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morphine-induced amnesia
Author’s Accepted Manuscript Neuromodulatory effects of the dorsal hippocampal endocannabinoid system in dextromethorphan/morphine-induced amnesia Zahra Ghasemzadeh, Ameneh Rezayof www.elsevier.com/locate/ejphar
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S0014-2999(16)30728-2 http://dx.doi.org/10.1016/j.ejphar.2016.11.025 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 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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 [email protected]
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, intraCA1 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 longterm 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
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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 dextromethorphaninduced 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 Δ9tetrahydrocannabinol (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
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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.
References Aimone, J.B., Wiles, J. & Gage, F.H., 2006. Potential role for adult neurogenesis in the encoding of time in new memories. Nat. Neurosci. 9(6), 723-7. Alijanpour, S., Rezayof, A., Zarrindast, M.R., 2013. Dorsal hippocampal cannabinoid CB1 receptors mediate the interactive effects of nicotine and ethanol on passive avoidance learning in mice. Addict. Biol. 18(2), 241-51. Bryner, J.K., Wang, U.K., Hui, J.W., Bedodo, M., MacDougall, C. & Anderson, I,B., 2006. Dextromethorphan abuse in adolescence: an increasing trend: 1999-2004. Arch. Pediatr. Adolesc. Med. 160(12), 1217-22. Carter, L.P., Reissig, C.J., Johnson, M.W., Klinedinst, M.A., Griffiths, R,R., Mintzer, M,Z., 2013. Acute cognitive effects of high doses of dextromethorphan relative to triazolam in humans. Drug. Alcohol. Depend. 128(3), 206-13. 15
Chavkin, C., 2000. Dynorphins are endogenous opioid peptides released from granule cells to act neurohumorly and inhibit excitatory neurotransmission in the hippocampus. Prog. Brain. Res.125, 363-7. de Oliveira, Alvares. L., Pasqualini, Genro. B., Diehl, F., Molina, V.A., Quillfeldt, J,A., 2008. Opposite action of hippocampal CB1 receptors in memory reconsolidation and extinction. Neuroscience. 154(4), 1648-55. Deng, W., Aimone, J.B. & Gage, F.H., 2010. New neurons and new memories: how does adult hippocampal neurogenesis affect learning and memory? Nat. Rev. Neurosci. 11(5), 33950. Douma, T.N., Borre, Y., Hendriksen, H., Olivier, B., Oosting, R.S, 2011. Simvastatin improves learning
and memory in control but not in olfactory bulbectomized rats. Psychopharmacology (Berl). 216(4):537-44. Eisch, A.J., Harburg, G., 2006. Opiates, psychostimulants, and adult hippocampal neurogenesis: Insights for addiction and stem cell biology. Hippocampus. 16(3), 271-86. Farahmandfar, M., Kadivar, M. & Naghdi, N., 2015. Possible interaction of hippocampal nitric oxide and calcium/calmodulin-dependent protein kinase II on reversal of spatial memory impairment induced by morphine. Eur. J. Pharmacol. 751, 99-111. Farahmandfar, M., Karimian, S.M., Naghdi, N., Zarrindast, M.R., Kadivar, M., 2010. Morphineinduced impairment of spatial memory acquisition reversed by morphine sensitization in rats. Behav. Brain. Res. 211(2), 156-63.
