Differential Rearing Conditions and Alcohol-Preferring Rats:

Consumption of and Operant Responding for Ethanol

Gerald A. Deehan, Jr., Matthew I. Palmatier, Mary E. Cain, and Stephen W. Kiefer
Kansas State University
Exposing rats to differential rearing conditions during early postweaning development has been shown
to produce changes in a number of behaviors displayed during adulthood. The purpose of the present
studies was to investigate whether rearing alcohol-preferring (P) and nonpreferring (NP) rats in an
environmental enrichment condition (EC), a social condition (SC), or an impoverished condition (IC)
would differentially affect self-administration of 10% ethanol. In Experiment 1, rats were tested for
consumption of 10% ethanol in limited- and free-access tests. For Experiment 2, rats were trained to
respond in an operant chamber for ethanol and then provided concurrent access to 10% ethanol and water.
Each solution was presented in a separate liquid dipper after meeting the schedule of reinforcement on
distinct levers. After concurrent access tests, the water lever/dipper was inactivated and a progressive ratio
(PR) schedule was initiated. Three successive solutions (10% ethanol, 15% ethanol, and 10% sucrose) were
tested under the PR. For P rats, rearing in an EC reduced ethanol consumption, preference, and motivation to
obtain ethanol, relative to P rats reared in an IC. Thus, exposure to a novel environment immediately after
weaning acted to decrease the reinforcing properties of ethanol in an animal model for alcoholism.
Keywords: differential rearing conditions, environmental enrichment, ethanol, alcohol-preferring rat
The determinants of alcohol use disorders are undoubtedly
complex, involving predispositional factors that arise from both
genetic and environmental sources. Hereditary mechanisms have been
linked to both ethanol consumption and alcohol use disorders in
human and animal models (Li, Lumeng, & Doolittle, 1993; Stacey,
Clarke, & Schumann, 2009). Similarly, environmental conditions
have been shown to influence ethanol intake as a variety of manipu-
lations effectively alter an organisms response to ethanol (Deather-
age, 1972; Ellison, 1981; Kulkosky, Zellner, Hyson, & Riley, 1980).
To date, few studies have investigated the interaction between hered-
itary factors and environmental influences on the proclivity to con-
sume and/or motivation to obtain ethanol in rodents.
Currently, there are several selectively bred rat lines that are
genetically predisposed to consume ethanol and are considered
useful as animal models of alcoholism. Selectively bred lines
consist of divergent groups of rodents that exhibit either a strong
preference for and high consumption levels of ethanol (preferring
line) or do not prefer and consume very little ethanol (nonprefer-
ring line). Of the selectively bred rat lines, the Indiana University
alcohol-preferring (P) and non-alcohol-preferring (NP) rat lines
have been studied extensively. Initially selected for high ethanol
consumption, the P rat line meets the major criteria for an animal
model of alcoholism (Li et al., 1993). For instance, when provided
24-hr free access to ethanol, P rats will consume over 5 grams of
ethanol per kilogram of body weight per day (Li, Lumeng,
McBride, & Murphy, 1987; Li, Lumeng, McBride, Waller, &
Murphy, 1986). Additionally, when given either limited-access or
24-hr free access to ethanol, P rats will consume enough ethanol to
establish blood alcohol levels (BACs) in the 5070 mg% range,
with some rats reported to establish a BAC up to 200 mg% (Bell,
Rodd, Lumeng, Murphy, & McBride, 2006; Rodd-Henricks, et al.,
2002). It has also been shown that P rats readily learn to respond
in an operant chamber for ethanol (Files, Samson, Denning, &
Marvin, 1998; Murphy, Gatto, McBride, Lumeng, & Li, 1989;
Oster et al., 2006; Penn, McBride, Lumeng, Gaff, & Li, 1978).
Although selectively bred rat lines represent a feasible way to
examine the contribution of genetic aspects to alcoholism, differ-
ential rearing environments provide a paradigm to examine the
effects of early environmental experience on alcohol drinking.
Much like humans, rodents undergo a period of development
(during adolescence) that is characterized by several neurological
and behavioral changes (Spear, 2000). During adolescence
(broadly estimated from immediately after weaning until Postnatal
Day 55), neurological development can be affected by a myriad of
external factors including environmental experience, which can in
turn affect several behaviors manifested later in life (Fone &
Porkess, 2008; Renner & Rosenzweig, 1987; Spear, 2000). For
example, rearing rats in an enriched environment (EC; typically
group housed with several novel objects and daily handling by
experimenters) compared with an impoverished environment (IC;
isolate housing in hanging metal cages without novel objects or
handling), has been shown to induce complex neurological and
behavioral changes in the rat in relation to drugs of abuse. Rats
exposed to an EC are more sensitive to the rewarding and stimu-
This article was published Online First January 31, 2011.
Gerald A. Deehan, Jr., Matthew I. Palmatier, Mary E. Cain, and Stephen
W. Kiefer, Department of Psychology, Kansas State University.
This work was partially supported by the Department of Psychology,
Kansas State University. We thank Tannis M. Sears for her assistance
during data collection.
Correspondence concerning this article should be addressed to Gerald A.
