GABA-cannabinoid interplays in the dorsal hippocampus and basolateral amygdala mediate morphine-induced amnesia

Journal Pre-proof GABA-cannabinoid interplays in the dorsal hippocampus and basolateral amygdala mediate morphine-induced amnesia Khadijeh Alsadat Sharifi, Ameneh Rezayof, Sakineh Alijanpour, Mohammad-Reza Zarrindast

PII:

S0361-9230(19)30613-6

DOI:

https://doi.org/10.1016/j.brainresbull.2020.01.012

Reference:

BRB 9845

To appear in:

Brain Research Bulletin

Received Date:

5 August 2019

Revised Date:

16 January 2020

Accepted Date:

17 January 2020

Please cite this article as: Sharifi KA, Rezayof A, Alijanpour S, Zarrindast M-Reza, GABA-cannabinoid interplays in the dorsal hippocampus and basolateral amygdala mediate morphine-induced amnesia, Brain Research Bulletin (2020), doi: https://doi.org/10.1016/j.brainresbull.2020.01.012

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GABA-cannabinoid interplays in the dorsal hippocampus and basolateral amygdala mediate morphine-induced amnesia

Khadijeh Alsadat Sharifi1, Ameneh Rezayof 2, Sakineh Alijanpour3, Mohammad-Reza Zarrindast4

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Department of Neuroscience, School of Advanced Technologies in Medicine, Tehran University of Medical Sciences, Tehran, Iran. 2

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Department of Animal Biology, School of Biology, College of Science, University of Tehran, Tehran, Iran. 3

Department of Biology, Faculty of Science, Gonbad Kavous University, Gonbad Kavous, Iran 4

M.R. Zarrindast,

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Correspondence to:

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Department of Neuroscience, School of Advanced Technologies in Medicine and Department of Pharmacology, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran; Institute for Studies in Theoretical Physics and Mathematics School of Cognitive Sciences (IPM), Tehran, Iran; Institute of Cognitive Sciences, Tehran, Iran; Iranian National Center for Addiction Studies, Tehran, Iran.

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Professor, Department of Pharmacology, School of Medicine,

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Tehran University of Medical Sciences, PO Box 13145-784, Tehran, Iran Fax: (+9821)- +9821 6640 2569 e-mail: [email protected]

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HIGHLIGHTS  Post-training administration of morphine induced amnesia.  Intra-CA1 microinjection of muscimol increased morphine-induced amnesia.  Activation of the BLA CB1 receptors enhanced muscimol/morphine-induced amnesia.

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 BLA CB1 receptors blockade reversed muscimol/morphine-induced amnesia.

Abstract

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The aim of the current study was to investigate the involvement of GABA

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neurotransmission in the CA1 region and endocannabinoid system in the basolateral amygdala (BLA) on morphine-induced memory impairment. We hypothesized that possible

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functional interaction between the GABAergic and cannabinoid systems in these brain regions would modulate morphine response in memory processing. Step-through type

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inhibitory avoidance paradigm was used for evaluating memory consolidation in adult male Wistar rats. Our results indicated that post-training systemic injection of morphine (3 and 5

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mg/kg, i.p.) impaired memory retrieval. The microinjection of a GABA-A receptor agonist, muscimol (0.01-0.03 µg/rat) into the CA1 region increased the response of an ineffective

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dose of morphine (0.5 mg/kg, i.p.) and induced memory impairment, suggesting a synergistic interaction between morphine and muscimol. Interestingly, the activation of the BLA CB1 receptors by the microinjection of WIN55,212-2 (0.05-0.1 µg/rat) increased the effect of ineffective doses of muscimol (0.01 µg/rat; intra-CA1) and morphine (0.5 mg/kg, i.p.), inducing amnesia. The obtained results also showed that microinjection of AM251, a cannabinoid CB1 receptor antagonist, (1-2 μg/rat) into the BLA reversed the synergistic 2

effect of muscimol and morphine, improving memory consolidation. It should be noted that the intra-CA1 microinjection of muscimol, intra-BLA microinjection of WIN55,212-2 or AM251 alone could not affect memory consolidation. Accordingly, it can be concluded that there may be a synergistic interaction between the CA1 GABAergic system and the BLA endocannabinoid neurotransmission with respect to the modulation of morphine-induced memory impairment.

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ABBREVIATIONS: ANOVA, analysis of variance; AP, Anterior-posterior; BLA,

basolateral amygdala; DV, Dorso-ventral; GABA, gamma-Aminobutyric acid; AM251, (N-(Piperidin-1-yl)-5-(4-iodophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-

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carboxamide); DMSO, dimethyl sulphoxide; S.E.M, standard error of mean.

