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Involvement of the ventral tegmental area (VTA) in morphine-induced memory retention in morphine-sensitized rats
Behavioural Brain Research 163 (2005) 100–106
Research report
Involvement of the ventral tegmental area (VTA) in morphine-induced memory retention in morphine-sensitized rats Mohammad-Reza Zarrindast a,b,c,∗ , Zahra Farajzadeh d , Parvin Rostami d , Ameneh Rezayof e , Parvaneh Nourjah d a
Department of Pharmacology and Iranian National Center for Addiction Studies, Tehran University of Medical Sciences, P.O. Box 13145-784, Tehran, Iran b School of Cognitive Science, Institute for Studies in Theoretical Physics and Mathematics, Tehran, Iran c Institute for Cognitive Science Studies, Tehran, Iran d Department of Biology, Teacher Training University, Tehran, Iran e Department of Biology, Faculty of Science, Tehran University, Tehran, Iran Received 8 March 2005; received in revised form 18 April 2005; accepted 18 April 2005 Available online 23 May 2005
Abstract In the present study, the effects of intra-ventral tegmental area (VTA) injections of morphine on memory retention of a one-trial passive avoidance task have been investigated in morphine-sensitized rats. Retrieval was examined 24 h after training and used as memory retention. Sensitization was obtained by subcutaneous (s.c.) injections of morphine, once daily for 3 and 5 days free of the opioid before training. Post-training administration of the both systemic (2.5, 5 and 7.5 mg/kg, s.c.) and intra-VTA (5 and 7.5 g/rat) of morphine, dose-dependently decreased memory retention. The response induced by post-training administration of intra-VTA morphine (7.5 g/rat) was significantly reversed in morphine-sensitized rats. The inhibition of morphine-induced amnesia in morphine-sensitized rats was decreased by once daily injections of naloxone (0.5, 1 and 2 mg/kg, s.c.), SCH 23390 (0.025, 0.05 and 0.1 mg/kg, s.c.) or sulpiride (25, 50 and 100 mg/kg, s.c.), during the sensitization. The results suggest that VTA has an important role in morphine-induced amnesia and morphine sensitization affects this process through opioid and dopamine receptors. © 2005 Elsevier B.V. All rights reserved. Keywords: Morphine; Ventral tegmental area; Sensitization; Naloxone; Dopamine receptor antagonists; Passive avoidance learning; Rat
1. Introduction A large body of evidence indicates that the ventral tegmental area (VTA) is the major source of dopamine (DA) neurons projecting to cortical and limbic regions involved in cognitive and motivational aspects of addiction [4]. Drugs of abuse influence neuronal plasticity in brain regions related to motivation and reward [12]. Other reports have shown that NMDA receptor-dependent long-term potentiation, which is widely believed to be a cellular basis of learning and memory, occurs at excitatory synapses in the VTA [2,15,18]. Recently it was ∗
Corresponding author. Tel.: +98 21 611 2801; fax: +98 21 640 2569. E-mail address: [email protected] (M.-R. Zarrindast).
0166-4328/$ – see front matter © 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.bbr.2005.04.006
reported that many abused drugs, including morphine and cocaine, induce long-term potentiation at glutamatergic receptors on VTA dopaminergic neurons [22]. Opiates are known to interact with DA systems by suppressing GABA inhibitory input to DA neurons in the VTA, thereby augmenting DA release [6,7]. It has also been shown that morphine and other opioidergic agents modulate learning and memory processes [14]. Morphine exerts amnestic effects in different models of memory [16,17,20]. In the step-down passive avoidance task, morphine impairs retention of memory, dose- and timedependently, when administered before or post-training [37]. Intermittent repeated exposure to opiates in various animal species can result in an enhancement of their behavioral effects [24,25]. This phenomenon, referred to
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as sensitization, is suggested to play an effective role in the psychopathology of drug abuse [21,23]. This enhanced response appears to be related to changes in the mesolimbic DA pathway, which arises in the VTA and projects mainly to the nucleus accumbens [10,11,32]. Behavioral sensitization has been shown to be involved in the locomotor stimulant, rewarding and discriminative effects of morphine [13]. Recently the importance of this process in learning and memory has also been realized [36]. Our previous study showed that amnesia induced by pretraining morphine was significantly inhibited in morphine-sensitized mice [36]. Thus, regarding the functional involvement of the VTA in cognitive and motivational aspects of addiction, and considering the influence of morphine sensitization on morphine-induced amnesia, it was postulated that the VTA may be involved in memorymodulating effects of morphine in morphine-sensitized rats. Accordingly, we examined the possible role of the VTA in the effect of morphine on memory retention using a stepthrough passive avoidance task in morphine-sensitized rats. If our hypothesis is correct, we also intend to investigate the possible role of dopamine receptor subtypes in this model.
