Developmental Psychobiology
Zachary E. Hurwitz
Andrew P. Merluzzi
Anthony L. Riley
Psychopharmacology Laboratory
Department of Psychology
American University, 4400 Mass. Ave.
NW, Washington, DC 20016
E-mail: zh7104a@american.edu
Age-Dependent Differences
in Morphine-Induced Taste
Aversions
ABSTRACT: Adolescence is a developmental period of particular importance
given the host of neurobiological changes that occur during this stage of development. Drug use and abuse is said to be a function of the balance of its rewarding
and aversive effects, and any age-dependent differences in morphine’s aversive
effects could impact drug intake. The present experiments examined the ability of
morphine sulfate (0, 3.2, 10, and 18 mg/kg) to induce taste aversions in adolescent and adult rats under high (20-min fluid access each day; Experiment 1A/B)
and low (50% of ad libitum access; Experiment 2A/B) deprivation conditions. In
both studies, adolescent and adult rats were given a novel saccharin solution to
drink and were subsequently injected with morphine. Independent of the deprivation condition, adults acquired stronger aversions than adolescents and did so at
a faster rate. On a subsequent two-bottle aversion test, all morphine-injected
subjects drank a significantly lower percentage of saccharin than vehicle-injected
controls with adults exhibiting stronger aversions than adolescents. These
age-dependent differences in morphine-induced CTAs extend the findings with
other drugs of abuse for which adolescents exhibit weaker aversions. The possible basis for and implications of these differences were discussed. ß 2012 Wiley
Periodicals, Inc. Dev Psychobiol
Keywords: adolescent; adult; morphine; taste aversion; development; deprivation
INTRODUCTION
Although the vulnerability to drug use and abuse is
multifaceted, one factor which has recently received
considerable attention is the age of drug exposure
(Spear, 2000). Assessments of adolescent drug use are
of particular importance given the host of neurobiological changes that occur during this stage of development
(Spear, 2000). Considerable evidence suggests these
changes may leave adolescents differentially sensitive
to the affective properties of drugs and thus more likely
Manuscript Received: 21 September 2011
Manuscript Accepted: 17 April 2012
Correspondence to: Zachary E. Hurwitz
Contract grant sponsor: Mellon Foundation ZEH; ALR; Contract
grant sponsor: Robyn Rafferty-Mathias Undergraduate Research
Award APM.
Article first published online in Wiley Online Library
(wileyonlinelibrary.com).
DOI 10.1002/dev.21046 ß 2012 Wiley Periodicals, Inc.
to engage in addictive behaviors (for reviews see
Doremus-Fitzwater, Varlinskaya, & Spear, 2010; Misanin,
Anderson, & Hinderliter, 2009). For example, Vastola,
Douglas, Varlinskaya, and Spear (2002) demonstrated
that adolescent rats exhibited a nicotine-induced place
preference at .6 mg/kg nicotine, while adult rats failed
to acquire a preference at this dose (for a review of
place preference conditioning, see Tzschentke, 1998,
2007). Similarly, Belluzzi, Lee, Oliff, and Leslie (2004)
reported that adolescents (PND38) acquired place preferences at .5 mg/kg nicotine whereas adults did not
(see also Brielmaier, McDonald, & Smith, 2007). Additional findings with ethanol (Philpot, Badanich, &
Kirstein, 2003), cocaine (Badanich, Adler, & Kirstein,
2006; Brenhouse & Andersen, 2008), and methamphetamine (Zakharova, Leoni, Kichko, & Izenwasser, 2009)
mirror those of nicotine in that adolescents typically
show preferences at doses lower than adults (if adults
exhibit a preference at all) with the exception of amphetamine in which no apparent age differences are
present (Mathews & McCormick, 2007).
2
Hurwitz, Merluzzi and Riley
Although assessments on the vulnerability to drug
use and abuse generally focus on the drug’s rewarding
effects, more recently it has been suggested that
another affective property of drugs, that is, its aversive
effect, is important in this vulnerability as well (Davis
& Riley, 2010; Doremus-Fitzwater et al., 2010; Riley,
2011; Spear & Varlinskaya, 2010). Specifically, drug
use is thought to be a function of the relative balance
of its aversive and rewarding effects (Riley, 2011) with
the drug’s aversive effects limiting overall drug intake
(Stolerman & D’Mello, 1981). Conditioned taste aversion (CTA) learning is one preparation that has been
used extensively to assay the aversive effects of various
drugs of abuse (Freeman & Riley, 2009; for an alternate explanation, see Grigson, 1997), and interestingly
such effects have been reported to differ as a function
of age (see Doremus-Fitzwater et al., 2010; Misanin
et al., 2009).
In one of the early reports comparing CTAs to drugs
of abuse in adolescent and adult rats, Infurna and Spear
(1979) reported that periadolescents (PND35) exhibited
weaker amphetamine-induced aversions than young
adult (PND52) rats, although the two age groups
did not differ in relation to the acquisition of an
amphetamine-induced place preference (Mathews &
McCormick, 2007). More recently, such differential
acquisition of drug-induced aversions (adolescents <
adults) has been reported with cocaine (SchrammSapyta, Morris, & Kuhn, 2006), THC (Schramm-Sapyta
et al., 2007), and ethanol (Anderson, Varlinskaya, &
Spear, 2010; Schramm-Sapyta et al., 2010; VetterO’Hagen, Varlinskaya, & Spear, 2009). Interestingly,
Shram, Funk, Li, and Lê (2006) demonstrated that adolescents exhibit weaker nicotine-induced aversions than
adults (see also Wilmouth & Spear, 2004) while
expressing stronger nicotine-induced place preferences
(Shram et al., 2006; see also Torres, Tejeda, Natividad,
& O’Dell, 2008), suggesting that adolescents are less
sensitive to the aversive properties of drugs and more
sensitive to their rewarding properties (see DoremusFitzwater et al., 2010). Although age differences are
consistently seen with a variety of drugs of abuse, the
findings with classical emetics such as LiCl are mixed,
that is, adolescents either show weaker CTAs or no
difference relative to adults (see Baker, Baker, &
Kesner, 1977; Balcom, Coleman, & Norman, 1981;
Guanowsky, Misanin, & Riccio, 1983; Klein, Domato,
Hallstead, Stephens, & Mikulka, 1975; Klein, Mikulka,
Domato, & Hallstead, 1977; Martin & Timmins, 1980;
Misanin, Blatt, & Hinderliter, 1985; Misanin, Greider,
& Hinderliter, 1988; Misanin, Guanowsky, & Riccio,
1983; Valliere, Misanin, & Hinderliter, 1988). Such
inconsistencies are likely a function of the dose used in
establishing aversions in that Schramm-Sapyta et al.