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Keywords: Morphine; Muscimol; Cannabinoid agents; Memory impairment; Rat(s)

1. Introduction

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Multiple abused drugs influence learning and memory processes (Kutlu and Gould, 2016; Poldrack and Packard, 2003; White and McDonald, 2002); amongst these, morphine is an

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important one (Kitanaka et al., 2015; Zarrindast et al., 2011). For many years, morphine has been widely used for pain relief, but the strong potential for dependence and addiction greatly

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limit its clinical practice. Furthermore, morphine addiction is considered to have a pathological effect on learning and memory (Hyman et al., 2006). Opioids exert an amnesic effect on memory formation, impairing neuronal plasticity (Lin et al., 2009; Yang et al., 2013). Although numerous findings have indicated that morphine agonists have a negative impact on memory processes (Drake et al., 2007; Tramullas et al., 2008), many other studies have reported the enhancing effects of morphine on memory retention (Bao et al., 2007; Niu 3

et al., 2009). While much research has been carried out on opioids, the exact neurotransmitter system and brain regions by which morphine modulate cognitive function have not been clearly established. Cannabis, an another frequently abused drug, alters many aspects of behavior such as emotion, motivation and cognition via the endocannabinoid system (Hölter et al., 2005; Zanettini et al., 2011). The physiological effects of cannabinoid agents are mainly mediated through the CB1 cannabinoid receptors, which have wide distribution in several brain regions such as the prefrontal cortex, the hippocampus and the amygdala (Chevaleyre et al., 2006;

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Pistis et al., 2004). The BLA is involved in the emotional memory system (Cahill, 2000; Richter-Levin and Akirav, 2003), suggesting the essential role of this part of the amygdaloid complex in post-training memory processing (Izquierdo et al., 1992b). The activation of the

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BLA has a remarkable key effect on hippocampal LTP activity and a lesion of the BLA impairs this process (Kim et al., 2001). BLA, as well as the other limbic systems associated

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with the BLA such as the CA1 region of hippocampus have, substantial impact in mediating certain species of cognition (Cammarota et al., 2008). Lisman (2005) reported a functional

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neurotransmitter circuit between the BLA and CA1 regions to adjust the information current into the long-term memory (LTP) (Lisman, 2005) indicating an impressive role of amygdala

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on hippocampus-dependent LTP.

The BLA, which has a wide distribution of GABA-A receptors (Lin et al., 2011), was

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previously suggested to be involved in memory consolidation via modulation of the

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GABAergic system (McGaugh, 2002). Pharmacological studies using GABAergic and adrenergic agonists and antagonists revealed that the BLA GABAergic system controls the release of norepinephrine (NE) to mediate memory consolidation in animal memory tasks (Berlau and McGaugh, 2006; Hatfield and McGaugh, 1999; Quirarte et al., 1998). Furthermore, there have been exciting findings regarding the role of amygdala glutamatergic NMDA receptors in memory modulation (Ben Mamou et al., 2006; Izquierdo et al., 1992a)

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which may indirectly regulate the activities of GABAergic system (Akirav, 2007). It was shown that GABAergic neurotransmission establishes the balance between the inhibitory and excitability conditions in the hippocampus (Paulsen and Moser, 1998). Endocannabinoid and GABAergic systems are mutually involved and interconnected in cognitive functioning (Monory et al., 2006; Witkin et al., 2005). For instance, it was shown that the regulation of the BLA GABAergic transmission via endocannabinoids imparts enhanced memory processing (McGaugh et al., 2002). On the other hand, the activation of cannabinoid CB1 receptors causes a reductive effect on GABAergic neurotransmission in the BLA (Ratano et

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al., 2014). These observations strongly suggest that GABA-A and CB1 receptors in the hippocampus and the BLA have a modulatory effect in learning and memory processes (Azad et al., 2004; Campolongo et al., 2009).

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Given that the CA1 region and the BLA have a high density of GABA-A and CB1 receptors (Chevaleyre and Castillo, 2003), and the functional interaction between these two

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brain regions are involved in the modulation of learning and memory (McDonald and Mott, 2017), the main purpose of the present study was to evaluate: 1) the effect of the activation of

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the CA1 region GABAergic system in memory impairment induced by morphine and also 2) the functional circuit between the CA1 GABA-A receptors and the BLA endocannabinoid

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system with respect to the amnestic effect of morphine administration in inhibitory avoidance

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paradigm.

2. Materials and methods

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2.1. Subjects

The experiments were performed on male adult Wistar rats (from Faculty of

Pharmacy, Tehran University of Medical Sciences) weighing between 220-240 g at the time of surgery. All rats were kept in groups of four per Plexiglas cage, in an animal room with controlled temperature (22±2 °C) and photoperiod 12-h light/12-h dark cycle (lights on at 07:00 AM). All animals also had freel access to food and water and were allowed to adapt to 5

the laboratory situation for at least 1 week prior to the experiments. Each animal was handled for 5 min a day during the adaptation period. Trial procedures were conducted during the light phase between 10:00 AM and 02:00 PM in a quiet environment without noise. Each group includes seven animals given that statistical studies (Arifin and Zahiruddin, 2017; Charan and Kantharia, 2013) suggest this to be an appropriate sample size in parametric data. All experimental procedures were approved by the guidelines for the care and use of laboratory animals observed at School of Medicine, Tehran University of Medical Sciences was in accordance with institutional guidelines for the care and use of laboratory animals

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(NIH, publication no. 85-23, revised 2010; European Communities Directive 86/609/EEC).

