Environmental Enrichment Ameliorates Psychological Dependence Symptoms and Voluntary Morphine Consumption in Morphine Withdrawn Rats Under Methadone Maintenance Treatment

Abstract

Introduction: Previous studies have shown that physical and psychological dependence and the vulnerability to relapse are still present during MMT. Thus, this study examined whether Enriched Environment (EE) would attenuate anxiety, depressive, and obsessive-compulsive-like behaviors, as well as voluntary morphine consumption following Methadone Maintenance Treatment (MMT) in morphine withdrawn rats.
Methods: The rats were injected bi-daily doses (10 mg/kg, 12-h interval) of morphine for 14 days. Then, the rats were reared in a Standard Environment (SE) or EE for 30 more days during morphine withdrawal, simultaneous with receiving MMT. The rats were tested for anxiety (the Elevated Plus Maze [EPM]) and depression (Sucrose Preference Test [SPT]), Obsessive-Compulsive Disorder (OCD) as grooming behavior, and voluntary morphine consumption using a Two-Bottle Choice (TBC) paradigm.
Results: The findings revealed that EE experience in morphine withdrawn rats under MMT significantly increased the EPM open-arm time and higher sucrose preference than SE rats. Also, we found that the EE decreased the self-grooming behavior and morphine preference ratio in morphine withdrawn rats receiving MMT compared to the SE group.
Conclusion: We conclude that exposure to EE decreased methadone-induced anxiety, depressive and OCD-like behaviors, and voluntary morphine consumption in morphine withdrawn rats under MMT. Thus, the EE seems to be one of the strategies for reducing MMT-induced behavioral dysfunction and the risk of relapse induced by morphine withdrawal.

Full text

Basic and Clinical
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September, October 2021, Volume 12, Number 5
Research Paper: Environmental Enrichment Ameliorates
Psychological Dependence Symptoms and Voluntary
Morphine Consumption in Morphine Withdrawn Rats
Under Methadone Maintenance Treatment
Marjan Lari1 , Hossein Miladi-Gorji2,3* , Mahmoud Naja1 , Ali-Mohammad Rezaei4
1. Department of Clinical Psychology, Semnan University, Semnan, Iran.
2. Research Center of Physiology, Semnan University of Medical Sciences, Semnan, Iran.
3. Department of Physiology, School of Medicine, Semnan University of Medical Sciences, Semnan, Iran.
4. Department of Educational Psychology, Semnan University, Semnan, Iran.
* Corresponding Author:
Hossein Miladi-Gorji, PhD.
Address: Research Center of Physiology, Semnan University of Medical Sciences, Semnan, Iran.
Tel: +98 (912) 5313069
E-mail: miladi331@yahoo.com
Introduction: Previous udies have shown that physical and psychological dependence and
the vulnerability to relapse are ill present during MMT. Thus, this udy examined whether
Enriched Environment (EE) would attenuate anxiety, depressive, and obsessive-compulsive-
like behaviors, as well as voluntary morphine consumption following Methadone Maintenance
Treatment (MMT) in morphine withdrawn rats.
Methods: The rats were injected bi-daily doses (10 mg/kg, 12-h interval) of morphine for 14
days. Then, the rats were reared in a Standard Environment (SE) or EE for 30 more days during
morphine withdrawal, simultaneous with receiving MMT. The rats were teed for anxiety
(the Elevated Plus Maze [EPM]) and depression (Sucrose Preference Te [SPT]), Obsessive-
Compulsive Disorder (OCD) as grooming behavior, and voluntary morphine consumption
using a Two-Bottle Choice (TBC) paradigm.
Results: The ndings revealed that EE experience in morphine withdrawn rats under MMT
signicantly increased the EPM open-arm time and higher sucrose preference than SE rats.
Also, we found that the EE decreased the self-grooming behavior and morphine preference
ratio in morphine withdrawn rats receiving MMT compared to the SE group.
