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Psychopharmacology
DOI 10.1007/s00213-014-3500-y
ORIGINAL INVESTIGATION
Effect of age on methylphenidate-induced conditioned taste
avoidance and related BDNF/TrkB signaling in the insular
cortex of the rat
B. Bradley Wetzell & Mirabella M. Muller &
Jennifer L. Cobuzzi & Zachary E. Hurwitz &
Kathleen DeCicco-Skinner & Anthony L. Riley
Received: 21 October 2013 / Accepted: 7 February 2014
# Springer-Verlag Berlin Heidelberg 2014
Abstract
Rationale Drug use and abuse is thought to be a function of
the balance between its rewarding and aversive effects, such
that the rewarding effects increase the likelihood of use while
the drug’s dissociable aversive effects limit it. Adolescents
exhibit a shift in this balance toward reward, which may
ultimately lead to increased use. Importantly, recent work
shows that adolescents are also protected from the aversive
effects of many abusable drugs as measured by conditioned
taste avoidance (CTA). However, such effects of methylphenidate (MPH, widely prescribed to adolescents with ADHD)
have not been characterized.
Objectives The effect of age on MPH-induced CTA was
assessed. In addition, MPH-induced changes in brainderived neurotrophic factor (BDNF) activity in the insular
cortex (IC) and central nucleus of the amygdala (CeA), known
to be important to CTA, were examined and related to CTAs in
adolescents and adults.
Methods CTAs induced by MPH (0, 10, 18, and 32 mg/kg)
were assessed in adolescent (n = 34) and adult (n = 33) male
Sprague Dawley rats. Following MPH CTA, IC and CeA tissue
This work was supported by a grant from the Mellon Foundation to ALR
and a Dean’s Graduate Research Grant to BBW.
B. B. Wetzell (*) : M. M. Muller : J. L. Cobuzzi : Z. E. Hurwitz :
A. L. Riley
Psychopharmacology Laboratory, Department of Psychology,
American University, 4400 Massachusetts Avenue NW, Washington,
DC 20016, USA
e-mail: bradley.wetzell@american.edu
A. L. Riley
e-mail: alriley@american.edu
K. DeCicco-Skinner : A. L. Riley
Department of Biology, American University, Washington,
DC 20016, USA
was probed for differences in BDNF and tropomyosin-related
kinase receptor-B (TrkB) using Western blots.
Results Blunted expression of MPH CTA was observed in the
adolescents versus adults, which correlated with generally
attenuated adolescent BDNF/TrkB activity in the IC, but the
drug effects ran contrary to the expression of CTA.
Conclusions Adolescents are protected from the aversive effects of MPH versus adults, but further work is needed to
characterize the possible involvement of BDNF/TrkB.
Keywords Methylphenidate . Adolescent . Age Effect . Rat .
CTA . BDNF . TrkB . Insular cortex . Western blot
Introduction
Evidence from the preclinical model indicates that the adolescent period of development plays a particularly significant
role in drug abuse liability (for a review, see Spear 2013).
Biochemical processes underlying developmental brain
changes during this period result in an enhanced response to
rewarding stimuli (for a review, see Spear 2011), which often
translates to an increased likelihood for drug use among
adolescents (see Doremus-Fitzwater et al. 2010 and
Schramm-Sapyta et al. 2009 for reviews). Although drug
intake is most often associated with reward, it is actually
thought to reflect an affective balance, such that a drug’s
rewarding effects increase, while its aversive effects limit,
the propensity to self-administer (Riley 2011; Wise et al.
1976). Interestingly, these two constructs are dissociable in
that manipulations that affect one often have no effect on the
other (Brockwell et al. 1991; King and Riley 2013), and many
abusable drugs produce both reward and aversion at the same
dose and route of administration (Hunt and Amit 1987; Riley
2011). Thus, variation in self-administration does not
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necessarily indicate a change in reward, as variation in the
drug’s aversive effects may also impact its use and/or abuse.
