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hikida_et_al_2003.pdf
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Acetylcholine enhancement in the nucleus accumbens
prevents addictive behaviors of cocaine and morphine
Takatoshi Hikida*, Yasuji Kitabatake*, Ira Pastan†, and Shigetada Nakanishi*‡
*Department of Biological Sciences, Kyoto University Faculty of Medicine, Kyoto 606-8501, Japan; and †Laboratory of Molecular Biology, Center for Cancer
Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892-4264
Contributed by Shigetada Nakanishi, March 26, 2003
D
rug addiction poses serious social, medical, and economic
problems, but effective treatments for drug addiction are
still limited (1, 2). The mesolimbic dopaminergic system serves
as a vital and fundamental role in pathological behavioral
changes that occur with repeated exposure of abusive drugs
(3–5). In the mesolimbic dopaminergic pathway, dopaminergic
neurons originate in the ventral tegmental area and project to
the nucleus accumbens (NAc), the ventral part of the striatum (6,
7). The NAc is a key neural substrate that is implicated in
reinforcement and addiction of cocaine and morphine (3–5).
These abusive drugs elevate dopamine levels in the NAc (8), and
the overwhelming actions of dopamine in the NAc lead to neural
adaptation that underlies reinforcement and addiction of cocaine and morphine (3, 4).
The activities of the principal ␥-aminobutyric acid-containing,
medium-sized spiny neurons in the NAc are modulated by not
only dopaminergic input but also cholinergic input (9). The
cholinergic input is derived from aspiny cholinergic interneurons
within the NAc (7, 10). Because acetylcholine (ACh) agonists or
antagonists generated global effects on many brain regions, the
role of ACh in reinforcement and addiction of abusive drugs was
not well understood (11–15). In our previous study, we investigated the role of ACh in the NAc circuit by selectively ablating
the NAc cholinergic neurons with use of immunotoxin (IT)mediated cell targeting techniques (16, 17). These investigations
revealed that ACh regulates the NAc circuit concertedly but
oppositely to dopamine and that cholinergic cell ablation enhances long-lasting behavioral changes of cocaine addiction (16,
17). ACh from cholinergic neurons in the NAc thus plays a
www.pnas.org兾cgi兾doi兾10.1073兾pnas.0631749100
pivotal role in neural responses and adaptation that underlie
cocaine reinforcement and addiction.
This investigation concerns whether ACh in the NAc commonly regulates morphine-induced behavioral changes and
whether enhancement of ACh in the NAc prevents behavioral
abnormalities of cocaine and morphine. To address the latter
question, we used acetylcholinesterase (AChE) inhibitors that
act on the brain AChE and elevate ACh levels in the striatum and
other brain regions (18–20). We report here that cholinergic cell
ablation in the NAc increases the sensitivity to morphine in both
its rewarding effects and negative reinforcements of morphine
withdrawal. We further report that centrally active AChE inhibitors block the induction and persistence of addictive behaviors of both morphine and cocaine via enhanced actions of ACh
in the NAc.
Materials and Methods
Animals and Drugs. Male C57BL兾6 mice (9–13 weeks) were
purchased from Japan SLC (Hamamatsu, Japan) and were used
as wild-type mice. The IG17 line of heterozygous transgenic mice
expressing the fusion protein of human IL-2 receptor ␣兾GFP
(21) and their wild-type littermates (9–13 weeks) were used for
the IT-mediated cell targeting experiments. Behavioral analysis
was carried out 2 weeks after IT injection (17). All procedures
were performed according to the guidelines of Kyoto University
Faculty of Medicine. The following drugs were obtained from
the following sources: morphine hydrochloride and cocaine
hydrochloride (both from Takeda, Osaka), naloxone hydrochloride (Sankyo), donepezil hydrochloride (Eisai, Tokyo), and
galanthamine hydrobromide (Sigma).