16
Ferraro, L., Tomasini, M.C., Gessa, G.L., Bebe, B.W., Tanganelli, S., Antonelli, T., 2001. The cannabinoid receptor agonist WIN 55,212-2 regulates glutamate transmission in rat cerebral cortex: an in vivo and in vitro study. Cereb. Cortex. 11(8), 728-33. Fiszman, M.L., Barberis, A., Lu, C., Fu, Z., Erdélyi, F., Szabó, G., Vicini, S., 2005. NMDA receptors increase the size of GABAergic terminals and enhance GABA release. J. Neurosci. 25(8), 2024-31. Ghasemzadeh, Z., Rezayof, A., 2016. Role of hippocampal and prefrontal cortical signaling pathways in dextromethorphan effect on morphine-induced memory impairment in rats. Neurobiol. Learn. Mem. 128, 23-32. Ghiasvand, M., Rezayof, A., Zarrindast, M.R., Ahmadi, S., 2011. Activation of cannabinoid CB1 receptors in the central amygdala impairs inhibitory avoidance memory consolidation via NMDA receptors. Neurobiol. Learn. Mem. 96(2), 333-8. Ghozland, S., Matthes, H.W., Simonin, F., Filliol, D., Kieffer, B.L., Maldonado, R., 2002. Motivational effects of cannabinoids are mediated by mu-opioid and kappa-opioid receptors. J. Neurosci. 22(3), 1146-54. Herkenham, M., Lynn, A.B., Johnson, M.R., Melvin, L.S., de Costa, B.R., Rice, K.C., 1991. Characterization and localization of cannabinoid receptors in rat brain: a quantitative in vitro autoradiographic study. J. Neurosci. 11(2), 563-83. Huang, E.Y., Liu, T.C., Tao, P.L., 2003. Co-administration of dextromethorphan with morphine attenuates morphine rewarding effect and related dopamine releases at the nucleus accumbens. Naunyn. Schmiedebergs. Arch. Pharmacol. 368(5), 386-92. 17
Katona, I., Freund, T.F., 2012. Multiple functions of endocannabinoid signaling in the brain. Annu. Rev. Neurosci. 35, 529-58. Liu, P., Bilkey, D.K., Darlington, C.L., Smith, P.F., 2003. Cannabinoid CB1 receptor protein expression in the rat hippocampus and entorhinal, perirhinal, postrhinal and temporal cortices: regional variations and age-related changes. Brain. Res. 979(1-2), 235-9. Ma, Y.L., Weston, S.E., Whalley, B.J., Stephens, G.J., 2008. The phytocannabinoid Delta(9)tetrahydrocannabivarin modulates inhibitory neurotransmission in the cerebellum. Br. J. Pharmacol. 154(1), 204-15. Maldonado, R., Valverde, O. & Berrendero, F., 2006. Involvement of the endocannabinoid system in drug addiction. Trends. Neurosci. 29(4), 225-32. Mansour, A., Fox, C.A., Burke, S., Meng, F., Thompson, R.C., Akil, H., Watson, S.J., 1994. Mu, delta, and kappa opioid receptor mRNA expression in the rat CNS: an in situ hybridization study. J. Comp. Neurol. 350(3), 412-38. Mao, J., Price, D.D., Caruso, F.S., Mayer, D.J., 1996. Oral administration of dextromethorphan prevents the development of morphine tolerance and dependence in rats. Pain. 67(2-3), 361-8. Marsicano, G., Lutz, B., 2006. Neuromodulatory functions of the endocannabinoid system. J. Endocrinol. Invest. 29(3 Suppl), 27-46. Mohammadmirzaei, N., Rezayof, A., Ghasemzadeh, Z., 2016. Activation of cannabinoid CB1 receptors in the ventral hippocampus improved stress-induced amnesia in rat. Brain Res. 1646:219-26. 18
Morris, R.G., 2006. Elements of a neurobiological theory of hippocampal function: the role of synaptic plasticity, synaptic tagging and schemas. Eur. J. Neurosci. 23(11), 2829-46. Nasehi, M., Sahebgharani, M., Haeri-Rohani, A., Zarrindast, M.R., 2009. Effects of cannabinoids infused into the dorsal hippocampus upon memory formation in 3-days apomorphine-treated rats. Neurobiol. Learn. Mem. 92(3), 391-9. Naghdi , N., Asadollahi, A., 2004. Genomic and nongenomic effects of intrahippocampal microinjection of testosterone on long-term memory in male adult rats. Behav. Brain. Res. 153(1):1-6. Parolaro, D., Rubino, T., Viganò, D., Massi, P., Guidali, C., Realini, N., 2010. Cellular mechanisms underlying the interaction between cannabinoid and opioid system. Curr. Drug. Targets. 11(4), 393-405. Paxinos, G., Watson, C., 2007. The rat brain in stereotaxic coordinates, 3rd edn. San Diego: Academic Press. Rice, O.V., Gordon, N., Gifford, A.N., 2002. Conditioned place preference to morphine in cannabinoid CB1 receptor knockout mice. Brain. Res. 945(1), 135-8. Robledo, P., Berrendero, F., Ozaita, A., Maldonado, R., 2008. Advances in the field of cannabinoid-opioid cross-talk. Addict. Biol.13(2), 213-24. Shen, M., Thayer, S.A., 1999. Delta9-tetrahydrocannabinol acts as a partial agonist to modulate glutamatergic synaptic transmission between rat hippocampal neurons in culture. Mol. Pharmacol. 55(1), 8-13. 19