2.2. Surgical procedures

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The mixture of ketamine hydrochloride plus xylazine (100 mg/kg and 5 mg/kg, respectively) was injected intraperitoneally (i.p.) for anesthetizing each animal. Then, each

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animal was placed in a stereotaxic apparatus (Stoelting Co., Wood Dale, Illinois, USA) to implant 22-gauge stainless steel guide cannulas unilaterally in the right hemisphere into the

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CA1 hippocampal region (intra-CA1) and the basolateral amygdala (intra-BLA) with the cannula tips 1 mm above the targets sites according to the atlas of Paxinos and Watson

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(Paxinos and Watson, 2007). Stereotaxic coordinates for the CA1 were AP: - 3.3 mm; ML: +2 mm; and DV: - 2.8 mm and for the BLA were AP: - 2.8 mm; ML: +5 mm; and DV: -8.5

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mm from the top of the skull. 27-gauge stainless steel stylets were inserted into the guide

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cannulas to keep them free of clot and debris. The animals were allowed 1 week to recover from the surgery and from the effect of the anesthetic agents. During this recovery period, each animal was handled for 5 min, twice a day prior to the behavioral testing to acclimate to the experimenters and experimental room by removing them from their cages. All experimental procedures were conducted during the light cycle.

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For microinjection of the drugs into the CA1 region and the BLA, each stylet was gently removed from the guide cannula and replaced by 27-gauge microinjection needle (1 mm below the tip of the guide cannulae) attached with a polyethylene tube to a 2-μl Hamilton syringe. The volume of the injected drugs in the CA1 and the BLA were 0.5 µl and 0.3 µl respectively, for over a 60 s period. In order to allow for the diffusion of the drugs and minimize the possibility of backflow of the solution through the needle track, the microinjection needle was kept at the microinjection site for an additional 1 min before

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removal.

2.3. Drugs and microinjections

The drugs used in the present experiments included morphine sulfate (Temad, Tehran, Iran), muscimol (Tocris, Bristol, UK) a GABA-A receptor agonist, WIN55,212-2 mesylate

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(Tocris Cookson, Bristol, UK) a cannabinoid CB1/CB2 receptor agonist and the AM251 (N-

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(Piperidin-1-yl)-5-(4-iodophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3carboxamide)(Tocris, Bristol, UK) a cannabinoid CB1 receptor antagonist. Morphine and

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muscimol were dissolved in sterile 0.9% saline. WIN55,212-2 and AM251 were diluted in Dimethyl sulfoxide (DMSO; up to 10% v/v), sterile 0.9% saline and a drop of Tween 80.

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Control groups received either saline or suitable vehicle. Morphine was administrated intraperitoneally (i.p.), muscimol was injected into the CA1 regions in a volume of 0.5 μl/rat,

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WIN55,212-2 and AM251 were unilaterally injected into the BLA in volumes of 0.3 μl/rat. There was a 5 min interval between the microinjection of the drugs. Dosing was chosen based

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on our pilot experiments and previous studies (Alijanpour and Rezayof, 2013; Alijanpour et al., 2013a; Stackman et al., 2016).

2.4. Inhibitory avoidance apparatus The animals were trained and tested in a step-through type inhibitory avoidance apparatus which consisted of two equal sized black and white opaque resin compartments (20 7

cm×20 cm×30 cm). The dark chamber contains a removable roof and having stainless steel grids (2.5 mm in diameter) were located at 1-cm intervals (distance between the centers of grids) in the floor for the administration of electric shocks (50 Hz, 3 sec, 1 mA intensity) via an insulated stimulator (Borj Sanat Co., Tehran, Iran). In an interesting study conducted by Eagle and co-workers (Eagle et al., 2016), it has been shown that the shock intensity ranging between 0.4–1.2 mA produced long-term memory. In our studies (Piri et al., 2013; Tirgar et al., 2018) and others (Narwal et al., 2012; Senik et al., 2012), a foot shock at 1 mA was appropriate to induce the avoidance response. A liftable guillotine-like door (7 cm×9 cm) was

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placed on the floor in the center of the partition which separated two compartments and could manually be lifted. The inhibitory avoidance processes have habituation, training and testing

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phases. After each phase, the compartments were cleaned and dried.

