Progress in Neuro-Psychopharmacology & Biological Psychiatry 66 (2016) 41–47

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Dorsal hippocampal NMDA receptors mediate the interactive effects of arachidonylcyclopropylamide and MDMA/ecstasy on memory retrieval in rats Marzieh Ghaderi a, Ameneh Rezayof b, Nasim Vousooghi a, Mohammad-Reza Zarrindast a,c,d,⁎ a

Department of Neuroscience, School of Advanced Technologies in Medicine, Tehran University of Medical Sciences, Tehran, Iran Department of Animal Biology, School of Biology and Center of Excellence in Phylogeny of Living Organisms, College of Science, University of Tehran, Tehran, Iran c Department of Pharmacology, School of Medicine and Iranian National Center for Addiction Studies, Tehran University of Medical Sciences, Tehran, Iran d School of Cognitive Sciences, Institute for Research in Fundamental Sciences (IPM), Tehran, Iran b

a r t i c l e

i n f o

Article history: Received 19 August 2015 Received in revised form 15 November 2015 Accepted 19 November 2015 Available online xxxx Keywords: ACPA MDMA Dorsal hippocampal NMDA receptors Memory formation Rat(s)

a b s t r a c t A combination of cannabis and ecstasy may change the cognitive functions more than either drug alone. The present study was designed to investigate the possible involvement of dorsal hippocampal NMDA receptors in the interactive effects of arachidonylcyclopropylamide (ACPA) and ecstasy/MDMA on memory retrieval. Adult male Wistar rats were cannulated into the CA1 regions of the dorsal hippocampus (intra-CA1) and memory retrieval was examined using the step-through type of passive avoidance task. Intra-CA1 microinjection of a selective CB1 receptor agonist, ACPA (0.5–4 ng/rat) immediately before the testing phase (pre-test), but not after the training phase (post-training), impaired memory retrieval. In addition, pre-test intra-CA1 microinjection of MDMA (0.5–1 μg/rat) dose-dependently decreased step-through latency, indicating an amnesic effect of the drug by itself. Interestingly, pre-test microinjection of a higher dose of MDMA into the CA1 regions significantly improved ACPA-induced memory impairment. Moreover, pre-test intra-CA1 microinjection of a selective NMDA receptor antagonist, D-AP5 (1 and 2 μg/rat) inhibited the reversal effect of MDMA on the impairment of memory retrieval induced by ACPA. Pre-test intra-CA1 microinjection of the same doses of D-AP5 had no effect on memory retrieval alone. These findings suggest that ACPA or MDMA consumption can induce memory retrieval impairment, while their co-administration improves this amnesic effect through interacting with hippocampal glutamatergic-NMDA receptor mechanism. Thus, it seems that the tendency to abuse cannabis with ecstasy may be for avoiding cognitive dysfunction. © 2015 Published by Elsevier Inc.

1. Introduction Patterns of polydrug use among young people are becoming increasingly alarming (Sala and Braida, 2005). Among poly-drug users, co-abuse of cannabis with drugs such as 3,4-methylenedioxymethamphetamine (MDMA or ecstasy) or cocaine is very common (Aberg et al., 2007; Schulz, 2011). A great deal of previous research Abbreviations: ACPA, Arachidonylcyclopropylamide; ANOVA, Analysis of variance; CA1, cornus ammonis; CB1, Cannabinoid receptor type 1; CB2, Cannabinoid receptor type 2; CP-55,940, (−)-cis-3-[2-Hydroxy-4-(1,1-dimethylheptyl)phenyl]-trans-4-(3hydroxypropyl)cyclohexanol; D-AP5, D-(−)-2-amino-5-phosphonopentanoic acid; Δ9–THC, Delta9-tetrahydrocannabinol; GABA, gamma-aminobutyric acid; GTP, Guanosine triphosphate; LTP, long-term potentiation; MDMA, 3, 4-methylenedioxy-Nmethylamphetamine; Nac, Nucleus accumbens; NMDA, N-methyl-D-aspartic acid; SEM, standard error of mean; VTA, Ventral tegmental area; WIN 55,212-2, WIN55,212-2 mesylate; 5-HT, 5-Hydroxytryptamine. ⁎ Corresponding author at: Department of Neuroscience, School of Advanced Technologies in Medicine and Department of Pharmacology, School of Medicine, Tehran University of Medical Sciences, P. O. Box 13145-784, Tehran, Iran. E-mail addresses: [email protected], [email protected] (M.-R. Zarrindast).

