we have observed dramatic increases in voluntary ethanol intake in
rats that were treated with high doses of ethanol during adolescence relative to those that
were treated with water during adolescence (Maldonado et al., 2009). Male and female rats
were exposed to ethanol (1.5, 3.0 or 5.0 g/kg/intragastric intubation) or water across
adolescence in four-day intervals (PND 2831, PND 3538, and PND 4345) with three
days between each administration in which animals were left undisturbed (PND 3234 and
PND 3941). Immediately following ethanol pretreatment during adolescence, all rats
underwent fourteen days of abstinence in which animals were left undisturbed (PND 4659).
Page 7
Finally, voluntary sweetened ethanol (0.5% saccharin/ 10% ethanol) intake was assessed in
young adulthood (PND 6069). According to preliminary data, when ethanol intake was
equated for body weight, females consumed significantly more ethanol than males
regardless of pretreatment with ethanol or water. Pretreatment with any dose of ethanol
enhanced this effect. However, when we equated for the innate sex differences in voluntary
ethanol consumption observed in adulthood, it appears males were more vulnerable to the
subsequent increase in voluntary ethanol consumption in adulthood. These data highlight the
importance of early ethanol history and the relative impact it has on later ethanol
consumption in adulthood when assessing vulnerability to subsequent ethanol-seeking and
consumption behaviors.
Long-term effects of adolescent ethanol exposure
Novelty preference has been extensively examined in the literature as a measure of

individual differences and its influence in responsivity to drugs of abuse. We have
established a paradigm that allows us to assess novelty preference in adolescent and adult
animals using a single novel object in a circular open field. Using this paradigm, we have
readily demonstrated adolescent rats display greater levels of novelty preference relative to
adult rats (Stansfield and Kirstein, 2006). Interestingly, using this paradigm, we found
chronic pretreatment (PND 3050) with a moderate dose of ethanol during adolescence (1.0
g/kg/ip) significantly increased novelty seeking behaviors manifested as a greater frequency
to approach a novel object in the center of the open field (Stansfield and Kirstein, 2007).
Given that novelty preference and risk-taking behaviors have repeatedly been associated
with an increased propensity to drug self-administration and greater drug intake (Bevins et
al., 1997), these data support the hypothesis that adolescent rats chronically exposed to
ethanol during adolescence may be at even greater risk to greater ethanol intake in
adulthood.
Differential biphasic effects of ethanol have been demonstrated for ethanol-induced motor
activity. Ethanol-induced locomotor activity has been shown to be increased among
adolescent male and female alcohol-preferring P rats (Rodd et al., 2004). Specifically, in
response to an acute dose of 0.50 or 0.75 g/kg ethanol, adolescent P female rats
demonstrated increased activity counts relative to animals administered saline or 0.25 g/kg
ethanol. Additionally, adolescent P males exhibited elevated activity counts when
administered 0.25, 0.50, or 0.75 g/kg ethanol relative to saline controls. In animals of both
sexes activity counts were significantly decreased at 1.50 g/kg ethanol. Because of the
heightened sensitivity to ethanol among adolescent P females, the present study used outbred female rats to assess the effects of chronic ethanol administration (PND 3446) during
adolescence to assess the dose-dependent effects of ethanol on locomotor activity to 0.75
and 1.50 g/kg/ip/b.i.d. ethanol (Maldonado and Kirstein, unpublished data). Rats underwent
abstinence from PND 47 to PND 59. Rats were then challenged with ethanol (0.75 or 1.50
g/kg/ip) or saline on PND 60.
The results of this study indicated significant long-term alterations in behavior among
adolescent female rats that were repeatedly exposed to ethanol during this period of
development. Adolescent females developed tolerance to the high dose of ethanol (1.50
Page 8
g/kg) during repeated exposure as evidenced by an initial acute suppression in locomotor