2.5. Behavioral testing

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2.5.1. Training phase:

All animals were allowed to habituate to the experimental room for at least 30 min

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before the experiments. In the habituation phase, each animal was gently placed into the white compartment and had free access to all parts of the apparatus; the guillotine door was

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opened after 5 s and the animal was allowed to enter the dark compartment. As soon as the animal crossed into the dark compartment with all four paws, the step-through latency was

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recorded. Once the animal entered with all four paws to the next compartment, the guillotine

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door was closed and the animal was taken into its home cage (habituation trial). An upper cut-off time of 100 s was set and each animal that delayed more than 100 s to enter to the dark compartment was excluded from the experiments. The acquisition trial was conducted 30 min after the habituation phase. In the training phase, each animal was placed again into the white compartment and after 5 s, the guillotine trap door was lifted and when the animal entered to the dark compartment, the door was closed and a foot shock (50 Hz, 3 sec, 1 mA)

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was immediately applied to the metal grid floor of the dark compartment. After 20 s, the animal was removed from the apparatus and returned temporarily into its home cage. After two minutes, the training trial was repeated again for each animal in the same way as the former trials. A successful acquisition of inhibitory avoidance response was recorded for the animals with 120 s latency to cross into the dark compartment. The training phase was finished when the animal stayed in the illuminated compartment for 120 consecutive seconds; otherwise, as soon as the animal entered the dark compartment (before 120 s) for a second time, the middle door was closed and the animal received the shock again. The number of

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trials (entries into the dark chamber) was recorded. All animals were taught with a maximum of 3 trials. When the rat had acquired passive avoidance successfully (after 2 or 3 trials), it was removed from the apparatus and immediately received post-training injection of the

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drugs.

2.5.2. Testing phase:

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The animals were tested 24 h after the training trial to evaluate memory retrieval. On the testing day, each rat was placed again in the white compartment and after 5 s, the middle

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guillotine door was opened. The step-through latency of entering the dark compartment was measured. An upper cut-off time of 300 s was set in this phase. The test phase was terminated

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when the animal crossed into the dark compartment and remained in the white compartment

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for 300 s. Test sessions were done without use of any foot shock.

2.6. Behavioral procedures Each experimental group consisted of seven animals. In all the experiments in which

the animals received two or three microinjections, the control groups received either saline or an appropriate vehicle microinjections (Nazari-Serenjeh and Rezayof, 2013). The sequential microinjections were conducted immediately after training with 5 min intervals. All 9

microinjections were made on the conscious freely moving animals 7 days after the surgery. During the recovery period and in order to minimize the microinjection-induced stress, each rat was handled every day and habituated to the microinjection conditions (Zaretsky et al., 2011). 2.6.1. Experiment 1. This experiment assessed the effect of post-training administration of morphine on inhibitory avoidance memory consolidation. Four groups of animals received saline (1 ml/kg, i.p., as a control group) or morphine (0.5, 3 and 5 mg/kg, i.p) immediately after successful training (Fig. 1). On the testing phase, the latency of step-through was

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recorded 24 h after the training session in each animal.

2.6.2. Experiment 2. The effect of post-training intra-CA1 microinjection of muscimol, a

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GABA-A receptor agonist, with or without morphine was evaluated on memory consolidation in eight groups of animals. Four groups received intra-CA1 microinjection of

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different doses of muscimol (0, 0.01, 0.02, and 0.03 µg/rat) plus saline (1 ml/kg; left panel of Fig. 2) immediately after successful training. In the other four groups, the animals received

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post-training intra-CA1 microinjections of the same doses of muscimol plus an ineffective dose of morphine (0.5 mg/kg, i.p) with 5 min interval (right panel of Fig. 2). Step-through

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latency was recorded for each animal 24 h after the training session.

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2.6.3. Experiment 3. In this experiment, the effect of intra-BLA microinjection of

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WIN55,212-2 with or without muscimol plus morphine was examined on memory consolidation in eight groups of animals. Immediately after the successful training, all experimental groups received different doses of WIN55,212-2 (0, 0.05, 0.075 and 0.1 μg/rat) into the BLA. Four groups of animals were injected with vehicle (0.5 µl/rat) into the CA1 region and after 5 min they received saline (1 ml/kg, i.p.; left panel of Fig. 3). The other four groups received the same doses of WIN55,212-2 (intra-BLA) plus muscimol (0.01 µg/rat;

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into the CA1) and morphine (0.5 mg/kg, i.p.; right panel of Fig. 3) with 5 min intervals. Latency of step-through for each animal was recorded 24 h later in the testing session. 2.6.4. Experiment 4. In this experiment, the effect of the blockade of the BLA cannabinoid CB1 receptors on co-administration of muscimol (intra-CA1) plus morphine was examined on memory consolidation in eight groups of animals. On the training session, all groups received the microinjections of different doses of the cannabinoid CB1 receptor antagonist, AM251 (0, 1, 1.5 and 2 μg/rat; left panel of Fig. 4) into the BLA. Four groups were injected with vehicle (0.5 µl/rat) into the CA1 and saline (1 ml/kg, i.p.). The other four animal groups

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received muscimol (0.03 µg/rat; intra-CA1) and morphine (0.5 mg/kg, i.p.; right panel of Fig. 4). All microinjections were performed in post-training manner with 5 min intervals. The

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latency of step-through for each animal was then recorded 24 h later in the testing phase.