http://dx.doi.org/10.1016/j.pnpbp.2015.11.008 0278-5846/© 2015 Published by Elsevier Inc.

indicated that various factors may be the reasons of co-abuse of cannabis and MDMA. For example, cannabis consumption has been reported to decrease MDMA-induced anhedonia, depression (Parrott, 2000; Parrott et al., 2004), somatic symptoms, aggressive behaviors (Milani et al., 2005) and acute hyperthermia (Morley et al., 2004). On the other hand, exposure to polydrug use has been shown to cause an additive dopamine release in the nucleus accumbens (Tizabi et al., 2007), which may be an important reason for the tendency toward the coabuse of the drugs. The mesolimbic dopaminergic reward system, which is the main target of abused drugs, originates from ventral tegmental area (VTA) and projects to the nucleus accumbens (Nac), the hippocampus and the amygdala (for review see Clay et al., 2008). A large body of evidence shows that the hippocampus plays a critical role in many aspects of the addictive processes (Eisch and Harburg, 2006) and drug-induced cognitive dysfunction may be related to the alterations in adult hippocampal neurogenesis (for a review, see Venkatesan et al., 2007). Cannabis is the most prevalent drug of abuse that exerts its effects via cannabinoid CB1 and CB2 receptors which are members of the

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GTP-binding protein coupled receptors (Demuth and Molleman, 2006). In view of the fact that the rat hippocampus has a high density of cannabinoid CB1 receptors (Iversen, 2003) and that the pre-synaptic cannabinoid CB1 receptors modulate different neurotransmitters release, it seems that hippocampal endogenous cannabinoid system plays a critical role in learning and memory processes (Han et al., 2012). The cannabinoid receptor agonists have been shown to impair long-term potentiation (LTP; a candidate mechanism for memory formation) in the rat hippocampal slices (Terranova et al., 1995; Han et al., 2012) probably via inhibition of glutamate release induced by the activation of pre-synaptic cannabinoid CB1 receptors (Schlicker and Kathmann, 2001). A considerable amount of literature has been published on the functional interaction between cannabinoid and other neurotransmitter systems including glutamatergic and serotonergic systems. For example, Rey et al. (2012) using mice lacking CB1 receptors showed that the biphasic effect of cannabinoids on anxiety responses may be mediated by the CB1 receptors on cortical glutamatergic terminal. Melis et al. (2004) also pointed to the involvement of endocannabinoids in presynaptic inhibition of glutamatergic transmission to the ventral tegmental area dopaminergic neurons which may prevent neuronal excitability and synaptic transmission. MDMA as an amphetamine derivative plays a modulatory role in different physiological functions such as memory formation (Sprague et al., 2003), anxiety (Maldonado and Navarro, 2000) and rewarding processes (Vidal-Infer et al., 2012). The effects of MDMA on the cognitive processes are various and depend on factors such as dose and the duration of exposure (Broening et al., 2001). It has generally been viewed as a monoaminergic agonist and modulates serotonin (5-hydroxytryptamine or 5-HT) release (Thomasius et al., 2003). To better understand the effect of MDMA on serotonergic neurotransmission, Mueller et al. (2013) showed that MDMA oral administration in a single dose can induce neurochemistry deficits of serotonin. This consumption will therefore cause long-term cognitive deficits (Schilt et al., 2008). It is important to note that MDMA produces an acute and rapid enhancement in the release of serotonin from the storage vesicles (Riegert et al., 2008; Capela et al., 2009) leads to a reduction in serotonin uptake sites and degeneration of serotonergic axons in certain brain areas (Ricaurte et al., 2000). Further research has shown that systemic administration of neurotoxic doses of MDMA decreases serotonergic axon density and aberrant swollen varicosities in the frontal cortex of rats (Adori et al., 2011). On the other hand, the enhancement of serotonin concentration in the hippocampus following MDMA exposure (Gudelsky and Nash, 1996) has been mediated via 5-HT1A transporters (Hasler et al., 2009) which are present in large numbers in the cortex, the hippocampus (Varnäs et al., 2004). Also, it has been shown that MDMA administration decreases GABA and increases glutamate release in the dorsal hippocampus (Anneken et al., 2013). It seems that MDMA-induced augmentation of hippocampal glutamate efflux may be mediated through a serotonergic mechanism (Anneken and Gudelsky, 2012). Considering that NMDA receptors mediate the acquisition of the conditioned rewarding effects of MDMA (García-Pardo et al., 2015), neurochemical MDMA effects on the brain may be explained, in part, by the interaction of MDMA with NMDA receptors. The main aims of this study were: (i) to examine the effects of microinjection of a selective CB1 receptor agonist (ACPA) or MDMA into the CA1 region of the dorsal hippocampus on memory formation in rats to show the possible role of this site in neurocognitive effects of cannabis or ecstasy consumption; (ii) to investigate the interactive effects of combined MDMA/ACPA administration on passive avoidance memory retrieval; and (iii) to assess whether this functional interaction between ACPA and MDMA can be affected by blockade of NMDA receptors in the dorsal hippocampus. Since the neurobiological basis of co-abuse tendency is poorly understood, this study makes a major contribution to research on memory performance induced by cannabis/ecstasy co-abuse.

