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Amylin Receptor Signaling in the Nucleus Accumbens
Negatively Modulates l-opioid-Driven Feeding
Sarah K Baisley
1
and Brian A Baldo*
,1,2
1
Neuroscience Training Program, University of Wisconsin-Madison, Medical Sciences Center, University Ave Madison, WI, USA;
2
Department of
Psychiatry University of Wisconsin-Madison, Research Park Blvd, Madison, WI, USA
Amylin is a peptide co-secreted with insulin that penetrates into the brain, and produces satiation-like effects via actions in the brainstem,
hypothalamus, and mesencephalon. Little is known, however, about the effects of amylin in the nucleus accumbens shell (AcbSh), where
a circumscribed zone of intense amylin receptor (AMY-R) binding overlaps reported mappings of a ‘hotspot’ for m-opioid receptor
(m-OR) amplification of food reward. Here, the ability of intra-AcbSh AMY-R signaling to modulate m-OR-driven feeding was explored.
Amylin (1–30 ng) was administered with the m-OR agonist, D-Ala2, N-MePhe4, Gly-ol]-enkephalin (DAMGO) (0.25 mg), directly into the
AcbSh of ad libitum-maintained rats. Amylin dose-dependently reversed DAMGO-induced hyperphagia; 3 ng of amylin reduced
DAMGO-mediated feeding by nearly 50%. This dose was, however, completely ineffective at altering DAMGO-induced feeding in the
anterior dorsal striatum. Intra-AcbSh amylin alone (3–30 ng) modestly suppressed 10% sucrose intake in ad libitum-maintained rats, and
chow in food-deprived rats, but only at the 30-ng dose. This result indicates that reversal of AcbSh DAMGO-induced feeding at a 10-fold
lower dose was neither due to malaise nor motoric impairment. Finally, intra-AcbSh infusion of the AMY-R antagonist, AC187 (20 mg),
significantly attenuated the ability of prefeeding to suppress DAMGO-induced food intake, with no effects in non-prefed rats. Hence,
AMY-R signaling negatively modulates m-OR-mediated appetitive responses at the level of the AcbSh. The results with AC187 indicate
that endogenous AMY-R transmission in the AcbSh curtails opioid function in the postprandial period, suggesting a novel pathway for
peripheral-central integration in the control of appetitive motivation and opioid reward.
Neuropsychopharmacology (2014) 39, 3009–3017; doi:10.1038/npp.2014.153; published online 13 August 2014
INTRODUCTION
Amylin, a 37-amino-acid peptide that belongs to the calci-
tonin gene-related peptide (CGRP) family (van Rossum et al,
1997), is co-secreted with insulin from pancreatic
beta cells in coordination with prandial stimuli (Butler
et al, 1990; Moore and Cooper, 1991; Ahren and Sundler,
1992). Once secreted, amylin modulates insulin’s effects
on glycogen synthesis and glucose uptake in muscle, and
therefore has an important role in glycemic control (Singh-
Franco et al, 2011). In addition to these metabolic effects,
amylin also modulates food intake via actions at multiple
levels of the central nervous system (CNS). Amylin penetrates
into the brain at least as well as insulin, and accumulates in
sites throughout the neural axis (Banks and Kastin, 1998).
Because CNS amylin receptors (AMY-Rs) show regional
differences and localization to discrete neural pathways and
structures, it is hypothesized that amylin and related peptides
have a role in neuroregulation (Beaumont et al, 1993; Sexton
et al, 1994; van Rossum et al, 1994; Christopoulos et al, 1995).
Accordingly, AMY-R ligands cause a satiation-like suppres-
sion of feeding when infused into the lateral ventricle, third
ventricle, hypothalamus, and ventral tegmental area (VTA)
(Chance et al, 1991; Morley and Flood, 1991; Bouali et al,
1995; Lutz et al,1998a;Rushinget al, 2000; Mietlicki-Baase
et al, 2013). Perhaps the most extensively studied site for
feeding-modulatory actions of amylin is the area postrema;
blockade of area postrema AMY-Rs and lesions specific to
the area postrema both attenuate the anorectic effect of
systemically administered amylin (Lutz et al, 1998b, 2001;
Mollet et al, 2004).