Deehan, Jr., Indiana School of Medicine, Institute for Psychiatric Research
(M-119), 791 Union Drive, Indianapolis, IN 46202. E-mail:
gdeehan@iupui.edu
Behavioral Neuroscience 2011 American Psychological Association
2011, Vol. 125, No. 2, 184193 0735-7044/11/$12.00 DOI: 10.1037/a0022627
184
lating effects of amphetamine than IC rats (Bardo et al., 1995;
Bowling & Bardo, 1994) by exhibiting decreased levels of operant
responding for amphetamine at low unit doses (Bardo, Klebaur,
Valone, & Deaton, 2001; Green, Gehrke, & Bardo, 2002). Simi-
larly, when differentially reared rats are provided access to cocaine
by means of operant responding and intravenous infusions, EC rats
respond significantly less for low unit doses of cocaine than their
IC counterparts (Ding, Kang, Li, & Ma, 2005; Yajie, Lin, Baom-
ing, & Lan, 2005).
The effect of differential rearing environments on ethanol self-
administration has been mixed. For example, Rockman and col-
leagues (Rockman, Borowski, & Glavin, 1986; Rockman, Gibson,
& Benarroch, 1989; Rockman, Hall, & Markert, 1988) demon-
strated that rats raised in an EC consumed significantly more
ethanol than rats raised in an IC. However, other laboratories have
reported that rats reared in an IC consumed more ethanol than rats
reared in an EC (Hall, Huang, Fong, Pert, & Linnoila, 1998;
Schenk, Gorman, & Amit, 1990). Recent research in our labora-
tory has agreed with the latter set of findingsrats reared in an IC
self-administer more ethanol than rats reared in an EC (Deehan,
Cain, & Kiefer, 2007; Deehan, Manion, Thomas, Cain, & Kiefer,
2008). Furthermore, rats reared in an IC were the only subjects to
show a clear preference for ethanol during a two-lever preference
test in which water and ethanol were available concurrently (Dee-
han et al., 2007). In a subsequent study measuring ethanol con-
sumption, IC rats consumed significantly more 10% ethanol (vol/
vol) during a limited-access consumption test than EC-reared
counterparts (Deehan et al., 2008).
In humans, the interaction between genetic predisposition and
environmental experience has been cited as of key importance in
the development of alcohol use disorders (Stacey, Clarke, &
Shumann, 2009). Therefore, the use of the selectively bred P line
(an established animal model of alcoholism with a genetic predis-
position to drink alcohol) in combination with the differential
rearing paradigm offers a way to examine the genetic/environment
interaction, with regard to alcohol use, in rodents. The purpose of
the present series of studies was to investigate the effect of differ-
ential rearing on consumption, preference, and motivation to ob-
tain ethanol in rats selectively bred for high (P) or low (NP)
ethanol consumption. After a 60-day rearing period, rats were first
tested for limited-access consumption of 10% ethanol. Immedi-
ately after limited-access testing, rats were evaluated for 24-hr
consumption and preference for ethanol (Experiment 1). In Exper-
iment 2, rearing conditions were the same, but rats were tested for
operant responding for ethanol at the conclusion of the rearing
period. We predicted that P rats would self-administer more eth-
anol and show greater motivation to obtain ethanol relative to NP
rats. However, we also predicted that rearing would moderate the
line difference; we anticipated that rearing rats in the EC condition
would decrease ethanol self-administration in both selectively bred
lines.
General Method
Subjects
Naive, male P and NP rats were obtained from Indiana Univer-
sity at 2130 days of age for both experiments. All rats were
provided ad libitum access to food and water throughout testing
except during limited access procedures (Experiment 1) and oper-
ant testing (Experiment 2). The animal colony room was on a 12-hr
lightdark cycle (lights on at 0700). All protocols and procedures
were approved by the Kansas State University Institutional Animal
Care and Use Committee.
Environmental Conditions
On arrival to the laboratory, each rat was randomly assigned to
one of three rearing conditions: EC, IC, or social condition (SC).
Once assigned, rats were exposed to their rearing condition for 60
days before testing began. The 60-day exposure period ensured
that rats experienced their respective conditions during the critical
period of neurological development. Rats reared in the EC were
housed in a large metal cage (60 120 45 cm) with cohorts (12
rats per cage). The EC contained 14 plastic objects (childrens
toys, large plastic bowls, etc.). Each day, all rats were handled and
removed from the EC cage so that 7 of the 14 objects could be
replaced with new objects and arranged in a novel configuration
with the remaining objects. SC rats were housed 2 per cage in a
standard shoebox cage with a wire rack top. SC rats were handled
once a week during the scheduled bedding change. Housing in the
SC condition was comparable with the suggested housing condi-
tions set forth in the Guide for the Care and Use of Laboratory
Animals (National Institutes of Health, 1996). Rats in the IC group
were housed 1 per cage in hanging metal cages (17 24 20 cm)
with a wire mesh floor and front panel and with solid metal sides
and back. The hanging metal cages were chosen for the present
study as food, water, and bedding changes were administered with
minimal contact with the rats for the entire 60-day rearing period.