Developmental Psychobiology
(2006) reported that adolescents exhibit weaker LiClinduced taste aversions at low-to-moderate doses, while
exhibiting no differences at higher doses, suggesting
that adolescents are less sensitive to the aversive effects
of LiCl but can acquire comparable aversions at higher
doses.
Although age-dependent differences in the acquisition of CTAs have been noted for LiCl and numerous
drugs of abuse (cocaine, nicotine, amphetamine, THC,
ethanol), direct comparisons of adolescents to adults in
regard to opioid-induced aversions have not yet been
investigated despite the abuse of such compounds by
adolescent populations (Johnston, O’Malley, Bachman,
& Schulenberg, 2010). In this context, morphine (like
amphetamine) is interesting in that it does not appear
to induce age-dependent place preferences, suggesting
that it is not differentially rewarding in adolescent and
adult rats (Campbell, Wood, & Spear, 2000). Given
that drug use and abuse may be a function of the balance of a drug’s rewarding and aversive effects, it
remains important to assess any age-dependent differences in morphine’s aversive effects that could impact
drug intake. To address this possibility, the present
experiments examined the ability of morphine sulfate
to induce taste aversions in adolescent and adult rats
under two different procedural variations. Specifically,
adolescent (Experiment 1A) and adult (Experiment 1B)
rats were initially restricted to 20-min daily fluid access
(high deprivation) before being given a novel saccharin
solution to drink and subsequently injected with various
doses of morphine to assess its ability to induce agedependent aversions. To address the possibility that restricted fluid access and the concomitant weight loss
could differentially influence the ability of adolescents
and adults to acquire and/or display aversions, different
groups of adolescents (Experiment 2A) and adults
(Experiment 2B) were examined for their ability to
acquire morphine-induced aversions under a low deprivation procedure in which water was restricted to 50%
of ad libitum consumption.
EXPERIMENT 1: METHODS
Subjects
Sixty-four experimentally naı̈ve male Sprague–Dawley
(Harlan Laboratories; Indianapolis, IN) rats arrived at the
facility weighing approximately 40 g (PND 21, n ¼ 32) and
90 g (PND 35, n ¼ 32). Food and water were available ad
libitum unless stated otherwise (see below). Procedures recommended by the National Research Council (1996), the
Committee on Guidelines for the Care and Use of Animals in
Neuroscience and Behavioral Research (2003), and the Institutional Animal Care and Use Committee at American University were followed at all times.
Developmental Psychobiology
Apparatus
Upon arrival to the animal colony, subjects were initially
handled and then group housed (four rats per bin) in polycarbonate bins (23 cm 44 cm 21 cm). All subjects were
maintained on a 12:12 light–dark cycle (lights on at 0800 hr)
and at an ambient temperature of 238C. All conditioning
and testing occurred during the light phase of the light–dark
cycle. During adaptation and conditioning, animals were
transferred to individual hanging wire-mesh (24.3 cm 19 cm 18 cm) test cages but were returned to their grouphousing bins after conditioning session (see details below).
Drugs
Morphine sulfate (generously supplied by the National Institute on Drug Abuse) was dissolved in sterile isotonic saline
(.9%) at a concentration of 5 mg/ml and was subsequently
filtered through a .2 mm filter to remove any contaminants
before being administered subcutaneously (SC) at a dose of
3.2, 10, or 18 mg/kg. Sterile isotonic saline was also filtered
before being administered to vehicle controls equivolume
to the highest dose of morphine administered (18 mg/kg).
Volume of the injection was manipulated in favor of concentration given the influence concentration has on the absorption/distribution of the drug.
Experiment 1: Procedure
Experiment 1A: Adolescents. Subjects were brought into the
facility at PND 21 and were maintained on ad libitum food
but were immediately water restricted. Subjects were weighed
and handled on this day (PND 21), and experimental procedures began on PND 22. On this day, subjects were removed
from their group-housed bins, weighed and placed in the individual test cages. A 50-ml graduated Nalgene tube containing
tap water was then affixed to each cage for 40 min. After
water access, bottles were removed and consumption was
recorded. Subjects remained in the test cages for an additional
20 min before being returned to their group-housed bins. This
procedure was repeated for 7 consecutive days with the exception that water access was restricted to 20 min daily after
the first day. On the day following adaptation, subjects were
weighed and handled as previously described and given
20-min access to a novel saccharin solution (1 g/L) in the test
cages after which they remained for an additional 20 min. At
this point, subjects (independent of their group-housed bin)
were assigned to one of four groups such that saccharin intake
was comparable among groups. Based on these group assignments, subjects were injected with either morphine (3.2, 10,
or 18 mg/kg SC) or vehicle (matched in volume to the highdose group) and then returned to their group-housed bins.
This procedure yielded Groups 0, 3.2, 10, and 18 for which
the number indicates the dose of morphine administered. The
following 2 days served as water-recovery days, and subjects
were treated as described during the 7 days of water adaptation. They received no injections on these days. This 3-day
cycle (conditioning/water-recovery) was repeated four times.