Less is known about feeding-modulatory effects of
amylin in the telencephalon, despite the fact that one of
the densest concentrations of high-affinity amylin-binding
sites, and expression of component genes encoding the
high-affinity AMY-R (Poyner et al, 2002) is found in the
medial nucleus accumbens shell (AcbSh) (Sexton et al, 1994;
van Rossum et al, 1994; Baisley et al, 2014). This zone of
intense AMY-R binding conforms remarkably well with the
circumscribed medial AcbSh area from which intense
feeding responses are elicited by GABA or m-opioid receptor
(m-OR) stimulation (Bakshi and Kelley, 1993; Stratford
and Kelley, 1997; Zhang and Kelley, 2000). Moreover,
the reported ‘hotspot’ for amplification of hedonic taste
reactions by m-OR stimulation (Pecina and Berridge, 2005)
*Correspondence: Dr BA Baldo, Department of Psychiatry, University
of Wisconsin-Madison, School of Medicine and Public Health, 6001
Research Park Blvd, Madison, WI 53719 USA. Tel: +1 608 263 4019,
Fax: +1 608 265 3050, E-mail: babaldo@wisc.edu
Received 20 March 2014; revised 16 June 2014; accepted 17 June
2014; accepted article preview online 24 June 2014
Neuropsychopharmacology (2014) 39, 3009 –3017
&
2014 American College of Neuropsychopharmacology. All rights reserved 0893-133X/14
www.neuropsychopharmacology.org
overlaps the AMY-R distribution. Because of this overlap,
AcbSh-localized AMY-Rs are well-positioned to modulate
food intake and hedonic taste reward by interacting with the
m-opioid system.
To date, only one study (Baldo and Kelley, 2001) has
investigated the role of AcbSh-localized AMY-Rs in control-
ling feeding behavior; this study showed that exogenously
administered amylin in the 30–100 ng range suppressed
feeding. Nevertheless, the interaction of AMY-Rs with other
Acb-localized neuromodulator systems, and, importantly,
the role of endogenous Acb AMY-R signaling in modulating
feeding behavior, remain unknown. Here, interactions
between AMY-Rs and m-ORs were studied, both in the
AcbSh where dense AMY-R binding is found, and the
anterior dorsal striatum (ADS), lacking high-affinity AMY-
R binding but where m-ORs also modulate feeding (Bakshi
and Kelley, 1993; DiFeliceantonio et al, 2012). We also
examined the effects of AMY-R blockade on m-OR-driven
feeding, during either a food-deprived state or immediately
after a prefeeding session (when circulating amylin levels
are high) (Alam et al, 1992; Arnelo et al, 1998), to explore
whether an endogenous ‘tone’ of AMY-R signaling at the
level of the AcbSh interacts with the behavioral functions
of m-ORs.
MATERIALS AND METHODS
Subjects
Subjects in all experiments were male Sprague-Dawley rats,
obtained from Harlan (Madison, WI), weighing 300–325 g
upon arrival at the laboratory. The rats were pair-housed
in clear polycarbonate cages (9.5-inch width 17-inch
length 8-inch height), with cob bedding, in a light- and
temperature-controlled vivarium. Animals were maintained
under a 12 : 12-h light–dark cycle (lights on at 7 : 00 AM).
Food and water were available ad libitum, except as
indicated for the various experiments. Animals were
handled daily to reduce stress. Testing occurred between
1200–1800 h. All facilities and procedures were in accor-
dance with the guidelines regarding animal use and care
put forth by the National Institutes of Health, and were
supervised and approved by the Institutional Animal
Care and Use Committee of the University of Wisconsin.
Surgical Procedure
Rats (weighing 300–325 g) underwent stereotaxic surgery
under isoflurane anesthesia according to standard techni-
ques (Perry et al, 2009). Bilateral stainless-steel cannulae
(10-mm long, 23 gauge) were aimed bilaterally either
at the AcbSh or at the ADS and anchored in place with
dental acrylic (New Truliner, Skokie, IL) and skull screws
(Plastics One, Roanoke, VA). Surgeries were conducted with
the nosebar set to 3.3 mm below interaural zero. For the
AcbSh, the coordinates from bregma were: þ3.2-mm
anteroposterior (AP); ±1.0-mm lateromedial (LM); and
5.2-mm dorsoventral (DV) (with injectors extending an
additional 2.5 mm beyond cannulae tips for a final DV
coordinate of 7.7). For ADS surgeries, the coordinates
from bregma were: þ1.6 mm AP; ±2.4 mm LM; and
1.7 mm DV (with injectors extending an additional
2.5 mm beyond cannulae tips for a final DV coordinate
of 4.2). Wire stylets (10-mm long, 30 gauge) were placed
in the cannulae to prevent blockage. Animals were given an
intramuscular injection of penicillin (0.3 ml of a 300 000 U/
ml suspension; Phoenix Pharmaceuticals, St Joseph, MO),
placed in a warm recovery cage, returned to their home
cages on awakening, and given a recovery period of no o5
days (with daily health checks) before behavioral testing
commenced.