Apparatus
Operant responding for ethanol (Experiment 2) was conducted
in standard two-lever operant chambers (Colbourn Instruments,
Allentown, PA) that were contained within sound-attenuated, ven-
tilated (by fan) environmental boxes. Each chamber contained a
house light (2.8 W) that remained illuminated for the duration of
the session. The inside of the chamber had a dimension of 28
21 21 cm, with Plexiglas walls on the front and back and with
aluminum ceiling and side walls. Two levers, 12-cm apart, were
located on the same aluminum side wall directly above their
corresponding dipper receptacle where a cup containing 0.1 ml of
fluid could be presented through an aperture approximately 0.5 cm
in diameter. Each dipper receptacle was approximately 2 cm from
the floor, allowing relatively unrestricted access to the aperture/
cup. On dipper presentation, a light (1 W) was illuminated within
the dipper trough for the duration of the dipper presentation (4 s).
A desktop computer controlled all operant chamber functions with
MedPC software and recorded all lever presses and dipper presen-
tations. For all sessions during ethanol and sucrose testing, the
response requirement was set at fixed ratio (FR) 1. The schedule
was incremented to FR 5 to habituate the rats to a partial rein-
forcement schedule before testing under the progressive ratio (PR)
schedule began. All operant testing sessions were 60 min long.
Solutions
Throughout all experiments, solutions were mixed fresh daily.
Ethanol concentrations were calculated as vol/vol (ethanol/
185
DIFFERENTIAL REARING CONDITIONS AND ETHANOL
deionized water), and sucrose concentrations were calculated as
wt/vol (grams of sucrose/deionized water).
Experiment 1
In the first experiment, both P and NP rats were raised in
differing environments for 60 days and then tested for ethanol
consumption, both in a mildly fluid-deprived state and under
conditions of continuous access. As noted earlier, outbred rats
raised in differential rearing environments differed in restricted
access consumption of ethanol (Deehan et al., 2008). Overall, there
has been a limited amount of research completed as to the effects
of differential rearing environments on ethanol consumption in
outbred rat lines, and even less research has focused on the effects
of differential rearing conditions on ethanol consumption in the
selectively bred P and NP rat lines. To fully examine the effects of
differential rearing environments on alcohol intake in both the P
and NP lines, the sucrose-fading procedure outlined by Samson
(1986) was implemented to provide indices of sweetened fluid
intake, ethanol intake, and more generally, fluid intake during a
limited-access testing procedure. Using the fading procedure in
conjunction with a limited-access paradigm allowed for a greater
sensitivity of increases and/or decreases in both sucrose and eth-
anol consumption in rat lines with a well-documented divergence
in the consumption of both sucrose and ethanol solutions. Further-
more, a 24-hr access test was also completed to assess the con-
sumption of ethanol under a non-fluid-deprived state.
Method
Subjects. A total of 19 male P and 19 male NP rats from the
65th generation (n 67 per rearing condition) were used for
Experiment 1.
Procedure. After the 60-day rearing period, consumption
testing took place in standard shoebox cages using a modified
sucrose-fading procedure similar to that outlined by Samson
(1986). Sucrose fading and alcohol testing consisted of: 10%
sucrose for 5 testing sessions, 10% sucrose/2% ethanol for 3
sessions, 5% sucrose/5% ethanol for 3 sessions, 2% sucrose/10%
ethanol for 3 sessions, and finally 10% ethanol for 10 sessions. At
the conclusion of 10% ethanol testing, all rats were faded in
reverse order back to 10% sucrose to observe any changes in
baseline sucrose consumption. The present research made use of a
limited-access paradigm whereby rats received a 15-min limited-
access test session each morning. Rats were provided 1 hr of water
access in the afternoon to ensure proper hydration.
At 0900 each day, all rats were weighed and placed, 1 per cage,
in standard shoebox cages. At 0930, after each rat had been
weighed and placed in its cage, 50-ml centrifuge tubes fitted with
rubber stoppers and stainless steel drinking spouts were filled with
the appropriate solution and attached to the cages. After 15 min of
drinking time, the tubes were removed and the amount of solution
consumed by each rat was recorded. After the final centrifuge tube
was removed, rats were placed back in their home environment. At
1430 each day during limited-access testing, water bottles were
placed on the cages for a 1-hr drinking period. Bottles were made
available in the same order every day, and the EC cage had 4
bottles fixed across the front of the cage so that all 12 rats were
provided adequate opportunity to drink during this period. Follow-
ing the 1-hr water access, bottles were removed and all rats were
deprived until the start of limited-access testing the following day.
This procedure was continued for the duration of limited-access
sucrose fading and 10% ethanol testing as described earlier.
When limited-access testing was completed, rats were removed
from their home environment and placed, 1 per cage, into standard
shoebox cages for the remainder of the experiment (10 days). In
this phase, testing occurred using a free-access paradigm with both
a bottle of water and a bottle of 10% ethanol available 24 hr/day.
