Pharmacology, Biochemistry and Behavior 201 (2021) 173106

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Pharmacology, Biochemistry and Behavior

journal homepage: www.elsevier.com/locate/pharmbiochembeh

Research article

Naloxone-induced conditioned place aversion score and extinction period

are higher in C57BL/6J morphine-dependent mice than in Swiss: Role of
HPA axis
Javier Navarro-Zaragoza *, E. Martínez-Laorden, F. Javier Teruel-Fernández,
Victoria Gómez-Murcia, Alberto Cánovas, María-Victoria Milanés, María-Luisa Laorden,
Pilar Almela
Department of Pharmacology, Faculty of Medicine, University of Murcia, Murcia, Spain

A R T I C L E I N F O A B S T R A C T

Keywords: Intense associative memories develop between drug-paired contextual cues and the drug withdrawal associated
Aversive memory aversive feeling. They have been suggested to contribute to the high rate of relapse. Our study was aimed to
CPA expression elucidate the involvement of hypothalamic-pituitary-adrenocortical (HPA) axis activity in the expression and
CPA extinction
extinction of aversive memory in Swiss and C57BL/6J (B6) mice. The animals were rendered dependent on
Morphine withdrawal
HPA axis
morphine by i.p. injection of increasing doses of morphine (10–60 mg/kg). The negative state associated with
naloxone (1 mg/kg s.c.) precipitated morphine withdrawal was examined by using conditioned place aversion
(CPA) paradigm. B6 mice obtained a higher aversion score and took longer to extinguish the aversive memory
than Swiss mice. In addition, corticosterone levels were increased after CPA expression. Moreover, corticosterone
levels were decreased during CPA extinction in Swiss mice without changes in B6 mice. Pre-treatment with the
selective CRF1 receptor antagonist CP-154,526 before naloxone, impaired morphine-withdrawal aversive
memory acquisition and decreased the extinction period. CP-154,526 also antagonized the increased levels of
corticosterone observed after CPA expression in Swiss mice, without any changes in B6 mice. These results
indicate that HPA axis could be a critical factor governing opioid withdrawal memory storage and retrieval, but
in a strain or stock-specific manner. The differences observed between Swiss and B6 mice suggest that the
treatment of addictive disorders should consider different individual predisposition to associate the aversive
learning with the context.

1. Introduction
Non-medical abuse and opioid abuse have reached epidemic pro­
portions in the United States in the last years (Upp and Waljee, 2020).
Although previous studies have shown that addictions are heritable
psychiatric diseases (Goldman et al., 2005; Ho et al., 2010; Ducci and
Goldman, 2012), the genetic substrates of opioid addiction are still
unknown. Differences between individuals have been reported
regarding opioid liking, finding people that show an obvious disliking,
what it has been hypothesized to have a deep genetic response (Angst
et al., 2012). Moreover, in the last years it has been proposed that ge­
netic factors are essential for variations in opioid liking/disliking,
influencing in the different development of opioid addiction in mice
(Kirkpatrick and Bryant, 2015).
Different animal models have been used previously, i.e., the induc­
tion of the acoustic startle reflex or the conditioned place aversion
(CPA), in order to assess the emotional component of opioid withdrawal
(Radke et al., 2013) and to imitate the negative consequences of the
withdrawal syndrome. Focusing on CPA, it has been tested before to
clear dysphoric or aversive responses of withdrawal (Tzschentke, 2007).
In rodents, the negative affective component of opioid dependence
could be reflected by CPA. In this protocol, opioid withdrawal is cue-
associated with the environment so, the negative symptoms that occur
during withdrawal syndrome are associated with the chamber where the
mice are suffering. Once the animals are re-exposed to the cue-
associated environment in a drug-free state, they usually avoid the
context paired with the aversive effects of drug withdrawal. There is
increasing evidence that suggests substrain differences in CPA with an

* Corresponding author at: Department of Pharmacology, Faculty of Medicine, Murcia, Spain.