2. Materials and methods 2.1. Animals Male Wistar rats (Pasteur Institute; Tehran, Iran) weighing 250–300 g at the time of surgery were used. The animals were kept in an animal house with a 12-h light/12-h dark cycle and controlled temperature (22 ± 2 ◦ C). They had ad libitum access to food and water. All animals were allowed to adapt to the laboratory conditions for at least 1 week before surgery and were handled for 5 min/day during this adaptation period. Each animal was used once only. Eight animals were used in each experimental group. All procedures were carried out in accordance with institutional guidelines for animal care and use. 2.2. Surgical and infusion procedures All surgical procedures were conducted under ketamine– xylazine (100 mg/kg ketamine–5 mg/kg xylazine) anaesthesia. Stainless steel, 22-gauge guide cannulas were implanted (bilaterally) 1.5 mm above the intended site of injection according to the atlas of Paxinos and Watson [19]. Stereotaxic coordinates for VTA were incisor bar (−3.3 mm), −5.8 mm posterior to the bregma, ±0.9 mm lateral to the sagittal suture and 8 mm from the top of the skull. Cannulae were secured to anchor jewellers screws with dental acrylic. To prevent clogging, stainless steel stylets (27 gauge) were placed in the guide cannulae until the animals were given the VTA injection. All animals were allowed 1 week to recover from surgery and clear anesthetic. For drug infusion, the animals were gently restrained by hand; the stylets were removed from the guide cannulae and replaced by 27-gauge injection needles (1.5 mm below the tip of the guide cannulae). Each injection unit was connected by polyethylene tubing to 1 l Hamilton syringe. The left and right VTA were infused with a 0.5 l solution on each side (1 l/rat) over a 60-s period. The injection needles were left in place additional
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60 s to allow diffusion and then the stylets were reinserted into the guide cannulae. 2.3. Passive avoidance apparatus A learning box consisted of two compartments, one light (white opaque resin, 20 cm × 20 cm × 30 cm) and the other dark (black opaque resin, 20 cm × 20 cm × 30 cm). A guillotine door opening (7 cm × 9 cm) was made on the floor in the center of the partition between the two compartments. Stainless steel grids (2.5 mm in diameter) were placed at 1-cm intervals (distance between the centers of grids) on the floor of the dark compartment to produce foot shock. Intermittent electric shocks (50 Hz, 1.5 s, 2.5 mA intensity) were delivered to the grid floor of the dark compartment by an insulated stimulator. 2.4. Training All animals were allowed to habituate in the experimental compartments for 30 min prior to testing. All training and testing was done between 08.00 and 14.00 h. All experimental groups were first habituated to the apparatus. Each animal was gently placed in the light compartment for 20 s, after which the guillotine door was lifted and the latency with which the animal crossed to the dark (shock) compartment was timed. Animals that waited more than 100 s to cross to the other side were eliminated from the experiment. Once the animal crossed with all four paws to the next compartment, the door was closed and the rat was taken from the dark compartment into the home cage. The habituation trial was repeated after 30 min and was followed after the same interval by the acquisition trial during which the guillotine door was closed and a foot shock (50 Hz, 1.5 s, 2.5 mA) was delivered immediately after the rat had entered the dark compartment. After 20 s, the rat was removed from the apparatus and placed temporarily into the home cage. Two minutes later, the rat was retested in the same way as before; if the rat did not enter the dark compartment during 120 s, successful acquisition of passive avoidance response was recorded. Otherwise, when the rat entered the dark compartment a second time, the door was closed and the rat received the same shock as above. After retesting, if the rat acquired acquisition of passive avoidance successfully, it was removed from the apparatus and injected with morphine subcutaneously or intra-VTA via the guide cannula. 