Conclusion: We conclude that exposure to EE decreased methadone-induced anxiety,
depressive and OCD-like behaviors, and voluntary morphine consumption in morphine
withdrawn rats under MMT. Thus, the EE seems to be one of the rategies for reducing MMT-
induced behavioral dysfunction and the risk of relapse induced by morphine withdrawal.
Article info:
Received: 13 Mar 2020
Fir Revision: 28 Jul 2020
Accepted: 22 Aug 2020
Available Online: 01 Sep 2021
Keywords:
Morphine-withdrawn rats,
Methadone, Enriched
environment, Anxiety,
Depression, Grooming,
Morphine preference
Citation:
Lari, M., Miladi-Gorji, H., Naja, M., & Rezaei, A. M. (2021). Environmental Enrichment Ameliorates Psychologi-
cal Dependence Symptoms and Voluntary Morphine Consumption in Morphine Withdrawn Rats Under Methadone Maintenance
Treatment. Basic and Clinical Neuroscience, 12(5), 607-616. http://dx.doi.org/10.32598/bcn.12.5.886.3
:
http://dx.doi.org/10.32598/bcn.12.5.886.3
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A B S T R A C T

Basic and Clinical
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September, October 2021, Volume 12, Number 5
1. Introduction
ethadone-Maintenance Treatment
(MMT) is a well-known approach to
treat opioid addiction (Saxon, Hser,
Woody, & Ling, 2013; Soyka et al.,
2011) and to improve addicts’ health and
social communication (Joseph, Stancli, & Langrod,
2000). Despite its ubiquitous use, considerable chal-
lenges have remained, such as the severity of physical
dependence signs, depression and anxiety (Alizadeh,
Zahedi-Khorasani, & Miladi-Gorji, 2018; Rounsaville,
Weissman, Kleber, & Wilber, 1982), Obsessive-Com-
pulsive Disorder (OCD) (Peles, Adelson, & Schreiber,
2009), voluntary morphine consumption (Alizadeh,
Zahedi-Khorasani, et al., 2018), locomotor sensitiza-
tion (Alizadeh, Khorasani, et al., 2018; Taracha, Chra-
pua, Lehner, Skórzewska, & Płaźnik, 2009) and the
vulnerability to relapse (Curran, Bolton, Wanigaratne,
& Smyth, 1999; Fareed et al., 2010). These issues have
been udied in animal and clinical udies. They may
reect persient changes induced by opioids in the brain
reward and motivational syem (Koob & Le Moal,
2008), which may hinder the eectiveness of MMT in
addicted individuals (Kauer & Malenka, 2007). Thus,
the reversal or prevention of MMT-induced behavioral
and mood disorders could be a helpful method for the
treatment of relapse in addicts during MMT. It seems
that the EE models by directly acting on the brain’s re-
ward syem (Thiel, Pentkowski, Peartree, Painter, &
Neisewander, 2010) may be eective in treating addicts
under MMT. An enriched cage is larger and contains
toys and running wheels (Hammami-Abrand Abadi &
Miladi-Gorji, 2016). We have previously shown that EE
attenuates the spontaneous morphine withdrawal signs,
the self-grooming behavior (Hammami-Abrand Abadi
& Miladi-Gorji, 2016), anxiety/depressive-like behav-
ior, and voluntary morphine consumption (Hammami-
Abrand Abadi, Miladi-Gorji, & Bigdeli, 2016) in mor-
phine withdrawn rats.
Given the well-known benecial eects of EE on mor-
phine-induced behavioral decits, it is expected that ex-
posure to the EE during MMT improves the behavioral
decits and relapse in addicted individuals. Thus, the
present udy aimed to inveigate whether EE would re-
duce anxiety, depressive, and obsessive-compulsive-like
behaviors, as well as voluntary morphine consumption
in morphine withdrawn rats under MMT.
2. Methods
Animals and induction of morphine dependence
and housing conditions
Adult male Wiar rats (average wright: 170±10 g)
were group-housed in cages (35×15×24 cm), 4 rats per
cage, under 12/12-h light/dark cycle (6 AM lights on – 6
PM lights o) at 22 ºC –24 ºC and had access to food
and water ad libitum. All experimental procedures were
conducted according to the National Initutes of Health
Guide for the Care and Use of Laboratory Animals. Mor-
phine sulfate (Temad, Iran) was injected subcutaneously
at a dose of 10 mg/kg, twice a day at 12 h intervals for 14
Highlights
● Methadone Maintenance Treatment (MMT) exacerbates psychological dependence in morphine-withdrawn rats.