E-mail address: jnavarrozaragoza@um.es (J. Navarro-Zaragoza).

https://doi.org/10.1016/j.pbb.2021.173106
Received 12 December 2019; Received in revised form 1 January 2021; Accepted 2 January 2021
Available online 12 January 2021
0091-3057/© 2021 Elsevier Inc. All rights reserved.
J. Navarro-Zaragoza et al.
inanimate object resembling a mouse (a toy mouse) (Pinheiro et al.,
2016) or in naloxone-induced CPA (Kirkpatrick and Bryant, 2015).
Nevertheless, the mechanism underlying these differences is currently
not well understood. However, an improved understanding of the ge­
netic and neurobehavioral basis of opioid aversion has therapeutic
relevance for prevention and treatment strategies in opioid dependence.
Most commonly abused drugs share a common action; they stimulate
the hypothalamic-pituitary-adrenocortical (HPA) axis when there is a
withdrawal through the activation of corticotropin-releasing factor
(CRF) in the paraventricular nucleus (PVN) of the hypothalamus,
resulting in a strong release of adrenocorticotropic hormone (ACTH) and
corticosterone (Ueno et al., 2011). Corticosterone mediates somatic and
negative affective-like components of withdrawal (Contarino and
Papaleo, 2005; Harris and Aston-Jones, 2007; Papaleo et al., 2007;
Koob, 2008). Although the role of HPA axis in addiction has been widely
studied, no literature has been published comparing strains differences
in morphine-withdrawn mice after a CPA paradigm. To address this
limitation, the present study was designed for two different mice
commonly used in research: Swiss and C57BL/6J (B6), which differ in
their locomotor responses to opioids (Brase et al., 1977). The B6 mouse
strain is one of the most widely used strains of inbred mice to assess
behavior and their response to different nociceptive stimuli is consid­
ered within the normal range (Mogil et al., 1999). Thus, we chose this
strain to be compared with Swiss as a prototype of outbred stock. Strain
surveys of common inbred and outbred mice are a useful way to gain
insight into the genetic contribution to complex disorders (Crabbe et al.,
1999).
The role of HPA axis in the aversive memories associated with
morphine-withdrawal in different strains has not been assessed. We
hypothesized a relationship between HPA axis activity and aversive
memory that will depend on the strain or outbred stock considered. To
support this hypothesis, we tested the role of HPA axis in the expression
and extinction of aversive effects observed after naloxone-induced
withdrawal using CP-154,526, selective CRF1 receptor antagonist.
Thus, the study of the possible differences in the memory processing of
drug aversion and cues could be important for understanding the basis of
individual vulnerability to drug addiction.
2. Methods
2.1. Animals
Adult (8–12 weeks old, n = 96) male Swiss ICR (CD-1) mice from or
C57BL/6J (B6) mice (n = 96) from the Animal Facilities of the Uni­
versity of Murcia weighing 25–30 g were housed 4–6/standard cage in a
temperature-controlled environment, received ad libitum access to food
and water and were maintained on a 12-h/12-h light/dark cycle. Mice
were habituated to the testing room for at least 1 week prior to the
experimental manipulations. All animals received human care according
to the guidelines provided by the European Communities Council
Directive of 22 September 2010 (2010/63/UE) and were approved by
Comité Ético de Experimentación Animal (CEEA, Universidad de Mur­
cia, RD 53/2013).
2.2. Drug treatment
Each strain or outbred stock of mice was randomly divided into two
groups: chronic saline-treated (n = 128) and chronic morphine-treated
(n = 64) mice. Morphine was injected i.p. starting on day 1, 10 mg/
kg; day 2, 30 mg/kg; day 3, 50 mg/kg and day 4, 60 mg/kg (only one
injection in the morning). This pattern of morphine administration,
which involves ascending drug doses, has been used extensively to study
opioid tolerance and dependence (García-Carmona et al., 2012; García-
Carmona et al., 2015a, 2015b; Goeldner et al., 2011; Liang et al., 2006,
2007; Martínez-Laorden et al., 2019; Valero et al., 2018; Ziólkowska
et al., 2012). The chronic saline-treated groups were administered with
2
Pharmacology, Biochemistry and Behavior 201 (2021) 173106