2.7. Histology

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After the completion of the behavioral sessions, each animal was euthanized with carbon dioxide. Subsequently, 0.3 μl and 0.5 μl of ink (1% aqueous Methylene Blue solution)

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were unilaterally injected into the BLA and the CA1 region respectively. After decapitation of each animal, the brain was expelled and fixed in a 10% formalin solution for 10 days

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before sectioning. Then, each brain was sliced to specify the accurate location of cannula in

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(2007).

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the targeted sites and each slice was verified using the rat brain atlas of Paxinos and Watson

2.8. Data analysis Considering that the obtained data from multiple-trial step-through passive avoidance

indicated normality of distribution and homogeneity of variance, the results were expressed as mean ± standard error of mean (S.E.M.). Analyzing the differences between the groups was carried out by One-way ANOVA, while two-way ANOVA was used to analyze the

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interaction between the two factors. Following a significant F-value, further analyses for between-group comparisons were carried out with post-hoc Tukey’s test and in all experiments, level of statistical significance was set at P < 0.05. Calculations were performed using SPSS statistical package.

3. Results 3.1. Effect of morphine administration on memory consolidation Fig. 1 shows the effect of post-training i.p. administration of different doses of

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morphine (0, 0.5, 3 and 5 mg/kg) on step-through latency in inhibitory avoidance paradigm. One-way ANOVA revealed that morphine administration inhibited memory retrieval [F (3, 24) = 45.267; P = 0.0001], suggesting an amnesic effect of morphine. Post-hoc analysis also

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showed the amnesic effect of the opiate at the doses of 3 and 5 mg/kg (P < 0.001).

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3.2. Effect of hippocampal GABA-A receptor activation on memory consolidation in animals receiving saline or morphine

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Fig. 2 shows the effect of post-training intra-CA1 microinjection of muscimol (0, 0.01, 0.02 and 0.03 µg/rat) alone or in combination with morphine (0.5 mg/kg, i.p.) on

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memory consolidation. Two-way ANOVA showed a significant difference between the groups of animals that received only muscimol and those that received muscimol plus

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morphine in memory formation [F treatment × dose (3,48)

= 9.854, P = 0.003; F

dose (3,48)

= 16.775, P =

= 18.269, P = 0.0001]. Further analysis revealed that the

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0.0001; F

treatment (1, 48)

microinjection of muscimol into the CA1 region, by itself, had no effect on memory consolidation [F (3, 24) = 1.707; P = 0.192; left panel of Fig. 2]. The analysis also indicated that the microinjection of muscimol, 5 min prior the administration of an ineffective dose of morphine (0.5 mg/kg, i.p.) impaired memory consolidation [F

(3, 24)

= 38.743, P = 0.0001;

right panel of Fig. 2], suggesting a synergistic interaction between muscimol and morphine.

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3.3. Effect of intra-BLA microinjection of CB1 receptor agonist on synergistic interaction between muscimol and morphine in memory consolidation impairment Fig.3 shows the effect of post-training microinjection of a cannabinoid CB1 receptor agonist, WIN55,212-2 (0, 0.05, 0.075 and 0.1 µg/rat) into the BLA with or without intra-CA1 microinjection of muscimol (0.01 µg/rat) plus morphine (0.5 mg/kg, i.p.) on memory consolidation. Two-way ANOVA revealed that there was a significant difference in memory consolidation between the animals that received intra-BLA microinjection of WIN55,212-2

treatment (1, 48)

= 32.781, P = 0.0001; F

dose (3, 48)

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alone and those that received the same doses of agonist plus muscimol and morphine [F = 18.421, P = 0.0001; F

treatment × dose (3, 48)

=

18.595, P = 0.0001]. Further analysis indicated that WIN55,212-2 by itself, had no effect on (3, 24)

= 0.327; P = 0.806; left panel of Fig. 3]. The analysis also

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memory consolidation [F

revealed that the intra-BLA microinjection of the same doses of WIN55,212-2 enhanced

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muscimol plus morphine response [F (3, 24) = 20.896, P = 0.0001; right panel of Fig. 3].