● An Enriched Environment (EE) decreases MMT-induced anxiety, depression, and obsessive-compulsive behavior
in morphine-withdrawn rats.
● EE decreases morphine preference ratio in morphine-withdrawn rats under MMT.
Plain Language Summary
Chronic adminiration of morphine is associated with increased physical and psychological dependence signs, in-
cluding anxiety, depression, OCD, and vulnerability to relapse, which are ill permanent under MMT. Previous nd-
ings indicate that exposure to Environmental Enrichment (EE), including physical, social, and cognitive enrichment,
can produce a range of plaic responses in the brain’s reward syem. Our ndings showed that exposure to EE
decreased methadone-induced anxiety, depressive and OCD, and voluntary morphine consumption in morphine with-
drawn rats under MMT. Our results may have a potential therapeutic application to prevent physical and psychological
dependence and relapse in opiate use disorder patients under MMT.
M
Lari, M., et al. (2021). Environmental Enrichment and Morphine-Dependence Following Methadone in Rats. BCN, 12(5), 607-616.

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days, and all injections were performed with a quantity
of 1 mL/kg. The control rats were injected similarly with
saline. Then, the rats were group reared over 30 days un-
der MMT during morphine withdrawal (n = 8 rats per
cage) in a Standard Environment (SE) or EE. Enriched
rats were placed in a large cage (96×49×38 cm) with
plaic tunnels, rope, swing, balls, ramp, ladder, shel-
ters, ep, cube and a running wheel, toys, which were
changed every two days to maintain its novelty. Con-
trol rats (SE) were placed in andard laboratory cages
(42×34×15 cm), as previously described by our labora-
tory (Hammami-Abrand Abadi & Miladi-Gorji, 20117)
(Figure 1; Timeline of the experiment).
Methadone-maintenance treatment
Methadone hydrochloride (Temad, Iran) was injected
subcutaneously once per day for four consecutive 2-day
periods with doses of 0.5, 1, 2, and 3 mg/kg, respective-
ly. The maintenance dose of 3 mg/kg was then adminis-
tered 6 days per week (to avoid toxicity) until day 30
of morphine withdrawal, as previously described by our
laboratory (Alizadeh et al., 2018). None of methadone
overdose symptoms (apnea, cyanosis, motionlessness,
and irritability) were observed during the r 72 h after
the injection in all our experiments.
Experimental protocol
Sixty-four rats were divided randomly into 8 groups
(n=8 rats per group) based on our experimental proto-
cols (Figure 1; timeline of experiment) as follows: sa-
line/saline/andard environment (Sal/Sal/SE), Sal/Sal/
enriched environment (Sal/Sal/EE), Sal/methadone/SE
(Sal/Meth/SE), Sal/Meth/EE, dependent/Sal/SE (D/Sal/
SE), D/Sal/EE, D/Meth/SE, and D/Meth/EE. To avoid
the bias of circadian rhythms, we conducted all behav-
ioral tes between 10:00 AM to 1:00 PM.
Anxiety Measurement in the Elevated Plus Maze
Test (EPM)
On day 46, all rats were teed with the EPM. The rats
were individually placed in the center of the EPM with
two open (50×10 cm) and two closed (50×10×40 cm)
arms, and a central platform (10×10 cm). They were al-
lowed to explore the apparatus for 5 min as described
previously (Alizadeh et al., 2018). Time spent in and
entries into open and closed arms were recorded during
each 5 min te by a tracking syem (EthoVision, Nol-
dus, The Netherlands). The apparatus was cleaned with
water after each trial.