saline (i.p.) using the same protocol. Another set of mice were acutely
pre-treated with the selective CRF1 receptor antagonist CP-154,526 [N-
butyl-Nethyl-2,5-dimethyl-7-(2,4,6-trimethylphenyl) pirrolo[3,2-e]
pyrimidin-4-amine] (30 mg/kg, i.p.) 30 min before the acute adminis­
tration of naloxone, in order to determine the involvement of CRF1 re­
ceptors on morphine withdrawal aversive memory. The dose and the
administration time of CRF1 receptor antagonist were selected based on
previous reports (Almela et al., 2012; García-Carmona et al., 2015b;
Valero et al., 2018). CP-154,526 was dissolved in 10% tween 80 and
given in a volume of 3 ml/kg body weight. In addition, previous studies
(Valero et al., 2018) from our laboratory have demonstrated that the
treatment with morphine+vehicle+saline, did not induce spontaneous
morphine withdrawal.
2.3. Naloxone-induced conditioned place aversion is a recognized
paradigm of negative affective learning
The CPA procedure results in place aversion when the animals con­
nect the chamber where they are placed with the negative effects of
morphine withdrawal syndrome (García-Carmona et al., 2012). Our
CPA equipment was composed of two identical boxes divided into three
polyvinylcarbonate (PVC) chambers (Valverde et al., 1996) connected to
a computer. It had two main chambers (20 length, 18 cm width and 25
cm height) separated by a smaller space (20 cm, length 7 cm width and
25 cm height). The larger chambers had a different wall painting and a
different floor texture (in our case, grey striped wall and a black smooth
floor for chamber 1 and black spotted wall together with a grey rough
floor for chamber 2) which allowed to relate the naloxone injections
with the different environment. Besides, all the three chambers were
separated by manual doors extracted during the test. This procedure has
been previously used by our laboratory (García-Carmona et al., 2012;
Gómez-Milanés et al., 2012; Valero et al., 2018). The protocol is divided
into five phases (Fig. 1): pre-test, drug treatment, conditioning, post-test
and extinction. At the beginning, (day 0), all the mice were placed one
by one in the smaller chamber with no restrictions to move themselves
between the three chambers for 15 min (pre-test). The computer named
above was able to record the time spent in each chamber by the animals,
being not considered as neutral in preference the animals that spent less
than 360 s in one chamber. These animals were excluded from further
study (n = 4). During days 1 to 4, animals were treated with morphine or
saline as described previously. On day 4 and 90 min after last morphine
injection, CP-154,526 (30 mg/kg, i.p.) or its vehicle (Tween 80, 10%)
were acutely injected to study the role of HPA axis, and 30 min later,
naloxone (1 mg/kg, s.c.) or its vehicle (saline, 1 ml/kg, s.c.) were
administered with the purpose of precipitating the morphine with­
drawal syndrome. The dose of naloxone used in this study was based in
previous studies from our laboratory to induce CPA in Swiss (Valero
et al., 2018; Martínez-Laorden et al., 2019) and B6 mice (García-Car­
mona et al., 2012; García-Carmona et al., 2015a, 2015b). Immediately
after, mice were confined to one of the chambers for 18 min using a
randomly unbiased design. On day 5, CPA expression was tested in a
drug-free state (post-test). This procedure was the same that during the
pre-test. CPA score is the result of deducting the time spent during the
testing phase in the drug naloxone-paired chamber minus the time spent
during the preconditioning phase.
Swiss and B6 mice undergoing morphine withdrawal displayed
characteristic withdrawal symptoms: wet-dog shakes, teeth chattering,
ptosis, tremor, piloerection, lacrimation, rhinorrhea, chromodiacryor­
rhea, spontaneous jumping, and diarrhea. In addition, we have quanti­
fied body weight loss after naloxone administration. Loss of body weight
was calculated as the difference between the body weight determined
immediately before saline or naloxone injection and a second determi­
nation made 20 min later, immediately after the conditioning.
J. Navarro-Zaragoza et al. Pharmacology, Biochemistry and Behavior 201 (2021) 173106