On the day following the completion of the fourth 3-day
Age Differences in Morphine CTA
3
cycle, subjects were removed from their group-housed bins
and placed in their respective test cages (as previously
described) and given access to two 50-ml Nalgene tubes (one
containing tap water and the other containing saccharin) for
20 min. Specifically, both the saccharin- and water-filled
Nalgene tubes were placed on the test cages simultaneously
with the placement of the tubes (left or right side) counterbalanced across subjects to prevent positioning effects. After
20 min, the bottles were removed, consumption was recorded,
and subjects were returned to their group-housed bins.
For the adolescent subjects, adaptation to the test cages
and restricted fluid access took place from PND 22 to 28,
saccharin pairings took place from PND 29 to 40, and the
two-bottle test was given on PND 41.
Experiment 1B: Adults. The procedures described above
were identical for the adult rats with the following exceptions.
Subjects were brought into the facility at PND 35 and maintained on ad libitum food and water until PND 77 to permit
the control of their developmental environment (housing
conditions, handling, and light/dark cycle). Subjects were
weighed and handled on PND 77 but had their bottles
removed so that experimental procedures began on PND 78.
Adaptation to the test cages and restricted fluid access took
place from PND 78 to 84, saccharin pairings took place from
PND 85 to 96, and the two-bottle test was given on PND 97.
Two subjects from the high-dose group died within 24 hr
after the second saccharin-drug pairing. All data from these
subjects were removed from the subsequent analyses.
Statistical Analysis
For each age group, saccharin consumption (ml) on the four
conditioning sessions was analyzed using a 4 (Dose) 4
(Session) mixed ANOVA. To determine if there were differences in saccharin consumption (ml) between conditioning
Sessions 1 and 4, Bonferroni-corrected t-tests were employed.
A 4 (Dose) 8 (Session) mixed ANOVA was used to analyze
water consumption during recovery days. One-way ANOVAs
and Tukey’s HSD post hoc tests were employed to evaluate
differences in the percent saccharin consumed between the
different dose groups on the two-bottle test. All statistical
analyses were based on significance level of a ¼ .05.
RESULTS
Experiment 1A: Adolescents
Acquisition. The 4 4 mixed ANOVA on saccharin
consumption (ml) during conditioning revealed significant effects of Dose [F(3, 84) ¼ 12.561, p < .05] and
Session [F(3, 84) ¼ 19.269, p < .05] as well as a significant Dose Session interaction [F(9, 84) ¼ 6.646,
p < .05] (Fig. 1A). A subsequent one-way ANOVA indicated significant differences in consumption between
the groups on Sessions 3 [F(3, 31) ¼ 8.903, p < .05]
and 4 [F(3, 31) ¼ 23.741, p < .05]. Tukey’s post hoc
4
Hurwitz, Merluzzi and Riley
A
Developmental Psychobiology
B
FIGURE 1 Mean (SEM) saccharin consumption (ml) by adolescents during acquisition
(A) and mean (SEM) percent saccharin consumed on the two-bottle test (B). For acquisition,
there were significant effects of Dose and Session as well as a significant Dose Session
interaction (p’s < .05). Subsequent multiple comparisons revealed that on Session 3 Groups
10 and 18 drank significantly less saccharin than Group 0. By conditioning Session 4, all
morphine-treated subjects drank significantly less saccharin relative to Group 0 with Groups
10 and 18 drinking less than Group 3.2. With regard to the two-bottle test, Groups 3.2, 10, and
18 all drank a significantly lower percent of saccharin than Group 0 with Group 18 drinking a
significantly lower percent relative to Group 3.2 (p’s < .05). Significant differences between
Group 0 and Groups 3.2, 10, and 18. ^Significant differences between Groups 3.2 and 18.
þ
Significant differences between Groups 3.2 and 10. #Significant differences between Groups
0 and 10/18 (p’s < .05).
analysis revealed that on Session 3 Groups 10 and
18 drank significantly less saccharin than Group 0
(p’s < .05). By Session 4, all morphine-injected subjects drank significantly less saccharin than Group 0
(p’s < .05) and Groups 10 and 18 drank significantly
less saccharin than Group 3.2 (p’s < .05). Subsequent
within-subject analyses (with Bonferroni corrections)
indicated that Groups 0 and 3.2 significantly increased
saccharin intake from Sessions 1 to 4 [t(7) ¼ 8.793,
p < .05 and t(7) ¼ 3.989, p ¼ .05, respectively].
Groups 10 and 18 exhibited no significant changes
in saccharin consumption over these sessions
[t(7) ¼ 1.033, p ¼ .336 and t(7) ¼ .120, p ¼ .908,
respectively].
There were no significant differences in water consumption among groups on the water-recovery day
immediately preceding each conditioning session (all
p’s > .05; data not shown).
Two-Bottle Test. A one-way ANOVA on the percent
saccharin consumed on the two-bottle test revealed significant differences among groups [F(3, 31) ¼ 16.171,
p < .05] (Fig. 1B). Specifically, all morphine-injected
subjects drank a significantly lower percent of saccharin than Group 0. In addition, Group 18 drank a significantly lower percent of saccharin than Group 3.2
(p’s < .05).
Experiment 1B: Adults
Acquisition. The 4 4 mixed ANOVA on saccharin
consumption (ml) during conditioning revealed significant main effects of Dose [F(3, 26) ¼ 22.919, p < .05]
and Session [F(3, 78) ¼ 42.608, p < .05] as well
as a significant Dose Session interaction [F(9,
78) ¼ 13.344, p < .05] (Fig. 2A). The subsequent
one-way ANOVA indicated significant differences in
consumption between the groups on Sessions 2 [F(3,
29) ¼ 10.104, p < .05], 3 [F(3, 29) ¼ 27.384,
p < .05], and 4 [F(3, 29) ¼ 31.097, p < .05]. Multiple
comparisons revealed that on Session 2 all morphineinjected subjects drank significantly less than Group 0
(p’s < .05). On Sessions 3 and 4, all morphine-injected
subjects drank significantly less saccharin than Group 0
(p’s < .05) and Group 18 drank significantly less
saccharin than Group 3.2 (p < .05). Subsequent withinsubject analyses (with Bonferroni corrections) indicated
that Group 0 increased consumption between Sessions 1 and 4 [t(7) ¼ 3.610, p < .05], while Group
3.2 showed no significant change over sessions
[t(7) ¼ 3.009, p > .05]. Groups 10 and 18 significantly
decreased saccharin consumption over these sessions
[t(7) ¼ 9.636, p < .05 and t(5) ¼ 6.089, p < .05,
respectively].