OCD-Like Behavior
Grooming behavior as an innate behavior in animals is
similar to OCD in humans. On day 47, the rats were indi-
vidually placed in a Plexiglas box (41 cm length×33 cm
height×41 cm width). Components of grooming behavior
included vibration, face and head washing, body groom-
ing, scratching, paw licking, head shaking, and genital
grooming. Grooming behavior was recorded at 15-s inter-
vals for 30 min. Thus, the maximum available score was
120 during the observation period, as previously described
(Hammami-Abrand Abadi & Miladi-Gorji, 2017)
.
Figure 1. Timeline of Experiments (see section 2 for details)
Rats were made dependent on morphine for 14 days. Then, the rats under Methadone Maintenance Treatment (MMT) were
reared in an Enriched Environment (EE) for 30 days of morphine abstinence. The rats were tested for the elevated plus maze
(EPM), OCD-like behavior, Sucrose Preference Test (SPT), and voluntary morphine consumption using a Two-Bottle Choice
(TBC) paradigm.
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Depressive-Like Behavior Using the Sucrose Pref-
erence Test (SPT)
All rats were kept individually in cages for 24 h before
teing. Then, they had free access to two bottles in each
cage for 48 h on days 48 and 49: one with 200 mL of 32%
sucrose (w/v) and the other with 200 mL of tap water.
The positions of the bottles were changed every 12 h to
avoid learning. Fluid intake and sucrose were measured
every day. At the end of 48 h, the bottles were removed,
and sucrose preference was calculated as 100%×sucrose
solution consumption (mL)/total uid consumption (mL)
as previously described
(Alizadeh et al., 2018)
.
Two-Bottle Choice (TBC) Paradigm
Each rat was housed in cages with two bottles over 12
days (from day 50 to 61). In one bottle, morphine sul-
fate was dissolved in 3% sucrose solution and also 3%
sucrose solution was in the control bottle as follows: on
days 1–4 (0.3 mg/mL morphine); 5–8 (0.5 mg/mL mor-
phine) and 9–12 (0.7 mg/mL morphine). The rats were
allowed to have continuous access to both bottles. The
positions of the bottles in the cage were changed at the
time of daily bottle weighing to avoid learning. Fluid
intake was measured by weighing the bottles between
9:00 and 10:00 AM every day. The body weights of rats
were measured at the art of each period as previously
described (Alizadeh et al., 2018). The average morphine
Figure 2. Effect of Environmental Enrichment (EE) on the Anxiety-Like Behavior in Morphine Withdrawn Rats Receiving MMT
A: The Percentages of Time Spent in Open Arm; and B: The Total Arm Entries (counts) of the EPM (n=8 rats per group).
Data are expressed as Mean±SEM. EE rats spent signicantly more time in the open arms in the Sal/Sal/EE, D/Sal/EE, and
D/Meth/EE groups.
*P<0.05 vs the Sal/Sal/SE group; ***P<0.0001 vs the Sal/Meth/SE group; ^^^P<0.0001 vs the D/Sal/SE group; ###P<0.0001 vs
the D/Meth/SE group.
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consumption and preference ratios (morphine solution
consumed [mL]/total consumed from both bottles [mL])
were evaluated during a 4-day intake period.
Statistical analysis
The data were expressed as the mean±SEM.
The data were analyzed using a 3-way Analy-
sis of Variance (ANOVA) with the xed factors of
dependence×methadone×housing and with repeated
measures as required. The po-hoc udy included
Tukey’s te. Statiical dierences were considered sig-
nicant at P<0.05.
3. Results
Anxiety-Like Behavior
The data of EPM are shown in Figure 2-A and B.
Three-way ANOVA showed the signicant eects of
methadone (F1,55=3.01, P=0.05), housing (F1, 55=169.38,
P=0.0001), methadone×housing (F1,55= 5.29, P=0.025),
and dependence×methadone×housing (F1,55=13.02,
Figure 3. Effect of Environmental Enrichment (EE) on the Depression and OCD-Like Behaviors in Morphine Withdrawn Rats
Receiving MMT
A: The Percentages of Sucrose Preference Using the SPT; and B: The Self-Grooming Sign (n=8 rats per group).