2. Materials and methods 2.1. Animals Male Wistar rats (from Faculty of Pharmacy, Tehran University of Medical Sciences) weighing 200–220 g at the time of surgery were used in this study. The animals were housed four per cage with free access to food and water. Cages were in a room with a temperature (22 ± 2 °C) and 12-h light/12-h dark cycle (lights on at 07:00 AM). All animals were allowed to adapt to the laboratory conditions for at least 1 week prior to the surgery and were handled for 5 min/day during this adaptation period. Trials were conducted between 10:00 AM and 02:00 PM. The experimental protocol was approved by the Research and Ethics Committee of the School of Medicine, Tehran University of Medical Sciences and was performed in accordance with institutional guidelines for the care and use of laboratory animals (NIH publications no. 80-23; revised 1996). 2.2. Surgical and microinjection Ketamine-Xylazine (50 mg/kg and 5 mg/kg, respectively) was used for deep anesthesia in the animals. Using a stereotaxic apparatus, each animal was bilaterally implanted with 22-gauge guide cannulas which were 1 mm above the CA1 regions according to the atlas of Paxinos and Watson (2007). Coordinates for the CA1 regions of the dorsal hippocampi were: AP: − 3.3; ML: ± 2; DV-2.8. The cannula was fixed to the skull with two jewelers screw and dental acrylic. To prevent clogging, the stainless steel stylets (27 gauge) were used in the guide cannulas until the animals were given the injection. The rats were given 1 week to recover from the surgical procedure. For drug injection, the stylets were slowly removed from the guide cannulas and replaced with 27-gauge injection needles (1 mm below the tip of the guide cannula). The injection unit was attached with a polyethylene tube to a 2-μl Hamilton syringe. The injection lasted about 60 s and the cannula was left in place for 60 s after each injection to allow for diffusion and the stylet was then reinserted into the guide cannula. 2.3. Drugs The drugs used in this study were ACPA (arachidonylcyclopropylamide; N-(2-cyclopropyl)-5Z, 8Z, 11Z, 14Z-eicosatertraenanmide), MDMA (3, 4-methylenedioxy-N-methylamphetamine) or ecstasy and D-AP5 [D-(_)-2-amino-5-phosphonopentanoic acid] (Tocris, Bristol, UK). ACPA dissolved in Tocrisolve™ (a soya oil and water emulsion) was obtained from Tocris and was diluted with sterile 0.9% saline. The other drugs were dissolved in sterile 0.9% saline just before the experiments and were bilaterally injected into the CA1 regions at a volume of 1 μl/rat (0.5 μl/each side). All control groups received sterile 0.9% saline, except for ACPA groups which received Tocrisolve™ with the same concentration as in the experimental solution (as vehicle). The time intervals of drug administrations and the drugs' doses were based on our pilot experiments and previous studies (Rezayof et al., 2011; Alijanpour et al., 2013; Tirgar et al., 2014). 2.4. Passive avoidance apparatus The animals were trained and tested in a step-through type passive avoidance apparatus which consisted of two compartments, one light (white compartment, 20 cm × 20 × 30 cm) and the other dark (black compartment, 20 cm × 20 × 30 cm). The chambers were separated by a guillotine door (7 cm × 9 cm) in the middle of the dividing wall. Stainless steel grids (2.5 mm in diameter) were placed at 1-cm intervals (distance between the centers of grids) on the floor of the dark compartment. Intermittent electric shocks (50 Hz, 3 s, 1 mA) were delivered to the floor of the dark compartment by an insulated stimulator (Borj Sanat Co., Tehran, Iran). It is important to note that the paradigm of