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3.4. Effect of intra-BLA microinjection of CB1 receptor antagonist on synergistic interaction between muscimol and morphine in memory consolidation impairment

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Fig. 4 shows the effect of the microinjection of a cannabinoid CB1 receptor antagonist, AM251 (0, 1, 1.5 and 2 µg/rat) into the BLA with or without intra-CA1

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microinjection of muscimol (0.03 µg/rat) plus morphine (0.5 mg/kg, i.p.) on memory

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consolidation. Two-way ANOVA revealed a significant difference in memory consolidation between the groups that received AM251 alone and those that received the same doses of AM251 plus muscimol and morphine [F treatment (1, 48) = 11.219, P = 0.002; F dose (3, 48) = 9.258, P = 0.0001; F

treatment × dose (3, 48)

= 16.078, P = 0.0001]. Further analysis indicated that the

microinjection of AM251 into the BLA, by itself, had no effect on memory consolidation [F (3, 24)

= 2.044, P = 0.134; left panel of Fig. 4]. The analysis also showed that the AM251

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reversed synergistic interaction between muscimol and morphine in memory impairment and improved memory consolidation [F (3, 24) = 21.613, P = 0.0001; right panel of Fig. 4].

4. Discussion The aim of the present study was to evaluate the possible involvement of the basolateral amygdala (BLA) cannabinoid system on memory impairment caused by coadministration of GABA-A receptor agonists and morphine in rats using a multi-trial stepthrough type of passive avoidance task. Our findings demonstrated: 1) post-training

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administration of morphine (3 and 5 mg/kg, i.p.) induced memory consolidation impairment in step-through passive avoidance paradigm, 2) post-training intra-CA1 microinjection of a selective GABA-A receptor agonist, muscimol, by itself had no effect on memory formation,

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but a higher dose of it (0.03 µg/rat) increased the memory impairment induced by administration of a non-effective dose of morphine (0.5 mg/kg, s.c.) and produced amnesia,

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3) post-training intra-BLA microinjection of a cannabinoid CB1/CB2 receptor agonist, WIN55, 212-2, had no impairing effect on memory consolidation by itself, but enhanced the

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memory impairment induced by co-administration of non-effective doses of muscimol (0.01 µg/rat, intra-CA1) plus morphine (0.5 mg/kg, s.c.), 4) post-training intra-BLA microinjection

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of a cannabinoid CB1 receptor antagonist, AM-251, 212-2, alone had no significant effect on memory formation, but reversed amnesia produced by muscimol (0.03 µg/rat, intra-CA1)

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plus morphine (0.5 mg/kg).

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Much of the literature emphasizes that the passive avoidance task is a valid and useful

paradigm in the investigation of the neurobiochemical basis of memory. To reduce stress as a confounder, a multi-trial type of passive avoidance and repetitive animal handlings before and during experimentation were carried out (Madjid et al., 2006). In the first set of experiments, in agreement with previous studies, we observed that post-training administration of morphine impaired memory formation (Hu et al., 2014; Niu et al., 2009). 14

Prior findings have noted the important coherence between morphine-induced amnesia and µopioid receptors, because administration of naloxone, a µ-opioid receptor antagonist, has a preventive role on morphine-induced amnesia (Bao et al., 2007; Kitanaka et al., 2015). Statedependent (STD) learning is defined as a phenomenon that memory retrieval is most efficient when an individual is in the same state as they were when the memory acquisition was formed (Shulz et al., 1990). Thus, one may suggest that one of the reasons for the observed memory deficit following post-training administration of morphine (5 mg/kg, i.p.) might be due to this phenomenon. Considering the existence a functional link between morphine

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administration and changes in brain neurotransmission such as GABA release (Sun et al., 2011) and the modulation of long-term potentiation through hippocampal GABA-A receptors (Ruiz et al., 2010), we assessed the possibility of the involvement of the CA1 GABAergic

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system with respect to the effect of morphine on memory function.

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The results revealed that intra-CA1 microinjection of a GABA-A receptor agonist muscimol, could accentuate the impairment the results from an ineffective dose of morphine,

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thus causing amnesia and pointing toward the existence of a functional interaction between opioidergic and GABAergic systems in memory formation. GABA receptors are expressed in

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the central nervous system (Engin et al., 2018). Located either on pre- (Han et al., 2009) or postsynaptic neurons (Levi et al., 2015; Xi and Akasu, 1996), they are responsible for

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modulating cognition (Mohler, 2009). Administration of GABA-A receptor agonist, muscimol, had a disruptive effect on neural activity (Mao and Robinson, 1998) and

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interrupted memory processing in some animal memory paradigms (Maruki et al., 2001; Ramirez et al., 2005). Muscimol administration impaired memory retention in the radial maze (Saito et al., 2010), Morris water maze (Morris et al., 2003) and object recognition paradigms (de Lima et al., 2006). Pharmacological evidence has demonstrated the implication of GABA receptors in the neurochemical effects and behavioral responses associated with morphine. For example, GABA-A receptor agents influence morphine-related behaviors in rodents 15

including self-administration (Yoon et al., 2007), antinociception and tolerance (Bobeck et al., 2014), as well as state-dependent memory (Rassouli et al., 2010). With regard to the inhibition of GABA-mediated neurotransmission following opioid application (Vaughan et al., 1997), one explanation may be that the muscimol probably activates more pre-synaptic GABA-A receptors to potentiate mu-opioid receptors function on the synaptic release of excitatory neurotransmitters. In the next series of experiments, we found that the combined negative effect of muscimol and morphine on memory consolidation was increased by intra-BLA

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microinjection of WIN55, 212-2, a CB1/CB2 cannabinoid receptor agonist. The BLA is one of the most substantial areas in the mesolimbic system which has a key involvement in memory formation (Nedaei et al., 2016) and retrieval (Beyeler et al., 2016) in laboratory

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animals. Furthermore, studies provide support for the notion that plasticity at excitatory synapses in the BLA is critical to associative memory formation (Ressler and Maren, 2019).