Data are expressed as Mean±SEM. The percentages of sucrose preference were lower in the D/Sal/SE and D/Meth/SE rats,
while the percentages were higher in the D/Sal/EE and D/Meth/EE groups. Also, the self-grooming scores were lower in the
D/Sal/EE and D/Meth/EE rats.
In A: ^P<0.05; ^^^P<0.0001; ^^P<0.003 vs the Sal/Sal/SE group; ***P<0.0001 vs the Sal/Meth/SE group; ###P<0.0001 vs the
D/Sal/SE group; $$$P<0.0001 vs the D/Meth/SE group.
In B: *P<0.02; ***P<0.0001 vs the Sal/Sal/SE group; ^P<0.047 vs the D/Sal/SE group; #P<0.04 vs the D/Meth/SE group.
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P=0.03) in the percentage of time spent in open arms
(Figure 2-A). Between-group comparisons revealed that
the percentages of time spent in the open arms were sig-
nicantly lower in the D/Sal/SE and D/Meth/SE (both,
P=0.049) groups compared to their control groups.
While the times were signicantly longer in the Sal/
Sal/EE (P=0.013), Sal/Meth/EE (P=0.0001), D/Sal/EE
(P=0.0001), D/Meth/EE (P=0.0001) groups than their
control rats. There was no signicant dierence in the
number of total arm entries among the groups as a mea-
sure of locomotion (Figure 2-B). Thus, EE completely
reversed the anxiogenic eects of MMT.
Depression and obsessive-like behavior
In the SPT te, 3-way ANOVA revealed the signicant
eects of dependence (F1,54=10.54, P=0.002), methadone
(F1,54=4.9, P=0.032), housing (F1,54=158.19, P=0.0001),
dependence×housing (F1,54=11.1, P=0.002), and
dependence×methadone×housing (F1,54=8.65, P=0.005).
Comparisons between groups showed that sucrose pref-
erence in the Sal/Meth/SE (P=0.05), D/Sal/SE (P=0.003),
and D/Meth/SE (P=0.0001) groups was less than that in
the Sal/Sal/SE group. While the sucrose preferences of the
Sal/Sal/EE (P=0.016), Sal/Meth/EE (P=0.0001), D/Sal/EE
(P=0.0001), D/Meth/EE (P=0.0001) groups were signi-
cantly higher than that in the control group (Figure 3-A).
The results of the self-grooming behavior te for assessing
OCD-like behavior (Figure 3-B) using a 3-way ANOVA
revealed the signicant eects of dependence (F1,56=21.57,
P=0.0001), methadone (F1,56=4.5, P=0.039), housing
(F1,56=16, P=0.0001), dependence×housing (F1,56=5.81,
P=0.019), and dependence×methadone×housing
(F1,56=3.3, P=0.049). Between-group comparisons showed
that the self-grooming scores in the D/Sal/SE (P=0.02) and
D/Meth/SE groups (P=0.001) were more than that in the
Sal/Sal/SE group, while rats of D/Sal/EE (P=0.047) and
D/Meth/EE groups (P=0.04) got signicantly lower scores
than the control rats. Thus, exposure to EE decreased de-
pressive/OCD-like behaviors in morphine withdrawn rats
under MMT.
Figure 4. Effect of Environmental Enrichment (EE) on Voluntary Morphine Consumption in Morphine Withdrawn Rats Under MMT
A: Water Intake; B: Morphine Consumption; C: The Morphine Preference Ratio (n=8 rats per group).
Data are expressed as Mean±SEM. The Sal/Meth/EE, D/Sal/EE, and D/Meth/EE groups exhibited a decrease in morphine
preference ratios during three periods.
In B and C: ^^^P<0.0001, ^^P< P=0.011, ^^^P< P=0.0001, ^^P< P=0.011, ^^^P<P=0.0001 vs the Sal/Sal/SE group; K
P<P=0.047, KKKP<P=0.0001, KKP<0.003, KKKP<0.0001vs the D/Sal/SE group; $P<0.04, $$P<0.011, $$$P<0.0001 vs the Sal/
Meth/SE group, +P<0.031, +++P<0.0001 vs the D/Meth/SE group, aaP<0.005, aaaP<0.0001, aaP<0.005 vs the D/Sal/EE group.
