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Effects of pre-training morphine on spatial memory acquisition and retrieval in mice
Physiology & Behavior 104 (2011) 754–760
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Physiology & Behavior j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / p h b
Effects of pre-training morphine on spatial memory acquisition and retrieval in mice Feng Zhu, Chun-xia Yan ⁎, Yan Zhao, Yang Zhao, Ping-ping Li, Sheng-bin Li ⁎⁎ Forensic Department, Xi'an Jiaotong University Medical College, Xi'an, PR China Key Laboratory of Ministry of Health for Forensic Sciences, Xi'an Jiaotong University, Xi'an, PR China Key Laboratory of Ministry of Education for Environment and Genes Related to Diseases, Xi'an Jiaotong University, Xi'an, PR China
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Article history: Received 19 January 2011 Received in revised form 13 June 2011 Accepted 8 July 2011 Keywords: Morphine Naloxone Morris water maze Memory acquisition Memory retrieval Spatial memory
a b s t r a c t The opioid system plays an important role in memory processess. Morphine mimics endogenous opioids by acting on opioid receptor in brain to regulate memory. However, the effects of morphine on spatial memory acquisition are controversial. Also, little evidence has suggested that morphine could affect the retrieval of spatial memory. In the current study, effects of pre-training morphine and naloxone on the acquisition vs. retrieval of spatial reference vs. working memory were examined using discrete water maze tasks in C57BL/6 mice. Pre-training morphine administration (7.5 and 15 mg/kg, i.p.) impaired the acquisition of both spatial reference memory and working memory. Motivation to escape from the water maze was not affected by morphine. Pre-test morphine also inhibited the retrieval of spatial working memory but not reference memory. The effects of morphine on the acquisition and retrieval of spatial working memory were eliminated by naloxone pretreatment (1 mg/kg). These results indicate that morphine could differentially modulate a variety of aspects of spatial memory and these effects are mediated by the mu-opioid receptor. © 2011 Elsevier Inc. All rights reserved.
1. Introduction Memory formation consists of three key phases: information encoding, storage, and retrieval. Based on temporal duration, memory can be classified as either working or reference memory. Within the context of visuospatial information, working memory behaves as a “sketchpad” to store perceived information for a short period of time [1]. Reference memory is the retention and retrieval of information for later use as a “reference”. Pharmacological and lesion studies have demonstrated that both spatial working memory (SWM) and spatial reference memory (SRM) occur in the hippocampus [2,3]. Memory retrieval is the process of recalling memory already established from previous experience. Most contemporary studies concerning the neurobiology of memory have focused on the mechanisms that govern memory acquisition [4]. However, the cellular and molecular mechanisms underlying memory acquisition differ significantly from that of memory retrieval [5]. Also, the mechanisms underlying the retrieval of working memory and reference memory are distinctive [6,7].
⁎ Correspondence to: C. Yan, Forensic Department, Xi'an Jiaotong University Medical College, 76 West Yanta Road, Xi'an, Shaanxi (710061), PR China. Tel.: + 86 29 82655117; fax: + 86 29 82655113. ⁎⁎ Correspondence to: S. Li, Forensic Department, Xi'an Jiaotong University Medical College, 76 West Yanta Road, Xi'an, Shaanxi (710061), PR China. Tel.: + 86 29 82656244; fax: + 86 29 82655113. E-mail addresses: [email protected] (C. Yan), [email protected] (S. Li). 0031-9384/$ – see front matter © 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.physbeh.2011.07.014
Memory processes are modulated by several neurotransmitter systems and one of the most important systems is opioid system [8,9]. It has been shown that mu-opioid receptor agonist morphine modulates learning and memory in both human and animal subjects [10–16]. Although it is well known that pre-training administration of morphine impairs various types of memory, such as fear memory [17,18], recognition memory [10] and spatial memory [11,14], it is unclear how morphine affects different phases of the memory. With regards to spatial memory, previous studies have primarily focused on the effects of morphine exposure on the acquisition of SRM; effects on the acquisition and retrieval of SWM remain unresolved. Beatty et al. [19] reported that neither pre- nor post-training morphine affects memory retention of rats in an 8-arm radial maze. In contrast, a recent study using a Y-maze paradigm showed that pretraining morphine impairs the acquisition of spatial recognition memory in mice [10]. With respect to the Morris water maze test (MWM), McNamara and Skelton [14,20] reported that repeated morphine administration impairs the acquisition of SRM of rats without affecting memory retention, and that this inhibitory effect is primarily due to motivation reduction rather than amnesia. Several other studies have shown that morphine impairs not only memory acquisition but also memory retention [21], without reducing the motivation to escape from the water maze task [21,22]. The endogenous opioid system is implicated in working memory. For example, opiate antagonists facilitate the recall of working memory in rats performing a 12-arm radial maze task [23]. Moreover, the depletion of endogenous morphine weakens the working memory performance in a passive avoidance task [24]. A recent study in human subjects also demonstrated that acute morphine exposure impairs
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working memory [16]. Although the influence of chronic morphine on SWM has been explored [11,12,25], little is known about the effects of pre-training morphine on SWM. Opioid receptors regulate the retrieval of fear memory and spatial memory [26–30]. Pre-test morphine facilitates the memory retrieval in morphine state-dependent learning [26]. Post-training morphine suppresses memory retrieval in a passive avoidance task [13]. Based on the above findings, we hypothesize that morphine may modulate the retrieval of spatial memory via mu-opioid receptors. In the present study, we used four water maze tasks (acquisition and retrieval of SRM vs. SWM) to investigate the effects of pretraining morphine and naloxone on the acquisition and retrieval of spatial memory in C57BL/6 mice.
