Author’s Accepted Manuscript

Neuromodulatory effects of the dorsal hippocampal

endocannabinoid system in
dextromethorphan/morphine-induced amnesia

Zahra Ghasemzadeh, Ameneh Rezayof

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PII: S0014-2999(16)30728-2
DOI: http://dx.doi.org/10.1016/j.ejphar.2016.11.025
Reference: EJP70930
To appear in: European Journal of Pharmacology
Received date: 13 June 2016
Revised date: 29 October 2016
Accepted date: 17 November 2016
Cite this article as: Zahra Ghasemzadeh and Ameneh Rezayof, Neuromodulatory
effects of the dorsal hippocampal endocannabinoid system in
dextromethorphan/morphine-induced amnesia, European Journal of
Pharmacology, http://dx.doi.org/10.1016/j.ejphar.2016.11.025
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Neuromodulatory effects of the dorsal hippocampal endocannabinoid system in

dextromethorphan/morphine-induced amnesia

Zahra Ghasemzadeh, Ameneh Rezayof*

Department of Animal Biology, School of Biology, College of Science, University of Tehran, Tehran,

Iran
*
Correspondence to. Department of Animal Biology, School of Biology, College of Science,
University of Tehran, P. O. Box 4155-6455, Tehran, Iran Fax: (+9821)-66405141 Tel: (+9821)-
61112483 rezayof@khayam.ut.ac.ir

Abstract

Dextromethorphan which is an active ingredient in many cough medicines has been

previously shown to potentiate amnesic effect of morphine in rats. However, the effect of

dextromethorphan, that is also a noncompetitive N-methyl-D-aspartate (NMDA) receptor

antagonist, in combination with morphine on hippocampus-based long term memory has not

been well characterized. The aim of the present study was to assess the possible role of

endocannabinoid system of the dorsal hippocampus in dextromethorphan /morphine-induced

amnesia. Our results showed that intraperitoneal (i.p.) injection of morphine (5 mg/kg) or

dextromethorphan (5-15 mg/kg) before testing the passive avoidance learning induced amnesia.

Combination of ineffective doses of dextromethorphan (7.5 mg/kg, i.p.) and morphine (2 mg/kg,

i.p.) also produced amnesia, suggesting the enhancing effects of the drugs. To assess the effect of

the activation or inhibition of the dorsal hippocampal cannabinoid CB1 receptors on this amnesia,

ACPA or AM251 as selective receptor agonists or antagonists were respectively injected into the

CA1 regions before systemic injection of dextromethorphan and morphine. Interestingly, intra-

CA1 microinjection of ACPA (0.5-1 ng/rat) improved the amnesic effect of dextromethorphan
1
/morphine combination. The microinjection of AM251 into the CA1 region enhanced the

response of the combination of dextromethorphan /morphine in inducing amnesia. Moreover,

Intra-CA1 microinjection of AM251 inhibited the improving effect of ACPA on

dextromethorphan /morphine-induced amnesia. It is important to note that intra-CA1

microinjection of the same doses of the agonist or antagonist by itself had no effects on memory

formation. Thus, it can be concluded that the dorsal hippocampal endocannabinoid system, via

CB1 receptor-dependent mechanism, may be involved in morphine/dextromethorphan -induced

amnesia.

Keywords: Dextromethorphan; Morphine; Dorsal hippocampal CB1 receptors; Passive

avoidance learning; Rat(s)

1. Introduction

Endocannabinoids play critical roles in numerous physiological and pathophysiological

processes (Katona and Freund, 2012). Endocannabinoids exert their effects through two types of

receptors, namely CB1 and CB2 receptors which belong to Gi/o-protein-coupled receptors

(Svíženská et al., 2008). CB1 receptors are widely expressed in the major brain regions including

the hippocampus (Liu et al., 2003) which is involved in learning and memory processes (Morris,

2006). Activation of CB1 receptors in the dorsal hippocampal CA1 regions also inhibited long-

term potentiation which may be associated with a G protein-dependent presynaptic inhibition of

glutamate transmission (Sullivan, 2000). In view of the fact that there is an overlapping

distribution of CB1 and mu-opioid receptors in the hippocampus (Robledo et al., 2008), it has

been suggested that a functional correlation between the endocannabinoid and opioid systems

mediate hippocampus-based memory formation (Parolaro et al., 2010). Since integrity of