activity in ethanol-treated (PND 35) rats followed by the development of similar levels of
locomotor activity between ethanol and saline-treated (PND 46) rats. However, this pattern
did not persist into adulthood following a washout period between adolescence and
adulthood, as ethanol-treated rats showed decreased locomotor activity in young adulthood
when challenged with the same dose of ethanol on PND 60. It is evident that the low dose of
ethanol produced a facilitation of locomotor activity when tested after repeated ethanol
exposure during adolescence, as rats treated with the lower (0.75 g/kg) dose of ethanol
showed increased locomotor activity (PND 46) following repeated ethanol treatment during
adolescence relative to saline-treated rats. This pattern persisted into young adulthood when
animals were challenged with the same moderate dose of ethanol on PND 60 (Maldonado
and Kirstein, unpublished data). Interestingly, behavioral sensitization has been observed in
Sprague Dawley rats to a lower dose of ethanol when animals were separated into high and
low responders to novelty (Hoshaw & Lewis 2001). These results again demonstrate
repeated alcohol exposure during adolescence results in persistent alterations in behavior
that last into young adulthood.

Adolescent exposure to ethanol also tended to alter responsivity in a conditioned place

preference paradigm in young adulthood. Adolescent male rats were chronically exposed to
saline or ethanol (0.75 g/kg/ip) during adolescence (PND 3050) followed by a 14-day
washout period and were conditioned with saline or ethanol (0.75 g/kg/ip) in young
adulthood in a two-chambered apparatus (Maldonado, Badanich and Kirstein, unpublished
data). In young adulthood (PND 6570), rats were conditioned with saline or ethanol.
Control rats had saline paired with each environment for a total of eight saline conditioning
trials. For rats conditioned with ethanol in adulthood, four pairings with saline and four
pairings with ethanol occurred. As shown in Figure 3, adolescent rats that were pretreated
with ethanol and conditioned with ethanol in young adulthood spent significantly more time
in the ethanol-paired chamber relative to those adolescent rats that were pretreated with
saline and conditioned with ethanol in young adulthood. Additionally, adolescent rats that
were treated with ethanol and subsequently conditioned with ethanol in young adulthood
demonstrated a greater amount of time spent in the ethanol-paired chamber relative to
animals pretreated with ethanol and conditioned with saline during young adulthood (Figure
3; Maldonado, Badanich, and Kirstein, unpublished data). These data indicate animals
exposed to ethanol during adolescence and re-exposed to ethanol in adulthood showed
greater ethanol-seeking behavior manifested as increased time spent in the drug-paired
chamber relative to animals exposed to ethanol for the first time in adulthood.
Taken together, all of these data indicate exposure to ethanol during adolescence does
indeed alter multiple behaviors relative to ethanol-nave animals. These data support the
hypothesis that the adolescent brain is sensitive to insult induced by ethanol exposure during
adolescence and these changes are manifested behaviorally as increased vulnerability to the
effects of ethanol in young adulthood. However, there are several areas in the brain that may
be uniquely susceptible to the long-term effects of early ethanol exposure, including the
mesolimbic dopaminergic system, which is one of the key systems that mediate the
behavioral changes we have observed in our work. We focus our neurochemical studies on
Page 9
the mesolimbic dopaminergic system given its involvement in reward, attention, novelty
preference and salience.
Neurochemistry
Alcohol consumption during adolescence is frequent and often involves heavy drinking
patterns such as binge drinking (Johnston, OMalley, Bachman and Schulenberg, 2006).
High rates of alcohol consumption have been reported for human (Bates and Labouvie,
1997) and rodent adolescents in comparison to adults (Doremus et al., 2005). Normal
development of neuronal pathways may be disturbed by alcohol consumption during
adolescence, consequently altering subsequent adult brain functioning. Therefore, it is
important to understand how the adolescent brain develops and also the impact of early
alcohol exposure on adult behavior and brain functioning.
Alcohol abuse research in rodents has focused on one particular pathway in the brain, the
mesolimbic dopamine (DA) pathway that originates in the ventral tegmental area (VTA) and
projects to the nucleus accumbens septi (NAcc; Dahlstrom and Fuxe, 1964). Due to the fact
that ethanol increases DA in the NAcc as measured by in vivo microdialysis (Yoshimoto et
al, 1992), examining ethanols impact on extracellular DA levels in adolescents would help
determine whether DA mediates adolescent vulnerability to high ethanol consumption rates
and alcohol dependency.
Our early work showed preadolescent rats (PND 25) had increased NAcc DA levels
following acute or repeated ethanol injections (1.0 g/kg/ip) relative to saline injected
controls with repeated ethanol inducing a leftward shift in peak DA levels (Philpot and
Kirstein, 1998). Therefore, ethanol-induced DA levels could be reliably obtained in the
young rat. The ability of ethanol to induce DA associated expectancy effects was also
measured. Preadolescent rats pretreated with ethanol but only given a saline injection during
DA sampling exhibited increased NAcc DA levels as well (Philpot and Kirstein, 1998).
These data suggest DA levels increased in anticipation or expectancy of ethanol
administration.
Recently, both conventional in vivo microdialysis and quantitative in vivo microdialysis