Drugs and Microinfusions
Amylin (Bachem, Torrance, CA) and ([D-Ala2, N-MePhe4,
Gly-ol]-enkephalin) (DAMGO) (Bachem) were dissolved in
sterile isotonic saline, whereas AC187 (Tocris Bioscience,
Ellisville, MO) was dissolved in sterile H
2
O. The 0.25 mg/
0.5 ml/side dose of DAMGO was chosen because it has been
shown to elicit robust feeding in satiated rats (eg, Perry
et al, 2009). The dose of the AMY-R antagonist AC187 (Hay
et al, 2005) was chosen because in our laboratory it altered
prepulse inhibition upon intra-AcbSh infusion (Baisley et al,
2014). In other literature, an AC187 dose of 30 mg but not
10 mg delivered into lower levels of the CNS increased food
intake in rats (Lutz et al, 1997; Mollet et al, 2004). Hence, the
present experiment used an AC187 dose of 20 mg/side. All
three drugs were infused directly into specific brain regions
in accordance with the experimental designs. For micro-
infusions, injectors (connected via tubing to a microdrive
pump) extended 2.5 mm past cannulae tips, and delivered
drugs at 0.32 ml/min over 1 min 33 s, with a 1-min post-
infusion period before reinsertion of stylets and placement
of rats into testing chambers.
Experimental Design
In all experiments, 30-min free-feeding test sessions were
conducted in wire-bottom polycarbonate cages with rat
chow pellets and water available, as previously described
(Baldo and Kelley, 2001). Prior to all the experiments, rats
were habituated to the testing cages to minimize stress.
Testing occurred on every other day.
Interactions between Amylin and DAMGO in the AcbSh
Effects of amylin were tested in two dose ranges (0, 1, 3 ng
and 0, 10, 30 ng), in separate groups of rats (n¼7 for the
low-dose range; n¼10 for the high-dose range) to minimize
the number of infusions per experiment. For both experi-
ments, rats were surgically implanted with cannulae aimed
at AcbSh. Rats were infused with saline or 0.25 mg/0.5 ml
DAMGO into the AcbSh 10 min before testing. Five minutes
before testing, rats received a second infusion of saline or
amylin into the AcbSh. DAMGO dose and amylin dose were
both within-subjects factors; hence, each rat received all
amylin doses with and without DAMGO (for a total of six
infusions). Treatments were counterbalanced across test
days according to Latin-square designs.
Interactions between Amylin and DAMGO in the ADS
The same basic protocol as Experiment 1 was followed,
except rats (n¼6) were implanted with cannulae aimed at
Intra-accumbens amylin/opioid interactions
SK Baisley and BA Baldo
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Neuropsychopharmacology
the ADS instead of the AcbSh. ADS coordinates were chosen
based on prior literature describing a m-opioid-sensitive
zone dorsal to the Acb (Bakshi and Kelley, 1993a;
DiFeliceantonio et al, 2012). Rats received a total of four
infusions.
Effects of Intra-AcbSh Amylin (Without DAMGO) on
Palatability- and Hunger-Induced Feeding
Eight rats were used to test the effects of intra-AcbSh
amylin upon sucrose intake in ad libitum-maintained rats.
Prior to experimentation, the rats were exposed daily for
2 days to a 10% sucrose solution until they consumed
a consistent amount over two 30-minute test sessions
spaced 4 h apart. Once stable intake was achieved, rats were
given intra-AcbSh infusion of amylin (0, 3, 10, 30 ng/0.5 ml/
side) administered according to a Latin-square design.
Amylin was infused 5 min before testing, whereupon rats
were placed into the testing cages for 30 min with free
access to chow, water, and a bottle containing 10% sucrose.
Seven of the eight rats in this experiment also underwent
testing for intra-AcbSh amylin effects on hunger-driven
chow intake. In this part of the experiment, rats were food-
deprived for 18 h prior to each testing period, given intra-
AcbSh amylin (0, 3, 10, 30 ng/0.5 ml) infusions, and placed
into the testing cages for 30 min with rat chow and water
present. The two experiments (sucrose intake and hunger-
driven chow intake) were performed in a counterbalanced
order, with half the rats receiving sucrose first, and the
other half, hunger/chow intake first (for a total of eight
infusions).