There were no significant differences in water
consumption among groups on the water-recovery day
Developmental Psychobiology
A
Age Differences in Morphine CTA
5
B
FIGURE 2 Mean (SEM) saccharin consumption (ml) by adults during acquisition (A) and
mean (SEM) percent saccharin consumed on the two-bottle test (B). For acquisition, there
were significant main effects of Dose and Session as well as a significant Dose Session
interaction (p’s < .05). Subsequent multiple comparisons revealed that on Session 2 all
morphine-treated subjects drank significantly less saccharin than Group 0 (p’s < .05). On conditioning Sessions 3 and 4, all morphine-treated subjects drank significantly less saccharin
relative to Group 0 with Group 18 drinking less saccharin than Group 3.2. With regard to the
two-bottle test, Groups 3.2, 10, and 18 all drank a significantly lower percent of saccharin than
Group 0 (p’s < .05). Significant differences between Group 0 and Groups 3.2, 10, and 18.
^Significant differences between Groups 3.2 and 18 (p’s < .05).
immediately preceding each conditioning session (all
p’s > .05; data not shown).
Two-Bottle Test. A one-way ANOVA on the percent
saccharin consumed on the two-bottle test revealed significant differences among groups [F(3, 29) ¼ 38.763,
p < .05] (Fig. 2B). Specifically, all morphine-injected
subjects drank a significantly lower percent of saccharin than Group 0 (p’s < .05).
Adolescent and Adult Comparison
An independent-samples t-test on saccharin consumption (ml) of the adolescent and adult vehicle groups
on the first saccharin conditioning session revealed
significant differences between adolescents and adults
[t(14) ¼ 11.092, p < .05]. To allow for a direct comparison between the adolescents and adults, consumption for the drug-injected groups was transformed to a
percent of the average consumption of Group 0 across
each age group for each conditioning session (Fig. 3A).
Specifically, for each session, consumption in each age
and dose group was calculated as a percent change
from the average absolute consumption of the vehicleinjected controls (Group 0) on that session. A 2
(Age) 3 (Dose) 4 (Session) mixed ANOVA on
these percent differences revealed significant main
effects of Age [F(1, 40) ¼ 51.498, p < .05], Dose
[F(2, 40) ¼ 6.412, p ¼ .05], and Session [F(3,
120) ¼ 126.982, p < .05] as well as significant
Session Age [F(3, 120) ¼ 24.016, p < .05] and
Dose Session [F(6, 120) ¼ 3.715, p < .05] interactions. There was no Age Dose [F(2, 40) ¼ .552,
p > .05] or Session Age Dose [F(6, 120) ¼ .815,
p > .05] interaction. Specifically, adults drank a significantly lower percentage of saccharin relative to adolescents. On Session 1, there were no age differences
in the percentage of saccharin consumed (relative to
vehicle controls). From Sessions 2 to 4, the percent
change in saccharin consumed was significantly greater
for adults than adolescents, reflective of the acquisition
of stronger aversions in the adult subjects.
Additionally, Bonferroni-corrected independent sample t-tests used to examine age differences in saccharin
preference during the two-bottle test (Fig. 3B) revealed
that adolescents consumed a significantly higher
percentage of saccharin relative to adults at 10 mg/kg
[t(14) ¼ 23.925, p < .0125] and 18 mg/kg [t(14) ¼
9.478, p < .0125] with no differences at 0 mg/kg
[t(14) ¼ 1.105, p > .05] or 3.2 mg/kg [t(14) ¼ 3.942,
p > .0125].
DISCUSSION: EXPERIMENT 1
Despite the documented abuse of opioids by human
adolescents (Johnston et al., 2010) and the importance
of the aversive affective response of drugs in shaping
drug intake (Stolerman & D’Mello, 1981), no report
has assessed potential age-dependent differences in the
6
Hurwitz, Merluzzi and Riley
A
Developmental Psychobiology
B
FIGURE 3 Mean (SEM) percent change in saccharin consumed (ml) from vehicle controls
over repeated conditioning sessions (A) and mean (SEM) percent saccharin consumed on the
two-bottle test (B) for adolescents (white bars) and adults (black bars). There were significant
main effects of Age, Dose, and Session, and significant interactions of Session Age and
Session Dose (p’s < .05), but no significant Age Dose or Age Dose Session interactions (p’s > .05). Specifically, adults drank a significantly lower percentage of saccharin
relative to adolescents. On conditioning Session 1, there were no differences in the percent
change in saccharin consumed from vehicle controls, while from Sessions 2 to 4 the percent
saccharin consumed by adults was significantly less than adolescents. For the two-bottle test,
adults in Groups 10 and 18 drank a significantly lower percentage of saccharin relative to
adolescents treated with the same dose (p’s < .05). Significant differences between adolescents and adults of the same dose group (p’s < .05).
ability of morphine to condition taste aversions. This
was addressed in Experiment 1, in which the acquisition of morphine-induced taste aversions was examined
in adolescent and adult rats. As described, adults
displayed significantly stronger aversions than the adolescents and acquired these aversions at a significantly
faster rate.