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Assessment of voluntary morphine consumption
Data of the voluntary morphine consumption during three
periods of the TBC te are shown in Figure 4. There was no
signicant dierence between groups in water consumption
(Figure 4-A). Three-way ANOVA with repeated measures
for the voluntary consumption of morphine during three pe-
riods exhibited the signicant eects of day (F2,110=148.67,
P=0.0001), dependence (F1,55=88.24, P=0.0001), metha-
done (F1,55=14.59, P=0.0001), housing (F1,55=78.51,
P=0.0001), dependence×methadone (F1,55=108.74,
P=0.0001), dependence×housing (F1,55=21.25, P=0.0001),
methadone×housing (F1,55=15.5, P=0.0001) and day×depe
ndence×methadone×housing (F2,110=3.42, P=0.036). Also,
3-way ANOVA with repeated measures for the morphine
preference ratio during three periods of intake revealed sig-
nicant eects of day (F2,110=6.11, P=0.003), dependence
(F1,55=146.4, P=0.0001), methadone (F1,55=35.79, P=0.0001),
housing (F1,55=230.45, P=0.0001), dependence×methadone
(F1,55=110.51, P=0.0001), dependence×housing (F1,55=70.1,
P=0.0001), methadone×housing (F1,55=31.12, P=0.0001),
dependence×methadone×housing (F1,55=8.4, P=0.005),
and day×dependence×methadone×housing (F2,110=4.1,
P=0.019). Between-group comparisons showed that mor-
phine preference ratios during three periods of intake
in morphine withdrawn rats receiving saline and MMT
housed in EE were signicantly lower than those in the D/
Sal/SE (P=0.003, P=0.0001, and P=0.0001), D/Meth/SE
groups (P=0.031, P=0.0001 and P=0.0001, respectively)
(Figure 4-C). Also, the morphine preference ratio of the
Sal/Meth/SE group was higher than that in the Sal/Sal/SE
group during three periods of intake (P=0.0001), which was
lower than in the Sal/Meth/EE group during periods 2 and
3 of intake (both, P=0.0001). Data related to the voluntary
consumption of morphine (Figure 4-B) were similar to that
observed in the morphine preference ratio and followed the
same pattern. Also, between-group comparisons showed
that the morphine consumption and preference ratio of the
D/Meth/EE rats were lower than those in the D/Sal/EE
group during periods 2 (P=0.007 and P=0.005, respective-
ly) and 3 (P=0.0001 and P=0.005, respectively) of intake.
Thus, EE decreased voluntary morphine consumption as an
animal model of relapse.
4. Discussion
The results of our udy showed that morphine-with-
drawn rats receiving saline and MMT reared in the SE
exhibited anxiety/depressive/OCD-like behaviors after
30 days of abinence from morphine. It seems that the
eects of morphine withdrawal are much longer, indi-
cating that the anxiety, depression, and OCD are persis-
tent behaviors after 30 days of abinence, even though
the rats simultaneously were under MMT. This nding
was consient with previous udies’ results, showing
the anxiety/depressive-like behaviors in morphine with-
drawn rats (Alizadeh et al., 2018) and patients (Brienza
et al., 2000; Lin et al., 2012) under MMT. Also, the Sal/
Meth/SE group showed an increase in depression-like
behavior.
It is likely that the mild persient of MMT-induced lo-
comotor sensitization in morphine-withdrawn rats
(Aliza-
deh et al., 2018)
and also, grooming behavior as a driver
of drug-seeking behavior
(Lubman, Yücel, & Pantelis,
2004)
in turn may intensify drug craving and relapse in
addicted individuals
(Boileau et al., 2006)
. The observed
behavioral dysfunction as a part of psychological depen-
dence can increase the risk of relapse and drug-seeking
after protracted abinence
(Lubman et al., 2004)
.