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2 only (n = 8 per group) to determine whether morphine affects the ability for mice to locate the visible platform after they have learned the task. 2.4. SRM task Mice received four trials per day for 6 consecutive days. The platform was placed in the center of the southwest quadrant and mice were released into the maze from randomly selected starting points [31]. Three spatial probe trials were performed 15 min after the last trial on days 1, 3, and 5 (as described in ref. [32]). Two spatial probe trials were performed on days 7 and 11 to test the retention and retrieval of memory. To study the different phases of SRM, the following two sets of experiments (Fig. 1, Exp. 3 and Exp. 4) were carried out.
2. Materials and methods 2.1. Animals and drugs Male C57BL/6 mice (8 weeks of age; 4 per cage; the Experimental Animal Center of Xi'an Jiaotong University Medical College) were maintained in a temperature-controlled (21–23 °C) environment with a 12/12-h light–dark cycle, with unlimited access to food and water. All mice were handled by the experimenters for 2 weeks prior to the experiments. All animal procedures were approved by the Animal Care and Use Committee of Xi'an Jiaotong University. Morphine hydrochloride (the First Pharmaceutical Factory of Shenyang, China) and naloxone hydrochloride (Huasu Pharmaceutical Factory of Beijing, China) were dissolved in 0.9% NaCl, and were injected intraperitoneally at a volume of 10 ml/kg body weight. 2.2. Morris water maze The apparatus is a circular pool (122 cm in diameter, 62.5 cm in height) with a black inner surface. A circular transparent platform (10 cm in diameter) was submerged 1 cm below the water surface and located in the center of one quadrant. Extra-maze cues were located in the test room. A video-computerized tracking system (SMART, Panlab SL, Barcelona, Spain) was used to record and analyze animal behavior. Mice were allowed 60 s to search for the platform and stay on the platform for 10 s in the training trial. If the mouse failed to locate the platform within 60 s, it was manually led to the platform by the experimenter. After each trial, mice were dried using a towel and returned to the home-cage until the next trial. The interval between trials varied from 15 s (for working memory acquisition experiments) to 5–15 min (for experiments of reference memory acquisition or retrieval and experiments of working memory retrieval). The platform was removed from the pool in the probe trials.
2.4.1. Acquisition of SRM Mice received four trials per day for 6 consecutive days, and were tested with four probe trials on days 1, 3, 5, and 7. Mice were treated with either morphine (7.5, 15 mg/kg) or saline 30 min before the first trial of each day (n = 8 per group for morphine treatment; n = 16 for saline treatment). 2.4.2. Retrieval of SRM Mice in the saline-treated group in the SRM acquisition experiment received a probe test 5 days after the training session. The mice were randomly treated with morphine (7.5 or 15 mg/kg) or saline at 30 min prior to the probe test. 2.5. SWM task The acquisition of working memory involved trial-dependent, matching-to-sample, learning of the platform location [31]. The daily SWM task contained a location trial (first trial) and a match trial (second trial), separated by 15 s. Both starting points and platform positions were varied on every set of the two trials. In the memory retrieval experiments, mice received five training trials with randomly selected starting points. The probe test was carried out at 75 min later. Two sets of experiments (Experiments 5 and 6 in Fig. 1) were carried out. 2.5.1. Acquisition of SWM Six groups of mice (n= 9 per group) were included to examine the effects of morphine, naloxone and morphine plus naloxone on working memory. Tests were conducted over a period of 3 consecutive days after the performance was stabilized (typically after 9 days). Mice received either morphine (7.5 and 15 mg/kg) or saline at 30 min before the first trial in the test session. A separate group of mice was pretreated with naloxone (1 mg/kg) at 20 min prior to morphine/saline.
2.3. Control task for sensorimotor function and motivation The platform was marked with a flag (12 cm above the water surface) to allow the mice to navigate by sight. The test consisted of four trials per day and over these trials, positions of the platform and starting points were quasi-randomized according to Vorhees and William [31]. To discriminate different components (escape motivation, swimming ability and the ability to learn a new task), two sets of experiments (Fig. 1, Exp. 1 and Exp. 2) were carried out. 2.3.1. Effects of pre-training morphine on cued learning The experiment was conducted over a period of six consecutive days. Mice received morphine (7.5, 15 mg/kg) or saline 30 min before the first trial on each day (n = 9 per group). 2.3.2. Effects of pre-training morphine on new task learning This experiment lasted for two consecutive days. Mice received morphine (7.5, 15 mg/kg) or saline 30 min before the first trial on day
2.5.2. Retrieval of SWM Six groups of mice (n= 8 per group) were used to examine the effects of morphine, naloxone and morphine plus naloxone on working memory retrieval. Mice received either morphine (7.5 and 15 mg/kg) or saline at 45 min after the last training trial. A spatial probe test was conducted at 30 min later. A separate group of mice was pretreated with naloxone (1 mg/kg) at 20 min prior to morphine/saline. The spatial probe test was conducted at 30 min after the morphine/saline injection. 2.6. Statistical analysis Data are expressed as mean± S.E.M, and were analyzed with oneway ANOVA followed by Dunnett's t-test or two-way ANOVA of repeated-measures (TW RM ANOVA) followed by Bonferroni post using the GraphPad Prism 5.0 software. In the TW RM ANOVA, the within-subject factor (the repeated factor) was training day and the between-subject factor was treatment. The Student's t-test was used for
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Fig. 1. Experimental design. Only key steps are shown. Probe tests on days 1, 3, and 5 of Experiments 3 and 4 are not shown. “N + M” in Exp. 5 denotes naloxone injection and morphine injection with a 20 min inter-injection interval.
