Vol.5, No.11A, 1-6 (2013) Health
http://dx.doi.org/10.4236/health.2013.511A1001
Further studies on the effects of acamprosate on
tolerance to the analgesic effects of morphine and
NO synthesis in the brain
Jacqueline Sepúlveda1*, Andrea Ortega1, Jorge Roa2, Enrique Contreras3
1Department of Pharmacology, School of Biological Sciences, University of Concepción, Concepción, Chile;
*Corresponding Author:
2Department of Physiology, School of Biological Sciences, University of Concepción, Concepción, Chile
3Department of Pre-Clinical and Clinical Sciences, School of Medicine, Catholic University of the Holy Conception, Concepción,
Chile
Received 18 July 2013; revised 19 August 2013; accepted 9 September 2013
Copyright © 2013 Jacqueline Sepúlveda et al. This is an open access article distributed under the Creative Commons Attribution
License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
ABSTRACT
The aim of this work was to investigate whether
acamprosate modifies the expression of the en-
zyme responsible for neuronal NO synthesis
(nNOS) in the nucleus accumbens (NAc) of mice
chronically treated with morphine and during
the abstinence syndrome induced by naloxone.
The enzyme was monitored by the NADPH dia-
phorase method. The number of cells stained for
NADPH diaphorase in the NAc of mice was
counted in 40 µm thick coronal brain slices at
40X. The intensity of the histochemical reaction
of stained cells from naive morphine plus saline
and morphine plus acamprosate treated mice
was analyzed by Image Pro Plus 4.5.1. Morphine
administered in a slow release preparation in-
creased the stain intensity of the positive neu-
rons. The increase in the NADPH staining per-
sisted after naloxone was given to mice chroni-
cally treated with morphine. Acamprosate an-
tagonized the effects induced by chronic mor-
phine treatment in the NAc of mice. These re-
sults indicate that up-regulation of nNOS in the
NAc is a consequence of the sust a ined effect s of
morphine stimulation, which, in turn, may result
from an increased in glutamate release during
the abstinence syndrome.
Keywords: Morphine; Nitric Oxide; Nucleus
Accumbens; Acamprosate
1. INTRODUCTION
Acamprosate is a taurine analog (calcium acetylho-
motaurinate) that has received considerable attention for
its ability to prevent relapse in abstinent alcoholics [1-5].
The mechanisms by which acamprosate decreases re-
lapse to alcohol users are still poorly understood. It has
been demonstrated that the drug reduces Ca2+ fluxes
through voltage-operated channels and that it interacts
with NMDA receptor-mediated glutamatergic neuron-
transmission in various brain regions with functional an-
tagonistic properties [6-8]. The drug also inhibits the
postsynaptic potential induced by glutamate in the neo-
cortex and the hippocampus [9]. Further studies suggest
that acamprosate may modify polyamine modulation of
the NMDA receptor, but does not alter receptor function
of the aminoacid [10].
The NAc is thought to be a determinant structure of
the brain for the expression of rewarding effects of opi-
ates [11,12]; additionally, neurochemical studies have
demonstrated that the acute administration of morphine
affects the concentrations of dopamine [13] acetylcholine
[14], and that chronic morphine administration in rats
augments glutamate release in the NAc, and in mor-
phine-tolerant rats following naloxone administration
[15].
There are now consisting evidences indicating that NO
modulates several actions of opioids [16]. In mice and
rats treated with morphine, the selective inhibitor of neu-
ronal nitric oxide synthase (nNOS) 7-nitroindazole (7NI)
has proven to suppress several actions induced by mor-
phine, e.g. the activation of the extracellular signal-regu-
lated kinase [17] and c-Fos expression in the striatum
[18].
On the other hand, glutamate effects are associated
with an increase in nitric oxide (NO) release [19,20]. Ac-
cordingly, an increase of glutamate effects must be ex-

J. Sepúlveda et al. / Health 5 (2013) 1-6
2
pected to induce a greater expression of nitric oxide syn-
thase (NOS), the enzyme responsible for NO release in
the central nervous system [21].