Bottles were placed on the cages at 0900 the day after the termi-
nation of limited-access testing. Every 24 hr, after the initial bottle
placement, bottles were removed from the cages and the rats and
bottles were weighed and the data recorded. After being weighed,
each rat was placed back into its cage and the bottles were replaced
with bottle positions alternating each day to control for place
preference. Free-access testing continued for a total of 10 days.
Data analysis. Mixed-design analyses of variance
(ANOVAs) were initially used to examine the consumption data,
with line and rearing as between-subjects variables and day as the
repeated-measure variable. Because the main effects and interac-
tions involving the day factor were nonsignificant for the omnibus
ANOVA, the data were collapsed across days to simplify analyses
and presentation. Ethanol preference during the two-bottle, free-
access test was calculated (amount of ethanol consumed divided
by total fluid consumed) for each rat. Because of differences in
body weight between EC and IC rats, consumption data were
expressed as grams of sucrose or ethanol consumed per kilogram
of body weight (g/kg). Estimates of effect size were computed with
partial eta squared (
p
2
). Tukeys post hoc tests were used to
examine significant treatment effects and to compare ethanol con-
sumption between the six groups. All analyses (for Experiment 1
and Experiment 2) were conducted with SPSS 12.0 for Windows.
Results
Ethanol fade in. Overall, P rats consumed more ethanol and
sucrose than NP rats. However, differential rearing decreased
self-administration of ethanol, as EC rats only drank less when the
solutions contained 510% ethanol. This was confirmed by
a significant main effect of line for all solutions, including when
sucrose was presented alone, Fs(1, 32) 9.17, ps .05; range of

p
2
0.240.41. As illustrated in Figure 1, P rats consistently
consumed more fluid than NP rats. However, the main effect of
rearing was significant when the ethanol concentration was 5% or
10%, Fs(2, 32) 4.18, ps .05;
p
2
0.20 and 0.24, respectively;
and was driven by differences between the IC and EC groups in the
NP line. Rearing did not significantly affect consumption when the
ethanol concentration was 2% or when the solution was only 10%
sucrose ( ps .05). Despite the significant rearing effect, group
comparisons, in the P line, for the 5% sucrose/5% ethanol solution
were statistically nonsignificant; similar comparisons for the 2%
sucrose/10% ethanol approached significance; For IC SC, p
.052; for IC EC, p .074.
10% Ethanol, limited access. Selectively bred P rats reared
in an EC exhibited decreased levels of ethanol consumption when
rats had limited access to 10% ethanol (no sucrose; see Figure 2).
This was confirmed by main effects of line, F(1, 32) 48.1, p
.05,
p
2
.60; and rearing, F(2, 32) 10.40, p .05,
p
2
.39.
The interaction term was not statistically significant. Not surpris-
186
DEEHAN, PALMATIER, CAIN, AND KIEFER
ingly, the significant effect of line was due to P rats consistently
consuming more ethanol than their NP counterparts. For the rear-
ing factor, P rats raised in the EC consumed significantly less
ethanol than did the IC rats ( p .05). The SC P group did not
differ significantly from either the IC P or EC P groups. There was
a similar trend of limited-access ethanol consumption across the
NP groups; however, the preplanned comparisons did not reach
statistical significance.
Ethanol fade out. Findings from the fade-out phase of the
experiment (see Figure 3) were comparable with those from the
fade-in phase. Basically, P rats consumed more of each solution,
and rearing was a significant factor only with 2% sucrose/10%
ethanol, as both P and NP rats reared in an EC consumed signif-
icantly less solution than rats reared in an IC. The interaction term
between line and rearing was not statistically significant in the
analyses for any of the solutions. The general pattern of the
limited-access data indicates that, when the concentration of eth-
anol was 5% or above, rats raised in the EC consumed less ethanol
than rats raised in the IC (this effect was strongest in the 10%
ethanollimited-access phase and the fade-out phase).
10% Ethanol, continuous access. Similar to the limited-
access tests, P rats consumed more ethanol and preferred ethanol
to a significantly greater degree than NP rats. The data from the
final test with free access to ethanol and water were calculated as
both grams of ethanol consumed/body weight in kilograms (Figure
4, top panel) and as a preference score (amount of ethanol con-
sumed divided by total ethanol and water consumed; see Figure 4,
bottom panel). Both characterizations of the data had similar
patterns and statistical analyses and the two data sets produced
similar outcomes. In each case, the line effect was statistically
significant, Fs 33.25, ps 0.01,
p
2
0.51, for each measure.
The effect of rearing and the Line Rearing interaction were not
statistically significant. However, post hoc tests revealed that IC P
rats consumed significantly more ethanol ( p .05) and preferred
ethanol over water to a significantly greater extent than the EC P
group ( p .05).
Experiment 2
The second experiment assessed the effect of differential rearing
environments on the motivation of P and NP rats to lever press for
ethanol. Rats were shaped to respond for ethanol and then succes-
sively tested with a continuous reinforcement schedule for con-
current access to ethanol and water, followed by a series of
progressive ratio schedules. As noted, previous research has shown
that outbred rats reared in an IC responded significantly more for
ethanol than rats reared in an EC or SC (Deehan et al., 2007).