Effects of AC187 on DAMGO-Induced Feeding, With or
Without Prefeeding
Seven rats were surgically prepared with cannulae aimed at
the AcbSh. After recovery, rats underwent behavioral testing
every other day for a total of eight test days. All rats were
food-deprived for 18 h before each testing day; however, on
each interim testing-free day, they had free access to food.
On each testing day, rats were either given a 30-min
‘prefeeding’ session, or given no prefeeding session, where-
upon they received intra-AcbSh infusions of DAMGO and
AC187. DAMGO (0, 0.25 mg/0.5 ml) was infused bilaterally
into the AcbSh 10 min before testing, followed 5 min later
by an infusion of AC187 (0, 20 mg/0.5 ml). For rats exposed
to the prefeeding session, infusions were given after the
prefeeding session, and the time between the prefeeding and
testing session was 15 min. The environmental contexts for
prefed and non-prefed rats were identical. Following drug
infusions, both prefed and non-prefed rats were tested in a
30-min feeding sessions. Each rat received all treatment
combinations (mock/mock, DAMGO/mock, mock/AC187,
DAMGO/AC187) under both prefed and non-prefed condi-
tions, according to a within-subjects, Latin-square design.
‘Mock’ infusions were used to limit the number of needle
insertions into the tissue of the AcbSh and therefore reduce
tissue damage. Ten-millimeter injectors that did not
protrude beyond the ends of the guide cannulae were
lowered into the cannulae for 2 min and 33 s to mimic a
saline infusion. Not including mock infusions, each rat
received a total of eight infusions.
Data Analysis
Multifactor ANOVAs were used for all experiments.
Significant main effects or interactions were followed by
Fisher’s PLSD post hoc. Experimenters blind to data and
treatments confirmed injector placements in Nissl-stained
sections. Final sample sizes exclude rats with placements
outside of target sites.
RESULTS
Figure 1 depicts histological verification of intra-tissue
injection placements. One rat was removed from Experi-
ment 1 owing to placements that fell outside of the targeted
region. Representative photomicrographs of injector place-
ments into the AcbSh and ADS of cannulated animals reveal
that cannulae and injector tracks are clearly visible with no
unusual damage to the targeted areas. For Acb placements,
although in some cases we would notice some damage to the
lateral ventricles induced by the guide cannulae, injector
tips were found always to be located within the cellular
neuropil of the AcbSh (not in the ventricles).
Amylin Potently Reduced Intra-AcbSh DAMGO-Induced
Feeding
As shown in Figure 2, DAMGO significantly elevated
feeding in both the low-dose and high-dose DAMGO/
amylin interaction studies (main effect of DAMGO:
F(1, 6) ¼50.7, Po0.001 for low-dose study; F(1, 9) ¼17.9,
Po0.01 for high-dose study). Post hoc comparison among
means with Fisher’s PLSD test confirmed that DAMGO-
associated levels of food intake were significantly elevated
relative to saline or to any of the amylin-alone doses
(Ps¼0.0001–0.05).
In both dose ranges tested, amylin significantly attenu-
ated DAMGO-induced hyperphagia (DAMGO amylin
interactions: F(2, 12) ¼4.8, Po0.05 for low-dose study;
F(2, 18) ¼6.6, Po0.01 for high-dose study). Post hoc
comparison among means revealed specific differences
between DAMGO/saline and DAMGO/amylin-3 ng, DAM-
GO/amylin-10 ng, and DAMGO/amylin-30 ng dose-combi-
nations (Figure 2a and b). Note that these doses of amylin
did not suppress feeding when tested in the absence of
DAMGO, as indicated by the lack of significant differences
between vehicle-treated rats and any of the amylin-alone
doses (although there was a small, nonsignificant trend at
the highest dose, 30 ng). Moreover, amylin (either alone or
in combination with DAMGO) did not affect water intake in
either the high-dose or low-dose experiment, as evidenced
by the lack of amylin main effects or amylin DAMGO
interactions (Fs¼0.23–2.5, not significant (NS)). Hence, the
potent reversal of DAMGO-driven feeding by amylin,
particularly at the low, 3-ng amylin dose, was unlikely the
result of nonspecific motor impairment or malaise. It
should be noted that for the group that received lower
doses of amylin, baseline saline/saline and DAMGO/saline
feeding values were higher relative to those for the group
that received higher doses of amylin. However, there
were no systematic differences in injector tip placements
or methodology across groups. These differing values may
Intra-accumbens amylin/opioid interactions
SK Baisley and BA Baldo
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Neuropsychopharmacology
have been due to normally occurring, between-cohort
differences across separate groups of rats.