NADPH diaphorase is responsible for the calcium/
calmodulin-dependent synthesis of the guanylylcyclase
activator nitric oxide from L-arginine. The enzyme selec-
tively labels a number of discrete populations of neurons
throughout the nervous system. The NADPH diaphorase
histochemical technique is used for labeling nNO syn-
thesis expression, considered to be a selective marker for
distinct neural populations widely distributed throughout
the CNS [22].
We have previously demonstrated that acamprosate re-
duces tolerance to the antinociceptive effect of morphine
and the intensity of the abstinence behavior in rats chro-
nically treated with the opiate [23]. Besides, it reduces
the high levels of the excitatory amino acid observed in
the nucleus accumbens (NAc) of morphine dependent
rats during the withdrawal period [24]. The present work
extends these studies and investigates whether acampro-
sate modifies the expression of NADPH diaphorase in
the NAc of mice chronically treated with morphine and
during the abstinence syndrome induced by naloxone,
the opiate antagonist.
2. MATERIALS AND METHODS
2.1. Animals and Drugs
Male adult albino Swiss Webster mice 12 - 15 weeks
of age, weighing 25 - 32 g from the animal reproduction
laboratory of the Department of Pharmacology of the
University of Concepcion were used in all experiments.
Mice were housed in groups of 10 and maintained on a
12/12-h light/dark cycles at a room temperature of 22˚C
± 2˚C with free access to food and water. All experiments
were performed in accordance with institutional guide-
lines for the use of experimental animals and with the
National Institutes of Health Guide for the Care and Use
of Laboratory Animals.
The drugs used were morphine HCl (May and Baker,
Dagenham, England), naloxone (Sigma, St. Louis, MO),
and acamprosate (Lipha, Lyon, France). All other chemi-
cal were purchased from Sigma, St. Louis, MO.
2.2. Induction of Morphine Dependence
For the induction of chronic morphine effects, the opi-
ate was administered in a suspension of the following
composition: morphine, as the base form, 300 mg, 4.2 ml
liquid paraffin, and 0.8 ml sorbital sesquioleate mixed
with 5 ml saline. After 30 h the animals were sacrificed
and destined for histological analyses.
For the induction of the withdrawal syndrome, mor-
phine (300 mg/kg) was administered 30 h before the i.p.
administration of 4 mg/kg naloxone. To test the effect of
acamprosate (100 mg/kg) on the expression of NADPH-
diaphorase in morphine-dependent mice, this drug was
i.p. administered during the chronic morphine treatment
according to the following schedule: 30 min before and
12 and 24 h after the priming dose of morphine. Control
groups were injected with the vehicle instead of acam-
prosate.
2.3. Histological Preparations
Mice destined for histological analyses were anesthe-
tized with chloral hydrate (10 ml/kg solution 5% w/v)
and after the induction of anesthesia; animals were in-
jected with heparin (500 UI). Initially, isotonic saline so-
lution (50 ml) for the intracardiac perfusion was used to
flush out the blood, after which a mixture containing 4%
(w/v) depolymerized paraformaldehyde in 0.1 M phos-
phate buffer (PBS), pH 7.3, was perfused for 20 min.
The brains were dissected out, cut into blocks, and
post-fixed in the same solution for 4 hr at 4˚C. Tissue
blocks were cryoprotected with 30% (w/v) sucrose in
PBS until they sank, after which they were quickly fro-
zen and stored at −80˚C. Tissue blocks were cut at 40 µm
along coronal planes with a freezing sliding microtome
(Leica SM 2000R, Germany) and collected in six series
in cold PBS. One of these series was stained with the
NADPH-diaphorase histochemical technique.
2.4. NADPH Histochemistry
Free-floating sections were rinsed in PBS (3 × 10 min)
and incubated at 37˚C in the darkness in a medium con-
taining 0.08% (w/v) Triton X-100, 0.8 M nitrobluetetra-
zolium, and 1 M
 -NADPH in 0.1 M Tris-HCl, pH 8.0,
for 90 - 120 min. The reaction was controlled under the
microscope to avoid undesirable formation of formozan
crystals, and stopped with cold PBS. Stained sections
were mounted onto gelatin-coated slides, dried overnight
at 37˚C, dehydrated, cleared with xylene, and cover slip-
ped with Entellan®. Controls for specificity of the NA-
DPH histochemistry were incubated without NADPH or
without chromogen. No reaction product was observed in
the tissue when incubated without NADPH or without
chromogen.