comparison between the location trial and the match trial in the working memory test. P-value of 0.05 was considered statistically significant for all analyses. 3. Results 3.1. Effects of pre-training morphine on cued learning
treatment. Morphine did not affect swimming speed (Fig. 3C, p N 0.05) or thigmotaxis (Fig. 3D, p N 0.05). 3.2.2. Probe test Both Treatment and Training Day had significant effects on the percent time in the goal quadrant (Treatment: F2,87 = 16.68, pb 0.0001; Training Day: F3,87 = 6.095, p b 0.001) (Fig. 3E). Pre-training morphine at
Morphine did not affect the performance in the 6-day cued learning task (Fig. 2; p N 0.05). On the first day, however, 15 mg/kg morphine significantly prolonged escape latency compared with 7.5 mg/kg of morphine (p b 0.01) and saline (p b 0.001). In the 2-day cued learning, pre-training morphine at 15 mg/kg did not affect the ability to locate visible platform when mice had learned the task. 3.2. Effects of pre-training morphine on the acquisition of SRM 3.2.1. Training day Both Treatment and Training Day had significant effects on latency (Fig. 3A; Treatment: F2,145 = 33.23, p b 0.0001; Training Day: F5,145 = 14.38, p b 0.0001) and swimming distance (Fig. 3B; Treatment: F2,145 = 21.70, p b 0.0001; Training Day: F5,145 = 17.66, p b 0.0001) in the acquisition task of SRM. Post-hoc analysis revealed that pre-training administration of morphine at both doses increased latency (Fig. 3A) and distance to the hidden platform (Fig. 3B) compared with saline
Fig. 2. Effects of pre-training morphine on escape latency in cued learning performance. Data are expressed as mean ± S.E.M. of the average of four trials for each mouse. *** p b 0.001: significant difference between 15 mg/kg morphine and saline. ++ p b 0.01: significant difference between the two morphine doses (7.5 and 15 mg/kg morphine).
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Fig. 3. Effects of pre-training morphine on the acquisition of SRM. Data are expressed as mean ± S.E.M. and represent the average of four trials for each mouse on each day per group. For the probe test, data are expressed as mean ± S.E.M. of all trials per group. (A) The latency during the training session. (B) Swimming distance during the training session. (C) Swimming speed during the training session. (D) Time spent swimming in the outer 20% of the pool (thigmotaxis). (E) Percent time spent in the goal quadrant in the 4 probe tests. (F) Percent time spent in the goal quadrant and opposite quadrant in the probe test of day 7. The dashed line denotes chance level. Δ p b 0.05, ΔΔ p b 0.01, and ΔΔΔ p b 0.001: significant difference between 7.5 mg/kg morphine and saline. * p b 0.05, ** p b 0.01, and *** p b 0.001: significant difference between 15 mg/kg morphine and saline. +++ p b 0.001: significant difference between the time in the goal quadrant and the opposite quadrant.
7.5 and 15 mg/kg significantly attenuated the increase of time in the goal quadrant over the training period (Fig. 3E). In the probe test on day 7, there was a significant Quadrant but not Treatment effect (Fig. 3F; Quadrant: F1,58 = 12.48, pb 0.001). There was a significant interaction between Treatment and Quadrant (Treatment×Quadrant: F2,58 = 19.58, p b 0.0001). A post-hoc analysis revealed morphine-treated mice spent significantly less time in the goal quadrant than saline-treated mice (Fig. 3F; p b 0.05 at 7.5 mg/kg; pb 0.001 at 15 mg/kg). 3.3. Effects of pre-training morphine on the retrieval of SRM Pre-test morphine did not affect the time spent in the goal quadrant (p N 0.05) in mice that had already acquired the task.
(Fig. 4A). There were significant differences in the latency in the match trials but not in the location trials among the treatment conditions (Fig. 4A; the match trial: F5,48 = 4.456, p b 0.01; the location trial: F5,48 = 0.6685, p N 0.05). A post-hoc analysis showed that morphine increased the latency in the match trial (p b 0.01 vs. saline). Naloxone treatment abolished the morphine effect. Naloxone alone did not affect the latency. Pre-training morphine (at both 7.5 and 15 mg/kg) significantly inhibited the reduction in the latency during the match trial; such effect was eliminated by naloxone (1 mg/kg). Effects of the treatment on swimming distance were similar (Fig. 4B). Swimming speed and thigmotaxis were not affected by any treatment (p N 0.05). 3.5. Effects of morphine on the retrieval of SWM
3.4. Effects of morphine on the acquisition of SWM The latency in the match trial was significantly shorter than that in the location trial in all mice groups not treated with morphine
The latency and swimming distance to the platform gradually decreased with the training but did not differ among groups in the training trials (p N 0.05, data not shown). Fig. 5A shows percent time
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Fig. 4. Effects of pre-training morphine, naloxone, and morphine plus naloxone on the acquisition of SWM. Data are expressed as mean ± S.E.M and indicate the average of the 3 location trials or 3 match trials for each animal during the 3-day test session per group (n = 9 per group). (A) The latency in location and match trials. (B) The swimming distances in location and match trials. * p b 0.05, ** p b 0.01: significant difference between latency in the location trial and the match trial. ++ p b 0.01: significant difference in latency between morphine and saline in the match trial .
spent in the goal quadrant and the opposite quadrant. There was a significant interaction between Treatment and Quadrant (F5,84 = 6.779, p b 0.0001) and a Quadrant effect (F1,84 = 21.66, p b 0.0001). A post-hoc analysis revealed that the pre-test morphine (both 7.5 and 15 mg/kg) significantly decreased the percent time spent in the goal quadrant. Naloxone treatment abolished the morphine effect. Naloxone treatment alone did not affect the time spend in the goal quadrant. Morphine treatment (7.5 and 15 mg/kg) also seemed to disrupt searching strategy (Fig. 5B).