Method
Subjects. In Experiment 2, subjects were 20 male P rats from
the 66th generation (n 67 per rearing condition) and 22 male
NP rats from the 65th generation (n 78 per rearing condition).
Procedure. All phases for Experiment 2 are outlined in Table
1. After the 60-day rearing period, all rats were deprived of water
Figure 2. Mean (plus or minus standard error of the mean) ethanol
(EtOH) consumed by the six groups of rats during testing with limited
access to a 10% ethanol solution. Rats were tested for 15 days, and their
data were collapsed into a single mean amount for analysis. An asterisk
signifies that rats from the enriched rearing condition (EC) consumed
significantly less 10% ethanol than those from the impoverished rearing
condition (IC), p .05. P alcohol-preferring rats; NP non-alcohol-
preferring rats; BW body weight; SC social rearing condition.
Figure 1. Mean (plus or minus standard error of the mean) amount of
ethanol (EtOH; left bars) and sucrose (right bars) consumed by rats from
the enriched rearing condition (EC; n 12), the social rearing condition
(SC; n 12) and the impoverished rearing condition (IC; n 14) in
Experiment 1. The various solutions used during the fade-in phase are
listed in order of presentation from top to bottom. Data were collapsed
across days of testing, and only an overall mean consumption measure was
used. Asterisks signify that the EC group consumed significantly less than
IC group ( p .05). P alcohol-preferring rats; NP non-alcohol-
preferring rats; BW body weight.
187
DIFFERENTIAL REARING CONDITIONS AND ETHANOL
for 16 hr before the first operant session. This marked the start of
magazine training, during which rats were placed in the operant
chambers without levers present and received noncontingent pre-
sentations of 6% ethanol randomly from both dippers. After 5 days
of magazine training, the levers were returned and all rats under-
went acquisition of operant responding for 6% ethanol on both
levers. A rat was considered to have successfully acquired operant
responding for 6% ethanol when it made at least 50 lever responses
in one session. After acquisition, all rats remained fluid deprived
while the concentration of ethanol was increased from 6% to 10%.
Briefly, rats received 3 days of 6% ethanol, 3 days of 8% ethanol,
and finally 10 days of 10% ethanol. After the final day of the
maintenance phase with 10% ethanol, all rats were provided water
in their home cages ad libitum for the remainder of the experiment.
After lever pressing acquisition, rats were provided concurrent
access to 10% ethanol and water for 39 days. Initially, ethanol was
earned with responses on the right lever, and water was earned
with responses on the left lever. After 25 days, the positions of the
ethanol lever and the water lever were switched. This was done to
ascertain whether the rats would track the ethanol solution and to
ensure that responding was not due to a lever bias.
After concurrent responding for ethanol and water, the water
lever was rendered inactive and only the ethanol lever remained
operable. Rats were given 3 days of training with the ethanol lever
alone. After these 3 days, the FR schedule on the ethanol lever was
increased from an FR 1 to an FR 2, and this was in effect for 3
days. The FR schedule was then increased to an FR 5 for 7 days.
Figure 3. Results from the fade-out phases during Experiment 1 showing
the mean (plus or minus standard error of the mean) amount of ethanol
(EtOH) and sucrose consumed. The format is similar to that of Figure 1,
but note that the ethanol/sucrose solutions were presented in reverse order
as ethanol was faded out (starting with 2% sucrose/10% ethanol). An
asterisk denotes that rats from the enriched rearing condition (EC) con-
sumed significantly lower amounts of 2% sucrose/10% ethanol than rats
from the impoverished rearing condition (IC), regardless of line ( p .05).
P alcohol-preferring rats; NP non-alcohol-preferring rats; BWbody
weight; SC social rearing condition.
Figure 4. Mean (plus or minus standard error of the mean) amount of
ethanol (EtOH) consumed (top panel) and mean ethanol preference (bot-
tom panel) as measured during the two-bottle free-access test in Experi-
ment 1. Preference was calculated as the amount of ethanol consumed
divided by total fluid (ethanol plus water) consumed. An asterisk indicates
that rats from the enriched rearing condition (EC) consumed and/or pre-
ferred ethanol significantly less than rats from the impoverished rearing
condition (IC). P alcohol-preferring rats; NP non-alcohol-preferring
rats; BW body weight; SC social rearing condition.
Table 1
Outline of Experiment 2
Training/testing phase Operant sessions
Magazine training/shaping (6% EtOH) Training (5 days)
Lever acquisition (6% EtOH) 14
Maintenance responding (Dep; 6%
10% EtOH) 520
Concurrent access (10% EtOH vs. water) 2159
FR schedule increase (FR 1FR 5) FR 1: 6062; FR 2: 6365;
FR 5: 6672
Shallow PR (10% EtOH) 7377
Exponential PR (10% EtOH) 7882
Exponential PR (15% EtOH) 8387
Exponential PR (10% sucrose) 8897
Note. EtOH ethanol, Fluid Deprivation Dep, Fixed Ratio FR.