Conditioned Place Preference (CPP), Conditioned Place Aversion (CPA),
and Morphine Withdrawal. The CPP test was performed as de-
scribed (17). Briefly, CPP was tested in a three-chamber apparatus (MED Associates, St. Albans, VT) in which the two large
side chambers were separated by a small middle chamber. The
two side chambers differed in floor and wall conditions. On day
0, mice were allowed to move freely in the three chambers for 30
min. On days 1–3, mice were confined to one large chamber for
20 min immediately after they had received saline. Four hours
later, they received morphine or cocaine and were confined to
the other side chamber for 20 min. On day 4, mice were placed
in the middle chamber and allowed to move freely in the three
chambers for 30 min. CPP was evaluated by calculating the time
difference in which the time mice spent in the saline-paired
chamber was subtracted from the time mice spent in the
drug-paired chamber. Doses of morphine and cocaine administered at each day were 5 and 10 mg兾kg, respectively, unless
otherwise stated. For CPA analysis, morphine dependence was
developed with twice daily i.p. morphine administration. Morphine administration was started with 10 mg兾kg on day 1 and
progressively increased with a 10-mg兾kg increment from day 2 to
Abbreviations: NAc, nucleus accumbens; ACh, acetylcholine; CPP, conditioned place preference; CPA, conditioned place aversion; AChE, acetylcholinesterase; IT, immunotoxin.
‡To whom correspondence should be addressed. E-mail: snakanis@phy.med.kyoto-u.ac.jp.
PNAS 兩 May 13, 2003 兩 vol. 100 兩 no. 10 兩 6169 – 6173
NEUROSCIENCE
Drug addiction poses serious social, medical, and economic problems, but effective treatments for drug addiction are still limited.
Cocaine and morphine elevate dopamine levels in the nucleus
accumbens (NAc), and the overwhelming actions of dopamine are
implicated in reinforcement and addiction of abusive drugs. In our
previous studies, we reported the regulatory role of acetylcholine
(ACh) in the NAc function by selectively ablating the NAc cholinergic neurons with use of immunotoxin-mediated cell targeting.
These studies indicated that ACh and dopamine acted convergently
but oppositely on the NAc circuit and that cholinergic cell ablation
enhanced long-lasting behavioral changes of cocaine addiction. In
this investigation, we showed that immunotoxin-mediated ablation of the NAc cholinergic neurons enhanced not only the sensitivity to morphine in conditioned place preference but also negative reinforcement of morphine withdrawal in conditioned place
aversion. Remarkably, acetylcholinesterase (AChE) inhibitors that
act on the brain AChE suppressed both cocaine- and morphineinduced conditioned place preference and blocked the induction
and persistence of cocaine-evoked hyperlocomotion. Importantly,
this inhibition was abolished by ablation of the NAc cholinergic
neurons. These results demonstrate that centrally active AChE
inhibitors prevent long-lasting behavioral abnormalities associated with cocaine and morphine addictions by potentiating the
actions of ACh released from the NAc cholinergic neurons. Centrally active AChE inhibitors could thus be approached as novel and
potential therapeutic agents for drug addiction.
day 4. Conditioning of naloxone-induced place aversion was
conducted on day 5 in the three-chamber apparatus described
previously. One hour after 50 mg兾kg morphine treatment, saline
was i.p. injected into the mice, and they were confined to one
chamber for 20 min. Four hours later, they were again treated
with 50 mg兾kg morphine, and 1 h later, they were i.p. injected
with 1 mg兾kg naloxone and confined to the other chamber for
20 min. On day 6, the mice were allowed to move freely for 30
min, and CPA was evaluated by calculating the time difference
in which the time mice spent in the saline-paired chamber was
subtracted from the time mice spent in the naloxone-paired
chamber. For examination of physical signs of morphine withdrawal, morphine dependence was developed over a period of 4
days as described previously. Saline or naloxone (1 mg兾kg) was
injected 1 h after 50 mg兾kg morphine treatment on day 5.
Jumping, rearing, and forepaw tremors were then counted
during a 20-min period.