Fig. 1. Schematic representation of the experimental protocol. On day 0, animals were allowed to freely explore the apparatus for 15 min. From day 1 to 4, animals
were treated with morphine or saline. On day 4, and 90 min after last morphine injection, CP-154,526 or its vehicle (Tween 80, 10%) were administered 30 min
before naloxone or saline and mice were immediately confined to one of the chambers during 18 min. The test was conducted on day 5 as in the preconditioning
phase (free access to each compartment for 15 min). 24 h after the post-training test, CPA extinction was performed for 21 consecutive days (day 6–27) under
identical conditions that the pre-test and the post-test.

2.4. Extinction of conditioned place aversion
24 h after the post-training test, the extinction training began. Then,
CPA extinction was performed for 21 consecutive days (day 6–27) under
identical conditions that the pre-test and the post-test. This protocol was
previously used in our laboratory (García-Carmona et al., 2015a).
2.5. Radioimmunoasay
Sixty minutes after the post-test or the CPA extinction finishes, mice
were decapitated at the same time (10:00–11:00 h) and blood samples
were collected. Plasma corticosterone concentrations were quantified by
using an available kit for mice (125 I-corticosterone radioimmunoassay;
MP Biomedicals, USA). The assay sensivity was 7.7 ng/ml for
corticosterone.
2.6. Drugs and reagents
Dilutions of morphine and naloxone to induce a withdrawal syn­
drome were prepared. Morphine HCl (Alcaliber, Madrid, Spain) and
naloxone HCl (Sigma Chemical, St. Louis, MO, USA), were dissolved in
physiological saline. CP-154,526 [N-butyl-Nethyl-2,5-dimethyl-7-
(2,4,6-trimethylphenyl) pirrolo[3,2-e] pyrimidin-4-amine], selective
CRF1 receptor antagonist was kindly provided by Pfizer (New York) and
was dissolved in Tween 80 (10%, Sigma Adrich).
2.7. Statistical analysis
Three-way ANOVA was applied to study weight loss and CPA
expression, specifically to compare three factors: acute treatment,
chronic treatment and strain. Two-way ANOVA was applied to analyse
the differences between groups of treatment for the corticosterone re­
sults. Moreover, repeated-measures ANOVA was performed for the
analysis of the extinction of aversion and post-hoc Bonferroni test was
analysed to obtain differences between the different treatment groups.
Unpaired Student’s t-test was used when comparisons were restricted to
two experimental groups. A correlation study between corticosterone
plasma levels and place aversion score was performed, including sal­
+veh+nx, sal+CP+nx, mor+veh+nx and mor+CP+nx groups. All sta­
tistical analyses were performed using GraphPad Prism 6 (GraphPad
Software Inc., San Diego, CA, USA). Statistical significance was set at α
level of p < 0.05 in order to provide validity to this study.
parameter in Swiss and B6 mice. Three-way ANOVA revealed significant
main effects on body weight loss for acute treatment (F(3,102) = 2234, p
< 0.0001), chronic treatment (F(1,102) = 5634, p < 0.0001), and for
strain (F(1,102) = 1427, p < 0.0001). Also showed main effects for the
following interactions: chronic treatment and acute treatment (F(3,102)
= 2266, p < 0.0001); strain and acute treatment (F(3,102) = 688, p <
0.0001); strain and chronic treatment (F(1,102) = 1442, p < 0.0001); and
finally, for the interaction of the three studied factors (strain, chronic
treatment and acute treatment, (F(3,102) = 719, p < 0.0001)). Naloxone
injection to morphine-treated mice produced a significant (p < 0.001)
increase in body weight loss in Swiss and B6 animals. However, the
weight loss in morphine-withdrawn B6 mice was significantly (p < 0.01)
higher than that observed in Swiss mice after naloxone-precipitated
morphine withdrawal suggesting more severe withdrawal in B6 mice.
The percentages of body weight loss were 3,3% for Swiss and 7,9% for
B6 mice. CP-154,526 administration antagonized the increased body
weight loss in Swiss mice without any changes in B6 mice. These results
indicate the involvement of CRF1 receptor in the body weight loss
observed in Swiss mice. In contrast, CRF1 receptor does not seem to be
involved in B6 mice (Fig. 2).
3.2. CPA to morphine withdrawal
Three-way ANOVA examined the effects of strain, morphine and
naloxone on place aversion induced by naloxone-precipitated morphine
withdrawal. We observed significant main effects for acute treatment
(F(3,106) = 93.62, p < 0.0001), chronic treatment (F(1,106) = 174.5, p <
0.0001), and for strain (F(1,106) = 547.5, p < 0.0001). Also showed main
effects for the following interactions: chronic treatment and acute
treatment (F(3,106) = 92.55, p < 0.0001); strain and acute treatment