188
DEEHAN, PALMATIER, CAIN, AND KIEFER
After the final day of FR 5 testing, rats were exposed to a PR
schedule of testing. The rats were first subjected to a shallow PR
for 10% ethanol by which the response requirement increased by
2 after every third ethanol delivery. That is, the rats started on an
FR 2 schedule and after three ethanol deliveries (six operant
responses) the schedule was increased to an FR 4. After 3 more
ethanol deliveries, the FR schedule was increased to an FR 6, and
so on. The shallow PR procedure was conducted for a total of 5
days. For the next 5 days, all rats were required to respond on a
steeper, exponential PR for 10% ethanol. For the exponential PR,
rats started at an FR 2 and, after each ethanol reinforcer, the
schedule increased according the formula published by Richardson
and Roberts (1996): [5exp
(R .012)
] 5. When testing on the
exponential PR for 10% ethanol was ended, the ethanol concen-
tration increased to 15% for 5 days. At the conclusion of PR testing
with 15% ethanol, there were an additional 5 days of PR testing
with 10% sucrose. For all PR testing, the session length was 60
min and the last response requirement that the rat successfully
completed was considered its breaking point.
Data analysis. A series of ANOVAs were used to analyze all
phases of the second experiment. Line (P and NP) and rearing (IC,
SC, & EC) were the between-subjects factors and, for the operant
responding with 10% ethanol, session was the repeated measure.
Separate analyses were conducted for the ethanol and water levers
when the rats were presented with both. The PR data for all four
phases were collapsed across sessions because the main effects and
interactions including session were not significant. Once again, we
computed effect sizes using
p
2
. Tukeys post hoc tests were used
to analyze significant main effects and interactions, with p .05
as the criterion for statistical significance.
Results
Responding for ethanol: Mild fluid deprivation. All groups
of rats displayed similar patterns of responding across the acqui-
sition and maintenance phases of the experiment. For each analy-
sis, the factors of line, rearing, session, and their interactions were
not statistically significant. On the final session of fluid-deprived
responding, the mean (plus or minus standard error of the mean)
number of total responses for 10% ethanol on both levers for each
group were as follows: IC P 117.29 7.96; IC NP 115.88
6.15; SC P 129.17 25.81; SC NP 102.67 7.62; EC P
114.71 10.72; EC NP 98.38 7.01.
Responding for ethanol and water: Nondeprived state.
Figure 5 shows the mean number of responses for ethanol and
water as a function of days for each of the six groups of rats. The
break in the data reflects the reversal of the ethanol and water
levers/dippers, which had little to no effect on responding. As
illustrated in Figure 5, P rats in the IC and SC were the only
subjects who responded more for ethanol than for water both
before and after the reversal manipulation. This conclusion was
confirmed by a mixed ANOVA, with significant main effects of
line, F(1, 36) 47.50, p .05,
p
2
.57; and rearing, F(2, 36)
14.40, p .05,
p
2
.44; as well as a significant Line Rearing
interaction, F(2, 36) 10.10, p .05,
p
2
.36. There were also
significant effects of day, F(38, 1368) 18.03, p .05,
p
2
.33;
Day Line, F(38, 1368) 10.54, p .05,
p
2
.23; Day
Rearing, F(76, 1368) 3.18, p .05,
p
2
.15; and Day
Line Rearing, F(76, 1368) 2.81, p .05,
p
2
.14. A similar
analysis of the water responding revealed a significant effect of
day, F(38, 1368) 2.35, p .05,
p
2
.06; and a significant
Rearing Day interaction, F(76, 1368) 1.76, p .05,
p
2
.09.
Initially, when the rats began responding for concurrent ethanol
and water, all groups displayed low levels of responding on both
levers (see Figure 5). Over the course of the 39 days of testing,
only the IC P and SC P rats showed significant changes in
responding. The IC P rats gradually increased responding on the
ethanol lever until there was a clear and significant preference for
this lever. Responding on the water lever remained low, although
there was a slight increase when the positions of the ethanol and
water levers were switched. The SC P rats displayed a similar
pattern of responding over days, although their overall ethanol
responding was significantly lower than the IC P rats ( p .05).
Rats in the EC P group showed little change for either ethanol or
water over the course of testing, which is similar to the patterns
seen in all three NP groups. Overall responding for ethanol was
significantly lower in the EC P rats relative to the IC P and SC P
rats ( p .05) but was not significantly different from any of the
NP groups. Finally, differential rearing conditions did not signif-
icantly affect ethanol lever responding in NP rats.
Responding for ethanol: PR tests. The data and the statis-
tical analyses from each of the four PR tests were quite similar (see
Figure 6). Analysis of each phase resulted in significant main
effects of line, Fs(1, 36) 19.65, ps .001, range of
p
2

0.350.63; and rearing, Fs(2, 36) 8.01, ps .01, range of

p
2

0.300.39; in addition, the interaction between line and rearing

was consistently significant, Fs(2, 36) 6.92, ps .05, range of

p
2
0.180.35. Post hoc comparisons with the Tukeys test also
were quite similar for each of the four phases. The IC P rats had
significantly higher break points than all the other groups ( ps
.05), with the exception of the SC P group. The SC P group had
significantly higher break points ( ps .05) than the EC P group
for the first two phases (shallow PR and exponential PR for 10%
ethanol). Finally, the EC P group was not significantly different
from any of the three NP groups for any of the phases (and the NP
groups never differed significantly from one another).