To this end, recent work has characterized age-dependent
variations in the aversive effects of various abusable drugs
(Doremus-Fitzwater et al. 2010; Schramm-Sapyta et al. 2009)
as measured by conditioned taste avoidance (CTA, a measure
of the aversive effects of a drug; see Freeman and Riley 2009;
Riley and Tuck 1985). Indeed, adolescent rats consistently
display attenuated CTA compared to adults with many drugs
of abuse, including cocaine (Schramm-Sapyta et al. 2006) and
amphetamine (Infurna and Spear 1979) among others
(Anderson et al. 2010, 2013; Cobuzzi et al. 2013; Hurwitz
et al. 2012, 2013; Merluzzi et al. 2013; Schramm-Sapyta et al.
2007; Shram et al. 2006).
One compound that has not been assessed for CTA age
effects is methylphenidate (MPH), the most widely prescribed
medication for attention deficit/hyperactivity disorder in children and young adults (Ritalin®; Volkow et al. 2001).
Interestingly, conditioned place preference (CPP) induced by
MPH is potentiated in adolescent spontaneously hypertensive
rats compared to adults (SHR, believed to model the ADHD
phenotype; see dela Peña et al. 2011 for MPH age
comparisons; see Sagvolden 2000 for a review of the SHR
model). Although this age effect is not evident in the Wistar
outbred strain, adolescents exhibit dose-dependent CPP, while
adult Wistars trend toward an attenuated, non-dose-dependent
response (dela Peña et al. 2011). Additionally, both adolescent
and adult rats self-administer MPH (dela Peña et al. 2011),
with no direct age comparisons reported. MPH produces dosedependent CTA in adult animals (Riley and Zellner 1978;
Wetzell and Riley 2012), and since adolescents display attenuated CTA to other psychostimulants, it is reasonable to
expect that the same will be true for MPH. Such a result could
indicate an enhanced abuse potential for MPH in adolescents
and should be examined further.
Although age differences in CTAs are generally well documented, their biochemical underpinnings have yet to be fully
characterized. Activity-dependent expression of brain-derived
neurotrophic factor (BDNF, a neuropeptide involved in
activity-dependent changes in protein expression related to
synaptic plasticity; see Barki-Harrington et al. 2009; Ohira
and Hayashi 2009) induces long-term potentiation (LTP) of
signaling in the insular cortex (IC; see Castillo and Escobar
2011), which is a central mechanism in the acquisition and
retention of CTA (Castillo and Escobar 2011; Castillo et al.
2006; Escobar and Bermúdez-Rattoni 2000; MoguelGonzález et al. 2008). Further, Ma et al. (2011) found CTAinduced secretion and expression of BDNF along with one of
its target receptors, the tropomyosin-related kinase receptor-B
(TrkB, see Fayard et al. 2005; Fryer et al. 1996) in the central
nucleus of the amygdala (CeA) and IC. Thus, variable signaling in this system may correlate with CTA age differences,
such that adults should exhibit stronger signaling relative to
adolescents, which could help further define age-related differences in the response to drugs of abuse and identify targets
for future research.
Thus, the present study assessed MPH-induced CTA and
related BDNF/TrkB signaling in the CeA and IC in adolescent
and adult rats. Specifically, 67 Sprague Dawley male rats (34
adolescents and 33 adults) were conditioned with three doses
of MPH or vehicle (VEH). Eight hours following the final
CTA test, brain tissue was collected and probed for BDNF,
TrkB and its activated form, phosphorylated TrkB (p-TrkB),
in the CeA and IC using Western blots. It was predicted that
adolescents would display attenuated MPH-induced CTA
compared to adults, which would correlate with agedependent variations in BDNF/TrkB signaling.
Methods
Experiment 1: CTA
Subjects
Sixty-seven experimentally naïve, male Sprague Dawley rats
(Harlan Laboratories, Indianapolis, IN) arrived at the facility on
postnatal day 21 (PND 21). Upon arrival, subjects were grouphoused in polycarbonate bins (23 × 44 × 21 cm, n = 3 per bin)
and maintained on a 12:12 light-dark cycle (lights on at
0800 hours) at an ambient temperature of 23 °C. Subjects were
weighed daily throughout the study, beginning 7 days immediately prior to CTA training. During CTA adaptation, conditioning and testing procedures (see below), animals were temporarily transferred to individual hanging wire-mesh test cages
(24.3 × 19 × 18 cm) located in an adjacent animal testing room.