4. Discussion MWM is a classic behavioral paradigm to assess spatial memory. The performance in MWM tasks, however, reflects not only the spatial memory, but also the motivation to escape and sensorimotor function [31]. Consistent with previous findings [21,22], we found that pretraining morphine impairs spatial memory acquisition without affecting visual-based learning. McNamara and Skelton [14,20] found that pretraining morphine (10 and 20 mg/kg) impairs the performance in a
Fig. 5. Effects of pre-test morphine, naloxone, and morphine plus naloxone on the retrieval of SWM. (A) Percent time spent in the goal quadrant and the opposite quadrant. Data are expressed as mean ± S.E.M. (n = 8 mice per group). (B) Representative trajectory. ++ p b 0.01, +++ p b 0.001: significant difference in the time spent in goal quadrant between morphine and saline. * p b 0.05, ** p b 0.01, *** p b 0.001: significant difference between percent time spent in the goal quadrant and opposite quadrant.
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MWM task with visual cues in rat. Our study also found a deficit in performance induced by pre-training morphine (15 mg/kg) at the beginning of the cued task, but performance improved over training trials. Since morphine did not affect performance in the visual-based task, it is unlikely that the effects of morphine are due to impaired sensorimotor function or motivation to escape. As has been reported [14,21] previously, our study showed that pretraining morphine impaired the acquisition of SRM and strongly inhibited memory retention. A recent study using MWM task showed that a single pre-training morphine injection (5 or 7.5 mg/kg) could impair spatial memory formation and attenuate memory retention 24 h after the last training trial in rats [22]. Taken together, our results indicated that pre-training morphine could impair both the acquisition and retention of SRM. The findings by McNamara and Skelton [14,20] that morphine did not affect memory retention in rats could probably be attributed to prolonged training (8 or 14 days consecutive training) in their paradigm. The difference may also reflect different animal species (rats vs. mice). The third major finding of the current study is that pre-training morphine could impair the acquisition of SWM. Previous studies have indicated that opiate antagonists enhance the working memory of rats in a radial maze [23]. In fact, acute opioid agonist treatment impairs working memory in humans [16]. Depletion of endogenous morphine disrupts the working memory of mice in a passive avoidance task [24]. Using a matching-to-sample paradigm for SWM, we found that pretraining morphine at both 7.5 and 15 mg/kg strongly inhibited the acquisition of working memory in a manner dependent on mu-opioid receptors. To our knowledge, this is the first direct evidence for inhibitory effects of pre-training morphine treatment on the acquisition of SWM of mice in a MWM task. Our results are supported indirectly by previous findings. In a study using a Y maze paradigm, pre-training morphine at 2.5, 5 and 10 mg/kg impaired the acquisition of recognition spatial memory after 1 or 2 h inter-trial interval [10]. Similarly, in a MWM study that included eight consecutive trials for training [22], pretraining morphine attenuated the reduction of the escape latency. Accumulating evidence suggests that SWM may not be a necessary step towards obtaining reference memory [33,34]. Thus, effects of morphine on SWM may not be the major cause of morphine-induced changes on SRM. Indeed, we found that pre-test morphine impairs the retrieval of SWM but not in SRM. Such results are in line with a previous study reporting that morphine has no effect on the retrieval of SRM [35]. These findings suggest that the opioid system is involved in the retrieval of SWM. These findings also support the notion that the mechanisms underlying the retrieval of SWM and SRM are different [6,7]. Because the half-life of morphine in rodent brain is approximately 1 h [36,37], we chose to perform most of the trials within the half-life of the drug. It is likely that the majority of morphine has crossed the blood–brain barrier when behavioral trails proceeded. Therefore, we suspect that the morphine-induced impairment of memory reflects acute effects of the morphine on neurons in memory-related brain areas. Our data indicate that pre-training morphine impairs multiple aspects of spatial learning and memory, including the acquisition of both SWM and reference memory, and the retrieval of working memory. Effects of morphine on the acquisition and retrieval of spatial memory are dependent on mu-opioid receptors since naloxone could block such effects. Mu-opioid receptors are abundant in the hippocampus [38,39], a key area in both consolidation and retrieval of spatial memory [6,40,41]. In fact, morphine has been shown to modulate the excitability of hippocampal pyramidal neurons [42,43]. Morphine also disrupts hippocampal long-term potentiation and long-term depression, the cellular basis for learning and memory [44–46]. The alterations of hippocampus underlying the morphine-induced impairment of memory should be clarified in further studies. Additionally, stress is an important consideration when interpreting behavioral results obtained using water maze task. Thus, our results may be in part caused by an interaction between
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stress and morphine. Experiments using behavioral paradigms that involve less stress (e.g., Barnes maze and radial arm maze) may help to further clarify the morphine action.
Acknowledgments We thank Drs. Ming Xu and Wanli Smith for thoughtful discussion and Ms. Guo-li Yan for manuscript revision. This research was supported by the Foundation of Shaanxi HR Department for Well-trained Personnel from Abroad (no. SLZ2008010), the Foundation of Xi'an Jiaotong University for New Frontier and Interdisciplinary Study (no. 201075) and Guanghua Medical Innovative Research (no. 0203417) for C.-X. Yan.