2.5. Evaluation of NADPH Histochemistry
The boundaries of the region of the NAc were deter-
mined, and NADPH stained cells of this nucleus were
counted. These planimetric analyses were performed in
four consecutive sections from the same series, corre-
sponding to Bregma levels ranging from 0.60 to 1.70 mm
[25]. Rectangular frames were captured with a 40X ob-
jective and positive neurons were counted in each frame.

J. Sepúlveda et al. / Health 5 (2013) 1-6 3
NADPH-histochemistry positive cells were counted only
if the soma and dendrites were clearly distinguished (Carl
Zeiss Series 237806 SNT Axioplan 2 Germany).
2.6. Quantitative Morphology
The population of cells stained for NADPH diaphorase
was counted in 40 µm thick coronal brain slices at 40X.
Seven toten fields of 2.8 µm2 area, from four consecutive
tissue sample sections, taken from 5 control animals and
from mice from each of the experimental groups, were
counted. Only cells with clear nuclear profiles, dendrites
and clear borders were considered.
To ensure reliable comparisons among the different
groups, one series from one animal from each group (i.e.,
series of sections from naive, morphine-, acamprosate- or
saline-treated mice) was included in a parallel staining
protocol using the same incubation medium. The inten-
sity of the histochemical reaction of stained cells was
analyzed by Image Pro Plus 4.5.1.
2.7. Statistical Analysis
The significance of the differences in the mean results
to the different treatments were determined by analysis
of variance (ANOVA) and confirmed with the Student-
Newman-Keuls test. A level of probability of 0.05 was
accepted as statistically significant.
3. RESULTS
3.1. NADPH Positive Neurons in the NAc
No significantly changes were found in the number of
NADPH positive neurons in the NAc of mice treated
with morphine, acamprosate, either alone or in combina-
tion with the opiate, when compared to the results ob-
served in naive mice or in control animals injected with
saline or vehicle. Similarly, no differences in the number
of NADPH positive neurons were observed in morphine
dependent mice at the end of the chronic morphine treat-
ment or after naloxone administration (Figure 1).
3.2. Stain Intensity of NADPH
Histochemistry
In contrast to the absence of differences in the number
of neurons, the stain of positive neurons was more in-
tense in the chronically morphine treated mice. Acam-
prosate significantly reduced the stain of neurons in ve-
hicle injected rats. The drug also decreased the intensity
of the stain with respect to values observed in mice
treated with morphine and injected with naloxone.
Naloxone administration (20 min before the mice were
sacrificed) did not change the morphine effects on the
intensity of the staining (Figure 2).
VEHICLE
MORPHINE
ACAMPROSATE
MORPHINE+SALINE+NALOXONE
MORPHINE+ACAMPROSATE+NALOXONE
0
20
40
60
80
AC
B
NADPH positi ve neur ons
(Number per mm
2
± S.E.M.)
(a) (b) (c)
Figure 1. Number of NADPH positive neu-
rons in the NAc. (a) Effect of morphine (300
mg/kg); (b) Effect of acamprosate (100 mg/kg);
(c) Effect of acamprosate in naloxone-precipit-
ated withdrawal of morphine treated-mice.
Morphine (300 mg/kg) was administered 30 h
before the i.p. administration of 4 mg/kg nalo-
xone. Acamprosate (100 mg/kg) was i.p. ad-
ministered 30 min before and 12 and 24 h after
morphine.
4. DISCUSSION
Since the original work of Bredt et al. [26], it is ac-
cepted that there is a relationship between the activity of
NOS and the increase of the expression of the enzyme.
The neuronal isoform of the enzyme generates NO, a
molecule recognized as a neurotransmitter in the central
nervous system (CNS) that induces the production of
cyclic GMP [27,28]. It has also been established that
NADPH-diaphorase histochemical staining is a selective
marker for distinct neural populations widely distributed
throughout the CNS [22]. Different approaches have re-
vealed that NADPH staining corresponds to nNOS ex-
pression [21].
In the present study, morphine induced an increase in
the intensity of the NADPH staining of neurons of the
NAc. The results suggest that the sustained effects of
morphine are responsible for the up-regulation of NO ge-
neration.