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passive avoidance learning is an accepted model to test long-term memory in a simple conditioning task (Sarter et al., 1992; Izquierdo et al., 1999; Almeida-Suhett et al., 2015). 2.5. Behavioral testing 2.5.1. Training phase The rats were allowed to habituate to the experimental room for at least 30 min prior to the experiments. Each animal was gently placed in the brightly lit compartment of the apparatus, after 5 s the guillotine door was opened and the animal was allowed to enter the dark compartment. The step-through latency of the animal to cross into the dark compartment with all four paws was recorded. The animals that waited more than 100 s to cross to the dark compartment were eliminated from the experiments. Once the animal crossed to the next compartment, the guillotine door was closed and the rat was taken into its home cage (habituation trial). In the training phase, after 5 s, the guillotine door was opened and as soon as the animal crossed to the dark compartment the door was closed and a foot shock (50 Hz, 1 mA, 3 s) was immediately delivered to the grid floor. After 20 s, the rat was removed from the apparatus and placed temporarily into its home cage. Two minutes later, the animal was retested in the same way as the prior trials; if the rat did not enter the dark compartment during 120 s, successful acquisition of passive avoidance response was recorded. Otherwise, when the animal entered the dark compartment (before 120 s) for a second time, the door was closed and the animal received the shock again. All animals learned after a maximum of 3 trials. 2.5.2. Testing phase 24 h after the training, a retrieval test was performed to assess memory retrieval. On the test day, the drugs were microinjected 5 min prior to the test (pre-test microinjection). After intra-CA1 injections of the drugs, each animal was gently placed in the light compartment, and after 5 s the door was opened and the step-through latency was measured for each animal. The testing process ended when the animal entered the dark compartment or remained in the light compartment for 300 s. During testing phase no electric shock was applied.

Fig. 1. Effects of post-training or pre-test intra-CA1 microinjection of ACPA on memory retrieval. Four groups of animals received intra-CA1 microinjection of vehicle (1 μl/rat) or different doses of ACPA (0.5, 2 and 4 ng/rat) immediately after the training phase (post-training microinjection). Another four groups of animals received vehicle (1 μl/rat) or the same doses of ACPA into the CA1 regions, 5 min before the testing phase (pre-test microinjection). Step-through latency of each rat was recorded 24 h after successful training. Each value represents the mean ± S.E.M. of seven rats. **P b 0.01 and ***P b 0.001 compared to pre-test vehicle (l μl/rat) control group.

animals. Successful acquisition of passive avoidance response was recorded in eight groups of animals in the training phase. On the test day, four groups received intra-CA1 microinjection of vehicle (1 μl/rat) plus different doses of MDMA (0, 0.5, 0.75 and 1 μg/rat; left panel of Fig. 2). The other four groups received pre-test intra-CA1 microinjection of the same doses of MDMA plus 4 ng/rat of ACPA with 5 min intervals (Right panel of Fig. 2). Step-through latency was recorded 5 min after the last injection.