Minimal use of cages with wire-mesh flooring is recommended
(National Research Council 2011) due to possible development
of foot lesions, which may occur with extended housing in such
conditions (Peace et al. 2001). Therefore, the use of wire-mesh
cages was restricted to experimental procedures, and subjects
were returned to their group-housed bins each day when testing
was complete. All procedures occurred during the light phase,
and unless otherwise stated, food and water were available ad
libitum. The study was approved by the Institutional Animal
Care and Use Committee at American University and followed
the National Research Council’s Guide for the Care and Use of
Laboratory Animals (2011) and the Guidelines for the Care and
Use of Mammals in Neuroscience and Behavioral Research
(2003).
Drugs and solutions
Methylphenidate hydrochloride (MPH, generously supplied
by NIDA) was dissolved in isotonic saline at 10 mg/ml, and
the solution was passed through a 0.2-μm filter to remove
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contaminants before being administered intraperitoneally (IP)
at doses of 10, 18, or 32 mg/kg. Saline vehicle equivolume to
the highest dose of MPH was also filtered before IP administration. Sodium saccharin (Sigma Aldrich) was prepared at
1 g/l in tap water for a 0.1 % (w/v) saccharin solution. All drug
weights are expressed as the salt form, and all drugs and
solutions were prepared daily.
CTA procedure
Adolescents Abbreviated CTA procedures designed to maintain proper growth rates in adolescent animals were utilized as
published previously (Hurwitz et al. 2013). Specifically, 34
subjects were deprived of water for 24 h prior to the start of
habituation, which began on PND 29. Following relocation to
test cages, animals received 45-min access to water presented
in graduated Nalgene tubes, after which they had ad libitum
access to water in their home bins for 23 h. This 2-day cycle,
beginning with deprivation, was repeated three more times to
ensure adaptation to test cage fluid consumption (final day on
PND 36).
For conditioning, animals were again deprived of water on
PND 37 and a novel saccharin solution was presented instead
of water on PND 38. Animals were immediately rank-ordered
according to saccharin consumption and assigned to one of
four groups [0 (n = 9), 10 (n = 8), 18 (n = 8), and 32 (n = 9),
where the group number indicates the dose of MPH], such that
mean saccharin intake among groups was comparable. Within
20 min of saccharin access, subjects were injected with drug
or vehicle and then given ad libitum water access in their
home bins for 23 h. This 2-day procedure was repeated three
more times for a total of four conditioning trials (final day on
PND 44).
For the two-bottle test on PND 45, subjects were given 45min access to two Nalgene tubes (one containing tap water
and the other containing saccharin solution) with placement
counterbalanced to control for positioning effects. Following
fluid access, animals were again injected with drug or vehicle
according to their group to facilitate biochemical analysis (see
below) and returned to their home bins with ad libitum water
access.
Adults The procedures for the adult replicate were identical,
with the following exceptions: Thirty-three subjects were
brought into the facility on PND 21 and maintained on ad
libitum food and water with no manipulations until PND 70
when daily weighing began. Water bottles were removed on
PND 77, and the habituation phase began on PND 78.
Adaptation to the procedure occurred more quickly in the
adults, requiring only two 2-day cycles (final day, PND 81).
The four CTA conditioning trials occurred from PND 82 to
89. Following the first conditioning trial, consumption was
rank-ordered, and group assignments made such that intake
was comparable among groups, yielding four groups [0
(n = 9), 10 (n = 8), 18 (n = 8), and 32 (n = 8) where the
number indicates the dose of MPH administered]. The final
two-bottle test was performed on PND 90.