2
hippocampal function is necessary for normal cognitive processes (Deng et al., 2010), the

amnesic effect of morphine seems to be related to the hippocampus-based memory system in

different animal learning models (Farahmandfar et al., 2010; Tirgar et al., 2014). It should also

be considered that the neuromodulatory role of opioids within hippocampal formation circuits

may be directly or indirectly associated with the high expression of mu opioid receptors in this

brain site (Stumm et al., 2004).

Ample evidence suggests that dextromethorphan which is a non-opioid cough

suppressant drug is frequently co-abused with other drugs (Wilson et al., 2011). Strong

motivations for the co-abuse of dextromethorphan with morphine are assumed to lie in the high

euphoric effect or the reduced side effects of the opiate (Mao et al., 1996). Although

dextromethorphan has been shown to be effective in reducing the rewarding properties, the abuse

liability of dextromethorphan is also reported among adolescents (Bryner et al., 2006). Drug

abuse seems to reduce the populations of hippocampal neurons via attenuating neurogenesis

(Eisch and Harburg, 2006) which is an important mechanism underlying memory formation

(Aimone et al., 2006). It is well known that morphine plays a critical role in the modulation of

hippocampal structure, physiology, and biochemistry (Simmons and Chavkin, 1996, Chavkin,

2000). Despite dextromethorphan’s long clinical success, our recent study showed that the use of

the drug alone or in combination with morphine induced amnesia via attenuating hippocampal

calmodulin-dependent protein kinase II (CAMKII) and cAMP-response-element-binding protein

(CREB) as critical mediators of memory formation (Ghasemzadeh and Rezayof, 2016).

gnorndisnoC that the effect of multi-drug abuse on learning and memory processes are not fully

understood and that the dorsal hippocampal endocannabinoid system affects memory formation

(de Oliveira Alvares et al., 2008), our aim was to investigate whether the CA1 endocannabinoid

3
system via CB1 receptors is involved in the effect of systemic co-administration of

dextromethorphan /morphine on memory recall.

2. Materials and methods

2.1. Animals

The experiments were carried out on male Wistar rats (weighing approximately 200–220

g) obtained from the animal house of the School of Biology, University of Tehran. The animals

were housed in groups of four per cage; they were maintained in a controlled temperature (22±2

°C), and a 12:12-h light–dark cycle (lights on at 7:00 h am) with ad libitum

access to food and water except during the test. All animals were allowed a week to adapt to the

laboratory conditions prior to the experiments and were handled daily. All procedures for the

treatment of animals were approved by the Research and Ethics Committee of the School of

Biology, University of Tehran and were done in accordance with the National Institute of Health

Guide for Care and Use of Laboratory Animals. Moreover, all efforts were made to minimize the

number of animals used and their suffering.

2.2. Surgery and microinjection procedures

Rats were anesthetized with an intraperitoneal injection of a mixture of ketamine

hydrochloride (50 mg/kg) plus xylazine (5 mg/kg). Using standard stereotaxic equipment, the

CA1 regions of the dorsal hippocampi were bilaterally implanted with guide cannulas (22

gauges) according to the atlas of Paxions and Watson (antero-posterior: -3 to -3.5 mm posterior

to the bregma, lateral: ±1.8–2 mm from midline, ventral: -2.8 to -3 mm relative to the dura;

Paxions and Watson, 2007). The guide cannulas were fixed to the skull with dental cement (1
4
mm above the CA1 region). During the 1-week recovery period, the animals were habituated to

the experimental room by being transferred to the experimental room and handled every day.

The microinjections into the CA1 regions were bilaterally performed with 2-µl Hamilton

microsyringe attached to the injection cannula via polyethylene tubing in a total volume of

1µl/rat (0.5 µl/each side). The solution was injected slowly (over 1 min) and the cannula was left

in place for an additional 60 s to reduce the backflow of the solution.