Amylin Did Not Alter Intra-ADS DAMGO-Induced
Feeding
It has been shown that, outside the Acb, a zone within the
ADS also subserves m-opioid-driven feeding (Bakshi and
Kelley, 1993; DiFeliceantonio et al, 2012). We replicated this
observation, obtaining a main effect of DAMGO in the ADS
(F(1, 5) ¼39.749, Po0.01) on food intake (see Figure 2b,
inset). In contrast to the AcbSh, there was no significant
main effect of 3-ng amylin in the ADS on food intake nor on
DAMGO amylin interaction (Fs¼1.5–2.0, NS). Amylin
also failed to alter water intake, as evidenced by lack of
an amylin main effect or amylin DAMGO interaction
(Fs¼0.6–0.7, NS). Therefore, the same dose of amylin that
reduced the effects of DAMGO on food intake by nearly
50% in the AcbSh was ineffective at reducing DAMGO-
induced feeding in the ADS.
In a Higher Dose Range, Intra-AcbSh Amylin Modestly
Decreased Hunger- or Palatability-Induced Feeding
(Without DAMGO)
There was no main effect of AcbSh amylin on sucrose intake
(F(3, 21) ¼1.9, NS), although a directed contrast showed a
significant difference between the saline condition and the
Amylin 30-ng condition, with the Amylin 30-ng condition
slightly suppressing sucrose intake (Po0.05, Figure 3a).
However, amylin failed to alter water intake in this experi-
ment (F(3, 21) ¼0.7, NS). AcbSh amylin had a significant
main effect on chow intake in food-deprived rats (F(3, 18) ¼
4.2, Po0.02) (see Figure 3b). Post hoc tests showed a
Figure 1 Injector placements for accumbens shell (AcbSh) and anterior dorsal striatum (ADS)-cannulated animals. Photomicrographs at the top of each
column show injector placements into the AcbSh (left) and ADS (right). Arrows indicate location of injector tips. Below the photomicrographs, line drawings
of coronal sections (with position of each section given in mm from bregma) show injection sites from rats with bilateral placements in AcbSh and ADS.
Each injector tip placement is represented by a dot color-coded by experiment; red and orange represent low-dose and high-dose amylin vs
DAMGO experiments, respectively; green represents the ADS study; blue represents hunger- and palatability-driven feeding; violet represents the AC187/
prefeeding study. ac, anterior commissure; cc, corpus callosum; LV, lateral ventricle. Line drawings were adapted from the atlas of Paxinos and Watson
(2007), with permission.
Intra-accumbens amylin/opioid interactions
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Neuropsychopharmacology
significant difference between the saline and amylin 30-ng
conditions (Po0.01), but not between saline and other
amylin doses. This was the only experiment in which
amylin affected water intake (F(3, 18) ¼3.3, Po0.05),
producing a significant (50%) decrease at the 30-ng dose
(Po0.008). No other dose significantly altered water intake.
These results further indicate that the reversal of DAMGO-
induced feeding by substantially lower amylin doses
(as observed in the aforementioned experiments) was not
the consequence of a nonspecific motivational or motoric
impairment.
Intra-AcbSh AMY-R Blockade Significantly Reversed
the Ability of Prefeeding to Suppress DAMGO-Induced
Food Intake
As expected, food-deprived rats that were given a 30-min
chow prefeeding session 15 min prior to the 30-min chow
testing session ate less than rats that were not prefed (main
effect of prefeeding: F(1, 6) ¼24.8, Po0.003). Also, DAMGO
had a significant main effect on food intake in both prefed
and non-prefed rats (F(1, 6) ¼268.2, Po0.0001). Again,
as expected, DAMGO-induced hyperphagia was lower after
prefeeding (Po0.0001, Figure 4). There was a significant
interaction between DAMGO and the AMY-R antagonist,
AC187 (F(1, 6) ¼6.1, Po0.05). Comparisons among means
revealed a significant difference between the prefed/
DAMGO condition compared with the prefed/DAMGO/
AC187 condition (Po0.05), with rats in the latter condition
eating more, thus demonstrating that blocking AMY-Rs
partly reverses the ability of prefeeding to diminish
m-opioid-driven food intake (Figure 4). Interestingly,
AC187 did not augment feeding in rats not treated with
DAMGO, suggesting that the modulatory effect of endo-
genous AcbSh AMY-R signaling exhibits some specificity
for excessive, mu-opioid-driven appetitive responses. For
additional means comparisons, see Figure 4 legend. For
water intake, there was no significant main effect of AC187,
AC187 DAMGO interaction, or feeding-status AC187
DAMGO interaction (Fs¼0.02–3.2, NS).