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Physiology & Behavior j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / p h b
Effects of pre-training morphine on spatial memory acquisition and retrieval in mice Feng Zhu, Chun-xia Yan ⁎, Yan Zhao, Yang Zhao, Ping-ping Li, Sheng-bin Li ⁎⁎ Forensic Department, Xi'an Jiaotong University Medical College, Xi'an, PR China Key Laboratory of Ministry of Health for Forensic Sciences, Xi'an Jiaotong University, Xi'an, PR China Key Laboratory of Ministry of Education for Environment and Genes Related to Diseases, Xi'an Jiaotong University, Xi'an, PR China
a r t i c l e
i n f o
Article history: Received 19 January 2011 Received in revised form 13 June 2011 Accepted 8 July 2011 Keywords: Morphine Naloxone Morris water maze Memory acquisition Memory retrieval Spatial memory
a b s t r a c t The opioid system plays an important role in memory processess. Morphine mimics endogenous opioids by acting on opioid receptor in brain to regulate memory. However, the effects of morphine on spatial memory acquisition are controversial. Also, little evidence has suggested that morphine could affect the retrieval of spatial memory. In the current study, effects of pre-training morphine and naloxone on the acquisition vs. retrieval of spatial reference vs. working memory were examined using discrete water maze tasks in C57BL/6 mice. Pre-training morphine administration (7.5 and 15 mg/kg, i.p.) impaired the acquisition of both spatial reference memory and working memory. Motivation to escape from the water maze was not affected by morphine. Pre-test morphine also inhibited the retrieval of spatial working memory but not reference memory. The effects of morphine on the acquisition and retrieval of spatial working memory were eliminated by naloxone pretreatment (1 mg/kg). These results indicate that morphine could differentially modulate a variety of aspects of spatial memory and these effects are mediated by the mu-opioid receptor. © 2011 Elsevier Inc. All rights reserved.
1. Introduction Memory formation consists of three key phases: information encoding, storage, and retrieval. Based on temporal duration, memory can be classified as either working or reference memory. Within the context of visuospatial information, working memory behaves as a “sketchpad” to store perceived information for a short period of time [1]. Reference memory is the retention and retrieval of information for later use as a “reference”. Pharmacological and lesion studies have demonstrated that both spatial working memory (SWM) and spatial reference memory (SRM) occur in the hippocampus [2,3]. Memory retrieval is the process of recalling memory already established from previous experience. Most contemporary studies concerning the neurobiology of memory have focused on the mechanisms that govern memory acquisition [4]. However, the cellular and molecular mechanisms underlying memory acquisition differ significantly from that of memory retrieval [5]. Also, the mechanisms underlying the retrieval of working memory and reference memory are distinctive [6,7].
⁎ Correspondence to: C. Yan, Forensic Department, Xi'an Jiaotong University Medical College, 76 West Yanta Road, Xi'an, Shaanxi (710061), PR China. Tel.: + 86 29 82655117; fax: + 86 29 82655113. ⁎⁎ Correspondence to: S. Li, Forensic Department, Xi'an Jiaotong University Medical College, 76 West Yanta Road, Xi'an, Shaanxi (710061), PR China. Tel.: + 86 29 82656244; fax: + 86 29 82655113. E-mail addresses: [email protected] (C. Yan), [email protected] (S. Li). 0031-9384/$ – see front matter © 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.physbeh.2011.07.014
Memory processes are modulated by several neurotransmitter systems and one of the most important systems is opioid system [8,9]. It has been shown that mu-opioid receptor agonist morphine modulates learning and memory in both human and animal subjects [10–16]. Although it is well known that pre-training administration of morphine impairs various types of memory, such as fear memory [17,18], recognition memory [10] and spatial memory [11,14], it is unclear how morphine affects different phases of the memory. With regards to spatial memory, previous studies have primarily focused on the effects of morphine exposure on the acquisition of SRM; effects on the acquisition and retrieval of SWM remain unresolved. Beatty et al. [19] reported that neither pre- nor post-training morphine affects memory retention of rats in an 8-arm radial maze. In contrast, a recent study using a Y-maze paradigm showed that pretraining morphine impairs the acquisition of spatial recognition memory in mice [10]. With respect to the Morris water maze test (MWM), McNamara and Skelton [14,20] reported that repeated morphine administration impairs the acquisition of SRM of rats without affecting memory retention, and that this inhibitory effect is primarily due to motivation reduction rather than amnesia. Several other studies have shown that morphine impairs not only memory acquisition but also memory retention [21], without reducing the motivation to escape from the water maze task [21,22]. The endogenous opioid system is implicated in working memory. For example, opiate antagonists facilitate the recall of working memory in rats performing a 12-arm radial maze task [23]. Moreover, the depletion of endogenous morphine weakens the working memory performance in a passive avoidance task [24]. A recent study in human subjects also demonstrated that acute morphine exposure impairs
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working memory [16]. Although the influence of chronic morphine on SWM has been explored [11,12,25], little is known about the effects of pre-training morphine on SWM. Opioid receptors regulate the retrieval of fear memory and spatial memory [26–30]. Pre-test morphine facilitates the memory retrieval in morphine state-dependent learning [26]. Post-training morphine suppresses memory retrieval in a passive avoidance task [13]. Based on the above findings, we hypothesize that morphine may modulate the retrieval of spatial memory via mu-opioid receptors. In the present study, we used four water maze tasks (acquisition and retrieval of SRM vs. SWM) to investigate the effects of pretraining morphine and naloxone on the acquisition and retrieval of spatial memory in C57BL/6 mice.