2.3. Drugs

The compounds used in this study were morphine sulfate (Temad, Tehran, Iran),

Dextrometorphan hydrobromide monohydrate (Sigma, USA), ACPA

(arachidonylcyclopropylamide; N-(2-cyclopropyl)-5Z, 8Z, 11Z, 14Z-eicosatertraenanmide;

Tocris, Bristol, UK) and AM 251 (N-(piperidin-1-yl)-5-(4-isodophenyl)-1-(2,4-dichlorophenyl)-

4-methyl-1H-pyrazole-3-arboxamide; Tocris, Bristol, UK). dextromethorphan and morphine

were dissolved in sterile 0.9% saline before use. ACPA was dissolved in Tocrisolve™ (a soya oil

and water emulsion) and was diluted with sterile 0.9% saline. In experiments where ACPA was

applied, the control solution contained Tocrisolve™ with the same concentration as in the

experimental solution (vehicle). AM251 was dissolved in dimethyl sulphoxide (DMSO; up to

10% v/v) and sterile 0.9% saline and a drop of Tween 80, which also was used as DMSO

(Mohammadmirzaei et al., 2016; Naghdi and Asadollahi, 2004). Morphine and

dextromethorphan were delivered intraperitoneally at a volume of 1 ml/kg. ACPA and AM251

were injected into the CA1 regions (intra-CA1) at a volume of 1 µl/rat.

5
2.4. Passive avoidance apparatus

A step-through passive avoidance apparatus (Borj Sanat, Tehran, Iran) was used for the

evaluation of memory performance. The apparatus consisted of two chambers (illuminated and

dark) separated by a guillotine door (20 × 20 × 30 cm high). The floor of the dark chamber

contained stainless steel rods (2.5 mm in diameter, 1 cm apart) that could deliver foot shocks.

2.5. Behavioral testing

2.5.1. Training

Animals were transported to the experimental room and allowed to habituate to the

experimental room for 60 min prior to the experiments. During the training trial, each animal

was placed in the illuminated compartment; After 5s, the guillotine door separating the chambers

was open. When the rat crossed into the black chamber, its latency to enter the black chamber

was measured. If any animal stayed on the illuminated chamber for over 120 s, it was excluded

from the experiments. After 30 min, the animal was placed in the illuminated compartment again

and after 5s, the guillotine door was opened. As soon as it entered the dark compartment, the

door was closed and the rat received an inescapable shock. After 2 min, the rat was transferred to

the illuminated compartment and the latency times for entering the dark compartment were

measured. An identical shock was delivered to animals entering the dark compartment before

120 s. If the animal did not enter the dark compartment during 120 s, successful acquisition of

passive avoidance response was recorded.

2.5.2. Recall test

6
 One day after the training trial, testing trial was done by placing the animal back in the

illuminated compartment and measuring its latency to enter the shock compartment. Foot shock

was not delivered on the testing trial, and the cut-off time limit was 300 s (Douma et al., 2011;

Tajik et al., 2016).

2.6. Experimental Design

2.6.1. Experiment 1. Dose-response curve of dextromethorphan, morphine or

dextromethorphan/morphine

In this experiment, six groups of animals were used for evaluating the effect of

dextromethorphan injection with or without morphine on memory recall. On the training day,

each animal was trained in a passive avoidance task. On the test day, six groups of animals

received morphine (0, 2 and 5 mg/kg) or dextromethorphan (5, 7.5 and 15 mg/kg). The other

three groups received the same doses of dextromethorphan plus an ineffective dose of morphine

(2 mg/kg) with 60 min interval. In all groups, the step-through latency was measured 30 min

after the last injection as an indicator of memory recall.

2.6.2. Experiment 2. The effect of intra-CA1 microinjection of ACPA (before the testing phase)

with or without systemic injection of dextromethorphan / morphine on memory recall

In this experiment, eight groups of animals were successfully trained. On the test day, the

animals received intra-CA1 microinjections of the different doses of a CB1 receptor agonist,

ACPA (0, 0.5, 0.75 and 1 ng/rat) and after 5 min they were intraperitoneally injected with an

effective dose of dextromethorphan (7.5 mg/kg)/morphine (2 mg/kg; Right panel of Fig. 2) or

7
saline (1 ml/kg)/saline (1 ml/kg; Left panel of Fig. 2). The animals were tested 30 min after the

last injection.