To explore the possibility of carry-over effects arising
from repeated exposure to food-restriction over the course
of the experiment, we conducted directed comparisons with
t-tests on sub-cohorts of rats receiving various treatments
either in the first half (days 1–4) or second half (days 5–8)
of the experiment (recall that the order of treatments was
counterbalanced across subjects). The following treatments
were analyzed with regard to possible differences in the first
vs second half: DAMGO, DAMGO þprefeeding, DAMGO þ
AC187, DAMGO þAC187 þprefeeding. These comparisons
revealed no effect of treatment order (ts ¼0.12–0.9, NS),
indicating a lack of carry-over effects over the duration of
the experiment.
DISCUSSION
These results show for the first time a potent modulatory
influence of AMY-R signaling on m-OR-mediated responses
at the level of the AcbSh. Our results demonstrate that
stimulating AMY-Rs with exogenously administered amylin
strongly reduces m-OR agonist-induced feeding at doses
considerably lower than those required to even modestly
diminish either hunger-associated chow intake or palatable
feeding (sucrose drinking). Moreover, blockade of AMY-Rs
partly reversed the ability of prefeeding to suppress intake
engendered by intra-AcbSh DAMGO. Together, these
results reveal a potent negative modulation of m-ORs by
both exogenous and endogenous AMY-R signaling, and
show for the first time a role of endogenous AMY-R ligands
in post-meal-feeding modulation at the level of the AcbSh.
The reversal of DAMGO-associated feeding seen in the
present study ranks among the most potent of the
behavioral effects of amylin obtained from anywhere in
the brain. The lowest dose of exogenously administered,
intra-AcbSh amylin to significantly reduce DAMGO-driven
feeding was 3 ng/side, or 6 ng/rat (1.52 pmol/rat). This dose
is similar to that required to suppress feeding upon infusion
into the third ventricle, immediately adjacent to the medial
basal hypothalamus (1 pmol/rat; Rushing et al, 2000), and
Figure 2 (a) The effects of intra-accumbens shell (AcbSh) amylin
(Vehicle (Veh), 1, or 3 ng) on chow intake elicited by intra-AcbSh DAMGO
(Veh or 0.25 mg). ***Po0.001 compared with Veh/Veh.
þþ
Po0.01
compared with Veh/DAMGO. Inset: Interaction between DAMGO (Veh
or 0.25 mg) and amylin (Veh or 3 ng) upon infusion of both compounds into
the anterior dorsal striaum (ADS). **Po0.01, main effect of DAMGO.
(b) Interaction between higher doses of amylin (Veh, 10, or 30 ng) and
DAMGO (Veh or 0.25 mg) upon infusion of both compounds into the
AcbSh. ***Po0.01, compared with Veh/Veh.
þ
Po0.05,
þþþ
Po0.001
compared with Veh/DAMGO. All testing sessions were 30-min long.
Error bars depict one SEM.
Intra-accumbens amylin/opioid interactions
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Neuropsychopharmacology
even lower than the dose required to reduce feeding in the
area postrema, where 10 pmol/rat amylin is effective but
1 pmol/rat is not (Mollet et al, 2004). We also found that the
3-ng/side amylin dose, which robustly suppressed DAMGO-
induced feeding in the AcbSh, was completely ineffective
at altering DAMGO-driven feeding in the ADS. It has been
shown that m-OR stimulation outside the Acb, in select
dorsal striatal regions, increases feeding (Bakshi and Kelley,
1993a; DiFeliceantonio et al, 2012). However, these striatal
territories possess neither AMY-R binding nor expression
of AMY-R-component genes (Sexton et al, 1994; van
Rossum et al, 1994; Baisley et al, 2014). Therefore, our
results indicate that DAMGO-induced hyperphagia is only
reduced when amylin is infused into striatal regions rich in
AMY-R receptors, suggesting that targeting this receptor
may represent a mechanism for modulating opioid effects in
the ventral striatum specifically.