Simmons, M.L., Chavkin, C., 1996. Endogenous opioid regulation of hippocampal function. Int. Rev. Neurobiol. 39, 145-96. Stumm, R.K., Zhou, C., Schulz, S., Höllt, V. 2004. Neuronal types expressing mu- and deltaopioid receptor mRNA in the rat hippocampal formation. J. Comp. Neurol. 469(1), 10718. Sullivan, J.M., 2000. Cellular and molecular mechanisms underlying learning and memory impairments produced by cannabinoids. Learn. Mem. 7(3), 132-9. Svízenská, I., Dubový, P., Sulcová, A., 2008. Cannabinoid receptors 1 and 2 (CB1 and CB2), their distribution, ligands and functional involvement in nervous system structures--a short review. Pharmacol. Biochem. Behav. 90(4), 501-11. Szabo, B., Schlicker, E., 2005. Effects of cannabinoids on neurotransmission. Handb. Exp. Pharmacol. (168), 327-65. Tajik, A., Rezayof, A., Ghasemzadeh, Z., Sardari, M., 2016. Activation of the dorsal hippocampal nicotinic acetylcholine receptors improves tamoxifen-induced memory retrieval impairment in adult female rats. Neuroscience. 327:1-9. Tirgar, F., Rezayof, A., Zarrindast, M.R., 2014. Central amygdala nicotinic and 5-HT1A receptors mediate the reversal effect of nicotine and MDMA on morphine-induced amnesia. Neuroscience. 277, 392-402.
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Wilson, M.D., Ferguson, R.W., Mazer, M.E., Litovitz, T.L., 2011. Monitoring trends in dextromethorphan abuse using the National Poison Data System: 2000-2010. Clin. Toxicol. (Phila). 49, 409–415. Zarrindast, M.R., Dorrani, M., Lachinani, R., Rezayof, A., 2010. Blockade of dorsal hippocampal dopamine receptors inhibits state-dependent learning induced by cannabinoid receptor agonist in mice. Neurosci. Res. 67(1), 25-32. Zarrindast, M.R., Ownegh, V., Rezayof, A., Ownegh, F., 2014. The involvement of dorsal hippocampus in dextromethorphan-induced state-dependent learning in mice. Pharmacol. Biochem. Behav. 116, 90-5.
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 21
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 22
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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PII: DOI: Reference:
S0014-2999(16)30728-2 http://dx.doi.org/10.1016/j.ejphar.2016.11.025 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 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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 [email protected]
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, intraCA1 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 longterm 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
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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
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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 dextromethorphaninduced 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 Δ9tetrahydrocannabinol (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.
References Aimone, J.B., Wiles, J. & Gage, F.H., 2006. Potential role for adult neurogenesis in the encoding of time in new memories. Nat. Neurosci. 9(6), 723-7. Alijanpour, S., Rezayof, A., Zarrindast, M.R., 2013. Dorsal hippocampal cannabinoid CB1 receptors mediate the interactive effects of nicotine and ethanol on passive avoidance learning in mice. Addict. Biol. 18(2), 241-51. Bryner, J.K., Wang, U.K., Hui, J.W., Bedodo, M., MacDougall, C. & Anderson, I,B., 2006. Dextromethorphan abuse in adolescence: an increasing trend: 1999-2004. Arch. Pediatr. Adolesc. Med. 160(12), 1217-22. Carter, L.P., Reissig, C.J., Johnson, M.W., Klinedinst, M.A., Griffiths, R,R., Mintzer, M,Z., 2013. Acute cognitive effects of high doses of dextromethorphan relative to triazolam in humans. Drug. Alcohol. Depend. 128(3), 206-13. 15
Chavkin, C., 2000. Dynorphins are endogenous opioid peptides released from granule cells to act neurohumorly and inhibit excitatory neurotransmission in the hippocampus. Prog. Brain. Res.125, 363-7. de Oliveira, Alvares. L., Pasqualini, Genro. B., Diehl, F., Molina, V.A., Quillfeldt, J,A., 2008. Opposite action of hippocampal CB1 receptors in memory reconsolidation and extinction. Neuroscience. 154(4), 1648-55. Deng, W., Aimone, J.B. & Gage, F.H., 2010. New neurons and new memories: how does adult hippocampal neurogenesis affect learning and memory? Nat. Rev. Neurosci. 11(5), 33950. Douma, T.N., Borre, Y., Hendriksen, H., Olivier, B., Oosting, R.S, 2011. Simvastatin improves learning
and memory in control but not in olfactory bulbectomized rats. Psychopharmacology (Berl). 216(4):537-44. Eisch, A.J., Harburg, G., 2006. Opiates, psychostimulants, and adult hippocampal neurogenesis: Insights for addiction and stem cell biology. Hippocampus. 16(3), 271-86. Farahmandfar, M., Kadivar, M. & Naghdi, N., 2015. Possible interaction of hippocampal nitric oxide and calcium/calmodulin-dependent protein kinase II on reversal of spatial memory impairment induced by morphine. Eur. J. Pharmacol. 751, 99-111. Farahmandfar, M., Karimian, S.M., Naghdi, N., Zarrindast, M.R., Kadivar, M., 2010. Morphineinduced impairment of spatial memory acquisition reversed by morphine sensitization in rats. Behav. Brain. Res. 211(2), 156-63.