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2 only (n = 8 per group) to determine whether morphine affects the ability for mice to locate the visible platform after they have learned the task. 2.4. SRM task Mice received four trials per day for 6 consecutive days. The platform was placed in the center of the southwest quadrant and mice were released into the maze from randomly selected starting points [31]. Three spatial probe trials were performed 15 min after the last trial on days 1, 3, and 5 (as described in ref. [32]). Two spatial probe trials were performed on days 7 and 11 to test the retention and retrieval of memory. To study the different phases of SRM, the following two sets of experiments (Fig. 1, Exp. 3 and Exp. 4) were carried out.
2. Materials and methods 2.1. Animals and drugs Male C57BL/6 mice (8 weeks of age; 4 per cage; the Experimental Animal Center of Xi'an Jiaotong University Medical College) were maintained in a temperature-controlled (21–23 °C) environment with a 12/12-h light–dark cycle, with unlimited access to food and water. All mice were handled by the experimenters for 2 weeks prior to the experiments. All animal procedures were approved by the Animal Care and Use Committee of Xi'an Jiaotong University. Morphine hydrochloride (the First Pharmaceutical Factory of Shenyang, China) and naloxone hydrochloride (Huasu Pharmaceutical Factory of Beijing, China) were dissolved in 0.9% NaCl, and were injected intraperitoneally at a volume of 10 ml/kg body weight. 2.2. Morris water maze The apparatus is a circular pool (122 cm in diameter, 62.5 cm in height) with a black inner surface. A circular transparent platform (10 cm in diameter) was submerged 1 cm below the water surface and located in the center of one quadrant. Extra-maze cues were located in the test room. A video-computerized tracking system (SMART, Panlab SL, Barcelona, Spain) was used to record and analyze animal behavior. Mice were allowed 60 s to search for the platform and stay on the platform for 10 s in the training trial. If the mouse failed to locate the platform within 60 s, it was manually led to the platform by the experimenter. After each trial, mice were dried using a towel and returned to the home-cage until the next trial. The interval between trials varied from 15 s (for working memory acquisition experiments) to 5–15 min (for experiments of reference memory acquisition or retrieval and experiments of working memory retrieval). The platform was removed from the pool in the probe trials.
2.4.1. Acquisition of SRM Mice received four trials per day for 6 consecutive days, and were tested with four probe trials on days 1, 3, 5, and 7. Mice were treated with either morphine (7.5, 15 mg/kg) or saline 30 min before the first trial of each day (n = 8 per group for morphine treatment; n = 16 for saline treatment). 2.4.2. Retrieval of SRM Mice in the saline-treated group in the SRM acquisition experiment received a probe test 5 days after the training session. The mice were randomly treated with morphine (7.5 or 15 mg/kg) or saline at 30 min prior to the probe test. 2.5. SWM task The acquisition of working memory involved trial-dependent, matching-to-sample, learning of the platform location [31]. The daily SWM task contained a location trial (first trial) and a match trial (second trial), separated by 15 s. Both starting points and platform positions were varied on every set of the two trials. In the memory retrieval experiments, mice received five training trials with randomly selected starting points. The probe test was carried out at 75 min later. Two sets of experiments (Experiments 5 and 6 in Fig. 1) were carried out. 2.5.1. Acquisition of SWM Six groups of mice (n= 9 per group) were included to examine the effects of morphine, naloxone and morphine plus naloxone on working memory. Tests were conducted over a period of 3 consecutive days after the performance was stabilized (typically after 9 days). Mice received either morphine (7.5 and 15 mg/kg) or saline at 30 min before the first trial in the test session. A separate group of mice was pretreated with naloxone (1 mg/kg) at 20 min prior to morphine/saline.
2.3. Control task for sensorimotor function and motivation The platform was marked with a flag (12 cm above the water surface) to allow the mice to navigate by sight. The test consisted of four trials per day and over these trials, positions of the platform and starting points were quasi-randomized according to Vorhees and William [31]. To discriminate different components (escape motivation, swimming ability and the ability to learn a new task), two sets of experiments (Fig. 1, Exp. 1 and Exp. 2) were carried out. 2.3.1. Effects of pre-training morphine on cued learning The experiment was conducted over a period of six consecutive days. Mice received morphine (7.5, 15 mg/kg) or saline 30 min before the first trial on each day (n = 9 per group). 2.3.2. Effects of pre-training morphine on new task learning This experiment lasted for two consecutive days. Mice received morphine (7.5, 15 mg/kg) or saline 30 min before the first trial on day
2.5.2. Retrieval of SWM Six groups of mice (n= 8 per group) were used to examine the effects of morphine, naloxone and morphine plus naloxone on working memory retrieval. Mice received either morphine (7.5 and 15 mg/kg) or saline at 45 min after the last training trial. A spatial probe test was conducted at 30 min later. A separate group of mice was pretreated with naloxone (1 mg/kg) at 20 min prior to morphine/saline. The spatial probe test was conducted at 30 min after the morphine/saline injection. 2.6. Statistical analysis Data are expressed as mean± S.E.M, and were analyzed with oneway ANOVA followed by Dunnett's t-test or two-way ANOVA of repeated-measures (TW RM ANOVA) followed by Bonferroni post using the GraphPad Prism 5.0 software. In the TW RM ANOVA, the within-subject factor (the repeated factor) was training day and the between-subject factor was treatment. The Student's t-test was used for
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Fig. 1. Experimental design. Only key steps are shown. Probe tests on days 1, 3, and 5 of Experiments 3 and 4 are not shown. “N + M” in Exp. 5 denotes naloxone injection and morphine injection with a 20 min inter-injection interval.