3. Results
3.1. Body weight loss after naloxone-precipitated morphine withdrawal
Since body weight loss induced by naloxone-precipitated morphine
withdrawal is an unbiased and accurate measurable sign of opioid
withdrawal (Becker and Hu, 2008; Wu et al., 2014), we evaluated this
Fig. 2. Changes in body weight. Effect of naloxone (nx, 1 mg/kg, s.c.) on body
weight loss in Swiss and C57BL/6 mice treated with morphine (10–60 mg/kg, i.
p.) or saline) every 12 h during four days. CP-154,526 or its vehicle (veh) was
administered 30 min before naloxone. Data are the mean ± SEM. N = 16 for
each group, ***p < 0.001 versus saline+veh+nx; +++p < 0.001 versus mor­
phine+veh+nx; ##p < 0.05 versus Swiss mice.

3
J. Navarro-Zaragoza et al.
(F(3,106) = 22.54, p < 0.0001); strain and chronic treatment (F(1,106) =
51.57, p < 0.0001); and finally, for the interaction of the three studied
factors (strain, chronic treatment and acute treatment, (F(3,106) = 25.28,
p < 0.0001). For the morphine treated groups, the aversion for the
naloxone-paired chamber was higher (p < 0.01) in B6 mice versus Swiss
mice. There was a significant difference between the scores exhibited by
morphine dependent mice receiving vehicle plus naloxone versus those
presented by Swiss mice receiving the CRF1 receptor antagonist before
vehicle plus naloxone (t14 = 3.165, p = 0.0069; Student t-test), indi­
cating that the blockade of CRF1 receptor before conditioning prevented
the avoidance behavior seen during naloxone-induced withdrawal in
Swiss mice. In contrast, CP-154,526 did not induce changes in B6 mice
(Fig. 3A).
We examined extinction of CPA score in withdrawn mice for aversion
from day 6 to 27 (Fig. 3B). ANOVA with repeated measures showed that
B6 mice extinguished their aversion much later than Swiss mice (F(1,15)
= 24,92, p < 0.0001). On day 15, B6 mice retained aversion whereas
Swiss mice significantly (p < 0.01) extinguished their aversion. These
results indicate that Swiss mice not only showed a lower aversion but
also it lasted much less time than in B6 mice. CP-154,526 administration
did not induce any changes in B6 mice whereas this selective CRF1 re­
ceptor antagonist decreases the extinction period in the Swiss mice but
not significantly (Fig. 3B).
3.3. Corticosterone plasma levels
We measured plasma corticosterone levels in Swiss and B6 mice after
Fig. 3. Morphine conditioned place aversion (CPA). A) CPA expression induced
by naloxone (nx, 1 mg/kg, s.c.) in Swiss and C57BL/6 mice treated with
morphine or saline. CP-154,526 or its vehicle (veh) was administered 30 min
before naloxone. The score was calculated for each mouse as the difference
between the postconditioning and the preconditioning time spent in the drug-
paired compartment. B) Extinction of CPA training. Aversion scores from day
5 to 27 are shown. Data are expressed as the mean ± SEM. N = 8 for each
group, ***p < 0.001 versus saline+veh+nx; ++p < 0.01 versus morphi­
ne+veh+nx; ## p < 0.01 versus Swiss mice.
4
Pharmacology, Biochemistry and Behavior 201 (2021) 173106
naloxone-induced CPA expression and CPA extinction (Fig. 4A,B). Two-
way ANOVA for corticosterone plasma levels after CPA expression
revealed a main effect of strain (F(1,84) = 12.87, p = 0.0006), treatment
(F(5,84) = 4.925, p = 0.0005) and strain x treatment interaction (F(5,84) =
5.530, p = 0.0002), including a significant positive correlation between
plasma corticosterone levels and the place aversion score (r2 = 0.3832,
p = 0.0002, Fig. 4C). With respect to corticosterone after CPA extinction,
two-way ANOVA revealed significant effect of strain (F(1,84) = 21.38, p
< 0.0001), treatment (F(5,84) = 6.289, p = 0.0001) and strain x treatment
interaction (F(5,84) = 8.475, p < 0.0001). As shown in Fig. 4A naloxone