2.5. Retention test Twenty-four hours after training, a retention test was performed to determine long-term memory. Each animal was placed in the light compartment for 20 s, the door was opened, and the step-through latency was measured for entering into the dark compartment. The test session ended when the animal entered the dark compartment or remained in the light compartment for 300 s (criterion for retention). During these sessions, no electric shock was applied. 2.6. Drugs The drugs used in the study were morphine sulfate (Temad Co., Teharan, Iran), naloxone hydrochloride (Tolid-Daru, Tehran, Iran), SCH 23390 and sulpiride (Sigma, St. Louis, CA, USA). All drugs were dissolved in sterile 0.9% saline, just before the experiment, except for sulpiride that was dissolved in one drop of glacial acetic
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acid with a Hamilton micro-syringe and made up to a volume of 5 ml with sterile 0.9% saline and was then diluted to the required volume. Control animals received either saline or vehicle. 2.7. Experimental procedure Eight animals were used in each experimental group. In experiments where animals received two or three injections, the control groups also received two or three saline or drug vehicle injections. The intervals of drug administration were based on previous studies in order to obtain a maximum response. 2.7.1. Experiment 1: effects of morphine on memory retention The experiment examined the effects of subcutaneous (s.c.) or intra-VTA administration of different doses of morphine on memory retention in rat. Four groups of animal received post-training administration of saline (1 ml/kg, s.c.) or morphine (2.5, 5 and 7.5 mg/kg, s.c.) (Fig. 1A). Another four groups of animals received intra-VTA injections of saline (1 l/rat) or morphine (2.5, 5 and 7.5 g/rat) just after training (Fig. 1B).
Fig. 2. The effect of bilateral intra-VTA injection of morphine on memory retention in the morphine-sensitized rats. In order to induce sensitization to morphine, the animals received morphine (5, 10 and 20 mg/kg, s.c.), once daily for 3 days, in the colony room. After 5 days, they received post-training morphine (7.5 g/rat, intra-VTA), immediately after the training session. Two control groups of rats received once daily injection of saline for 3 days, in the colony room. Five days later, they received saline (1 l/rat) or morphine (7.5 g/rat, intra-VTA), immediately after the training session. The animals were tested 24 h after training. Data are expressed as mean ± S.E.M. of eight animals per group. *** P < 0.001 different from the saline/saline group. + P < 0.05, +++ P < 0.001 different from saline/morphine group.
2.7.2. Experiment 2: effects of intra-VTA injection of morphine on memory retention in morphine-sensitized rats The effect of bilateral intra-VTA injection of morphine on memory retention in the morphine-sensitized rats has been examined. In order to induce sensitization to morphine, the animals received either saline or morphine (5, 10 and 20 mg/kg, s.c.), once daily for 3 days (days 1–3) in the colony room. After 5 days (no drug treatment), all groups of animals received intra-VTA morphine (7.5 g/rat), just after training (Fig. 2). 2.7.3. Experiment 3: inhibition of morphine sensitization by naloxone administration and effect of intra-VTA administration of morphine on memory retention The effects of bilateral intra-VTA injection of morphine on memory retention in animals that had previously received a 3-day morphine treatment regimen in combination with naloxone have been examined. Four groups of animals received once daily injections of saline or the opioid receptor antagonist naloxone (0.5, 1 and 2 mg/kg, s.c.), 30 min prior to s.c. injections of morphine (20 mg/kg/day × 3 days). After 5 days (no drug treatment), all groups of animals received intra-VTA morphine (7.5 g/rat), just after training (Fig. 3).
Fig. 1. The effects of post-training administration of morphine on memory retention. In panel A, animals received post-training systemic administration of saline (1 ml/kg, s.c.) or morphine (2.5, 5 and 7.5 mg/kg, s.c.). In panel B, animals received intra-VTA injections of saline (1 l/rat) or morphine (2.5, 5 and 7.5 g/rat) just after training. Animals were tested 24 h after training. Data are expressed as mean ± S.E.M. of eight animals per group. ** P < 0.01, *** P < 0.001 different from the control group.