2.6. Behavioral procedures Seven animals were placed in each experimental group. In all the experiments in which the animals received two or three microinjections, the control groups also received two or three saline or vehicle microinjections (Nazari-Serenjeh and Rezayof, 2013). Consecutive injections were performed with 5 min intervals. All microinjections were made on the conscious freely moving rats one week after the surgery. In order to minimize the microinjection-induced stress, each animal was handled every day and habituated to the microinjection conditions during recovery period (Zaretsky et al., 2011). 2.6.1. Experiment 1 The effects of post-training (immediately after the training phase) and pre-test (prior to the testing phase) microinjection of ACPA, a cannabinoid CB1 receptor agonist, on memory of passive avoidance task were examined in eight groups of animals. One control group received vehicle (1 μl/rat, intra-CA1) immediately after training (post-training). Three groups of animals received post-training intra-CA1 microinjection of different doses of ACPA (0.5, 2 and 4 ng/rat; left panel of Fig. 1). In another four groups of animals, the animals received pretest intra-CA1 microinjection of vehicle (1 μl/rat) or the same doses of ACPA (Right panel of Fig. 1). Step-through latency of each rat was recorded 24 h after successful training. 2.6.2. Experiment 2 The effect of pre-test intra-CA1 microinjection of MDMA with or without ACPA was evaluated on memory retrieval in eight groups of

Fig. 2. Effects of intra-CA1 microinjection of MDMA with or without ACPA on memory retrieval. Four groups of animals received pre-test intra-CA1 microinjection of saline (1 μl/rat, intra-CA1) or different doses of MDMA (0.5, 0.75 and 1 μg/rat) plus vehicle (1 μg/rat). Another four groups of animals received the same doses of MDMA plus ACPA (4 ng/rat, intra-CA1) with 5 min interval. Step-through latency was recorded 5 min after the last injection. Each value represents the mean ± S.E.M. of seven rats. ***P b 0.001 compared to pre-test saline/vehicle control group. +++P b 0.001 compared to pre-test saline/ACPA control group.

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2.6.3. Experiment 3 In this experiment, the effect of pre-test intra-CA1 microinjection of D-AP5, a selective NMDA receptor antagonist, with or without MDMA plus ACPA was evaluated on memory retrieval. Successful acquisition of passive avoidance response was recorded in eight groups of animals in the training phase. On the test day, different doses of D-AP5 (0, 0.5, 1, and 2 μg/rat) plus saline (1 μl/rat) and ACPA vehicle (1 μl/rat) were injected into the CA1 regions in four groups of animals with 5 min intervals (left panel of Fig. 3). The other four groups received intra-CA1 microinjection of the same doses of D-AP5. After 5 min, MDMA (1 μg/rat) and ACPA (4 ng/rat; right panel of Fig. 3) were injected into the CA1 regions with a 5 min interval and step-through latency of each animal was then recorded. 2.7. Histology After the completion of behavioral testing, each animal was killed with carbon dioxide. Subsequently, 1 μl of a 1% methylene-blue solution was bilaterally injected into the CA1 regions (0.5 μl/side). This procedure was done by a 27-gauge injection needle which projected a further 1 mm ventral to the tip of the guide to aid in histological verification. Then each rat was decapitated and its brain was removed and placed in a 10% formalin solution. After 10 days, the sections were examined to determine the location of the cannula which targeted the CA1 regions. The sites of injections were verified according to the atlas of Paxinos and Watson (2007). 2.8. Statistics The data are expressed as mean ± standard error of mean (S.E.M.). The statistical analysis was performed using a one- and two-way analysis of variance (ANOVA). One-way ANOVA was used for analyzing the