under transient conditions were performed to determine whether age-related differences in
basal DA exist in the NAcc. Preadolescent (PND 25), early adolescent (PND 35), late
adolescent (PND 45) and young adult (PND 60) rats were implanted with cannula and
microdialysis probes into the NAcc. Using the conventional approach, artificial
cerebrospinal fluid was perfused through the probe and DA was recovered (Philpot and
Kirstein, 2004). Using the quantitative approach (adopted from Olson & Justice, 1993)
differing concentrations of DA were perfused and the dialysate analyzed to determine levels
based on the concentration put in and the concentration recovered (Badanich, Adler and
Kirstein, 2006). The concentration that reached steady state (DA in = DA out) was
considered the basal level. Both approaches yielded similar age results, with late adolescent
animals (PND 45) having significantly higher basal DA levels than adults, but with the
conventional method (Philpot and Kirstein, 2004), dialysate DA levels obtained in
adolescents were higher than those obtained using the quantitative method (Figure 4;
Badanich et al., 2006). Additionally, the quantitative approach revealed lower basal DA
Page 10
levels for early adolescent (PND 35) rats. It should be noted the conventional method
reported dialysate DA levels that were not corrected for probe recovery. These results
indicate ontogenetic differences in basal DA. Furthermore, different findings for PND 35
rats between the two methods suggest that reuptake and/or metabolic differences for early
adolescent rats given that the quantitative method indirectly assesses group differences in
reuptake and transporter functioning. These data characterized basal DA levels in naive
adolescent rats and suggest basal inherent differences in DA during development may likely
impact neurochemical reactivity to early life alcohol exposure and produce long-term
consequences in neuronal functioning in adulthood.

Early alcohol use is highly associated with more severe alcohol dependency as an adult
(Grant and Dawson, 1997) and mesolimbic DA mediate the rewarding properties of alcohol
in adult rodents (Engleman et al., 2000; Ericson, Blomqvist, Engel and Soderpalm, 1998;
Gonzales, Job and Doyon, 2004; Katner and Weiss, 2001; Weiss, Lorang, Bloom and Koob,
1993; Weiss et al., 1996). Given these data, it was hypothesized that adult rats pretreated
with ethanol during adolescence would have different basal DA concentrations than salinetreated controls. Adolescent rats were injected daily from PND 3050 with either 0.75
g/kg/ip ethanol or saline followed by an ethanol-abstinent period from PND 5165. Changes
in extracellular DA levels in the NAcc were quantified on PND 70. Extracellular DA levels
were greater in rats chronically treated with ethanol during adolescence in comparison to
saline-exposed controls (Figure 5; Badanich, Maldonado and Kirstein, 2007). These data
support similar reports in the adult literature (Weiss et al., 1996); however, a recent
adolescent alcohol report (Sahr, Thielen, Lumeng, Li and McBride, 2004) suggested there
were no differences in DA for rats selectively bred to drink alcohol during adolescence.
Differences between the previous adolescent report and the present study may certainly be
attributable to differences in route of administration whereas the previous report
incorporated voluntary self-administration and the present study incorporated passive
experimenter administration of alcohol injections. Voluntary self-administration of alcohol
produces different neurochemical responses than involuntary or passive administration of
alcohol (Ericson et al., 1998; Weiss et al., 1993; Imperato and Di Chiara, 1986; Kiianmaa,
Nurmi, Nykanen and Sinclair, 1995; Yoshimoto, McBride, Lumen and Li, 1992). DA results
may have differed had rats in the present study been allowed to voluntarily drink alcohol
during adolescence. Further, differences between the previous report and the present study
may be attributable to genetic differences between rodent strains particularly because rats
used in the previous report were selectively bred for high ethanol consumption while rats in
the present study were out-bred rats.
These data provided insight into how the adolescent brain responds to ethanol insult during
adolescence and that these effects persist into adulthood. These data contribute to an
understanding of how the human adolescent brain may develop and also how alcohol use
during adolescence effects normal brain development. Clinical and experimental research
should continue to focus on the development of the adolescent brain so to better understand
how to treat adolescent substance use/abuse. Future research should investigate whether
elevated basal DA impacts ethanol-seeking behavior differently in adolescent and adult rats
as DA has been shown to mediate drug-seeking behavior in adult rodents.
Page 11
Summary and Future Directions