Experiment 2: BDNF/TrkB protein analysis
Expression of BDNF and TrkB protein levels were assessed
through Western blotting. Eight hours following injections
after the two-bottle test (in accordance with the findings of
Ma et al. 2011), subjects were rapidly decapitated, and brain
tissue was immediately extracted and flash frozen in ice-cold
methylbutane. Four samples from each age and dose group
were randomly selected and cold-sectioned. The CeA and IC
were located by cross-referencing maps from Paxinos and
Watson (2005) and Palkovits and Brownstein (1988) and were
isolated using a variant of the micropunch procedure described by the latter. Samples were placed in 70 μl of room
temperature sucrose lysis buffer (containing 2.2 g sucrose,
2 ml 10 % SDS, and 100 μl 1 M HEPES in 18 ml distilled
water) with protease inhibitors (Thermo Scientific #78442)
and ultrasonically homogenized. After incubating on ice for
an additional 45 min, they were cold-centrifuged for 15 min at
13,500 RPMs.
Western blot analyses were performed as described previously (Kohut et al. 2012). Following electrophoresis and transfer, membranes were blocked in 5 % milk, probed with antiBDNF (Abcam, ab46176; 1:1,000), anti-TrkB (Abcam,
ab51190; 1:1,000), anti-phospho-TrkB antibody (Abcam,
ab75173; 1:2,000), and anti-β-actin as a loading control (Cell
Signaling; 1:2,000; all diluted in 5 % BSA or milk) and then
incubated with secondary antibody (anti-rabbit HRP; Cell
Signaling; 1:2,000). Membranes were stripped, washed, and
re-blocked with milk prior to proceeding to the next primary
antibody. Bands were developed using Pierce West Dura
chemiluminescence substrate (Thermo Scientific) and visualized on a UVP Biosystem Imaging system. Densitometry was
performed using Image-J software (NIH, Bethesda).
Statistical analyses
Since adolescents drank less saccharin than their adult counterparts on the initial conditioning trial (mean of 5.3 vs. 9.1 ml,
respectively), saccharin consumption for each subject on all
subsequent trials was transformed to percent shift from its
own baseline consumption (trial 1) and was analyzed with a
2 × 4 × 4 repeated measures ANOVA with between-subject
factors of age (adolescent or adult) and dose (0, 10, 18, and 32)
and a within-subject factor of trial (1–4). In the case of a threeway interaction, simple effects of trial at each age and dose
(multivariate analysis) and the effects of age at each dose and
trial (univariate analysis) were assessed with Bonferronicorrected multiple comparisons as warranted.
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Results
CTA acquisition and test
a
100
‡
% Shiftfrom Trial 1
50
^
^
0
‡
‡
-50
^
‡
‡
1
2
3
4
Trial
0
b
10
18
100
32
‡
50
0
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^‡
^‡
-50
‡
‡
‡
‡
‡
Percent shifts in saccharin consumption from baseline for
adolescents and adults are represented in Fig. 1a, b, respectively. The 2 × 4 × 4 repeated measures ANOVA revealed
significant effects of age [F (1, 59) = 4.063], dose [F (3, 59) =
18.708], and trial [F (3, 177) = 39.358], as well as an age ×
dose × trial interaction [F (9, 177) = 2.370]. The tests for
simple effects of trial at each age and dose were significant
[adolescents: group 0 F (3, 57) = 7.674, group 10 F (3, 57) =
12.137, group 18 F (3, 57) = 7.736, group 32 F (3, 57) =
9.266; adults: group 0 F (3, 57) = 5.337, group 10 F (3, 57) =
9.302, group 18 F (3, 57) = 8.079, group 32 F (3, 57) =
15.214]. Adjusted multiple comparisons revealed that the
adolescent group 0 drank significantly more saccharin on trial
2 than trial 1 (see Fig. 1a), while there were no differences
from trial 1 to trial 2 for any of the adolescent drug-treated
groups. By trial 3, all of the adolescent drug-treated groups
drank significantly less saccharin than their trial 1 baseline and
groups 18 and 32 maintained significant suppression of saccharin consumption on trial 4. Conversely, all adult MPHtreated groups drank significantly less saccharin than on trial 1
on all subsequent trials (see Fig. 1b). Additionally, group 0
adults drank significantly more on trial 4 than on trial 1. The
analyses of simple effects of age at each dose revealed that
adolescent group 10 drank significantly more saccharin than
adult group 10 on trial 2 [F (1, 59) = 6.695], and adolescent
group 32 exhibited increased consumption compared to their
^
‡
-100
% Shiftfrom Trial 1
On the two-bottle CTA test, saccharin and water consumption were recorded and percent saccharin of total fluid consumption (i.e., saccharin/saccharin + water) was then tested
with a 2 × 4 factorial ANOVA with factors of age (adolescent
or adult) and dose (0, 10, 18, and 32). A two-way interaction
was followed by univariate analyses for simple effects at each
level of age and dose and followed by Bonferroni-corrected
pairwise comparisons as needed.