differences between the groups and two-way ANOVA was used to analyze the interaction between the two factors. Post-hoc comparison of the means was carried out with the Tukey test for multiple comparisons. The level of statistical significance was set at P b 0.05. Calculations were performed using SPSS statistical package. 3. Results 3.1. Post-training and pre-test intra-CA1 microinjection of ACPA alters memory formation As shown in the left panel of Fig. 1, intra-CA1 microinjection of different doses of ACPA (0.5, 2 and 4 ng/rat) immediately after the training phase (post-training microinjection) had no effect on memory consolidation [One-way ANOVA: F (3, 24) = 1.74, P N 0.05. On the other hand, one-way ANOVA showed that the microinjection of ACPA into the CA1 regions before the testing phase (pre-test microinjection) impaired memory retrieval in a dose dependent manner [(3, 24) = 30.6, P b 0.05; right panel of Fig. 1]. The maximum effect was obtained with 4 ng/rat of ACPA. 3.2. Effect of pre-test intra-CA1 microinjection of MDMA plus ACPA on memory retrieval Fig. 2 shows the effect of intra-CA1 microinjection of MDMA with or without ACPA on memory retrieval. Two-way ANOVA revealed a significant difference in memory retrieval between the groups of animals that received MDMA (0.5, 0.75 and 1 μg/rat, intra-CA1) and those that received the same doses of MDMA plus ACPA (4 ng/rat, intra-CA1) in memory retrieval [for treatment, F (1, 48) = 40.5, P b 0.001; dose, F (3, 48) = 1.0, P N 0.05; and treatment × dose interaction, F (3, 48) = 56.5, P b 0.001]. Further analysis also indicated that intraCA1 microinjection of 1 μg/rat of MDMA impaired memory retrieval, suggesting an amnesic effect of the drug by itself [One-way ANOVA: F (3, 24) = 14.4, P b 0.001]. One-way ANOVA also showed that the microinjection of the same doses of MDMA into the CA1 regions, 5 min before intra-CA1 microinjection of ACPA (4 ng/rat) reversed ACPA-induced memory impairment [F (3, 24) = 78.1, P b 0.001]. By comparing the step-through latency obtained for MDMA (1 μg/rat) alone with that obtained for the same dose of MDMA plus ACPA (4 ng/rat), a reversal of the MDMA effect by the presence of ACPA can be suggested. 3.3. Pre-test intra-CA1 microinjection of D-AP5 inhibited the improving effect of MDMA on ACPA-induced memory impairment

Fig. 3. Effects of pre-test intra-CA1 microinjection of D-AP5 on memory formation induced by co-administration of MDMA plus ACPA. Different doses of D-AP5 (0, 0.5, 1, and 2 μg/rat) plus saline (1 μl/rat) and vehicle (1 μl/rat) were injected into the CA1 regions of four groups of animals with 5 min intervals. The other four groups received intra-CA1 microinjection of the same doses of D-AP5. After 5 min, MDMA (1 μg/rat) and ACPA (4 ng/rat) were injected into the CA1 regions with 5 min interval and then the step-through latency of each animal was recorded. **P b 0.01 and ***P b 0.001 compared to pre-test saline/ MDMA/ACPA control group.

Fig. 3 shows the effect of intra-CA1 microinjection of D-AP5 with or without MDMA plus ACPA on memory retrieval. Two-way ANOVA revealed a significant difference in memory retrieval between the groups of animals that received D-AP5 (0.5, 1, and 2 μ g/rat, intra-CA1) and those that received the same doses of D-AP5 plus MDMA (1 μg/rat) and ACPA (4 ng/rat) [for treatment, F (1, 48) = 21.5, P b 0.001; dose, F (3, 48) = 9.4, P b 0.001; and treatment × dose interaction, F (3, 48) = 8.6, P b 0.001]. Further analysis indicated that intra-CA1 microinjection of D-AP5 alone at different doses (0.5, 1, and 2 μg/rat) had no effect on memory retrieval [One-way ANOVA: F (3, 24) = 0.2, P N 0.05]. One-way ANOVA also showed that intra-CA1 microinjection of D-AP5 inhibited the improving effect of MDMA on ACPA-induced memory impairment in passive avoidance task [F (3, 24) = 20.3, P b 0.001; right panel of Fig. 3]. Post-hoc analysis showed that the maximum effect was obtained with 2 μg/rat of D-AP5. 4. Discussion The results of our study showed that the activation of CB1 receptors in the dorsal hippocampus by the microinjection of an agonist,