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Zubedat et al. (2017) showed that the BLA is implicated in morphine-related associative learning and memory processes (Zubedat and Akirav, 2017). Previous literature emphasize

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the crucial role of the BLA endocannabinoid system in synaptic plasticity (Azad et al., 2004) and processing of the different stages of memory (Campolongo et al., 2009; Leão et al.,

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2016). Singly cerebral or intraperitoneal administration of CB1 receptor agonists has impairing (Laviolette and Grace, 2006), facilitatory (Misner and Sullivan, 1999) or no

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(Alijanpour et al., 2013b; Zarrindast et al., 2012) effect on learning and memory processes in

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several avoidance tasks. It seems that this discrepancy in results is because of the modulatory effect of the CB1 cannabinoid receptors, which is dependent on the brain regions and the neuronal circuitry between these sites (Pazos et al., 2005; Puighermanal et al., 2009). Also, several studies have shown the vigorous involvement of the BLA CB1 receptors in regulation of either rewarding or aversive associative memory formation (Atsak et al., 2015; Yoshida et al., 2011). Former behavioral and pharmacological studies indicate the existence of a

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functional cross-talk between opioidergic and endocannabinoid systems, which may affirm some drug roles e.g. analgesia (Altun et al., 2015; Welch, 2009) and reward-related behavior (Baysinger, 2016; Costanzi et al., 2003). Yuan et al. (2017) revealed that morphine can potentiate glutamatergic synaptic transmission from BLA to the nucleus accumbens (Yuan et al., 2017). Wilson et al. (2015) in an electrophysiological study illustrated that blockade of CB1 receptors via AM-251 increased the frequency of spontaneous miniature IPSCs in periaqueductal grey neurons of morphine-treated rats. This group suggested that morphine administration likely inhibits GABA release through induction of endogenous cannabinoid

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transmission affecting on presynaptic CB1 receptors (Wilson‐Poe et al., 2015). Suppression of reward-related behaviors induce morphine following blockade of CB1 receptors of the nucleus accumbens (Karimi et al., 2013) as well as BLA (Haghparast et al., 2014) has been

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well demonstrated. Prior studies suggested that the CB1 receptor neurotransmission plays a modifying role on drug-evoked plasticity at the excitatory synapses of dopaminergic neurons

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in the CA1 region (Davies et al., 2002; Wilson et al., 2001). Accordingly, it is predictable that the increasing dopamine-mediated BLA plasticity via activation of GABAergic transmission

of muscimol/morphine.

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in the CA1 regions has an amended effect on amnesia induced by concurrent administration

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Given that the inhibition of the BLA CB1 receptors reversed the amnesic effect produced by co-administration of intra-CA1 muscimol/morphine, two assumptions may be

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raised regarding these findings. One would be that BLA is an important part of the loop that

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involved in memory consolidation under drug administration. Another possibility would be that there may be a dynamic correlation between the BLA and the CA1 region for adjusting the information into memory. It has been previously shown that the activation of GABA-A receptors and PKA signaling pathway causes synaptic plasticity in the CA1 hippocampal region, which is essential element for reward-related learning (Cavalier et al., 2015; Jappy et al., 2016). Several studies also show the eminent role of the BLA in different stages of

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learning and memory procedures by communicating with the main sites of memory formation such as hippocampus (Zinn et al., 2016).

Conclusion Considering the obtained results of the present study in conjunction with preexisting data, it can be concluded that the BLA-CA1 circuit has a fundamental effect in modulating memory consolidation procedures with respect to inhibitory avoidance. Furthermore, our current findings also showed that the BLA and CA1 regions have a mutual functional

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interaction, which modulate the memory impairment induced by muscimol/morphine administration; additionally, both CB1 and GABA-A receptors play a crucial role in this effect. Nevertheless, it seems supplementary studies are needed in order to address the exact

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mechanisms of the cannabinoid and GABAergic neurotransmission and their implication at various phases of memory consolidation in inhibitory avoidance learning paradigm between

Author Statement

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the different brain regions.

M.R. Zarrindast and A. Rezayof designed the experiments and supervised the research. K.A.

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Sharifi performed experiments. All of the authors managed the literature searches and analyses, undertook the statistical analyses, and wrote the first draft of the manuscript. All

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authors contributed and approved the final manuscript.