To assess differences in protein expression, densitometric
data for each probe within each brain region were normalized
to the respective individual’s β-actin result. Normalized
values were divided by the mean for group 0 for that age
group to arrive at the dependent variable, fold change, such
that group 0’s mean fold change for each age group was
always 1. Fold change data were analyzed with a 2 × 4
factorial ANOVA with factors of age (adolescent or adult)
and dose (0, 10, 18, and 32). Given an interaction, simple
effects of age and dose at all levels of each were assessed with
univariate analyses followed by Bonferroni-corrected
pairwise comparisons where indicated. Significance for all
tests was set to α = 0.05.
‡
^
^
-100
1
2
3
4
Trial
Fig. 1 CTA acquisition data for a adolescents and b adults represented as
percent shift from trial 1 ‡p<0.05 from respective trial 1, ^p<0.05
between ages
adult counterparts on trials 2–4 [trial 2: F (1, 59) = 10.685, trial
3: F (1, 59) = 6.011, and trial 4: F (1, 59) = 7.540].
The 2 × 4 factorial ANOVA for percent saccharin consumed during the two-bottle test indicated significant effects
of age [F (1, 59) = 37.665] and dose [F (3, 59) = 61.907],
including an age × dose interaction [F (3, 59) = 5.571; see
Fig. 2]. Tests for effects of dose at each age indicated significant differences [adolescents: F (3, 59) = 23.132; adults:
F (3, 59) = 44.239], and multiple comparisons indicated that
all groups receiving MPH for both ages drank a significantly
lower percentage of saccharin than their respective group 0.
Adolescent groups 10 and 32 drank a significantly higher
percentage of saccharin than their adult counterparts [group
10: F (1, 59) = 35.255; group 32: F (1, 59) = 13.618].
BDNF/TrkB expression
Analyses of the densitometric data from BDNF, TrkB, and pTrkB probes of samples dissected from the CeA in both age
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100
a
1.5
Adolescents
Adults
80
*
60
^
40
*
*
1.0
^
(From Group 0)
Fold Change
% Saccharin
^
*
*
*
*
0.5
20
*
18
32
Adol.
10
0
28 kDa
Adult
0
0.0
*
28 kDa
14 kDa
- act
*
0
Adol.
10
18
10
18
32
14 kDa
Group
Fig. 2 CTA two-bottle test data normalized to percent saccharin of total
fluid consumed (e.g., saccharin/saccharin+water); *p<0.05 from respective group 0, ^p<0.05 between ages
Adult
0
32
MPH Dose Group
Adolescents
Adults
1.5
(From Group 0)
Fold Change
1.0
0.5
0.0
Adol.
0
10
18
10
18
32
28 kDa
Adult
28 kDa
14 kDa
- act
14 kDa
Adol.
Adult
0
32
MPH Dose Group
c
3
^
(FromGroup0)
Fold Change
*
^
2
*
^
1
*
*
Adol.