Other Behavioral Analyses. The hot-plate test was conducted by
placing mice on a hot plate at 55°C. The time at which the mice
showed the first hind-paw licking or jumping was measured with
a cut-off time of 30 sec. The tail-immersion test was carried out
by immersing a distal half of the mouse tail in water at 53°C. The
time at which the mice showed the first tail movement was
measured with a cut-off time of 20 sec. Morphine was injected
20 min before the tests. Locomotor activity was measured with
an infrared activity monitor (MED Associates) for a 10-min
period immediately after cocaine injection with and without
10-min pretreatment of donepezil (1 mg兾kg) or galanthamine
(1 mg兾kg).
Data Analysis. Data are expressed as means ⫾ SEM. Behavioral
Fig. 1. Effects of cholinergic cell elimination on morphine-induced CPP and
naloxone-induced CPA. (A) CPP was developed by repeated administration of
indicated doses of morphine for 3 days (n ⫽ 7–12). Cholinergic cell-eliminated
transgenic mice spent significantly more time at the morphine-paired chamber
after conditioning with 1 mg兾kg morphine (*, P ⬍ 0.05). (B) CPA was developed
by naloxone injection (1 mg兾kg) after establishment of morphine dependence
(n ⫽ 8 each). Cholinergic cell-eliminated transgenic mice spent significantly less
time at the naloxone-paired chamber (*, P ⬍ 0.05).
data were subjected to ANOVA, and post hoc comparisons were
made with a Scheffé test.
Results
Cholinergic neurons in the NAc were selectively ablated by
IT-mediated cell targeting techniques (IMCT; ref. 17). In the
IMCT techniques, we generated transgenic mice in which the
fusion protein of human IL-2 receptor ␣兾GFP (hIL-2R兾GFP)
Fig. 2. Effects of cholinergic cell elimination on naloxone-induced withdrawal behaviors and morphine antinoception. (A) Physical signs of morphine withdrawal
were monitored during a 20-min period after naloxone injection in morphine-dependent mice (n ⫽ 8 each). After naloxone treatment, jumping, but not rearing or
forepaw tremor, was most frequently induced in cholinergic cell-eliminated mice than IT-treated wild-type littermates (*, P ⬍ 0.05). No abnormal physical behaviors
were observed in both genotypes by saline injection. (B and C) IT-treated wild-type and transgenic mice were treated with indicated doses of morphine, and latencies
of antinociceptive responses in the hot-plate (B) and tail-immersion (C) tests were measured 20 min after morphine administration (n ⫽ 7– 8). Cholinergic cell-eliminated
mice showed a significantly prolonged latency of antinociceptive responses at 10 mg兾kg morphine in the hot-plate test (**, P ⬍ 0.01).
6170 兩 www.pnas.org兾cgi兾doi兾10.1073兾pnas.0631749100
Hikida et al.
Fig. 3. Effects of donepezil on morphine-induced CPP. (A) Wild-type mice
were pretreated with donepezil (1 or 3 mg兾kg) or saline 20 min before
place-conditioning with 5 mg兾kg morphine for 3 days (n ⫽ 7–11). Both doses
of donepezil significantly reduced morphine-induced CPP (**, P ⬍ 0.01). (B)
IT-treated wild-type and transgenic mice were place-conditioned by using the
same protocol described in A (n ⫽ 7–9). Donepezil significantly reduced
morphine-induced CPP in IT-treated wild-type mice but failed to suppress
morphine-induced CPP in IT-treated transgenic mice (**, P ⬍ 0.01).
was driven by the promoter function of metabotropic glutamate
receptor subtype 2 (16, 17, 21, 22). In these mice, hIL-2R兾GFP
was specifically expressed in cholinergic neurons within the cell
population of the NAc (16, 17). The IT is composed of the Fv
portion of a mAb reacting with hIL-2R fused to a 38-kDa
fragment of Pseudomonas exotoxin (21). IT was injected at a
single site on both sides of the NAc (17). The IT injection
selectively eliminated ⬎70% of the cholinergic neurons in the
NAc cell population of transgenic mice (17). No such ablation
was observed in the NAc of IT-treated wild-type mice (17). Two
weeks after IT injection, we examined the effects of cholinergic
cell elimination on CPP developed with repeated morphine
administration (Fig. 1A). In the CPP paradigm, mice learn to
associate the rewarding effect of morphine with a drug-paired
environment (11). Before conditioning, both IT-treated wildtype and transgenic mice showed no preference in visiting drugand saline-paired chambers that differed visually and textually.