2.6.3. The effect of intra-CA1 microinjection of AM251 (before the testing phase) with or without

systemic injection of dextromethorphan / morphine on memory recall

In this experiment, eight groups of animals were successfully trained in a passive

avoidance task. On the test day, the animals received intra-CA1 microinjections of different

doses of a CB1 receptor antagonist, AM251 (0, 30, 40 and 50 ng/rat) and after 5 min they were

intraperitoneally administrated with an ineffective dose of dextromethorphan (5

mg/kg)/morphine (2 mg/kg; Right panel of Fig. 3) or saline (1 ml/kg)/saline (1 ml/kg; Left panel

of Fig. 3). The animals were tested 30 min after the last injection.

2.6.4. The effect of intra-CA1 microinjection of AM251 on ACPA improvement of the amnesia

induced by dextromethorphan /morphine combination

In this experiment, seven groups of animals were successfully trained in a passive

avoidance task. On the test day, five group received intra-CA1 microinjection of different doses of

AM251 (0, 10, 15, and 20 ng/rat) followed by vehicle (1 µl/rat) or ACPA (1 ng/rat) with 5 min

interval. After 5 min, they received dextromethorphan (7.5 mg/kg; i.p.) plus morphine (2 mg/kg;

i.p.). Two control groups were used in this experiment. One group received intra-CA1

microinjections of DMSO and vehicle (1 µl/rat) with 5 min interval, and after 5 min they

received interaperitoneal injection of saline (1 ml/kg; i.p.) plus saline (1 ml/kg; i.p.) with 60 min

interval. Another group received intra-CA1 microinjections of DMSO and vehicle (1 μl/rat) with

8
5 min interval, and after 5 min they were injected with saline (1 ml/kg; i.p.) plus morphine (2

mg/kg, i.p.). The latency times were measured 30 min after the last injection in each animal.

2.7. Histology

At the end of the experiments, the animals were deeply anesthetized with carbon dioxide,

and 1% methylene-blue solution was bilaterally injected into the CA1 regions of the dorsal

hippocampi (0.5 µl /side). The rats were decapitated and the brains were removed and fixed in

formaldehyde (10%). After several days, sectioning was done using a vibratome and the sections

were examined under a stereomicroscope according to the rat brain atlas of Paxinos and Watson

(2007). Only the data from animals with correct cannula implants were included in the statistical

analysis.

2.8. Data analysis

The effects of the drug treatments on memory during the test were analyzed by one-way

ANOVA, using SPSS software. Significance was set as P<0.05. All data values are expressed as

mean ± S.E.M. Two-way ANOVA was used to analyze the interaction between the drugs.

3. Result

3.1. The effect of dextromethorphan with or without morphine on memory recall

Fig. 1 shows the effect of pre-test injection of dextromethorphan and/or morphine on

latency time. Two-way ANOVA showed a significant difference between the effects of

dextromethorphan alone and dextromethorphan /morphine (2 mg/kg) on memory recall [for

Treatment, F (1, 48) = 4.08, P < 0.05; Dose, F (3, 48) = 18.18, P < 0.001; and Treatment×Dose

9
interaction, F (3,48)=5.4, P < 0.01]. One-way ANOVA [F (8, 54) = 10.24, P < 0.001] revealed

that injection of dextromethorphan (15 mg/kg, i.p.) or morphine (5 mg/kg, i.p.) before the testing

phase disrupted memory recall. The analysis also indicated that the combination of 7.5 and 15

mg/kg of dextromethorphan with an ineffective dose of morphine (2 mg/kg, i.p.) induced

amnesia, showing the enhancing effect of dextromethorphan on morphine response in passive

avoidance learning.