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arachidonylcyclopropylamide (ACPA) into the CA1 regions (intra-CA1) altered memory formation. Pre-test (before testing phase), but not post-training (immediately after training phase), intra-CA1 microinjection of ACPA decreased step-trough latency and induced the impairing effect of ACPA on memory retrieval, but not on memory consolidation. In agreement with our results, previous investigations showed that the activation of endocannabinoid system especially via stimulating cannabinoid CB1 receptor signaling pathways has a disruptive effect on learning and memory processes in different types of animal models. For example, pre-test systemic or intra-hippocampal administration of cannabinoids such as delta9-tetrahydrocannabinol (Δ9-THC) or CP55,940 was reported to impair working memory in the eight-arm radial-maze paradigm (Lichtman et al., 1995). Suenaga and Ichitani (2008), Suenaga et al. (2008) also reported that pre-test intrahippocampal microinjection of WIN55,212-2, a CB1/CB2 receptor agonist, decreased memory formation in radial arm- and object recognition-mazes. In contrast, the knock-out/knock-down of CB1 receptor gene or the inhibition of endocannabinoid transmission facilitates hippocampal memory processes (Basavarajappa et al., 2014; Lichtman et al., 2002). In view of the fact that CB1 receptors are highly expressed at pre-synaptic terminals to inhibit neurotransmitter release, it has been suggested that pre-synaptic suppression of glutamatergic (Maier et al., 2012) and cholinergic transmission (Gessa et al., 1997) may be involved in cannabinoid-induced impairment of hippocampalbased memory processing. It should be considered that the present results indicated that intra-CA1 microinjection of ACPA immediately after training phase had no effect on memory consolidation, while post-training intra-central amygdala (Ghiasvand et al., 2011) or -nucleus accumbens shell (Rasekhi et al., 2014) administration of ACPA impaired passive avoidance memory consolidation in rats. A possible explanation for this is that the endocannabinoid system in rats' hippocampus may not be essential for memory consolidation of passive avoidance task. However, more research on the role of CB1 receptors in hippocampus-dependent memory consolidation needs to be undertaken in the future. Another important finding was that pre-test microinjection of a higher dose of MDMA (1 μg/rat) into the CA1 regions impaired memory retrieval of passive avoidance task. As reported in many basic and clinical studies, MDMA (ecstasy) administration can cause cognitive deficits both in laboratory animals (McAleer et al., 2013) and humans (Gouzoulis-Mayfrank et al., 2000). With respect to this effect, it was found that intraperitoneal administration of MDMA-induced neurodegeneration in various rat brain areas such as the hippocampus (Riezzo et al., 2010) which may play a critical role in MDMA-induced cognitive dysfunction. The disruptive effects of MDMA administration on verbal memory using a spatial memory task in humans (van Wel et al., 2011) and passive avoidance learning in rats (Shariati et al., 2014) have also been previously reported. As mentioned in the introduction, MDMA administration affects serotonergic system and thus alters the serotoninrelease, reuptake or transporter function (Verkes et al., 2001; Taffe et al., 2002; Parrot, 2012). The dopaminergic and cholinergic neurotransmission have also been reported to change (Gudelsky and Yamamoto, 2008) by MDMA consumption. MDMA administration is also associated with promoting glutamate efflux in the hippocampus which may lead to neurotoxicity (Sanchez et al., 2001; Verrico et al., 2007) and neuroadaptive alterations in gene expression for NMDA receptor subunits (Kindlundh-Högberg et al., 2008). Therefore, it seems that memory retrieval impairment under MDMA administration may depend on these neurotransmitter imbalances. Further study using microdialysis technique is however required to establish the validity of this hypothesis in the future. Although the use of cannabis among young people is commonly accompanied by the use of MDMA, little is known about the combined effect of these drugs on learning and memory processes. Thus, another objective of this study was to assess whether there is a functional interaction between ACPA and MDMA in passive avoidance memory