0
0
10
18
10
18
32
220kDa
Adult
Fig. 3 Densitometry data for BDNF bands at a 14 kDa, b 28 kDa, and c„
220 kDa; *p<0.05 from respective group 0, ^p<0.05 between ages
b
220 kDa
- act
groups revealed no significant differences between age or
drug groups (data not shown). The BDNF probe in samples
dissected from the IC in both age groups resulted in two bands
at the expected molecular weights of 14 and 28 kDa (see
Figs. 3a, b). Pro-BDNF is a homodimer weighing 28 kDa,
while mature BDNF is a 14-kDa monomer, each of which is
capable of binding to, and activating, target receptor proteins
(Fayard et al. 2005; Kolbeck et al. 1994). However, a third
band also appeared at approximately 220 kDa in the IC
samples of each age group but not in the CeA (see Fig. 3c).
Similar bands at this weight have been reported from probes
for p75, a second receptor protein to which BDNF may bind,
yet they have remained unexplained (Djakiew et al. 1994;
Pflug et al. 1992). After conducting additional probes for
potential protein candidates that might complex with BDNF
(e.g., p75 and Kidins220; see discussion below), tissue lysates
were twice more processed and re-probed with the original
antibodies, yielding the same results.
The 2 × 4 factorial ANOVA for fold change in 14 kDa
BDNF indicated no main effect of age or dose, but an age ×
dose interaction was present [F (3, 24) = 3.171; see Fig. 3a].
The test for simple effects of dose at each age indicated
significant differences for both [adolescents: F (3, 24) =
20.759; adults: F (3, 24) = 7.177], and the adjusted multiple
comparisons revealed that groups 18 and 32 in both age
groups exhibited lower expression of 14 kDa BDNF than their
respective group 0 controls. Analyses of age at each dose
showed that adolescent group 32 expressed less 14 kDa
BDNF than their adult counterparts [F (1, 24) = 9.773], while
no other comparisons reached significance. The 2 × 4 factorial
ANOVA for fold change in 28 kDa BDNF between age and
Adol.
Adult
0
MPH Dose Group
32
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1.5
1.0
(From Group 0)
Fold Change
a
0.5
0
10
18
10
18
32
Adult
92 kDa
92 kDa
-a c t
Adol.
0.0
Adol.
Adult
0
32
MPH Dose Group
Adolescents
Adults
b
2 .0
^
1 .5
^
^
(From Group 0)
Fold Change
dose groups indicated no main effect of age or dose nor
an age × dose interaction (see Fig. 3b).
For the 220-kDa BDNF band, the 2 × 4 factorial ANOVA
for fold change between age and dose groups indicated no main
effect of age or dose, with an age × dose interaction
[F (3, 24) = 32.954; see Fig. 3c]. The tests of dose at
each age indicated differences for both ages [adolescents: F (3, 24) = 6.296; adults: F (3, 24) = 38.847], and
multiple comparisons revealed that adolescent groups 18 and
32 expressed less 220 kDa BDNF than their respective group
0 (p < .05 for each), while the adult groups 18 and 32
expressed significantly more than the adult group 0. Further,
the univariates for effects of age at each dose indicated that all
adolescent groups treated with MPH expressed significantly
less of the 220-kDa BDNF band than their adult counterparts
[group 10: F (1, 24) = 9.644; group 18: F (1, 24) = 65.936;
group 32: F (1, 24) = 170.874], while there were no age
differences in group 0.
The 2 × 4 factorial ANOVA for fold change in TrkB
expression between age and dose groups indicated no effect
of age, nor dose, nor an age × dose interaction (see Fig. 4a).
The 2 × 4 factorial ANOVA for the activated form of TrkB (pTrkB) revealed no effect of age or dose, but an age × dose
interaction [F (3, 24) = 5.727; see Fig. 4b]. Tests for simple
effects of dose at each age showed that such effects occurred
only in the adolescents [F (3, 24) = 5.743], and multiple
comparisons indicated that adolescent group 32 expressed less
activated TrkB than their respective group 0 (p < .05). Tests
for dose at each age revealed that all adolescent MPH groups
expressed less p-TrkB than their adult counterparts [group 10:
F (1, 24) = 5.865; group 18: F (1, 24) = 19.569; group 32: F
(1, 24) = 29.332].
1 .0
0 .5
*
0 .0
Adol.