16
Ferraro, L., Tomasini, M.C., Gessa, G.L., Bebe, B.W., Tanganelli, S., Antonelli, T., 2001. The cannabinoid receptor agonist WIN 55,212-2 regulates glutamate transmission in rat cerebral cortex: an in vivo and in vitro study. Cereb. Cortex. 11(8), 728-33. Fiszman, M.L., Barberis, A., Lu, C., Fu, Z., Erdélyi, F., Szabó, G., Vicini, S., 2005. NMDA receptors increase the size of GABAergic terminals and enhance GABA release. J. Neurosci. 25(8), 2024-31. Ghasemzadeh, Z., Rezayof, A., 2016. Role of hippocampal and prefrontal cortical signaling pathways in dextromethorphan effect on morphine-induced memory impairment in rats. Neurobiol. Learn. Mem. 128, 23-32. Ghiasvand, M., Rezayof, A., Zarrindast, M.R., Ahmadi, S., 2011. Activation of cannabinoid CB1 receptors in the central amygdala impairs inhibitory avoidance memory consolidation via NMDA receptors. Neurobiol. Learn. Mem. 96(2), 333-8. Ghozland, S., Matthes, H.W., Simonin, F., Filliol, D., Kieffer, B.L., Maldonado, R., 2002. Motivational effects of cannabinoids are mediated by mu-opioid and kappa-opioid receptors. J. Neurosci. 22(3), 1146-54. Herkenham, M., Lynn, A.B., Johnson, M.R., Melvin, L.S., de Costa, B.R., Rice, K.C., 1991. Characterization and localization of cannabinoid receptors in rat brain: a quantitative in vitro autoradiographic study. J. Neurosci. 11(2), 563-83. Huang, E.Y., Liu, T.C., Tao, P.L., 2003. Co-administration of dextromethorphan with morphine attenuates morphine rewarding effect and related dopamine releases at the nucleus accumbens. Naunyn. Schmiedebergs. Arch. Pharmacol. 368(5), 386-92. 17
Katona, I., Freund, T.F., 2012. Multiple functions of endocannabinoid signaling in the brain. Annu. Rev. Neurosci. 35, 529-58. Liu, P., Bilkey, D.K., Darlington, C.L., Smith, P.F., 2003. Cannabinoid CB1 receptor protein expression in the rat hippocampus and entorhinal, perirhinal, postrhinal and temporal cortices: regional variations and age-related changes. Brain. Res. 979(1-2), 235-9. Ma, Y.L., Weston, S.E., Whalley, B.J., Stephens, G.J., 2008. The phytocannabinoid Delta(9)tetrahydrocannabivarin modulates inhibitory neurotransmission in the cerebellum. Br. J. Pharmacol. 154(1), 204-15. Maldonado, R., Valverde, O. & Berrendero, F., 2006. Involvement of the endocannabinoid system in drug addiction. Trends. Neurosci. 29(4), 225-32. Mansour, A., Fox, C.A., Burke, S., Meng, F., Thompson, R.C., Akil, H., Watson, S.J., 1994. Mu, delta, and kappa opioid receptor mRNA expression in the rat CNS: an in situ hybridization study. J. Comp. Neurol. 350(3), 412-38. Mao, J., Price, D.D., Caruso, F.S., Mayer, D.J., 1996. Oral administration of dextromethorphan prevents the development of morphine tolerance and dependence in rats. Pain. 67(2-3), 361-8. Marsicano, G., Lutz, B., 2006. Neuromodulatory functions of the endocannabinoid system. J. Endocrinol. Invest. 29(3 Suppl), 27-46. Mohammadmirzaei, N., Rezayof, A., Ghasemzadeh, Z., 2016. Activation of cannabinoid CB1 receptors in the ventral hippocampus improved stress-induced amnesia in rat. Brain Res. 1646:219-26. 18