3.2. The effect of intra-CA1 microinjection of ACPA (before the testing phase) with or without

systemic injection of dextromethorphan / morphine on memory recall

Fig. 2 (left panel) shows the effect of intra-CA1 microinjection of ACPA (before the testing

phase) on memory recall. Two-way ANOVA revealed a significant difference between the

effects of ACPA alone and ACPA plus dextromethorphan (5 mg/kg) plus morphine (2 mg/kg)

on memory recall [for treatment, F (1, 48)= 57.09, P < 0.001; Dose, F (3,48)= 2.89, P < 0.01;

and treatment×dose interaction, F (3, 48)= 4.42, P < 0.01]. As can be seen in Fig. 2 (left

panel), one-way ANOVA showed no significant change in the recall latencies of the animals that

were tested following the microinjection of different doses of ACPA (0.5, 0.75 and 1 ng/rat,

intra-CA1), compared to the vehicle control group [F (3, 24) = 0.25, P> 0.05]. However, Fig. 2

(right panel) shows the effects of pre-test injection of the same doses of ACPA on

dextromethorphan - induced enhancement of morphine amnesia. Post-hoc analysis indicated that

injection of ACPA (1ng/rat, intra-CA1) reversed dextromethorphan - induced enhancement of

morphine amnesia [F (3, 24) = 5.33, P < 0.01].

3.3. The effect of intra-CA1 microinjection of AM251 (before the testing phase) with or without

systemic injection of dextromethorphan / morphine on memory recall

10
 Fig. 3 (left panel) shows the effect of intra-CA1 microinjection of AM251 (before testing

phase) on memory recall. Two-way ANOVA revealed a significant difference between the

effects of AM251 alone and AM251 plus dextromethorphan (5 mg/kg) plus morphine (2 mg/kg)

on memory recall [for treatment, F (1, 48)= 15.07, P < 0.001; Dose, F (3,48)= 4.83, P < 0.01;

and treatment×dose interaction, F (3, 48)= 4.32, P < 0.01]. One-way ANOVA showed that

pre-test AM251 (30, 40, and 50 ng/rat) had no effect on memory recall of a passive avoidance

task by itself [F (3, 24) = 1.73, P> 0.05]. As can be seen in fig. 3 (right panel), one-way ANOVA

revealed that the microinjection of same doses of AM251 along with ineffective doses of

dextromethorphan (5 mg/kg, i.p.) and morphine (2 mg/kg, i.p.) reinforced dextromethorphan

effect on memory disrupting effect induced by morphine [F (3, 24)= 7.95, P<0.01].

3.4. The effect of intra-CA1 microinjection of AM251 on ACPA improvement of the amnesia

induced by dextromethorphan /morphine combination

Fig. 4 shows the effect of the blockade of CB1 receptors via intra-CA1 microinjection of

different doses of AM251 on the inhibitory effects of ACPA on dextromethorphan -induced

enhancement of morphine response. One-way ANOVA indicated that intra-CA1 microinjection

of different doses of AM251 (10, 15 and 20 ng/rat) before the testing phase altered the inhibitory

effect of the microinjection of ACPA (1 ng/rat, intra-CA1) on dextromethorphan- induced

enhancement of morphine amnesia [F (6, 42)= 13.34, P<0.001]. Post-hoc analysis showed that

only 20 ng/rat of AM251 significantly reversed the effect of ACPA on dextromethorphan-

induced enhancement of morphine response in passive avoidance learning.

4. Discussion

11
 Dextrometorphan and morphine are often co-abused in humans. Considering that a

functional relationship between the NMDA receptors and mu opioid receptors has previously

been reported in cognitive processes (see the introduction), different doses of dextromethorphan

(a NMDA receptors antagonist) were intraperitoneally injected with or without morphine. Since

there is a potentiative effect between dextromethorphan and morphine which can affect passive

avoidance memory (Ghasemzadeh and Rezayof, 2016), studying the possible role of the CA1

endocannabinoid system in the effect of dextromethorphan/morphine co-injection on memory

formation may further our understanding of the drugs’ mechanisms of action. The obtained data

from experiment 1 showed that amnesia can be produced by systemic injection of morphine or

dextromethorphan in a passive avoidance learning task. There is an increasing amount of

evidence arising from studies on humans and rodents that indicate that morphine or

dextromethorphan injection impairs memory in different learning tasks (Farahmandfar et al.,

2015; Carter et al., 2013; Zarrindast et al., 2014). To investigate the existence of a relevant

interaction between the drugs, we used an ineffective dose of morphine (2 mg/kg) with different

doses of dextromethorphan. Interestingly, our results showed that systemic co-injection of the

drugs induced amnesia.