After conditioning with morphine for 3 days, cholinergic celleliminated transgenic mice exhibited a significant preference to
a morphine-paired chamber with a low dose of morphine (1
mg兾kg; Fig. 1 A). The result indicates that ACh in the NAc
Hikida et al.
controls long-lasting actions of morphine in a manner similar to
that reported for cocaine (17).
We next examined withdrawal responses of morphine addiction by using the CPA paradigm. Morphine dependence was
developed by twice daily i.p. administration of morphine with a
gradual increment from 10 to 40 mg兾kg morphine during days
1–4. On day 5, the mice were place-conditioned by i.p. injection
with saline in one chamber and then with the morphine antagonist naloxone in the other chamber, each injected 1 h after
morphine administration (50 mg兾kg). On day 6, CPA was tested
by allowing the mice to visit freely the saline- and naloxonepaired chambers. Wild-type and transgenic mice showed CPA
but cholinergic cell-eliminated transgenic mice were more aversive to naloxone than wild-type mice (Fig. 1B). Negative reinforcement with morphine withdrawal is also enhanced by elimination of cholinergic neurons in the NAc.
Mice also develop a physical morphine dependence after
repeated morphine exposure. When the physical signs of naloxone-induced withdrawal symptoms were analyzed, both wildtype and cholinergic cell-eliminated mice showed jumping, forepaw tremor, and enhanced rearing (Fig. 2A). Jumping behavior
is regarded as a dominant physical sign of morphine withdrawal
(23). This behavior was significantly enhanced in cholinergic
cell-eliminated mice as compared with wild-type mice (Fig. 2 A).
These findings indicate that not only a psychological but also a
physical dependence of chronic morphine exposure is enhanced
by cholinergic cell elimination.
We next tested for antinociceptive responses to morphine. In
the hot-plate test, antinociceptive effects became stronger at a
higher dose of morphine in cholinergic cell-eliminated mice than
wild-type mice (Fig. 2B). In contrast, no difference was observed
between two types of mice in the tail-immersion test (Fig. 2C).
This difference between the two tests is interesting, because it
has been generally accepted that the hot-plate response involves
supraspinal analgesia, whereas the tail-immersion response
mainly occurs at the level of the spinal cord (24). In the hot-plate
and tail-immersion tests, both wild-type and cholinergic celleliminated mice developed comparative antinociceptive tolerance by daily administration of 10 mg兾kg morphine for 5 days
(data not shown). The results indicate that cholinergic cell
elimination influences supraspinal antinociceptive responses to
PNAS 兩 May 13, 2003 兩 vol. 100 兩 no. 10 兩 6171
NEUROSCIENCE
Fig. 4. Effects of donepezil on cocaine-induced CPP. Wild-type mice were
pretreated with donepezil (1 or 3 mg兾kg) or saline 20 min before placeconditioning with 10 mg兾kg cocaine for 3 days (n ⫽ 7–9). Both doses of
donepezil significantly reduced cocaine-induced CPP (**, P ⬍ 0.01).
Fig. 5. Effects of donepezil on cocaine-induced locomotor sensitization. (A) Wild-type mice were pretreated with donepezil (1 mg兾kg) or saline (n ⫽ 10 each)
10 min before daily cocaine administration (10 mg兾kg). Locomotor activities were counted during a 10-min period immediately after cocaine administration.