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retrieval. Surprisingly, we observed that pre-test intra-CA1 microinjection of MDMA reversed ACPA-induced memory impairment, suggesting an interactive effect between MDMA and cannabis. Since pre-test intraCA1 microinjection of MDMA (1 μg/rat) by itself induced memory impairment, it is likely that there is a reversal of the MDMA effect by the presence of ACPA. Therefore, it seems that MDMA and ACPA reverse each other's effects. This interaction was previously suggested by Schulz et al., 2013 using the object recognition task. They reported a CB1 receptor-induced reversal of the MDMA effect on working memory. In contrast to these findings, it has been shown that the blockade of hippocampal cannabinoid CB1 receptors suppressed recognition memory impairment during withdrawal from repeated administration of MDMA (Nawata et al., 2010). In a clinical study, Dumont et al. (2011) proposed that cognitive disruption induced by the co-abuse of acute MDMA and THC is similar to the impairment observed with the administration of each drug alone. Further research is therefore suggested to better clarify the possible effects of polydrug abuse on cognition by focusing on the interactive effects of the drugs. This study also aimed at assessing the possible role of NMDA receptors in the CA1 region of the hippocampus in the interactive effects of MDMA and ACPA on memory retrieval. The results showed that intra-CA1 microinjection of D-AP5 (a selective NMDA receptor antagonist), reversed the improving effect of MDMA on ACPA-induced memory impairment respectively. Since MDMA and ACPA reverse each other's effects, it is possible that D-AP5 inhibits the improving effect of ACPA in MDMA-induced memory impairment. A great deal of previous research into the functional interactions between these drugs has focused on the expression of cannabinoid CB1 receptors at excitatory pre-synaptic sites in different brain areas. Using electrophysiological and immunohistochemical techniques, it was found that the release of both the glutamate and serotonin be, directly or indirectly, modulated by specific presynaptic cannabinoid CB1 receptors in the hippocampus (Kawamura et al., 2006) and the cortical areas (Domenici et al., 2006; Ferreira et al., 2012), suggesting that coadministration of MDMA and ACPA may induce alterations in hippocampal glutamate neurotransmission related to memory formation. A strong relationship between NMDA receptors and MDMA has also been reported in the literature. For example, it has been shown that memantine as an NMDA receptor antagonist inhibits MDMAinduced serotonergic neurotoxicity (Chipana et al., 2008) and blocks the acquisition of MDMA-induced conditioned place preference (García-Pardo et al., 2015). Moreover, the hippocampal reduction of synaptic NMDA receptor subunits may be involved in MDMA-induced memory impairment in the passive avoidance task (Moyano et al., 2004). Since microdialysis studies have shown that MDMA administration enhances the extracellular concentration of glutamate in the rat hippocampus (Anneken and Gudelsky, 2012), one may suggests that the blockade of NMDA receptors in the CA1 regions of the dorsal hippocampus via inhibiting glutamatergic neurotransmission could alter the co-administrative effect of MDMA plus ACPA on memory formation. Although it has been shown that the injections of NMDA receptor antagonists into the hippocampus impair learning and memory in a variety of behavioral tasks (McHugh et al., 2008; Yamada et al., 2015), pre-test intra-CA1 microinjection of the doses of D-AP5 used in the present study did not have any effect by itself on the step-through latencies of passive avoidance learning. Considering that the microinjection of the higher doses of D-AP5 into the dorsal hippocampus induced memory deterioration in previous studies, it seems that the effect of the antagonist may depend on its dose. It could be concluded that the dorsal hippocampus, considering the effects of bilateral intra-CA1 microinjection of MDMA when coadministered with ACPA, plays a pivotal role in improving the effect of MDMA on ACPA-induced memory impairment. Involvement of NMDA receptor mechanisms in this brain site can also be suggested in the MDMA-induced improvement of ACPA amnesia in passive avoidance task.

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M. Ghaderi et al. / Progress in Neuro-Psychopharmacology & Biological Psychiatry 66 (2016) 41–47

Acknowledgments None.

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ecstasy on memory retrieval in rats.

A combination of cannabis and ecstasy may change the cognitive functions more than either drug alone. The present study was designed to investigate th...
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