induced a dramatic increase (p < 0.001) in corticosterone plasma levels
after CPA expression in morphine-treated Swiss mice versus the sali­
ne+vehicle+naloxone group (p < 0.001) or B6 mice treated with mor­
phine+vehicle+naloxone (p < 0.01). However, no changes in
corticosterone plasma levels were observed after CPA expression in B6
mice. CP-154,526 significantly (p < 0.001) antagonized the increased
expression of corticosterone observed in Swiss mice after CPA expres­
sion (Fig. 4A). Extinct morphine-withdrawn Swiss mice showed a
decreased (p < 0.001) in corticosterone plasma levels versus the same
strain treated with saline and B6 mice treated with morphine. The se­
lective CRF1 receptor antagonist, significantly (p < 0.001) decreased the
corticosterone plasma levels versus the group treated with morphi­
ne+vehicle+naloxone. There were no changes in the corticosterone
levels in B6 mice after conditioned-naloxone withdrawal or after CP-
154,526 administration (Fig. 4B).
4. Discussion
The current study employed two different mice (B6 and Swiss) to
model various strategies able to deal with an aversive event. The two
selected mice represented different phenotypes with different response
to well validated anxiolytic screening tests. Swiss mice showed inter­
mediate levels of anxiety-related behaviors while B6 mice responded
with high levels of emotional reactivity (da Silva et al., 2016). Our re­
sults demonstrated that body weight loss, a prominent somatic sign of
opioid withdrawal, was higher in B6 mice versus Swiss mice. These re­
sults suggest that B6 mice are more sensitive to naloxone-induced
morphine withdrawal, what would also explain the basal differences
found in CPA expression. These findings are consistent with reports
showing that Swiss mice are less sensitive to opioid withdrawal from
morphine than B6 mice, as measured by abrupt withdrawal jumping
incidence (Brase et al., 1977). It is commonly accepted that affective
drug withdrawal symptoms have a major motivational significance in
contributing to relapse and continued drug use; thus, it is important to
understand the mechanisms that mediate affective behaviors during
morphine withdrawal. A previous work has suggested that morphine
associated with negative affective states and place aversion to previous
neutral environmental stimuli, could represent a motivational compo­
nent for the maintenance of drug abuse (Budzynska et al., 2012). In the
present study, we further investigated the mechanism underlying strain-
specific differences in CPA expression and extinction.
Our study confirms that naloxone triggers CPA in morphine-
dependent mice but not in naïve mice as previously shown (Maldo­
nado et al., 2004; Rothwell et al., 2009; Gamage et al., 2015; García-
Carmona et al., 2015a, 2015b; Valero et al., 2018; Martínez-Laorden
et al., 2019; Solecki et al., 2019). In contrast, some studies described
aversive property of naloxone administration in drug-naïve animals
(Mucha and Walker, 1987; Skoubis et al., 2001; Shoblock and Maid­
ment, 2006; Kirkpatrick and Bryant, 2015). Since naloxone blocks the
action of enkephalins at the mu opioid receptor (Skoubis et al., 2001,
2005), this discrepancy suggests differences in the release of endogenous
opioids depending on the stress levels during CPA. A previous study
(García-Carmona et al., 2015a, 2015b) described aversive effects
induced by naloxone in morphine-dependent mice together with with­
drawal signs such as body weight loss, jumping, rearing, rubbing,
grooming, diarrhea, freezing and time to first immobility.
J. Navarro-Zaragoza et al.
Fig. 4. Corticosterone plasma levels after the expression (A) and extinction (B) of CPA training. C) Correlation between corticosterone and conditioned place