comparison between the location trial and the match trial in the working memory test. P-value of 0.05 was considered statistically significant for all analyses. 3. Results 3.1. Effects of pre-training morphine on cued learning
treatment. Morphine did not affect swimming speed (Fig. 3C, p N 0.05) or thigmotaxis (Fig. 3D, p N 0.05). 3.2.2. Probe test Both Treatment and Training Day had significant effects on the percent time in the goal quadrant (Treatment: F2,87 = 16.68, pb 0.0001; Training Day: F3,87 = 6.095, p b 0.001) (Fig. 3E). Pre-training morphine at
Morphine did not affect the performance in the 6-day cued learning task (Fig. 2; p N 0.05). On the first day, however, 15 mg/kg morphine significantly prolonged escape latency compared with 7.5 mg/kg of morphine (p b 0.01) and saline (p b 0.001). In the 2-day cued learning, pre-training morphine at 15 mg/kg did not affect the ability to locate visible platform when mice had learned the task. 3.2. Effects of pre-training morphine on the acquisition of SRM 3.2.1. Training day Both Treatment and Training Day had significant effects on latency (Fig. 3A; Treatment: F2,145 = 33.23, p b 0.0001; Training Day: F5,145 = 14.38, p b 0.0001) and swimming distance (Fig. 3B; Treatment: F2,145 = 21.70, p b 0.0001; Training Day: F5,145 = 17.66, p b 0.0001) in the acquisition task of SRM. Post-hoc analysis revealed that pre-training administration of morphine at both doses increased latency (Fig. 3A) and distance to the hidden platform (Fig. 3B) compared with saline
Fig. 2. Effects of pre-training morphine on escape latency in cued learning performance. Data are expressed as mean ± S.E.M. of the average of four trials for each mouse. *** p b 0.001: significant difference between 15 mg/kg morphine and saline. ++ p b 0.01: significant difference between the two morphine doses (7.5 and 15 mg/kg morphine).
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Fig. 3. Effects of pre-training morphine on the acquisition of SRM. Data are expressed as mean ± S.E.M. and represent the average of four trials for each mouse on each day per group. For the probe test, data are expressed as mean ± S.E.M. of all trials per group. (A) The latency during the training session. (B) Swimming distance during the training session. (C) Swimming speed during the training session. (D) Time spent swimming in the outer 20% of the pool (thigmotaxis). (E) Percent time spent in the goal quadrant in the 4 probe tests. (F) Percent time spent in the goal quadrant and opposite quadrant in the probe test of day 7. The dashed line denotes chance level. Δ p b 0.05, ΔΔ p b 0.01, and ΔΔΔ p b 0.001: significant difference between 7.5 mg/kg morphine and saline. * p b 0.05, ** p b 0.01, and *** p b 0.001: significant difference between 15 mg/kg morphine and saline. +++ p b 0.001: significant difference between the time in the goal quadrant and the opposite quadrant.
7.5 and 15 mg/kg significantly attenuated the increase of time in the goal quadrant over the training period (Fig. 3E). In the probe test on day 7, there was a significant Quadrant but not Treatment effect (Fig. 3F; Quadrant: F1,58 = 12.48, pb 0.001). There was a significant interaction between Treatment and Quadrant (Treatment×Quadrant: F2,58 = 19.58, p b 0.0001). A post-hoc analysis revealed morphine-treated mice spent significantly less time in the goal quadrant than saline-treated mice (Fig. 3F; p b 0.05 at 7.5 mg/kg; pb 0.001 at 15 mg/kg). 3.3. Effects of pre-training morphine on the retrieval of SRM Pre-test morphine did not affect the time spent in the goal quadrant (p N 0.05) in mice that had already acquired the task.
(Fig. 4A). There were significant differences in the latency in the match trials but not in the location trials among the treatment conditions (Fig. 4A; the match trial: F5,48 = 4.456, p b 0.01; the location trial: F5,48 = 0.6685, p N 0.05). A post-hoc analysis showed that morphine increased the latency in the match trial (p b 0.01 vs. saline). Naloxone treatment abolished the morphine effect. Naloxone alone did not affect the latency. Pre-training morphine (at both 7.5 and 15 mg/kg) significantly inhibited the reduction in the latency during the match trial; such effect was eliminated by naloxone (1 mg/kg). Effects of the treatment on swimming distance were similar (Fig. 4B). Swimming speed and thigmotaxis were not affected by any treatment (p N 0.05). 3.5. Effects of morphine on the retrieval of SWM
3.4. Effects of morphine on the acquisition of SWM The latency in the match trial was significantly shorter than that in the location trial in all mice groups not treated with morphine
The latency and swimming distance to the platform gradually decreased with the training but did not differ among groups in the training trials (p N 0.05, data not shown). Fig. 5A shows percent time
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Fig. 4. Effects of pre-training morphine, naloxone, and morphine plus naloxone on the acquisition of SWM. Data are expressed as mean ± S.E.M and indicate the average of the 3 location trials or 3 match trials for each animal during the 3-day test session per group (n = 9 per group). (A) The latency in location and match trials. (B) The swimming distances in location and match trials. * p b 0.05, ** p b 0.01: significant difference between latency in the location trial and the match trial. ++ p b 0.01: significant difference in latency between morphine and saline in the match trial .
spent in the goal quadrant and the opposite quadrant. There was a significant interaction between Treatment and Quadrant (F5,84 = 6.779, p b 0.0001) and a Quadrant effect (F1,84 = 21.66, p b 0.0001). A post-hoc analysis revealed that the pre-test morphine (both 7.5 and 15 mg/kg) significantly decreased the percent time spent in the goal quadrant. Naloxone treatment abolished the morphine effect. Naloxone treatment alone did not affect the time spend in the goal quadrant. Morphine treatment (7.5 and 15 mg/kg) also seemed to disrupt searching strategy (Fig. 5B).