Repeated ANOVA showed that donepezil prevented cocaine-induced locomotor sensitization (F1,18 ⫽ 20.7, P ⬍ 0.001). The locomotor activity was significantly
reduced when compared at each day (*, P ⬍ 0.05; **, P ⬍ 0.01). (B) Wild-type mice daily received cocaine (10 mg兾kg) from day 1 to day 5. On day 6, the mice
received saline (n ⫽ 12), donepezil (1 mg兾kg, n ⫽ 7), or galanthamine (1 mg兾kg, n ⫽ 5) 10 min before cocaine administration (10 mg兾kg). The locomotor activities
of both donepezil- and galanthamine-treated mice were significantly reduced as compared with that of saline-treated mice (***, P ⬍ 0.001; **, P ⬍ 0.01). (C)
Wild-type and cholinergic cell-eliminated mice daily received cocaine (10 mg兾kg) for 5 days. On day 6, the animals were pretreated with saline or donepezil (1
mg兾kg) 10 min before cocaine administration (n ⫽ 7–11). Cholinergic cell-eliminated mice showed resistance to donepezil-mediated inhibition (*, P ⬍ 0.05). (D)
Wild-type mice daily received cocaine (10 mg兾kg) for 6 days. Five days after a cocaine-free interval (day 12), the animals were challenged with cocaine (10 mg兾kg)
10 min after saline or donepezil (1 mg兾kg) injection (n ⫽ 6). The locomotor activity on day 12 was significantly higher than that on day 1 (***, P ⬍ 0.001) and
this high locomotor activity on day 12 was abolished by pretreatment with donepezil (***, P ⬍ 0.001).
acute challenge with a high dose of morphine but has no effect
on morphine-induced desensitization.
Ablation of intrastriatal cholinergic neurons significantly reduces ACh levels in the striatum (16). We addressed whether
elevation of ACh in the NAc prevents behavioral changes
associated with abusive drugs. We used donepezil, a selective,
centrally active AChE inhibitor (18, 19) that elevates ACh levels
in the striatum and other brain regions (20). Wild-type mice were
i.p. injected with donepezil or saline 20 min before placeconditioning with daily administration of morphine (5 mg兾kg)
for 3 days. Pretreatment with donepezil strikingly reduced
development of morphine-induced CPP (Fig. 3A).
6172 兩 www.pnas.org兾cgi兾doi兾10.1073兾pnas.0631749100
We then examined the effects of donepezil in cholinergic
cell-eliminated transgenic mice to address whether ACh derived
from the NAc cholinergic neurons was required for reduction of
CPP by donepezil. Remarkably, cholinergic cell-eliminated
transgenic mice failed to respond to donepezil and showed
significant morphine-induced CPP comparable with salinetreated mice (Fig. 3B). The result indicates that ACh released
from NAc cholinergic neurons is essential as a target of the
AChE inhibitor for preventing morphine-induced CPP.
Our analysis of donepezil was extended to behavioral changes
associated with cocaine addiction. We first examined the effects
of donepezil on the development of cocaine-induced CPP.
Hikida et al.
provide rich connections with the principal medium-sized spiny
neurons throughout the NAc. ACh released from these neurons
acts concertedly but oppositely to dopamine on the principal
medium-sized spiny neurons in the NAc (9, 16, 17). The convergent interactions between dopamine and ACh would thus
contribute to regulation of neural responses and adaptation in
the NAc circuit. However, earlier studies with ACh agonists and
antagonists failed to indicate the regulatory role of ACh in
abusive drugs because these agents exhibited global effects on
many other brain regions (11–15). The behavioral studies combined with cholinergic cell ablation now reveal that ACh from
cholinergic neurons plays a pivotal role in reinforcement and
addiction of both cocaine and morphine. Importantly, centrally
active AChE inhibitors blocked the development and persistence
of behavioral changes associated with addiction of these drugs.
This inhibition is derived from enhancement of ACh in the NAc,
because depletion of ACh sources by cholinergic cell elim …
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