The results of experiment 2 revealed that the activation of the dorsal hippocampal CB1

receptors via the microinjection of ACPA significantly decreased the enhancing effect of

dextromethorphan on morphine response. In view of the fact that the stimulation of CB1

receptors reduced glutamate release in cultured hippocampal neurons (Shen and Thayer 1999), it

has been suggested that there is a functional interaction between endocannabinoid system and

glutamatergic neurotransmission (see for review Szabo and Schlicker, 2005). In contrast, Ferraro

et al. (2001) reported that glutamate level in the prefrontal cortex (PFC) increased following the
12
injection of cannabinoid receptor agonist. Thus, it seems that the activity of endocannabinoid

system may have various effects on the neurotransmission in different brain regions. It has

previously been reported that there is a cross-talk between opioidergic and endocannabinoid

systemr. Cannabinoid CB1 receptors as well as mu opioid receptors are both presented in high

density in the dorsal hippocampus (Herkenham et al., 1991; Mansour et al., 1994). Rewarding

effect of opioids has been shown to be related to CB1 cannabinoid receptors (see for review

Maldonado et al., 2006). For example, morphine injection could not produce conditioned place

preference in CB1 receptors knockout mice (Rice et al., 2002) and the injection of Δ9-

tetrahydrocannabinol (THC) did not produce the rewarding effect in mu receptors knockout mice

(Ghozland et al., 2002). Huang et al. (2003) showed that the co-administration of

dextromethorphan with morphine induced attenuation in morphine rewarding effect. Since THC

injection increased dopamine release (Ferraro et al., 2001), one may suggest that the improving

effect of ACPA on the amnesia induced by the combination of dextromethorphan/morphine may

be directly or indirectly associated with the dopaminergic mechanism of the CB1 cannabinoid

receptor agonist which can affect memory formation. Further studies with more focus on this

hypothesis are therefore suggested. In addition, our results also showed that intra CA1

microinjection of the same doses of ACPA before the testing phase had no effect on memory

recall. In support of the present results, it has been reported that intra-CA1 microinjection of

ACPA before the testing phase could not induce any significant changes in memory formation

(Alijanpour et al., 2013). Although these results are consistent with some published studies, they

differ from those which suggested that endocannabinoid agonists disrupted memory formation

(Nasehi et al., 2009; Zarrindast et al., 2010). For example, Ghiasvand et al. (2011) reported that

microinjection of ACPA after the training phase into the central amygdala disrupted memory

13
consolidation. The modulatory role of cannabinoids on memory formation may be dependent on

the brain site and the neuronal circuit (Marsicano and Lutz, 2006). Moreover, the dose size, the

animals’ strain and the route of injection are among the most important factors which may

influence the results.

The current findings also showed that intra-CA1 microinjection of a selective CB1

receptor antagonist, AM251, before the testing phase had no effect on memory recall while the

microinjection of the same doses of the antagonist reinforced the enhancing effect of

dextromethorphan on the response of an ineffective dose of morphine in the passive avoidance

task. Using single-cell and network-level recordings, Ma et al. (2008) reported that CB1 receptor

antagonists increased inhibitory neurotransmission at interneuron-Purkinje cell synapses. Our

results may be related to the enhancing effect of AM251 on GABAergic transmission which is

reinforced in the presence of NMDA antagonists (Fiszman et al., 2005), leading to an

enhancement in dextromethorphan-induced potentiation of morphine response. To support the

hypothesis that endocannabinoid-dependent mechanisms may be involved in the enhancing

effect of dextromethorphan on morphine-induced amnesia, AM251 was injected into the CA1

regions before the microinjection of ACPA. The results obtained from the last experiment

showed that bilateral intra CA1 microinjection of AM251 significantly reversed the effect of

ACPA on dextromethorphan-induced enhancement of morphine response. However, our findings

may suggest that the enhancement of morphine-induced amnesia by dextromethorphan is

mediated through a CB1 cannabinoid receptor mechanism.

