Basic Research Journal of Pharmaceutical Science Vol. 1(1) pp. 08-14 May 2013Available online http//www.basicresearchjournals.orgCopyright 2012 Basic Research Journal
The effect of propranolol and metoprolol on memory performance during morphine withdwal in mice Azadeh Mesripour*, Mahmoud Rafieian-kopaei, Roya Ansari
Medical Plants Research Centre, Shahrekord University of Medical Sciences, Shahrekord, IRAN
*Corresponding author email: [email protected] or [email protected]; Tel: +98 381 3335652; Fax: +98 381 3330709. Beta blockers were used to see if they could prevent the destructive effects of GCs on memory acts. Cognition was evaluated using the object recognition task, the difference in the exploration time between a familial (F) object and a novel (N) object was taken as an index of recognition (RI). Male mice were made dependent by increasing doses of morphine twice daily for three days; RI was assessed after naloxone precipitated withdrawal on the third day. Propranolol and metoprolol were used before the test trial in morphine withdrawn animals. These drugs were also administered as a 3 day protocol. Memory performance was impaired in morphine dependent animals, however neither propranolol nor metoprolol single dose, or three days protocol showed no improvement in memory performance. Beta blockers effects on other aspects of recognition for instance sedation and anxiety may augment recognition loss. Keywords: morphine; withdrawal; memory; β-blocker; object recognition task INTRODUCTION
Several lines of evidence indicate that opiates modify
noradrenergic activity of the basolateral amygdala is
learning and memory processes (Peart, Gross et al.,
critical for enabling glucocorticoid effects on memory
2005; Bodnar and Klein 2005). It is also well known that
consolidation and retrieval (Roozendaal et al., 2003).
learning and memory are critically involved in the
Due to a rich and widespread projection of adrenergic
morphine dependence and relapse (Robbins and Everitt
axons, it is thought that norepinephrine (NE) plays a
1999). Morphine impaired memory acquisition in the
modulatory role in some aspects of brain function
radial maze (Spain and Newsom 1991) and water maze
(Jurgens et al., 2005). Evidently NE plays an important
(Li, et al., 2001). Using the object recognition task it was
role in regulating memory storage through a beta
also observed that recognition memory was impaired in
adrenergic receptor-mediated mechanism (Ferry et al.,
The effect of beta receptors on memory performance
Morphine withdrawal is associated with activation of the
after morphine withdrawal, and the elevated level of
hypothalamic pituitary adrenocortical axis (Morley, 1981).
corticosterone concentration are not clear yet. Therefore
Glucocorticoid measurements in serum and brain
in the present study the effects of propranolol (β1, 2
samples after morphine withdrawal also showed
antagonist) and metoprolol (β1 selective antagonist) were
considerable increase in glucocorticoid concentration
compared with controls (Rabbani et al., 2009).
dependent animals after withdrawal. Since beta blockers
Findings demonstrate that after memories are
have proved to be useful in controlling stress disorders,
consolidated, the efficacy or accuracy of the information
therefore they may also be useful to decrease
retrieved is vulnerable to glucocorticoid influences at the
corticosterone induced memory impairment following
morphine withdrawal in dependant animals. If such these
results could open new windows toward improving drug
therapy following morphine withdrawal in patients.
increasing doses of morphine sulfate (Temade co.,Tehran, Iran), twice daily with 12 hour intervals; 30 and45 mg/kg at the first day, 60 and 90 mg/kg at the second
MATERIALS AND METHODS
day, and 90 mg/kg the last day (Mesripour et al., 2008) . Time interval elapsed between the last morphine dose
Animals. Male Balb/C mice weighing 25-30 g were
and naloxone injection was 3 h, naloxone (Tolid Daru
housed in cages of six at 21± 2 oC in a 12 h light-dark
Co., Tehran, Iran) was injected after T1 (in the morphine+
cycle with the lights on at day time 6 am-6 pm. Tap water
naloxone and naloxone alone groups). Twenty minutes
and standard food pellets were available ad libitum. Tests
after naloxone injection mice were tested in the memory
were performed only after the mice had acclimated to the
apparatus (T2). Propranolol and metoprolol (Darou-
above environment for at least 2 days. In order to
pakhsh, Tehran, Iran) (2 mg/kg) were injected 40 min
minimize circadian rhythm influence, all experiments
before T2 and control animals received saline.
were conducted between 08:00 and 13:00 h, in a special
The effect of 3 days propranolol and metoprolol (2
noise-free room with controlled illumination. Minimum of
mg/kg) were also determined on memory performance
six mice were used for each treatment group. All
after morphine withdrawal. In this regard animals
procedures were approved by the Ethical Committee of
received a single dose of propranolol, metoprolol or
the University of Medical Sciences, and conducted in
saline each day 2 hour after morphine injection. The
accordance with the ‘Principles of Laboratory Animal
timing of injections at the last day was as mentioned
Care’ (National Institutes of Health publication no. 86-23,
earlier. The effect of propranolol and metoprolol alone on
memory performance was also determined.