We have assessed sex-differences in voluntary ethanol consumption during adolescence and

adulthood and the influence of binge ethanol exposure during adolescence. We have
demonstrated that males are sensitive to passive social influences that mediate voluntary
ethanol consumption and that early ethanol exposure induces long-term changes in
responsivity to ethanol in adulthood. Neurochemically, we have demonstrated exposure to
moderate doses of ethanol during adolescence produced alterations in DA during
adolescence and later in adulthood. All of these data indicate the adolescent brain is
sensitive to the impact of early ethanol exposure during this critical developmental period. It
is important to note that these effects may be limited by the age of ethanol exposure and
dose administered. Therefore, it is important to assess if these effects observed in our work
are specific to exposure during adolescence or if similar effects would be observed if
exposure was initiated in adulthood.

Our work has primarily focused on characterizing alcohol consumption patterns,

socialization with intoxicated peers and alcohol-induced changes in NAcc DA levels of
adolescent and adult rats. Many questions regarding sensitivity to alcohol during
adolescence still need to be addressed including how long do these age-related changes in
behavioral and dopaminergic responsivity persist? Are changes in sensitivity to alcohol
throughout development dependent on dose or exposure pattern? Does early alcohol
exposure induce cognitive deficits that persist into adulthood such as changes in behavioral
inhibition, decision-making, memory and attending to salient stimuli. Future experiments
should utilize behavioral measures dependent on cortical activity such as the effects of
chronic alcohol exposure on working memory, set-shifting, impulsivity and reversal
learning. It would be interesting to explore whether alcohol use and/or dependency during
adolescence exacerbates deficits in performance on cognitive tasks such as slowing the
acquisition rate or by increasing the perserveration rate on a cortical-dependent task. While
recently we have begun to examine sex differences in the impact of early ethanol exposure
on behavior, future work should be extended to examine sex differences in NAcc DA
following early ethanol exposure. Given that research has been conducted on the interaction
of glutamate and dopamine following chronic exposure to ethanol exclusively in adult
rodents (Kapasova and Szumlinski, 2008; Szumlinski et al, 2007; Piepponen et al, 2002), it
would be interesting to evaluate these same neurochemical interactions in male and female
adolescent rats and to determine if any alterations persist into adulthood. It is plausible that
both DA and glutamate interact to induce long-lasting changes in the mesolimbic pathway
and cortical areas since ethanol has been shown to alter synaptic efficacy of both
glutamatergic and dopaminergic pathways (Carpenter-Hyland and Chandler, 2007; Yim and
Gonzales, 2000; Smith and Weiss et al, 1999; Philpot et al, 1998; Badanich et al, 2007).
Taking these approaches will allow us to determine underlying neurochemical mechanisms
mediating changes in sensitivity to alcohol consumption and other alcohol-related behaviors.
Acknowledgments
This work was funded by NIAAA AA11820, the University of South Florida Neuroscience Health Program, the
University of South Florida College of Arts and Sciences, the Department of Psychology and the University of
South Florida Honors College.
Page 12
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Page 16
Figure 1.
The diagram is a schematic of the experimental protocol used in the social interaction
experiment.