General Discussion
The general hypothesis that rearing environment would affect
the response to ethanol in selectively bred rats was supported by
the present experiments. The specific patterns of outcomes varied
as a function of rearing environment, rat line, ethanol concentra-
tion, and method of testing. During limited-access tests, P rats
consistently consumed greater amounts of ethanol, sucrose, and
the combination solutions than NP rats. During limited-access
testing, EC rats consumed significantly less ethanol than the IC
rats, with SC rats generally between the other two groups. This
finding may be specific to ethanol intake, as all rearing conditions
consumed similar quantities of solutions with high sucrose con-
centrations (10%) and low ethanol concentrations (02%). Fur-
thermore, differential rearing conditions significantly affected
24-hr two-bottle choice ethanol consumption and preference as P
rats reared in an EC consumed significantly less ethanol and
preferred ethanol significantly less to water compared with P rats
reared in an IC.
In tests that directly measured the reinforcing properties of
ethanol, we observed an interaction between rearing environment
189
DIFFERENTIAL REARING CONDITIONS AND ETHANOL
and selective breeding. At the initiation of operant tests, all groups
responded at very low levels for 10% ethanol. As training pro-
gressed, EC P rats maintained low levels of operant responding for
ethanol relative to the other groups and did not differ significantly
from the three NP groups. However, P rats that were reared in an
IC increased responding significantly and consistently relative to
the other groups. The SC P rats also exhibited an increase in
responding over sessions but never achieved similar levels as the
IC P rats. These observations would suggest that the reinforcing
properties of ethanol were directly affected by the complexity of
the rearing environment in which the rats were raised. This notion
is further supported by the observation that switching the ethanol
and water levers had little or no effect, as both IC P and SC P
groups continued the same level of responding for ethanol. Al-
though previous research has shown that rats reared in an EC
significantly outperform both SC and IC rats on complex learning
tasks (Pena, Prunell, Rotlant, Armario, & Escorihuela, 2009;
Schrijver, Bahr, Weiss, & Wurbel, 2002), the lack of change in
responding after the lever switch precludes such commonly ob-
served learning differences between the EC, SC, and IC rats as a
potential underlying cause for differences in responding for etha-
nol. The general pattern of responding between the various groups
Figure 5. Average lever responding during Experiment 2 when rats from the enriched rearing condition (EC;
n 15), the social rearing condition (SC; n 12), and the impoverished rearing condition (IC; n 15) had
access to two levers, 10% ethanol (EtOH) and water, for 39 days. The break in the data reflects the switching
of the ethanol and water levers. Shown is the mean (plus or minus standard error of the mean) number of
responses on each lever as a function of test days. P alcohol-preferring rats; NP non-alcohol-preferring rats;
BW body weight.
190
DEEHAN, PALMATIER, CAIN, AND KIEFER
was also found during the PR testing (e.g., IC P rats consistently
had higher break points for both ethanol and sucrose).
Given past research, it was anticipated that P rats would con-
sistently consume more ethanol than NP rats. The present findings
show that P rats also consumed more sucrose solution than NP rats
during the fade-in and fade-out procedures and during the PR tests.
The coexistence of high ethanol and sucrose consumption by P
rats, compared with NP rats, has been reported previously (Stew-
art, Russell, Lumeng, Li, & Murphy, 1994). With regard to the
effect of differential rearing conditions on alcohol self-
administration, the results for the present research (utilizing the P
rat line) are in line with the results of previous research in our
laboratory using the LongEvans outbred rat line (Deehan et al.,
2007; Deehan et al., 2008). The present research utilized the same
rearing and testing paradigm as was used previously in our labo-
ratory. However, future research will be focused on exploring the
effect of differential rearing conditions on specific aspects of
ethanol consumption in the P and NP lines. For example, separate
analyses of sucrose and ethanol consumption in distinct popula-
tions of both P and NP rats will more fully characterize the effects
of differential environmental experience on ethanol consumption,
both during limited-access and 24-hr access.
Some authors have argued that IC rearing or isolate housing
causes increases in ethanol consumption due to higher levels of
isolation stress or anxiety (Parker & Radow, 1974; Roske, Baeger,
Reinhard, & Oehme, 1994). For example, a recent study explored
the effects of differential housing conditions in P rats and reported
that isolate, compared with social housing caused increases in
ethanol consumption in the P line during both limited- and free-
access tests (Ehlers, Walker, Pian, Roth, & Slawecki, 2007). For
the present studies, however, the pattern of ethanol responding and
consumption among the different rearing groups in the P line offer
support for the hypothesis that EC rearing decreases ethanol con-
sumption and ethanol responding relative to IC rearing. For ex-
ample, during testing, P rats reared in an IC condition did not
exceed ethanol consumption and responding levels that have been
thoroughly documented in the literature (Bell et al., 2006; Li et al.,
1986; Li et al., 1987). Even more compelling evidence for this
argument is the observation that P rats reared in the EC responded
significantly less for ethanol than P rats reared in either the IC, as
well as those reared in the SC. Although the P rats reared in an EC
did not consume significantly less ethanol than those reared in an
SC, the EC did provide an added benefit as P rats in the SC and IC
did not significantly differ in ethanol consumption either. Further-
more, it is believed that the trend observed for both limited- and
free-access consumption testing, although not significant, may
have been more evident with a larger sample size.