Adult
The present study evaluated age effects in CTA induced by
MPH in adolescent and adult animals. In accordance with
previous research, MPH induced robust taste avoidance of
saccharin (Riley and Zellner 1978; Wetzell and Riley 2012),
and in support of our hypothesis, the effect was attenuated in
adolescent animals compared to adults. Adolescents were generally slower to acquire avoidance, as none of the MPH groups
suppressed consumption until trial 3, while all three of the adult
MPH groups had done so by trial 2. Further, the MPH-treated
adults in groups 10 and 32 drank a lower percentage of saccharin on the two-bottle test than their adolescent counterparts.
These results are in line with previous reports of attenuated
adolescent CTA induced by abusable compounds and suggest
that adolescents are similarly protected from the aversive effects of MPH relative to adults (see “Introduction”).
Some explanations for the age-dependent variation of CTA
include that adolescents are generally deficient in mechanisms
0
92 kDa
92 kDa
- act
Discussion
10
18
10
18
32
Adol.
Adult
0
32
MPH Dose Group
Fig. 4 Densitometry data for a TrkB and b p-TrkB bands *p<0.05 from
respective group 0, ^p<0.05 between ages
of memory and learning, have blunted taste reactivity, or
enhanced motivation to drink compared to adults. If a general
adolescent learning deficit were present, then this age group
should exhibit attenuated conditioning in other associative
preparations as well. Yet, CPP induced by many drugs of
abuse are enhanced as a function of increases in adolescent
reward sensitivity (Badanich et al. 2006; Belluzzi et al. 2004;
Philpot et al. 2003; Vastola et al. 2002; Zakharova et al. 2009).
Further, adolescents exhibit comparable CTA to adults when
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induced by emetics that lack reinforcing effects (i.e., lithium
chloride; see Cobuzzi et al. 2013 and Hurwitz et al. 2012 for
discussions on this topic), indicating similar learning, as well
as taste reactivity. Finally, it has been demonstrated that the
differences in taste avoidance induced by morphine (adol <
adults) are unaffected by variations in the deprivation level,
suggesting that, at least for this compound, changes in motivation to drink unlikely impact the strength of avoidance
learning (Hurwitz et al. 2012).
Another possibility for the observed age effects concerns
the possible impact of stress on the acquisition of CTAs.
Specifically, adolescents were less removed from the stresses
related to shipping than the adults. Both groups arrived at the
facility on PND 21, and although the adolescents were
allowed 7 days to acclimate, the adults had close to 50. Such
differential stress may have mediated, in part, the behavioral
differences reported. Although possible, it should be noted
that there is no consistent evidence that stress impacts CTAs
(see Hurwitz et al. 2012 for a discussion) and two direct
assessments of such effects, as a function of age, have found
none (Anderson et al. 2010, 2013). Further work is needed to
conclusively demonstrate a roll for stress, or lack thereof, on
CTA in adolescent versus adult animals.
As well, we hypothesized that CTA age differences would
correlate with changes in IC and CeA BDNF/TrkB activity,
which are associated with memory processes during CTA (Ma
et al. 2011) and are generally considered central to taste
avoidance acquisition and retention (Castillo and Escobar
2011; Castillo et al. 2006; Martínez-Moreno et al. 2011;
Moguel-González et al. 2008). We found no effects of age
or drug on BDNF, TrkB, or p-TrkB in the CeA. However,
there was a drug-dependent decrease in the expression of
mature BDNF (14 kDa) in the IC of both age groups and the
adolescent group 32 exhibited greater attenuation than their
adult counterparts. The same adolescent group also exhibited
a drug-induced decrease in p-TrkB, and expression of this
activated receptor was blunted compared to the adults at all
three doses of MPH. Although these results suggest attenuated
IC BDNF activity in the adolescent animals compared to
adults, in partial support of our hypothesis, the blunted expression of mature IC BDNF induced by MPH is inconsistent
with the dose-dependent CTA in both ages and does not likely
represent a mechanism for CTA age differences.