4. Discussion MWM is a classic behavioral paradigm to assess spatial memory. The performance in MWM tasks, however, reflects not only the spatial memory, but also the motivation to escape and sensorimotor function [31]. Consistent with previous findings [21,22], we found that pretraining morphine impairs spatial memory acquisition without affecting visual-based learning. McNamara and Skelton [14,20] found that pretraining morphine (10 and 20 mg/kg) impairs the performance in a
Fig. 5. Effects of pre-test morphine, naloxone, and morphine plus naloxone on the retrieval of SWM. (A) Percent time spent in the goal quadrant and the opposite quadrant. Data are expressed as mean ± S.E.M. (n = 8 mice per group). (B) Representative trajectory. ++ p b 0.01, +++ p b 0.001: significant difference in the time spent in goal quadrant between morphine and saline. * p b 0.05, ** p b 0.01, *** p b 0.001: significant difference between percent time spent in the goal quadrant and opposite quadrant.
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MWM task with visual cues in rat. Our study also found a deficit in performance induced by pre-training morphine (15 mg/kg) at the beginning of the cued task, but performance improved over training trials. Since morphine did not affect performance in the visual-based task, it is unlikely that the effects of morphine are due to impaired sensorimotor function or motivation to escape. As has been reported [14,21] previously, our study showed that pretraining morphine impaired the acquisition of SRM and strongly inhibited memory retention. A recent study using MWM task showed that a single pre-training morphine injection (5 or 7.5 mg/kg) could impair spatial memory formation and attenuate memory retention 24 h after the last training trial in rats [22]. Taken together, our results indicated that pre-training morphine could impair both the acquisition and retention of SRM. The findings by McNamara and Skelton [14,20] that morphine did not affect memory retention in rats could probably be attributed to prolonged training (8 or 14 days consecutive training) in their paradigm. The difference may also reflect different animal species (rats vs. mice). The third major finding of the current study is that pre-training morphine could impair the acquisition of SWM. Previous studies have indicated that opiate antagonists enhance the working memory of rats in a radial maze [23]. In fact, acute opioid agonist treatment impairs working memory in humans [16]. Depletion of endogenous morphine disrupts the working memory of mice in a passive avoidance task [24]. Using a matching-to-sample paradigm for SWM, we found that pretraining morphine at both 7.5 and 15 mg/kg strongly inhibited the acquisition of working memory in a manner dependent on mu-opioid receptors. To our knowledge, this is the first direct evidence for inhibitory effects of pre-training morphine treatment on the acquisition of SWM of mice in a MWM task. Our results are supported indirectly by previous findings. In a study using a Y maze paradigm, pre-training morphine at 2.5, 5 and 10 mg/kg impaired the acquisition of recognition spatial memory after 1 or 2 h inter-trial interval [10]. Similarly, in a MWM study that included eight consecutive trials for training [22], pretraining morphine attenuated the reduction of the escape latency. Accumulating evidence suggests that SWM may not be a necessary step towards obtaining reference memory [33,34]. Thus, effects of morphine on SWM may not be the major cause of morphine-induced changes on SRM. Indeed, we found that pre-test morphine impairs the retrieval of SWM but not in SRM. Such results are in line with a previous study reporting that morphine has no effect on the retrieval of SRM [35]. These findings suggest that the opioid system is involved in the retrieval of SWM. These findings also support the notion that the mechanisms underlying the retrieval of SWM and SRM are different [6,7]. Because the half-life of morphine in rodent brain is approximately 1 h [36,37], we chose to perform most of the trials within the half-life of the drug. It is likely that the majority of morphine has crossed the blood–brain barrier when behavioral trails proceeded. Therefore, we suspect that the morphine-induced impairment of memory reflects acute effects of the morphine on neurons in memory-related brain areas. Our data indicate that pre-training morphine impairs multiple aspects of spatial learning and memory, including the acquisition of both SWM and reference memory, and the retrieval of working memory. Effects of morphine on the acquisition and retrieval of spatial memory are dependent on mu-opioid receptors since naloxone could block such effects. Mu-opioid receptors are abundant in the hippocampus [38,39], a key area in both consolidation and retrieval of spatial memory [6,40,41]. In fact, morphine has been shown to modulate the excitability of hippocampal pyramidal neurons [42,43]. Morphine also disrupts hippocampal long-term potentiation and long-term depression, the cellular basis for learning and memory [44–46]. The alterations of hippocampus underlying the morphine-induced impairment of memory should be clarified in further studies. Additionally, stress is an important consideration when interpreting behavioral results obtained using water maze task. Thus, our results may be in part caused by an interaction between
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stress and morphine. Experiments using behavioral paradigms that involve less stress (e.g., Barnes maze and radial arm maze) may help to further clarify the morphine action.
Acknowledgments We thank Drs. Ming Xu and Wanli Smith for thoughtful discussion and Ms. Guo-li Yan for manuscript revision. This research was supported by the Foundation of Shaanxi HR Department for Well-trained Personnel from Abroad (no. SLZ2008010), the Foundation of Xi'an Jiaotong University for New Frontier and Interdisciplinary Study (no. 201075) and Guanghua Medical Innovative Research (no. 0203417) for C.-X. Yan.
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