Another possibility is that differential rearing conditions may
affect appetitive and consummatory behaviors, with regard to
ethanol responding and ethanol consumption, to a different extent.
Appetitive and consummatory behaviors have been shown to be
separable and distinct (for a review, see Cunningham, Fidler, &
Hill, 2000), and it is possible that differential rearing environments
have a greater effect on appetitive rather than consummatory
behavior in the P rat line. However, this is unlikely as a recent
study found that differences in early environmental experiences
affect both appetitive and consummatory behaviors in a similar
manner in outbred rats (McCool & Chappell, 2009). The present
data indicate that rearing P rats in an EC immediately after wean-
ing reduces reinforcing properties of ethanol to levels comparable
Figure 6. Average break points (plus or minus standard error of the mean) for each of the six groups during
the four progressive ratio tests (in order from top left to bottom right). Asterisks indicate that P rats from an
enriched rearing condition (EC) exhibited significantly lower break points than those from either the impover-
ished rearing condition (IC) or the social rearing condition (SC), p .05. Carets denote that EC-reared P rats
displayed a significantly lower break point than IC-reared P rats ( p .05). PR progressive ratio; EtOH
ethanol; P alcohol-preferring rats; NP non-alcohol-preferring rats.
191
DIFFERENTIAL REARING CONDITIONS AND ETHANOL
with those found in rats selectively bred not to consume, prefer, or
respond for alcohol. Taken as a whole, the data support the
hypothesis that rearing P rats in a complex and novel environment
acts to decrease genetically predisposed behaviors associated with
ethanol.
The present findings are consistent with those of other studies
showing that EC rearing protects rats from an increased intake of
various drugs of abuse (Bardo & Dwoskin, 2004; Bardo et al.,
2001; Bozarth, Murray, & Wise, 1989; Ding et al., 2005; Green et
al., 2002). Speculation as to how EC rearing offers protection from
increased drug intake has focused on neurotransmitter systems that
are affected by rearing and that also underlie drug self-
administration. Many of the neurotransmitter systems shown to be
affected by differential rearing conditions have also been impli-
cated in the high ethanol intake levels observed in the P rat line. A
prime candidate is the mesolimibic dopamine (DA) system, which
has been shown to underlie the rewarding properties of ethanol and
virtually every other drug of abuse (Spanagel & Weiss, 1999; Wise
& Rompre, 1989). Past research established that the P line pos-
sessed decreased levels of DA in the reward pathway, compared
with NP rats, which is believed to contribute to their high ethanol
intake (McBride & Li, 1998). Rearing rats in an EC may counter-
act the lower DA levels in P rats, as it has been shown to decrease
levels of DA transporter within the medial prefrontal cortex
(mPFC; Zhu, Apparsundaram, Bardo, & Dwoskin, 2005; Zhu,
Green, Bardo, & Dwoskin, 2004), increasing DA in the mPFC and
possibly affecting DA levels at other downstream structures in the
reward pathway. Other possible candidates for neurochemical sys-
tems that may be affected by EC rearingwhich may, in turn,
affect ethanol self-administration in the P ratinclude the gamma
aminobutyric acid, serotonergic, and opioid systems.
Research with humans has identified several hereditary factors
in the development of alcohol abuse or alcoholism (Gelernter &
Kranzler, 2009). With the development of several alcohol-
preferring and nonpreferring rat lines, research on the effects of
genetic predisposition and the underlying physiological and be-
havioral traits that accompany this predisposition has served to
identify many facets of alcohol addiction and alcoholism (for a
review, see McBride & Li, 1998). More recently, clinical research
has demonstrated a substantial role of environmental experience in
the etiology of alcoholism (Stacey et al., 2009). In fact, the
contribution of genetic, compared with environmental, factors
toward the development of alcoholism has been estimated close to
a 50:50 ratio, with the interaction between the two cited as being
of key importance (Stacey et al., 2009). Findings from the present
research complement such clinical findings in that rats that were
selectively bred to exhibit a genetic predisposition to consume,
prefer, and respond for ethanol exhibited a reduction in all three
behaviors to the levels of rats that have been selectively bred to
avoid ethanol. It is acknowledged that the EC used for the present
experiments represents an extreme in one direction, whereas the IC
represents an extreme in the other; these do not fully account for
the nearly infinite variations in environmental conditions experi-
enced by the human population. However, the present research can
be considered a useful approximation of the effects of early envi-
ronmental experience on the development of alcohol abuse or
alcoholism in a population that is susceptible to high ethanol
intake. Future research investigating the interaction between he-
redity and early environmental experience may serve to identify
therapeutic targets on which to develop novel treatments and/or
interventions for populations at risk of not just alcoholism but
several other drug addictions.
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Received July 13, 2010
Revision received December 8, 2010
Accepted December 13, 2010
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