Leza et al. have demonstrated that chronic morphine
administration increases the calcium-dependent NOS
activity in several areas of the brain [29]. These findings
and a further study of the same laboratory [30] reporting
an increase in NO synthase immunoreactivity suggest an
up-regulation of the enzyme after morphine administra-

J. Sepúlveda et al. / Health 5 (2013) 1-6
4
VEHICLE
MORPHINE
SALINE
ACAMPROSATE
MORPHINE+SALINE+NALOXONE
MORPHINE+ACAMPROSATE+NALOXONE
0.0
0.1
0.2
0.3
0.4
0.5
0.6
*
****
ABC
*
S t ain Int ens i t y
(picoc urie ± S. E . M )
(a) (b) (c)
Figure 2. Stain intensity of NADPH histochemistry of
the neurons of NAc in mice. (a) Effect of chronic mor-
phine administration. Morphine (300 mg/kg) was ad-
ministered as a slow release preparation. (b) Effect of
acamprosate treatment. Acamprosate (100 mg/kg) was
i.p. administered 30 min before and 12 and 24 h after
the vehicle. (c) Effect of acamprosate in naloxone-pre-
cipitated withdrawal of morphine treated-mice. Mor-
phine (300 mg/kg) was administered 30 h before the i.p.
administration of 4 mg/kg naloxone. Acamprosate (100
mg/kg) was i.p. administered 30 min before and 12 and
24 h after morphine. *Significantly higher than values
observed in vehicle injected mice (P < 0.05). **Signi-
ficantly lower than values observed in saline injected
mice (P < 0.05). ***Significantly lower than values ob-
served in the group injected with morphine, saline and
naloxone (P < 0.001) and significantly higher than val-
ues observed in mice treated only with acamprosate (P
< 0.05).
tion. More recently, it was reported that NO synthase
inhibition decreases tolerance development to morphine
[31] and attenuates opioid withdrawal syndrome [32].
The exact mechanism by which morphine administra-
tion increases the expression of NADPH-diaphorase and
NO synthesis is unknown, however, it has been suggested
that the elevated intracellular concentration of calcium
activates phospholipase C and protein kinase C [33]. The
latter enzyme may enhance NMDA mediated calcium
entry through glutamate, the crucial molecule involved in
NO production [34].
Although the high glutamate levels during the with-
drawal period are promptly decreased as a result of a
counterbalance adaptation, the elevated expression of
nNOS, which involves complex reactions in neuronal cells,
may explain the persistence of some symptoms following
the discontinuation of the drug administration. In addi-
tion, despite the increase of glutamate release observed
during the abstinence period induced by naloxone [24], it
is reasonable to expect that the induction of the absti-
nence syndrome by naloxone administration should not
affect the intensity of the number of the stained neurons
or the stain intensity because the animals were sacrificed
20 min after the induction of the syndrome, an interval
not conclusive to increase gene expression and, concur-
rently, to enhance NO synthase activity.
Acamprosate per se, or administered in morphine
treated animals, reduced the intensity of the stained neu-
rons, suggesting a decrease in the expression of neuronal
NADPH diaphorase. Effects of acamprosate per se were
also observed in other areas of the brain implicated in
physical dependence of morphine, including frontal cor-
tex, midbrain and cerebellum. The drug also reduced the
intensity of the staining in the group of animals treated
with morphine and injected with naloxone to precipitate
a withdrawal syndrome. Therefore, it seems reasonable
to suppose that acamprosate does not affect glutamate
release but rather that its effects are consistent with a
postsynaptic interference on glutamate-NMDA receptors,
perhaps modulating the spermidine site of the NMDA
receptor complex as has been suggested [35,36]. Conse-
quently, the final result of acamprosate, administered in
doses that decrease the intensity of morphine dependence,
is a decrease in the synthesis of NO.
The present results suggest that glutamate and NO
play a role in the neurochemical adaptation, which oc-
curs in the brain during the administration of morphine,
in particular on the NAc, a structure related with chronic
drug consumption. The results also support the notion
that acamprosate’s effects on chronic morphine responses
may be partially assigned to the interaction with gluta-
mate responses.
5. ACKNOWLEDGEMENTS
This work was supported by grant 203.032.011-1.0 from Vice-rectoría
de Investigación y Desarrollo (VRID), Universidad de Concepción.
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