Although suggestive of age-dependent differences in
the aversive effects of morphine (for similar differences
with other drugs of abuse, see Anderson et al., 2010;
Infurna & Spear, 1979; Schramm-Sapyta et al., 2006,
2007, 2010; Shram et al., 2006; Vetter-O’Hagen et al.,
2009; Wilmouth & Spear, 2004), there are other possible interpretations. For example, the water-deprivation
procedure used to induce fluid consumption during
conditioning, that is, 20-min daily access, may have
differentially affected the motivation to drink in the
adolescents and adults. This possibility is supported by
the fact that the restricted fluid schedule impacted body
weights differently in the two age groups. Specifically,
adolescent and adult vehicle-injected controls weighed
90 and 319 g, respectively, at the end of the experiments (after 20 days of restricted fluid access). This
resulted in adolescent and adult subjects weighing
approximately 52% and 80% of age-matched subjects
maintained under free-feeding procedures (Harlan Laboratories, Indianapolis, IN). These differences in body
weight suggest that the adolescent rats were impacted
greater by the deprivation procedure than the adults,
possibly increasing the motivation to drink. Given that
deprivation is a factor in the induction and expression
of taste aversions (weaker aversions under more
restricted deprivation; see De la Casa & Lubow, 1995;
Domjan, 1972; Peck & Ader, 1974; Revusky, Pohl,
& Coombes, 1980; Sengstake & Chambers, 1979;
Sengstake, Chambers, & Thrower, 1978), it is possible
that these potential differences in motivation may be
responsible for the behavioral differences and not any
differential sensitivity to the drug’s aversive effects. To
address this possibility, morphine-induced taste aversions were examined in separate groups of adolescent
and adult subjects maintained under a low deprivation
condition (i.e., 50% of ad libitum consumption; see
Anderson et al., 2010).
EXPERIMENT 2: METHODS
Subjects
The subjects were 32 adolescent and 34 adult naı̈ve rats of
the same age, sex, and supplier as those described above in
§2.1. Unless otherwise specified, they were maintained under
the same conditions as described in Experiment 1.
Experiment 2: Procedure
Experiment 2A: Adolescents. Subjects were brought into the
laboratory on PND 21. For the first 7 experimental days,
Developmental Psychobiology
subjects were maintained on ad libitum food and water and
weighed and handled daily. Over the next 2 days (Days 8 and
9), water consumption for each group-housed bin was
recorded to the nearest tenth of a milliliter. On Day 10, the
amount of water available for each bin was reduced to 50%
(plus 5 ml for inaccessible water in the bottle) of the average
of the previous 2 days’ drinking levels to encourage consumption of water that was presented in the individual test cages
on the next day. On the following day, subjects were removed
from their group-housed bin, weighed, and placed into individual test cages where they were given 45-min access to tap
water in graduated 50-ml Nalgene tubes. After 45 min, the
bottles were removed, consumption was recorded, and subjects remained in the hanging cages for an additional 20 min
before being returned to their group-housed bin and given ad
libitum water for the next 22.5 hr. On the next day, the
amount of water available for each bin was again reduced to
50% (as described above) with the exception that individual
test cage consumption was also factored into the previous
22.5 hr of consumption. On the subsequent day, subjects were
again weighed and handled, placed into the test cages, and
given 45-min access to tap water before being returned to
their group-housed home cages with ad libitum water for the
next 22.5 hr. Water available to subjects was again reduced
(as described above) before undergoing saccharin conditioning in the test cages. On this conditioning session, subjects
were weighed and handled as previously described and given
45-min access to a novel saccharin solution (1 g/L) in the test
cages after which they remained for an additional 20 min. At
this point, subjects (independent of their group-housed bin)
were assigned to one of the four groups such that saccharin
intake was comparable among groups. Based on these group
assignments, subjects were injected with either morphine
(3.2, 10, or 18 mg/kg SC) or vehicle (matched in volume to
the high-dose group), and then returned to their group-housed
bins and given ad libitum water for the next 22.5 hr. This
procedure yielded Groups 0, 3.2, 10, and 18 for which the
number indicates the dose of morphine administered. On the
next day, subjects in each bin had their fluid consumption
reduced (as described above) before the subsequent saccharin
conditioning session. This procedure (saccharin—22.5-hr
recovery—50% deprivation) was repeated four times. On the
day following the last conditioning session and determination
of 50% deprivation, subjects were transferred to test cages
where two 50-ml Nalgene tubes (one containing tap water
and the other containing the .1% sodium saccharin solution)
were affixed to the cage for 45 min and consumption of both
solutions was recorded. Placement of the bottle was counterbalanced across groups to prevent positioning effects. After
the 45 min, the bottles were removed, consumption recorded,
and subjects were then returned to their home cages where
water was available ad libitum.
Experiment 2B Procedure: Adults. The procedures described
above were identical for the adult rats with the following
exceptions. Subjects were brought into the facility at PND 35
and maintained on ad libitum food and water until PND 77
to permit the control of their developmental environment
Age Differences in Morphine CTA
7
(housing conditions, handling, and light/dark cycle). Subjects
were weighed and handled on PND 77–83 at which point water adaptation and aversion conditioning began (as described
above). During PND 87–95, subjects were given 45-min
access to tap water in the individual test cages. From PND 97
to 103, subjects were given four saccharin-drug (or vehicle)
pairings until the two-bottle test, which was administered on
PND 105. Two subjects from the high-dose group died within
2 hr after the first saccharin-drug pairing. All data from these
subjects were removed from the subsequent analyses. Complications associated with opioid-induced hyperphagia were
observed in these two subjects. Specifically, excessive intake
of woodchips and food blocks were observed which has been
reported in subjects administered buprenorphine (a m-opioid
agonist) and maintained on hardwood bedding (see Clark
Jr., Myers, Goelz, Thigpen, & Forsythe, 1997; Jacobson,
2000). This effect was not observed in subjects maintained on
grid floors or on paper during the first 9 hr postinjection
(Jablonski, Howden, & Baxter, 2001). In the present assessment, morphine-treated subjects that exhibited these complications postdrug administration were lavaged with 1 ml of tap
water to clear the material from their mouths.
Statistical Analysis
For each age group, saccharin consumption (ml) on the four
conditioning sessions was analyzed using a 4 (Dose) 4
(Session) mixed ANOVA. To determine if there were differences in saccharin consumption (ml) between conditioning
Sessions 1 and 4, Bonferroni-corrected t-tests were employed.