Interestingly, we found that intra-AcbSh amylin was
considerably less potent in its modulation of sucrose
drinking, compared with its effects on DAMGO-driven
feeding; a 30-ng amylin dose was required to produce a
small reduction in sucrose intake, 10-fold higher than that
required to significantly reverse DAMGO-associated feed-
ing. The 30-ng dose is within the parameters reported in the
only prior study of intra-Acb amylin infusion on hunger-
associated chow intake (Baldo and Kelley, 2001), and is
also consistent with results shown in the present study
for hunger-driven feeding. Considering the evidence that
m-opioid signaling in the Acb robustly modulates palatable
feeding (Zhang and Kelley, 1997; Pecina and Berridge, 2005;
Woolley et al, 2006), our initial hypothesis was that
amylin would reverse sucrose intake in a dose range closer
to that observed for the negative modulation of DAMGO
effects. It is worth considering, however, that whereas
intra-AcbSh DAMGO infusions affect m-ORs only in that
structure, sucrose drinking may recruit m-opioid transmis-
sion in multiple redundant sites (Koch et al, 1995; Kim et al,
2004; Smith and Berridge, 2007; Denbleyker et al, 2009).
Therefore, amylin actions (in the dose range tested) in the
AcbSh may not be sufficient to reduce sucrose solution
intake beyond the modest degree seen here. Accordingly,
Kelley et al (1996) found that intra-Acb infusions of
naloxone or naltrexone effectively reduced sucrose drink-
ing, but only by about 20%. Moreover, whereas intra-AcbSh
naloxone did not significantly reduce chow intake, there
was a trend towards a reduction of about 15%. Hence, the
present results with amylin are not inconsistent with these
opioid antagonist findings, in the sense that both intra-Acb
stimulation of AMY-Rs, and blockade of opioid receptors,
reduced modestly, but did not eliminate, both sucrose
intake and hunger-driven feeding.
To explore the role of endogenous AMY-R signaling, we
tested the ability of prefeeding to suppress AcbSh DAMGO-
induced hyperphagia either with or without intra-AcbSh
infusions of the AMY-R antagonist, AC187 (Hay et al, 2005).
Intra-AcbSh AC187 significantly reversed the ability of
prefeeding to suppress DAMGO-induced food intake;
however, this treatment did not alter food intake in non-
DAMGO-treated rats, nor did it elevate DAMGO-induced
feeding in non-prefed rats. These results suggest some
Figure 3 (a) The effects of intra-accumbens shell (AcbSh) amylin (Amy), (vehicle (Veh), 3, 10 or 30 ng) on intake of a 10% sucrose solution. *Po0.05,
compared with Veh condition. (b) Effects of intra-AcbSh Amy (Veh, 3, 10, or 30 ng) in 18-h food-deprived rats during a 30-minute testing session. **Po0.01
compared with Veh condition. DAMGO was not given in either experiment. All testing sessions were 30-min long. Error bars depict one SEM.
Figure 4 The effects of intra-accumbens shell (AcbSh) infusions of
DAMGO (0.25 mg) plus AC187 (20 mg) combinations on chow intake in
grams (g) during 30 min testing sessions. All rats were food-deprived for
18 h. Non-prefed rats were given either drug or ‘mock’ infusions (see text)
directly before the 30 min feeding test session. Prefed rats ate chow in a
30 min prefeeding session, were given drug infusions, and then were tested
in a second 30-min feeding session. See text for further methodological
details. Values represent means±SEM. *Po0.05, ***Po0.001 compared
with Non-Prefed/DAMGO/Mock condition.
þ
Po0.05 between the
Prefed/DAMGO/Mock and Prefed/DAMGO/AC187 conditions.
Intra-accumbens amylin/opioid interactions
SK Baisley and BA Baldo
3014
Neuropsychopharmacology
degree of specificity of AMY-R modulation for m-opioid
function. One possible explanation for these effects is
that the AMY-R ligands that negatively modulate m-OR
responses fluctuate according to prandial stimuli, with the
highest levels occurring in the postprandial period. One
candidate ligand is peripherally secreted amylin, which is
co-released with insulin in response to feeding and
macronutrient flux (Ogawa et al, 1990; Arnelo et al, 1998).
According to this hypothesis, prefed rats could have higher
amylin levels than the non-prefed rats and this elevated
amylin ‘tone’ may underlie the reduction of opioid-driven
feeding in the early postprandial period. Given that the non-
prefed, food-deprived rats likely had lower levels of amylin,
the lack of AC187 effect in non-prefed rats (DAMGO-treated
or otherwise) could reflect a paucity of endogenous ligand
in the Acb, and, consequently, negligible levels of endo-
genous AMY-R signaling to block. The question arises,
however, as to whether sufficient levels of amylin cross the
blood-brain barrier to enact behavioral effects. Studies with
radiolabeled peptides showed that intact amylin accumu-
lates in multiple brain structures, including the striatum,
after systemic injection in mice, possibly via a saturable
transport mechanism. Indeed, amylin showed greater over-
all brain penetrance that insulin (Banks and Kastin, 1998).