Morris, R.G., 2006. Elements of a neurobiological theory of hippocampal function: the role of synaptic plasticity, synaptic tagging and schemas. Eur. J. Neurosci. 23(11), 2829-46. Nasehi, M., Sahebgharani, M., Haeri-Rohani, A., Zarrindast, M.R., 2009. Effects of cannabinoids infused into the dorsal hippocampus upon memory formation in 3-days apomorphine-treated rats. Neurobiol. Learn. Mem. 92(3), 391-9. Naghdi , N., Asadollahi, A., 2004. Genomic and nongenomic effects of intrahippocampal microinjection of testosterone on long-term memory in male adult rats. Behav. Brain. Res. 153(1):1-6. Parolaro, D., Rubino, T., Viganò, D., Massi, P., Guidali, C., Realini, N., 2010. Cellular mechanisms underlying the interaction between cannabinoid and opioid system. Curr. Drug. Targets. 11(4), 393-405. Paxinos, G., Watson, C., 2007. The rat brain in stereotaxic coordinates, 3rd edn. San Diego: Academic Press. Rice, O.V., Gordon, N., Gifford, A.N., 2002. Conditioned place preference to morphine in cannabinoid CB1 receptor knockout mice. Brain. Res. 945(1), 135-8. Robledo, P., Berrendero, F., Ozaita, A., Maldonado, R., 2008. Advances in the field of cannabinoid-opioid cross-talk. Addict. Biol.13(2), 213-24. Shen, M., Thayer, S.A., 1999. Delta9-tetrahydrocannabinol acts as a partial agonist to modulate glutamatergic synaptic transmission between rat hippocampal neurons in culture. Mol. Pharmacol. 55(1), 8-13. 19
Simmons, M.L., Chavkin, C., 1996. Endogenous opioid regulation of hippocampal function. Int. Rev. Neurobiol. 39, 145-96. Stumm, R.K., Zhou, C., Schulz, S., Höllt, V. 2004. Neuronal types expressing mu- and deltaopioid receptor mRNA in the rat hippocampal formation. J. Comp. Neurol. 469(1), 10718. Sullivan, J.M., 2000. Cellular and molecular mechanisms underlying learning and memory impairments produced by cannabinoids. Learn. Mem. 7(3), 132-9. Svízenská, I., Dubový, P., Sulcová, A., 2008. Cannabinoid receptors 1 and 2 (CB1 and CB2), their distribution, ligands and functional involvement in nervous system structures--a short review. Pharmacol. Biochem. Behav. 90(4), 501-11. Szabo, B., Schlicker, E., 2005. Effects of cannabinoids on neurotransmission. Handb. Exp. Pharmacol. (168), 327-65. Tajik, A., Rezayof, A., Ghasemzadeh, Z., Sardari, M., 2016. Activation of the dorsal hippocampal nicotinic acetylcholine receptors improves tamoxifen-induced memory retrieval impairment in adult female rats. Neuroscience. 327:1-9. Tirgar, F., Rezayof, A., Zarrindast, M.R., 2014. Central amygdala nicotinic and 5-HT1A receptors mediate the reversal effect of nicotine and MDMA on morphine-induced amnesia. Neuroscience. 277, 392-402.
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Wilson, M.D., Ferguson, R.W., Mazer, M.E., Litovitz, T.L., 2011. Monitoring trends in dextromethorphan abuse using the National Poison Data System: 2000-2010. Clin. Toxicol. (Phila). 49, 409–415. Zarrindast, M.R., Dorrani, M., Lachinani, R., Rezayof, A., 2010. Blockade of dorsal hippocampal dopamine receptors inhibits state-dependent learning induced by cannabinoid receptor agonist in mice. Neurosci. Res. 67(1), 25-32. Zarrindast, M.R., Ownegh, V., Rezayof, A., Ownegh, F., 2014. The involvement of dorsal hippocampus in dextromethorphan-induced state-dependent learning in mice. Pharmacol. Biochem. Behav. 116, 90-5.
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 21
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 22
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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