One-way ANOVAs and Tukey’s HSD post hoc tests were
employed to evaluate differences in the percent saccharin
consumed between the different dose groups on the two-bottle
test. All statistical analyses were based on significance level
of a ¼ .05.
RESULTS
Experiment 2A: Adolescents
Acquisition. The 4 4 mixed ANOVA on saccharin
consumption (ml) revealed significant effects of Dose
[F(3, 28) ¼ 3.916, p < .05] and Session [F(3, 84) ¼
6.242, p < .05] as well as a significant Dose Session
interaction [F(9, 84) ¼ 3.908, p < .05] (Fig. 4A).
The subsequent one-way ANOVA indicated significant differences in consumption between the groups on
Sessions 3 [F(3, 31) ¼ 7.237, p < .05] and 4 [F(3,
31) ¼ 4.922, p < .05]. Tukey’s post hoc analysis
revealed that on Sessions 3 and 4 all morphine-injected
subjects drank significantly less saccharin than Group 0
(p’s < .05). Subsequent within-subject analyses (with
Bonferroni corrections) indicated that Groups 0, 3.2,
and 10 displayed no significant changes in saccharin
intake from Sessions 1 to 4 [t(7) ¼ 1.839, p > .05,
t(7) ¼ 2.005, p > .05, and t(7) ¼ 2.089, p > .05,
8
Hurwitz, Merluzzi and Riley
A
Developmental Psychobiology
B
FIGURE 4 Mean (SEM) saccharin consumption (ml) by adolescents during acquisition
(A) and mean (SEM) percent saccharin consumed on the two-bottle test (B). For acquisition,
there were significant effects of Dose and Session as well as a significant Dose Session
interaction (p’s < .05). Subsequent multiple comparisons revealed that on Sessions 3 and 4 all
morphine-treated subjects drank significantly less saccharin than Group 0. With regard to the
two-bottle test, Groups 3.2, 10, and 18 all drank a significantly lower percent of saccharin than
Group 0 (p’s < .05). Significant differences between Group 0 and Groups 3.2, 10, and 18
(p’s < .05).
respectively]. Group 18 exhibited a significant decrease
in saccharin consumption over these sessions [t(7) ¼
5.446, p < .05].
Two-Bottle Test. A one-way ANOVA on the percent
saccharin consumed on the two-bottle test revealed significant differences among groups [F(3, 31) ¼ 13.929,
p < .05] (Fig. 4B). Specifically, all morphine-injected
subjects drank a significantly lower percent of saccharin than Group 0 (p’s < .05).
Experiment 2B: Adults
Acquisition. The 4 4 mixed ANOVA on saccharin
consumption (ml) revealed significant effects of Dose
[F(3, 28) ¼ 56.512, p < .05] and Session [F(3,
84) ¼ 20.018, p < .05] as well as a significant Dose Session interaction [F(9, 84) ¼ 12.617, p < .05]
(Fig. 5A). The subsequent one-way ANOVA indicated
significant differences in consumption between groups
on Sessions 2 [F(3, 31) ¼ 24.267, p < .05, 3 [F(3,
31) ¼ 7.237, p < .05], and 4 [F(3, 31) ¼ 52.688,
p < .05]. Tukey’s post hoc analysis revealed that on
Sessions 2–4 all morphine-injected subjects drank significantly less saccharin than Group 0 (p’s < .05). Subsequent within-subject analyses (with Bonferroni
corrections) indicated that Group 0 displayed no significant changes in saccharin intake from Sessions 1 to 4
[t(7) ¼ 3.053, p > .0125]. Group 3.2 [t(7) ¼ 7.392,
p < .0125], 10 [t(7) ¼ 7.400, p < .0125], and 18
[t(7) ¼ 5.452, p < .0125] exhibited a significant decrease in saccharin consumption over these sessions.
Two-Bottle Test. A one-way ANOVA on the percent
saccharin consumed on the two-bottle test revealed significant differences among groups [F(3, 31) ¼ 112.286,
p < .05] (Fig. 5B). Specifically, all morphine-injected
subjects drank a significantly lower percent of saccharin than Group 0 with Group 18 drinking a significantly
lower percent than Group 3.2 (p’s < .05).
EXPERIMENT 2: ADOLESCENT–ADULT
COMPARISONS
Consumption for the drug-injected groups was transformed to a percent of the average consumption of
Group 0 across each age group for each conditioning
session (Fig. 6A). Specifically, for each session consumption in each age and dose group was calculated as
a percent change from the average absolute consumption of the vehicle-injected controls (Group 0) on that
session. A 2 (Age) 3 (Dose) 4 (Session) mixed
ANOVA on these percent differences revealed significant main effects of Age [F(1, 42) ¼ 49.389, p < .05]
and Session [F(3, 126) ¼ 109.104, p < .05], as well
as a significant Session Age [F(3, 126) ¼ 16.522,
p < .05] interaction. There was no effect of Dose
[F(2, 42) ¼ .083, p > .05] and no significant Dose Session [F(6, 126) ¼ .514, p > .05], Age Dose
[F(2, 42) ¼ .633, p > .05], or Session Age Dose
[F(6, 126) ¼ 310, p > .05] interactions. Specifically,
adults drank a significantly lower percentage of saccharin relative to adolescents. On Session 1, there were
no age differences in the percentage of saccharin
Developmental Psychobiology
A
Age Differences in Morphine CTA
9
B
FIGURE 5 Mean (SEM) saccharin consumption (ml) by adults during acquisition (A) and
mean (SEM) percent saccharin consumed on the two-bottle test (B). For acquisition, there
were significant main effects of Dose and Session as well as a significant Dose Session
interaction (p’s < .05). Subsequent multiple comparisons revealed that on Sessions 2–4 all
morphine-treated subjects drank significantly less saccharin than Group 0 (p’s < .05). With
regard to the two-bottle test, all morphine-treated subjects drank a significantly lower percent
of saccharin than Group 0 with Group 18 drinking a significantly lower percent relative to
Group 3.2 (p’s < .05). Significant differences between Group 0 and Groups 3.2, 10, and 18
(p’s < .05). ^Significant differences between Groups 18 and 3.2 (p < .05).
consumed (relative to vehicle controls). From Sessions
2 to 4, the percent change in saccharin consumed was
significantly greater for adults than adolescents, reflective of the acquisition of stronger aversions in the adult
subjects.