aversion (CPA). Data are expressed as the mean ± SEM. N = 8 for each group, ***p < 0.001 versus saline+veh+nx; ++p < 0.01, +++p < 0.001 versus morphi­
ne+veh+nx, ###p < 0.001 versus B6 mice.
On the other hand, Swiss mice developed less aversion and took less
time to extinguish aversive memory, together with a lower body weight
loss, than B6 mice. These results indicate that Swiss mice present a
weaker withdrawal syndrome. The greater sensitivity of B6 strain could
be explained by strain differences in the unconditioned response to
naloxone. These results are consistent with previously published data
indicating strain differences for the aversive properties of naloxone in
morphine-treated mice (Kirkpatrick and Bryant, 2015). In accordance
with these data, it has been described that B6 mice are more susceptible
to cocaine-induced locomotor activation that other inbred mouse strains
(Wang et al., 2014; Roberts et al., 2018).
On the other hand, there is much information about the neurobio­
logical mechanisms of extinction or reward memory of drug taking
(Feltenstein and See, 2007; Hsu and Packard, 2008; Torregrossa et al.,
2010). However, little information is known about extinction of aversive
memory of drug withdrawal (Myers and Carlezon, 2010). We found
differences between Swiss and B6 mice for CPA extinction. Thus, CPA
expression in B6 mice was showed until day 27 while Swiss mice
extinguished their associative learning on day 18. Memory impairment
during drug withdrawal is a complex phenomenon. It requires an un­
derstanding of the mechanisms underlying extinction of aversive
memories. This further research could lead to pharmacological ap­
proaches to shorten the extinction period and might facilitate the
treatment of drug addiction. In the present study, we investigated the
mechanism underlying the aversive memory associated with CPA
expression and extinction in morphine-withdrawn mice. Our data
demonstrated that naloxone injection into morphine-treated Swiss mice
produced significant CPA. However, CP-154,526 interrupts naloxone-
induced CPA in morphine-treated animals. It suggests that the consoli­
dation process of opioid-withdrawn associated aversion could be linked
to CRF1 receptor activation. These results agree with previous studies
performed in CFR1 knockout mice (Contarino and Papaleo, 2005; Gar­
cía-Carmona et al., 2015a). In addition, CP-154,526 administration
abolished the acquisition of morphine conditioned place preference
(CPP) (Lasheras et al., 2015; García-Carmona et al., 2015b). Altogether,
these studies indicate that CRF through CRF1 receptor plays an impor­
tant role in the consolidation and expression of either aversive or
rewarding drug-related memories in Swiss mice. However, CRF1 re­
ceptor is not involved in aversive memory observed in B6 mice. It
5
Pharmacology, Biochemistry and Behavior 201 (2021) 173106
suggests marked differences according to the strain or stock considered.
This study is the first to specifically investigate the function of HPA axis
in CPA expression and extinction in two different mice. Our findings
demonstrated that the acquisition of CPA was accompanied by increased
corticosterone plasma levels, which was blocked by CP-154,526 sug­
gesting that HPA axis could contribute through CRF1 receptor to the
formation of aversive memory by increasing corticosterone concentra­
tions. However, the CPA expression observed in B6 mice was not
accompanied by increases in plasma corticosterone levels. These last
results clearly confirm that different mechanisms are involved depend­
ing on the strain or stock considered in the withdrawal syndrome
conditioned by naloxone. Moreover, it is possible that CPA paradigm in
B6 mice results in a reduced induction of HPA axis, with a lack of in­