In line with this assumption, we saw that the D/Sal/SE,
D/Meth/SE, and Sal/Meth/SE groups exhibited an in-
crease in the voluntary morphine consumption and mor-
phine preference ratios during three intake periods with
the Sal/Sal/SE group. Thus, the rats under MMT are ill
at risk for relapse, although the morphine preference ra-
tio was signicantly lower in the D/Meth/SE group than
the D/Sal/SE group. A heightened response to drug cues
probably occurs in the medial prefrontal cortex and the
extended limbic syem in patients under MMT (Lan-
gleben et al., 2008), which drives further drug-seeking
behavior.
We found that exposure to EE decreased anxiety/de-
pressive/OCD-like behaviors. These ndings are in line
with previous udies showing that exposure to EE re-
duces the severity of physical and psychological depen-
dence on morphine (Hammami-Abrand Abadi & Mila-
di-Gorji, 2017; Hammami-Abrand Abadi et al., 2016;
Pooriamehr, Sabahi, & Miladi-Gorji, 2017), behavioral
sensitization (Bardo, Robinet, & Hammer, 1997) and
rewarding and reinforcing properties of morphine (Xu,
Hou, Gao, He, & Zhang, 2007). Thus, EE, because of its
positive life experiences and anti-ress eects, may pre-
vent the development of drug addiction (Solinas, Thiri-
et, Chauvet, & Jaber, 2010). This nding is supported
further by previous udies showing that EE decreased
repetitive behavior (Lewis, Tanimura, Lee, & Bodsh,
2007), ereotyped behavior (Turner, Lewis, & King,
2003), ress level, and locomotor activity (Xu, Sun,
Xue, & Li, 2014) in animal udies.
We also showed that the morphine consumption and
preference were lower in the D/Meth/EE group than
the D/Sal/EE group during periods 2 and 3 of intake,
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September, October 2021, Volume 12, Number 5
which was consient with an earlier udy that MMT
may improve craving for heroin in drug abusers (Wang
et al., 2014). Therefore, exposure to EE in the morphine
withdrawal rats receiving MMT may exert an adjunctive
protective role on the risk of relapse after protracted ab-
inence. Our nding may sugge some additive eects
between MMT and EE on the craving and drug-seeking
behavior in morphine withdrawn rats.
Presently, it is unclear how the EE can reduce MMT-
induced behavioral dysfunction and the risk of relapse
in morphine-withdrawn rats. Our ndings cannot answer
this queion. Previous results indicate that exposure to
an EE enhanced hippocampal neurogenesis (Simpson &
Kelly, 2011) and Insulin‐Like Growth Factor 2 (IGF‐2)
(Li et al., 2014), higher serotonin levels in the frontal
cortex (Leger et al., 2015), and metabolic activity in the
cortex and riatum (Turner, Yang, & Lewis, 2002) are
probably involved in reducing anxiety/depression and
OCD. Also, it seems that the modulation of neurotrans-
mitter syems and plaic changes resulting from the EE
in the hippocampus, the frontal cortex, and the riatum
(Simpson & Kelly, 2011) may account for the decrease
in the morphine preference ratio and behavioral dysfunc-
tion in morphine-withdrawn rats under MMT. Future
udies should examine the neurobiological mechanisms
caused by EE in morphine-withdrawn rats under MMT.
5. Conclusion
This udy indicates that morphine dependence and
MMT are associated with increased anxiety/depression/
OCD-like behaviors in parallel with morphine preference
in rats. It provides novel evidence that exposure to the EE
attenuates anxiety/depression/OCD-like behaviors and
morphine preference ratio in morphine withdrawn rats
under MMT. Therefore, exposure to the EE during mor-
phine withdrawal and receiving MMT may be a proper
therapeutic rategy for preventing relapse in addicts.
Ethical Considerations
Compliance with ethical guidelines
There were no ethical considerations to be considered
in this research.
Funding
This research did not receive any grant from funding
agencies in the public, commercial, or non-prot sectors.
Authors' contributions
All authors equally contributed to preparing this article.
Conict of interest
The authors declared no conict of intere.
Acknowledgments
We thank the Physiology Research Center, School of
Medicine, Semnan University of Medical Sciences, for
providing research facilities.
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