In order to evaluate the effect of morphine withdrawal
Data processing and statistical analysis. The following
induced stress on the adrenal, animals were euthanized
parameters were measured: time required to achieve 20
using di-ethyl ether for less than 3 min and rapidly after
s of object exploration on T1 (duration of T1), time spent
laparotomy their adrenals were collected and placed on
in active exploration of the familiar (F) or novel (N) object
water absorbent paper to absorb the extra blood and they
on T2. Recognition memory was evaluated using a
were weighed rapidly and measured as mg/g body
recognition index (RI) calculated for each animal using
the formula: (N−F/N+F)×100 corresponding to the
Object recognition task. The object recognition task
difference between the time exploring the novel and the
was performed as described previously (Mesripour et al.,
familiar object, corrected for total time exploring both
2008). Briefly, square wooden open-field (35×35×40 cm)
objects (Bertaina-Anglade et al., 2006). Positive values
with the inside painted in dark black and a white floor was
indicate a good discrimination performance, while
used. It was placed in a dark room with a uniform dim
negative values or those around zero indicate very poor
At the first day, animals were submitted to a habituation
Results were expressed as group mean ± SEM. All
session in the open field and allowed to freely explore the
results were analyzed by a one-way analysis of variance
arena in the presence of two objects for at least 15 min.
(ANOVA), followed by Duncan’s multiple comparison
On experimental day, animals were submitted to two
tests, P values less than 0.05 were considered
trials spaced by an intertrial interval (20 min). During the
significant. The software used for data analyzing and
first trial (acquisition trial, T1), animals were placed in the
making graphs was the GraphPad Prizm 5.
arena containing two identical objects for an amount oftime necessary to explore the objects for 20 s. Anymouse not exploring the objects for 20 s within the 12-
min period was excluded from experiments. Explorationis defined as the animal directing the nose within 2 cm of
The effect of treatments on the learning procedure. At the
the object while looking at, sniffing, or touching it. For the
first trial or the learning period the time required for 20 s
second trial (test trial, T2), one of the objects presented in
the first trial was replaced by new object, animals were
recognize the objects rapidly (T1= 2.6 min ± 0.42).
placed back in the arena for 5 min and total time spent in
Propranolol and metoprolol were used in normal
exploration of each object was determined. Animals
animals and the results were compared with vehicle
behavior were recorded by using a web camera mounted
(Table 1). At the first trial although propranolol and
above the experimental apparatus, records were
metoprolol increased the recognition time (T1) to 3.3min
± 0.3 and 3.2min ± 0.7 respectively but it was not
Figure 1. The effect of propranolol single dose (sc, 40 min before T2) alone together with its effect on memory performance after naloxone (0.1mg/kg, ip) precipitated morphine withdrawal on day 3. This figure also shows the effect of naloxone alone on RI. Memory is expressed as recognition index RI= (N−F/N+F) ×100, in the novel object recognition task. Vehicle treated animals received saline. Results are expressed as group mean ± SEM and analyzed by ANOVA followed by Duncan’s multiple comparison tests. *P< 0.05 compared with control values, that received only saline (blank bar). Number of animals in each group was 6.
considerably differed compared with control. Morphine
observed in the vehicle group (RI= -2.4% ± 5, P< 0.05).
dependent animals also required more time to recognize
the objects but this was not significant (T1= 5min ± 1.6). The effect of beta antagonists alone on memory
withdrawal in morphine dependent animals. performance. Propranolol or metoprolol (2 mg/kg) were
The effect of 3 days injection of beta antagonists on RI
injected 40 min before T2. These drugs affected memory
during withdrawal. Figure 3 demonstrates the effect of
performance at the second trial. As can be depicted in
daily propranolol treatment on the RI during morphine
Figure 1, RI- significantly reduced to 3.3% ± 0.33
withdrawal. The simultaneous daily administration of
(P<0.05) in animals treated with 2 mg/kg propranolol, that
propranolol 2 mg/kg with morphine did not improve RI
is animals did not distinguish between the new and the
familial object. Treating animals with 2 mg/kg metoprolol
The effect of daily metoprolol on the RI is shown in
also caused a drop in RI (-2.9% ± 8.4) compared with
figure 4. The concurrent daily administration of metoprolol
saline values accounting for RI= 34% ± 3.6, P<0.05 (Fig.