crease in plasma corticosterone levels at 1 h-time point following the
CPA paradigm. Corticosterone plasma levels decreased in Swiss mice
after CPA extinction without any changes in B6 mice. It is known that
glucocorticoids may have different effects on memory depending on the
dose and timing of administration (de Quervain et al., 2009). Gluco­
corticoids promote the consolidation of memory processing and, at the
same time, impair the retrieval of memory of emotionally arousing ex­
periences (Wolf et al., 2015). We found that CPA expression in Swiss
mice increases corticosterone levels, which were decreased after CPA
extinction. In contrast, B6 mice showed a much higher CPA expression
and a longer period of extinction of the aversive memory than Swiss
mice. These results suggest that HPA activation could impair retrieval of
aversive memory and would promote a decrease in the response to the
conditioned context. These results are in accordance with previous
studies that showed that dexamethasone, a glucocorticoid receptor
agonist, could impair the consolidation of fear memory (Sawamura
et al., 2016; Szklarczyk et al., 2016). In addition, a previous study in our
laboratory showed that the pre-treatment with a selective CRF1 receptor
antagonist interrupts naloxone-induced CPA in morphine treated ani­
mals indicating a role for CRF1 receptor (Valero et al., 2018). Moreover,
present results demonstrated a relationship between CPA and HPA
response, mediated by CRF1 receptor activation, that is dependent on
strain or stock used. The differences found in Swiss and B6 in the
establishment and extinction of aversive memory could be due to
changes in the expression of CRF1 receptors. This hypothesis is sup­
ported by previous studies that have demonstrated strain differences in
J. Navarro-Zaragoza et al.
stress-induced changes in central CRF1 receptor expression (O’Malley
et al., 2014).
Comparisons between Swiss and B6 mice revealed that different
neurobiological mechanisms may be involved in the retrieval and
extinction of aversive memory. Differences in the behavioral response
may be based, in part, in intrinsic differences in the ability to associate
(learning) and recall (memory) aversive events. It has been demon­
strated significant strain differences in different learning tasks (Y-maze,
object recognition and passive avoidance), using C57 and BALB animals
(García and Esquivel, 2018). Although, these data support the idea that
genetic background influences addiction-related behaviors, the extent of
cognitive differences in the ability to store and remember aversive
events remains to be elucidated.
5. Conclusions
We concluded that CPA expression and extinction induces neuro­
adaptive changes in the HPA axis, but in a strain-specific manner. CPA
expression and extinction induced changes in the activity of HPA axis
through CRF/CRF1 receptor in Swiss mice, which could be responsible
of the differences in aversion and the shortening of the extinction period
observed in this stock. Thus, the genetic variability in the memory
processing of drug aversion and cues could be a consequence of different
individual vulnerability to drug addiction.
CRediT authorship contribution statement
JNZ, PA and MLL designed the study. MVM wrote the protocol. JNZ,
EML, FJTF, AC and PA conducted the experiments. MLL performed the
statistical analysis. JNZ, MLL and PA wrote the manuscript. All the au­
thors read and approved the final version of the manuscript.
Acknowledgements
Funding for this study was provided by grants from Ministerio de
Ciencia e Innovación (SAF/FEDER 2017-85679-R), Fundación Séneca
(20847/PI/18), Murcia, Spain; and Red de Trastornos Adictivos (RTA;
RD12/0028/0003; Instituto de Salud Carlos III), Madrid, Spain.
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