Taken together, our observations may support the idea that the CA1 region of the

hippocampus is a target site for dextromethorphan effects on memory recall. Moreover, the

14
cannabinoid system of the CA1 regions of the dorsal hippocampus may be effectively involved

in the enhancing effect of dextromethorphan on morphine amnesia.

Statement of Interest

None.

Acknowledgment

None.

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Fig. 1. The effects of the injection of dextromethorphan before the testing phase with or

without morphine on memory recall. Nine groups of animals were trained in a passive avoidance

task. On the test day, two groups received morphine (2 and 5 mg/kg) and were tested after 30

min (left panel). Three groups received dextromethorphan (5, 7.5 and 15 mg/kg) and after 90

min, they were tested (middle panel). The other three groups were administrated with the same

doses of dextromethorphan plus an ineffective dose of morphine (2 mg/kg, i.p.) and were tested

after 30 min (right panel). Each value represents mean± S.E.M. **P<0.01, ***P<0.001

compared with the control saline group. ++ P <0.01, +++P<0.001 compared with the morphine

(2 mg/kg) group.

Fig. 2. The effects of intra-CA1 microinjection of ACPA before the testing phase with or

without systemic injection of dextromethorphan plus morphine on memory recall. Animals were

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trained and on the test day, they received intra-CA1 microinjection of ACPA (0, 0.5, 0.75 and 1

ng/rat; left panel). The other four groups received intra-CA1 microinjection of the same doses of

ACPA and after 5 min they received ineffective doses of dextromethorphan (7.5 mg/kg; i.p.) plus

morphine (2 mg/kg; i.p.). After 30 min, they were tested for evaluating latency times. Test

session latency times are expressed as mean ± S.E.M. *** P <0.001 compared with the control

vehicle group, ++ P < 0.01, compared to vehicle/ dextromethorphan / morphine group.

Fig. 3. The effects of intra-CA1 microinjection of AM251 before the testing phase with

or without systemic injection of dextromethorphan plus morphine on memory recall. Four groups

of animals were trained and on the test day, they received intra-CA1 microinjection of AM251

(0, 30, 40 and 50 ng/rat; left panel). The other four groups received intra-CA1 microinjection of

the same doses of AM251 and after 5 min they received ineffective doses of dextromethorphan

(5 mg/kg; i.p.) and morphine (2 mg/kg; i.p.). After 30 min, they were tested for evaluating

latency times. Test session latency times are expressed as mean ± S.E.M. * P < 0.05, ** P < 0.01

compared to DMSO/ dextromethorphan / morphine group.

Fig. 4. The effect of blockade of CB1 receptors via intra-CA1 microinjection of different

doses of AM251 on ACPA-reversed dextromethorphan-induced enhancement of morphine

response. Animals were trained and after 24h, they received intra-CA1 microinjection of

different doses of AM251 (0, 10, 15, and 20 ng/rat) followed by vehicle (1 µl/rat) or ACPA (1

ng/rat) with 5 min interval. After 5 min, they received dextromethorphan (7.5 mg/kg; i.p.) plus

morphine (2 mg/kg; i.p.). Two control groups were used in this experiment. One group received

intra-CA1 microinjections of DMSO and vehicle (1 µl/rat) with 5 min interval, and after 5 min

they received interaperitoneal injection of saline (1 ml/kg; i.p.) plus saline (1 ml/kg; i.p.) with 60

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min interval. Another group received intra-CA1 microinjections of DMSO and vehicle (1 μl/rat)

with 5 min interval, and after 5 min they were injected with saline (1 ml/kg; i.p.) plus morphine

(2 mg/kg, i.p.). Data are expressed as mean±S.E.M. of seven animals per group. *** P < 0.001

compared to DMSO/ vehicle/ saline/ morphine group. + P <0.05 compared with DMSO/ vehicle/

dextromethorphan/ morphine group. ## P <0.01 compared with DMSO/ ACPA/

dextromethorphan / morphine group.

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