However, the 220-kDa BDNF results indicate a dosedependent increase in the adults, similar to their expression
of p-TrkB, which is in line with the adult MPH CTA and
consistent with the position that IC BDNF correlates directly
with strength of CTA learning. Further, the dose-dependent
decrease of the adolescent 220-kDa band mirrors that group’s
patterns of mature BDNF and p-TrkB, but still contrasts the
dose-dependent increase in CTA in adolescents (albeit attenuated compared to adults). As stated, similar bands have been
reported with probes for p75 in rat testicular tissue (Djakiew
et al. 1994), as well as human prostate (Pflug et al. 1992),
neither of which has been identified nor explained.
Accordingly, our samples were probed with anti-p75
(Abcam, ab8874; 1:1,000), yielding no evidence that the
protein was present (data not shown). Additionally, there have
been recent reports of kinase-D-interacting substrate of
220 kDa (Kidins220; see Kong et al. 2001) which is known
to complex with TrkB and p75 and modulate their signaling
pathways following activation by BDNF (Chang et al. 2004;
Neubrand et al. 2012). Although there is no evidence of
BDNF binding directly to Kidins220, we also probed with
anti-Kidins220 (Abcam, ab34790; 1:1,000) and again found
no evidence that it was present (data not shown). Our methods
incorporate sodium dodecyl sulfate (SDS) to linearize proteins
and disassemble complexes, yet it is known that some complexes exhibit remarkable stability that can impart SDS resistance (among the more well-characterized of which are
SNARE complexes; see Hayashi et al. 1994). In these cases,
Western probes for any of the constituents of the complex can
result in multiple bands over a wide weight range (Kubista
et al. 2004). Thus, although impossible to interpret presently,
it is conceivable that the 220-kDa band represents BDNF
bound in a stable, as-yet-unidentified complex that is SDS
resistant. However, future work will need to assess other
candidates for a BDNF complex in the 220-kDa range and
determine whether their presence has functional significance.
That we did not see the expected drug effects on CeA
BDNF/TrkB in either age group or in the IC of the adolescents, as reported by Ma et al. (2011), remains unexplained,
but could relate to parametric differences. Ma et al. assessed
secretion and synthesis of BDNF and TrkB after two conditioning trials, whereas we conducted five total trials. From
trials 1 to 3, suppression of saccharin consumption was undergoing dramatic changes in both ages. Yet, from trials 3 to 4,
suppression became asymptotic, suggesting that learning had
occurred between trials 1 and 3. Thus, by the time of our
assessment following trial 5, BDNF/TrkB activity related to
CTA learning in the CeA and IC may have subsided. Future
research will need to assess age differences in BDNF/TrkB
while CTA learning is still occurring and whether the activity
is dose-dependent.
In conclusion, we assessed age differences in the expression of MPH CTA, as well as related BDNF/TrkB signaling in
the IC and CeA. Our results demonstrate a blunted MPH CTA
in adolescents compared to adults, which correlated with
generally reduced IC BDNF/TrkB activity. However, while
adult BDNF/TrkB activity appeared consistent with their behavioral results, MPH induced a dose-dependent attenuation
of protein expression in the adolescent animals, contrary to
their expression of CTA. Thus, the present results do not likely
represent a general mechanism for CTA age differences.
Perhaps the most parsimonious explanation for the present
CTA results is that adolescents are simply less sensitive to the
Author's personal copy
Psychopharmacology
aversive effects of MPH. The interaction of rewarding and
aversive effects of abusable drugs likely results in a complex
cluster of subjective stimuli that mediates the overall affective
response and influences the propensity for continued use (see
Verendeev and Riley 2012 for a recent review and
interpretation for mechanisms of CTA with abusable drugs).
For this reason, it is important to establish the physiological
mediation of CTA age differences and establish targets for
treatment and prevention of compulsive drug use in
adolescents.
Acknowledgements The authors would like to thank Nick Watson and
Gervaise Henry from the Department of Biology, American University
for their input and technical assistance with the Western blot analyses.
Conflict of interest There are no conflicts of interest.
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