Additionally, Bonferroni-corrected independent sample t-tests used to examine age differences in saccharin
preference during the two-bottle test (Fig. 6B) revealed
that adolescents consumed a significantly higher percentage of saccharin relative to adults at 3.2 mg/kg
A
[t(14) ¼ 12.281, p < .0125] and 10 mg/kg [t(14) ¼
25.847, p < .0125] with no differences at 0 mg/kg
[t(14) ¼ 4.909, p > .0125] and 18 mg/kg [t(14) ¼
.003, p > .0125].
DISCUSSION: EXPERIMENT 2
To assess the possible effects of differences in motivation on the differences in morphine-induced taste
B
FIGURE 6 Mean (SEM) percent change in saccharin consumed (ml) from vehicle controls
over repeated conditioning sessions (A) and mean (SEM) percent saccharin consumed on the
two-bottle test (B) for adolescents (white bars) and adults (black bars). There were significant
main effects of Age, Session, and a significant Session Age interaction (p’s < .05), but no
significant effects of Dose or significant Age Dose, Dose Session, or Age Dose Session interactions (p’s > .05). Specifically, adults drank a significantly lower percentage of
saccharin relative to adolescents. On conditioning Session 1, there were no differences in the
percent change in saccharin consumed from vehicle controls, while from Sessions 2 to 4, the
percent saccharin consumed by adults was significantly less than adolescents. For the twobottle test, adults in Groups 3.2 and 10 drank a significantly lower percentage of saccharin
relative to adolescents treated with the same dose (p’s < .05). Significant differences between
adolescents and adults of the same dose group (p’s < .05).
10
Hurwitz, Merluzzi and Riley
aversions between the two age groups (as reported in
Experiment 1), morphine-induced taste aversions
were assessed under a low deprivation procedure
(Experiment 2). Specifically, adolescent and adult
animals were given 50% of their ad libitum levels
of fluid consumption prior to aversion conditioning
with morphine (see Anderson et al., 2010). Unlike
Experiment 1, the two groups displayed minimal loss
of body weight under these deprivation conditions
and further did not differ from each other. Adolescent
and adult vehicle-injected controls weighed approximately 95% and 94% of free-feeding rats, respectively
(as compared to Experiment 1 where adolescents and
adults were 52% and 80% of free-feeding).
Although body weight changes in response to deprivation were comparable for adolescents and adults
under the low deprivation schedule of Experiment 2,
adults acquired the aversions significantly faster and
exhibited stronger aversions than adolescents. That the
adults continued to display significantly greater morphine-induced aversions than the adolescents under low
deprivation where the two groups did not differ in their
body weight changes suggests that these differences are
a function of differential sensitivity to the aversive
effects of morphine and not an artifact of differences in
motivation.
GENERAL DISCUSSION
Considerable evidence suggests that the host of neurobiological changes which occur during adolescence
may leave individuals in this age group differentially
sensitive to the affective properties of drugs and
thus more likely to engage in addictive behaviors
(for reviews see Doremus-Fitzwater et al., 2010;
Misanin et al., 2009). Given the abuse of opioid
compounds by adolescent populations (Johnston et al.,
2010) and the role the aversive properties play in
shaping overall drug intake (Stolerman & D’Mello,
1981), any age differences in the aversive effects
of morphine may leave adolescents more sensitive to
its use and abuse. To address this issue, the present
report assessed the ability of morphine to induce
aversions in both adolescents and adults in high- and
low-deprivation conditions. As described, both adolescents and adults acquired morphine-induced taste
aversions with adolescents taking longer to acquire
aversions that were weaker than adults. These agedependent differences with morphine parallel those
reported in a number of studies assessing age differences in the affective properties of other drugs of
abuse (Anderson et al., 2010; Infurna & Spear, 1979;
Schramm-Sapyta et al., 2006, 2007, 2010; Shram
Developmental Psychobiology
et al., 2006; Vetter-O’Hagen et al., 2009; Wilmouth &
Spear, 2004).
The present data, when taken in conjunction with
these previous reports, suggest some general developmental phenomenon that generalizes across a number
of drugs and procedural variations, possibly a differential sensitivity to the aversive effects of such drugs.
Although possible, there are several other interpretations for the reported differences in aversion learning
between the two age groups. Given that the taste aversion design is one that is based in conditioning (see
Garcia, McGowan, Ervin, & Koelling, 1968), any agedependent differences in taste processing, learning,
and/or retention could mediate the reported differences.
Work with place preference conditioning (often with
the same compounds), however, has shown that adolescents acquire such preferences more rapidly and at
lower doses (Belluzzi et al., 2004; Brielmaier et al.,
2007; Vastola et al., 2002; though see Campbell et al.,
2000 for a report of no differences with morphine and
cocaine). Any general deficits in learning and memory
should be reflected in weaker effects in adolescents.
Further, although taste aversions are generally weaker
in adolescents than adults, when LiCl is used as the
aversion-inducing compound it is often reported that
there are no age-dependent differences (see Balcom
et al., 1981; Guanowsky et al., 1983; Klein et al., 1977;
Misanin et al., 1983; Valliere et al., 1988), revealing
that adolescents are capable of processing the taste
stimulus and learning and retaining the association
comparably to adults (for age differences in LiClinduced CTA see Baker et al., 1977; Klein et al., 1975;
Martin & Timmins, 1980; Misanin et al., 1985, 1988).