Page 17

Figure 2.
As depicted in the figure adolescent male alcohol nave observer rats that socially interacted
with demonstrators administered the highest dose of ethanol showed an increase in time
spent in the chamber paired with social interaction with an alcohol-intoxicated demonstrator
after conditioning relative to all other groups. Adolescent alcohol nave female observers
showed no change in time spent in the chamber paired with social interaction with an
alcohol-intoxicated demonstrator, regardless of dose. Data presented are mean +/ SEM
difference in seconds spent in the paired chamber at test minus baseline presented as the
change in time spent in the chamber paired with social interaction in the adolescent alcohol
naive observers initially least preferred chamber. * indicates significant difference from all
other sex- and dose-matched groups. The bars indicate the type of demonstrator the alcohol
nave adolescent observer socially interacted with (0.0 (clear bars)- social interaction with a
sober peer; 0.5 (light grey) and 1.5 (dark grey)- social interaction with an alcoholintoxicated peer). Female 0.0 n = 13; Female 0.5 n = 14; Female 1.5 n = 13; Male 0.0 n = 8;
Male 0.5 n = 11; Male 1.5 n = 10.

Page 18
Figure 3.
As depicted in the figure, animals that were pretreated with ethanol during adolescence and
conditioned with ethanol in young adulthood spent more time in the ethanol-paired chamber
relative to rats that were pretreated saline and conditioned with ethanol in young adulthood
and also those that were pretreated with ethanol during adolescence and conditioned with
saline in adulthood. Data presented are mean +/ SEM of the change in time spent in the
paired chamber in the initially least preferred chamber (test-baseline). The bars indicate
conditioning in young adulthood with saline (clear bars) or ethanol (grey bars). Saline-Saline
n = 9; Saline-Ethanol n = 10; Ethanol-Saline n = 9; Ethanol-Ethanol n = 9.

Page 19
Figure 4.
In both panels, the x-axis denotes Age and the y-axis denotes extracellular DA levels (nM).
Both approaches yielded similar age results [quantitative: F(2, 12)=14.01, P < 0.05;
conventional: F(3, 279)=17.171, P < 0.05] with late adolescent animals (PND 45) having
significantly higher basal DA levels than adults, but with the conventional method (Philpot
and Kirstein, 2004), levels obtained in adolescents were higher than those obtained using the
quantitative method (Badanich, Adler & Kirstein, 2006). Additionally, the quantitative
approach revealed lower basal DA levels for early adolescent (PND 35) rats. For panel A, #
= differs from PND 60; ## = differs from PND 45 and PND 60. For panel B, diamonds =
differs from PND 45 and PND 60; stars = differs from all other ages. It should be noted the
conventional method in panel B shows dialysate DA levels that were not corrected for probe
recovery. Figures reproduced with permission from Philpot and Kirstein, 2004 and Badanich
et al, 2006).

Page 20

Figure 5.
The amount of DA in the microdialysis perfusate (DAin) is on the x-axis while the
difference between DAin and recovered DA (DAout) is on the y-axis (DAin DAout). The
broken horizontal line depicts the point of no net flux of DA between the brain and the
microdialysis perfusate. The x-intercept denotes the extracellular DA concentration in the
NAcc for ethanol-treated (solid line; n=4) and saline control (broken line n=4) rats. Basal
DA was greater for rats pretreated with ethanol during adolescence (6.5 nM DA) than for
saline controls (3.6 nM DA); [t(6)= 2.458, P < 0.05]. The slope of the regression line
denotes the extraction fraction (Ed), an indirect measure of DA reuptake, for ethanol-treated
(87%) and saline control (68%) rats. Each data point denotes mean and SEM. This figure
has been reproduced with permission from Badanich et al., 2007.


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