2 mg/kg with morphine also could not have any useful
2). Therefore memory performance was influenced by the
impact on the corresponding values (RI=5.5% ± 7).
above drugs in the NOR task. As it is presented in figures
memory performance was not improved according to the
3 and 4, three days treatment with propranolol or
metoprolol (2 mg/kg) also impaired memory performance
Adrenal gland weights. After animals were euthanized
(RI=-5.5% ± 4.3, RI=-8.5% ± 3.1 respectively, P<0.05).
adrenal glands were excised and measured as mg/g
The effect of beta antagonists’ single dose on RI during
body weight. As presented in table 1 after naloxone
withdrawal. Figure 1 illustrates RI scores following the
precipitated morphine withdrawal there was no difference
concurrent treatments. Memory index in animals that
between adrenal weights in either of the groups.
received 2 mg/kg propranolol was -2.2% ± 9.9 that alikevehicle values (RI= -12% ± 4.5) indicate memorydamage, these values significantly differed with the
control values (RI= 34% ± 3.6, P< 0.05).
As can be depicted in Figure 2, administration of
Based on our experiments memory performance was
metoprolol 2 mg/kg following withdrawal did not improve
impaired in morphine dependent animals compared to
memory performance (RI= 3.5% ± 11) same was
control values. Neither Propranolol nor metoprolol single
Figure 2. The effect of metoprolol single dose (sc, 40 min before T2) alone together with its effect on memory performance after naloxone (0.1mg/kg, ip) precipitated morphine withdrawal on day 3. Memory is expressed as recognition index RI= (N−F/N+F) ×100, in the novel object recognition task. Vehicle treated animals received saline. Results are expressed as group mean ± SEM and analyzed by ANOVA followed by Duncan’s multiple comparison tests. *P< 0.05 compared with control values, that received only saline (blank bar). Number of animals in each group was 6. Figure 3. The effect of 3 days propranolol injections alone together
precipitated morphine withdrawal on day 3. Memory is expressed asrecognition index RI= (N−F/N+F) ×100, in the novel object recognitiontask. Vehicle treated animals received saline. Results are expressedas group mean ± SEM and analyzed by ANOVA followed byDuncan’s multiple comparison tests. *P< 0.05 compared with controlvalues, that received only saline (blank bar). Number of animals ineach group was 6. Figure 4. The effect of 3 days metoprolol injections alone together with its effect on memory performance after naloxone (0.1mg/kg, ip) precipitated morphine withdrawal on day 3. Memory is expressed as recognition index RI= (N−F/N+F) ×100, in the novel object recognition task. Vehicle treated animals received saline. Results are expressed as group mean ± SEM and analyzed by ANOVA followed by Duncan’s multiple comparison tests. *P< 0.05 compared with control values, that received only saline (blank bar). Number of animals in each group was 6. Table 1. Effect of beta blockers on adrenal weight and on learning performance propranolol metoprolol Morphine Morphine Morphine +propranolol +metoprolol +vehicle
The effect of naloxone (0.1 mg/kg, ip) precipitated morphine withdrawal on adrenal weight in morphine dependentanimals. The amount of time necessary to explore the objects for 20 s was measured as the T1 value. Vehicletreated animals received saline. Results are expressed as group mean ± SEM and analyzed by ANOVA followed byDuncan’s multiple comparison tests. There were no difference concerning adrenal weight or learning performanceamong groups. Number of animals in each group was 6.
dose, or daily dose showed no improvement in memory
these experiments memory was impaired after withdrawal
performance, compared to vehicle group in morphine
In order to evaluate the validity of this memory
paradigm scopolamine (0.5 mg/kg) was used. Learning
Norepinephrine or the β-adrenoceptor agonist clenbuterol
did not differ from control animals but memory
infused into the BLA immediately post training enhanced
performance was impaired. These experiments proved
the memory of many emotionally arousing training tasks,
that in our experimental condition this memory apparatus
including inhibitory avoidance (Ferry et al., 1999),
could show the memory performance in mice. As shown
contextual fear conditioning (Huff et al., 2005) and water-
previously, naloxone 0.1 mg/kg did not impair memory by
maze spatial training (Hatfield and McGaugh 1999). In
its own (figure 1) and the effect of naloxone observed in
contrast, post training intra-BLA infusions of β-
morphine dependent animals is due to induction of
adrenoceptor antagonists impaired the consolidation of
withdrawal (Mesripour et al., 2008). Since zero or values
memory for emotionally arousing training experiences
of RI under zero indicate memory impairments, thus in
(Miranda et al., 2003). Therefore noradrenergic activities
in brain regions remain important also for memory
Memory impairment observed following morphine
withdrawal in mice, did not recover after using a single
dose of propranolol or metoprolol at the last day of the
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