Another possible explanation for the present data
could be due to age-dependent differences in morphine’s pharmacokinetics. Interestingly, younger rats
(PND 26, 32, 42) are reported to have significantly
lower plasma levels of morphine relative to adults (see
Jøhannesson & Becker, 1973; Spratto & Dorio, 1978;
Wang et al., 2005). Further, Spratto and Dorio (1978)
have reported that the levels of morphine in the brain
are lower in adolescents relative to adults (Spratto &
Dorio, 1978; though see Jøhannesson & Becker, 1973).
These reports seem to suggest inherent developmental
differences in the absorption, distribution, metabolism,
and/or excretion of the drug (see also Wang et al.,
2005). In addition to these pharmacokinetic differences,
age-dependent differences in the pharmacodynamics of
morphine have also been reported that could mediate
the reported differences in aversion learning. For example, opiate receptor subtypes differentially develop in
the rat (see Auguy-Valette, Cros, Gouarderes, Gout, &
Pontonnier, 1978; Clendeninn, Petraitis, & Simon,
1976; Petrillo, Tavani, Verotta, Robson, & Kosterlitz,
Developmental Psychobiology
1987; Spain, Roth, & Coscia, 1985) and could potentially be involved in the differential expression of aversions. Although the receptor subtypes differentially
develop, it should be noted that binding affinities at
these receptors do not vary with age (Auguy-Valette
et al., 1978; Clendeninn et al., 1976; Petrillo et al.,
1987). These pharmacokinetic and pharmacodynamic
differences may contribute in part to the reported agedependent differences in aversion learning. Until such
differences have been examined concurrently with the
behavioral demonstrations (i.e., under similar parametric conditions), it remains unknown to what extent the
behavioral effects are a function of these differences.
Although the two age groups were treated similarly
in relation to the induction of morphine-induced aversions, it is important to note that there were procedural
differences in the assessments with the adolescents and
adults that could underlie the reported differences in
aversion learning. For example, in both Experiments 1
and 2 adolescent subjects were brought into the animal
facility and immediately adapted to the deprivation
schedule prior to the initiation of conditioning. Adult
subjects, on the other hand, were brought into the facility at a young age (PND 35) to allow for experimental
control of their developmental environment (e.g., housing conditions, handling, light/dark cycle) with adaptation not starting until PND 78. It is possible that this
differential adaptation may have generated different
degrees of stress (adolescent > adults) that impacted
conditioning. Although possible, it is important to note
that the work assessing the role of stress in aversion
learning is actually quite mixed with the vast majority
of studies illustrating that stress has no effect on the
acquisition and/or expression of taste aversions (Anderson, Hinderliter, & Misanin, 2006; Bourne, Calton,
Gustavson, & Schachtman, 1992; Bowers, Amit, &
Gringras, 1996; Misanin, Kaufhold, Paul, Hinderliter,
& Anderson, 2006; Revusky & Reilly, 1989). Importantly, a majority of this work is with adult animals
using LiCl so it is difficult to conclude that adolescents
would not be affected (or affected in a manner different
from adults) with regard to aversions induced by drugs
of abuse. In one assessment, which systematically examined the role of different stressors (isolation housing,
restraint stress) on age-dependent drug-induced aversion learning, there were no significant effects on adolescent or adult ethanol-induced CTAs (Anderson et al.,
2010), although the authors argued that water deprivation may have masked such effects. It is clear that additional assessments of the influence stress may have on
the acquisition of drug-induced aversions in adolescence are merited.
The differences in saccharin consumption reported
here are discussed in the context of differential
Age Differences in Morphine CTA
11
sensitivity to the aversive effects of morphine; however,
there are other interpretations of the suppression of
consumption of drug-associated tastes. One such interpretation is anticipatory contrast (AC; Grigson, 1997).
This model states that the reduction in consumption of
a taste paired with a drug is not due to its aversive
properties but rather the drug’s rewarding effects. That
is, rats avoid drinking the drug-paired taste in anticipation of the drug’s greater rewarding properties.
Although an interesting position, the actual support for
this model is somewhat limited. In fact, recent work in
a number of areas (see Schramm-Sapyta et al., 2006;
Turenne, Miles, Parker, & Siegel, 1996) provides data
contrary to this hypothesis. One specific area that has
been problematic for the AC position is in aversion
work with adolescents. According to the AC model,
there should be a direct correlation between the ability
of a drug to induce an aversion and the drug’s rewarding effects (given that it is these effects which are
responsible for conditioned avoidance). Interestingly,
for a number of compounds adolescents display greater
place preferences (indicative of the drug’s rewarding
effects) yet weaker taste aversions. Further, Verendeev
& Riley, 2011) have recently demonstrated no correlation between morphine- or amphetamine-induced
taste aversions and place preferences in individual adult
animals trained in a concurrent CTA/CPP procedure.
Thus, it is not clear to what extent (if any) AC underlies the differences reported here between adults and
adolescents in morphine-induced taste aversions.
Independent of the specific mechanism underlying
the age-dependent differences reported here with
morphine, it is clear that adolescents display weaker
morphine-induced aversions than adults, a finding consistent with several other drugs of abuse within this preparation. This effect was also maintained regardless of the
level of deprivation employed during conditioning.
Given that adolescence is characterized by a lack of
behavioral inhibition with regard to risk-taking and
novelty-seeking (Spear, 2000), this relative insensitivity
to the aversive effects of drugs of abuse argues that
adolescence is a period of increased vulnerability to
drug use. It is important to continue to characterize the
drugs for which such differences exist and to determine
the factors that might impact this relative sensitivity, for
example, sex, drug history, stress. An examination of the
neurobiological and neurochemical mediation of this
differential sensitivity to the aversive effects of such
drugs may also be important in understanding any possible individual or genetic susceptibility to abuse. Examination of age differences in the aversive effects of drugs
should parallel those made on drug reward. Such combined assessments may provide greater insights into
vulnerability of use and abuse (see Riley, 2011).
12
Hurwitz, Merluzzi and Riley
NOTES
This work was supported in part by grants from the Mellon
Foundation to A.L.R. and Z.E.H. and a Robyn RaffertyMathias Undergraduate Research Award to A.P.M.
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