Nevertheless, caution should be used in interpreting these
results, as only a small fraction of the systemically adminis-
tered amylin reached the brain and the striatum was among
the sites showing relatively lower levels of amylin accumu-
lation. A more definitive answer awaits detailed analysis
of real-time amylin flux in the Acb, using sensitive mass
spectrometry-based methods. Another possibility (though
not mutually exclusive) is that the endogenous AMY-R
ligand is CGRP. There are appreciable densities of
CGRP-like immunoreactive fibers in the Acb, and relatively
high densities of CGRP binding (Kruger et al, 1988; van
Rossum et al, 1997). CGRP binds to the Acb-localized
AMY-R, albeit with less affinity than amylin (Beaumont
et al, 1993). Hence, it is possible that either CGRP, amylin,
or a combination of both ligands participate in postprandial
m-OR modulation through AMY-Rs. Further studies are
needed to clarify this issue. Regardless, the present results
clearly indicate for the first time that there is a negative-
modulatory interaction between endogenous AMY-R and
m-opioid systems at the level of the AcbSh; this interaction
is revealed immediately following a meal.
Note that the lack of AC187-induced feeding augmenta-
tion in DAMGO-treated, non-prefed rats could be attributed
to a ceiling effect. However, close examination of intake
levels in individual rats shows that roughly half of the rats
ate more during the food deprivation þDAMGO þAC187
condition relative to food deprivation þDAMGO—includ-
ing the rat exhibiting the highest intake score under
DAMGO alone—whereas the other half ate less (data not
shown). This pattern would tend to argue against the idea
that there was no room to move upward under the non-
prefed-DAMGO þAC187 condition.
Presently, the mechanism underlying AMY-R and m-OR
interaction is unknown. However, it is interesting to consi-
der that the high-affinity AMY-1 receptor is a G-protein
coupled receptor that increases intracellular cAMP levels,
and that m-ORs are coupled to G(i)-proteins, which decrease
intracellular cAMP levels (Morfis et al, 2008; Williams et al,
2013). Therefore, it is possible that the AMY-Rs may
negatively modulate m-ORs via interactions between post-
receptor cAMP-dependent transduction pathways.
Clinically, our results may be relevant to disorders such
as binge-eating disorder and bulimia nervosa. Mu-opioid
signaling in the CNS is implicated in both disorders;
accordingly, there is some evidence that opioid-blocking
drugs (including selective m-OR antagonists) ameliorate at
least some symptoms of these disorders, and an association
has been reported between binge-eating disorder and a
gain-of-function polymorphism of the m-OR gene (Marrazzi
et al, 1995; Davis et al, 2009; Berner et al, 2011; Ziauddeen
et al, 2013). A theoretical framework has been proposed
stating that intra-Acb m-OR signaling acts to extend
feeding (particularly on palatable foods) beyond physiolo-
gical need, resulting in excess caloric intake (Kelley et al,
2005). Hence, in addition to its established clinical role in
the regulation of Type 2 diabetes mellitus, the FDA-
approved amylin analog, Pramlintide, may be useful
treatment for excessive, m-opioid-driven non-homeostatic
palatable feeding, as occurs putatively in pathological
conditions such as binge-type eating disorders and obesity.
Beyond feeding, AMY-R-based drugs may have therapeutic
effects in opiate and alcohol craving, conditions in which
both the Acb, and m-OR transmission, have been implicated
(O’Brien, 2005).
In summary, this is the first study to examine interactions
between AcbSh m-ORs and amylin. We find that AMY-R
signaling enacts robust negative modulation over m-OR-
mediated responses, highlighting a novel receptor-based
mechanism with which to modulate central m-OR signal-
ing in multiple ‘disorders of appetitive motivation,’ includ-
ing, but not limited to, psychiatric disorders with binge
features.
FUNDING AND DISCLOSURE
The authors declare no conflict of interest.
ACKNOWLEDGEMENTS
This work was supported by R21 MH093824 (BAB), and
SKB was supported by training grant T32 GM007507. We
are grateful to Ken Sadeghian and Ryan Selleck for technical
assistance. Facilities and procedures complied with animal
use and care guidelines from the National Institutes of
Health of the USA, and were approved by the Institutional
Animal Care and Use Committee of the University of
Wisconsin.
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