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Conflict of interest

The authors declare no conflict of interest.

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Legends

Fig. 1. Effect of post-training intraperitoneal administration of morphine on memory consolidation in inhibitory avoidance task. Four groups of animals were used. One group received post-training administration of saline (1 ml/kg) while the other three groups received various doses of morphine (0.5, 3 and 5 mg/kg, i.p.) in post-training manner. Each value

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represents the mean ± SEM of seven animals per group. ***P < 0.001 compared with the saline control group.

Fig. 2. Effect of post-training intra-CA1 microinjection of muscimol with or without morphine (0.5 mg/kg, i.p.) on memory consolidation in inhibitory avoidance task. Eight groups of animals were used. Four groups received post-training intra-CA1 microinjection of different doses of muscimol (0, 0.01, 0.02, and 0.03 µg/rat) plus saline (1 ml/kg, i.p.; left panel of Fig. 2) and the other four groups also received post-training intra-CA1

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microinjection of the same doses of muscimol, 5 min prior the administration of morphine (0.5 mg/kg, i.p.; right panel of Fig. 2). Each value represents the mean ± SEM of seven rats

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per group. ***P < 0.001 compared with the vehicle/morphine control group.

Fig. 3. Effect of post-training microinjection of WIN55,212-2 (0, 0.05, 0.075 and 0.1 μg/rat)

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into the BLA alone or in combination with muscimol (0.01 µg/rat; intra-CA1) plus morphine (0.5 mg/kg, i.p.) on memory consolidation in inhibitory avoidance task. Eight groups of

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animals were used. Four groups received post-training microinjection of different doses of WIN55,212-2 (0, 0.05, 0.075 and 0.1 μg/rat; left panel of Fig. 3) into the BLA plus

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microinjection of vehicle (0.5 µl/rat; intra-CA1) and saline (1 ml/kg, i.p.) with 5 min intervals. The other four groups also received microinjection of the same doses of

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WIN55,212-2 into the BLA plus microinjection of muscimol (0.01 µg/rat; intra-CA1) and

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morphine (0.5 mg/kg, i.p.; right panel of Fig. 3) with 5 min intervals, immediately after the training phase. Each value represents the mean ± SEM of seven rats per group. ***P < 0.001 compared with the vehicle/muscimol/morphine control group.

Fig. 4. Effect of post-training microinjection of AM251 into the BLA, with or without muscimol (0.03 µg/rat; intra-CA1) plus morphine (0.5 mg/kg, i.p.) on memory consolidation

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in inhibitory avoidance task. Eight groups of animals were used. Four groups of animals received post-training intra-BLA microinjection of different doses of AM251 (0, 1, 1.5 and 2 μg/rat; left panel of Fig. 4) plus intra-CA1 microinjection of vehicle (0.5 µl/rat) and saline (1 ml/kg, i.p.) with 5 min intervals. The other four groups also received intra-BLA microinjection of the same doses of AM251 plus muscimol (0.03 µg/rat; intra-CA1) and morphine (0.5 mg/kg, i.p.; right panel of Fig. 4) with 5 min intervals. All microinjections were done immediately after the training. Each value represents the mean ± SEM of seven animals per group. ***P < 0.001 compared with the vehicle/vehicle/saline control group. +++P

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< 0.001 compared with the vehicle/muscimol/morphine control group.

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re

250

lP

***

200

150

100

ur

50

***

na

Step-through latency (S)

300

0

0.5

3

Morphine (mg/kg)

Jo

0

25

5

26

ro of

-p

re

lP

na

ur

Jo Fig. 1

350

Step-through latency (S)

300

250

200

150

*** 100

0

0

0.01

0.02

0.03

0

ro of

50

0.01

0.02

0.03

Intra-CA1 microinjection of Muscimol (µg/rat) Saline (1 ml/kg)

Jo

ur

na

lP

re

-p

Morphine (0.5 mg/kg)

Fig. 2

27

350

Intra-CA1 microinjection of vehicle (0.5 µl/rat)

Intra-CA1 microinjection of Muscimol (0.01 µg/rat)

250

200

150

***

100

50

0

0

0.05

0.075

0.1

0

ro of

Step-through latency (S)

300

0.05

0.075

0.1

Intra-BLA microinjection of WIN55,212-2 (µg/rat)

Morphine (0.5 mg/kg)

Jo

ur

na

lP

re

-p

Saline (1 ml/kg)

28

Fig. 3

Intra-CA1 microinjection of vehicle (µl/rat)

Intra-CA1 microinjection of Muscimol (0.03 µg/rat)

ro of

350

+++ ***

250

+++ ***

+++ ***

-p

200

150

re

***

100

lP

Step-through latency (S)

300

50

0 0

1

1.5

2

0

1

1.5

2

na

Intra-BLA microinjection of AM251 (µg/rat)

Saline (1 ml/kg)

Jo

ur

Morphine (0.5 mg/kg)

29