2.7.4. Experiment 4: inhibition of morphine sensitization by SCH 23390 or sulpiride administration and effects of intra-VTA administration of morphine on memory retention The effect of bilateral intra-VTA injection of morphine on memory retention in animals that had previously received a 3-day morphine treatment regimen in combination with SCH 23390 or sulpiride have been examined. Four groups of animals received once daily injections of saline or the dopamine D1 receptor antagonist, SCH 23390 (0.025, 0.05 and 0.1 mg/kg, s.c.) 30 min prior to s.c. injections of morphine (20 mg/kg/day × 3 days; Fig. 4). Another four
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Fig. 3. The effect of bilateral intra-VTA injection of morphine on memory retention in morphine-sensitized rats in the presence or absence of naloxone. The animals received once daily injections of saline or the opioid receptor antagonist, naloxone (0.5, 1 and 2 mg/kg, s.c.), 30 min prior to s.c. injections of morphine (20 mg/kg/day × 3 days). After 5 days, all groups received morphine (7.5 g/rat, intra-VTA) immediately after training and tested 24 h after training. Data are expressed as mean ± S.E.M. of eight animals per group. ** P < 0.01, *** P < 0.001 different from the control group.
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Fig. 5. The effect of bilateral intra-VTA injection of morphine on memory retention in morphine-sensitized rats in the presence or absence of SCH 23390. The animals received once daily injections of saline or the dopamine D2 receptor antagonist, sulpiride (25, 50 and 100 mg/kg, s.c.), 90 min prior to s.c. injections of morphine (20 mg/kg/day × 3 days). After 5 days, all groups received morphine (7.5 g/rat, intra-VTA) immediately after training and tested 24 h after training. Data are expressed as mean ± S.E.M. of eight animals per group. ∗ P < 0.05, *** P < 0.001 different from the control group.
2.8. Histology groups of animals received once daily injections of vehicle or the dopamine D2 receptor antagonist, sulpiride (25, 50 and 100 mg/kg, s.c.) 90 min prior to s.c. injections of morphine (20 mg/kg/day × 3 days; Fig. 5). After 5 days (no drug treatment), all groups of animals received intra-VTA morphine (7.5 g/rat), just after training.
After completion of behavioral testing, each animal was killed with an overdose of chloroform. Animals received a 0.5 l/side injection of ink (1% aquatic methylene blue solution) into the VTA. The brains were then removed and fixed in a 10% formalin solution for 10 days before sectioning. Sections were examined to determine location of the cannulae aimed at the VTA. The cannulae placements were verified using the atlas of Paxinos and Watson [19]. Data from animals with injections sites located outside the VTA region were not used in the analysis. 2.9. Statistics Comparisons between groups were made with one-way analysis of variance (ANOVA) following Tukey test. A difference with P < 0.05 between experimental groups was considered statistically significant. Calculations were performed using the SPSS statistical package.
3. Results 3.1. Effects of morphine on memory retention
Fig. 4. The effect of bilateral intra-VTA injection of morphine on memory retention in morphine-sensitized rats in the presence or absence of SCH 23390. The animals received once daily injections of saline or the dopamine D1 receptor antagonist, SCH 23390 (0.025, 0.05 and 0.1 mg/kg, s.c.), 30 min prior to s.c. injections of morphine (20 mg/kg/day × 3 days). After 5 days, all groups received morphine (7.5 g/rat, intra-VTA) immediately after training and tested 24 h after training. Data are expressed as mean ± S.E.M. of eight animals per group. ** P < 0.01, *** P < 0.001 different from the control group.
Fig. 1A shows the effects of post-training systemic administration of morphine on step-through latency. One-way ANOVA revealed that post-training systemic morphine (2.5, 5 and 7.5 mg/kg, s.c.) dose dependently reduced the step-through latency in the one-trial passive avoidance task [F(3,28) = 34.7, P < 0.001]. Fig. 1B also shows that post-training intra-VTA injections of morphine (5 and 7.5 g/rat) decreased step-through latency [F(3,28) = 20.39,
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P < 0.001]. Both systemic and intra-VTA injections of morphine attenuated memory retention and induced amnesia. 3.2. Effects of intra-VTA administration of morphine on memory retention in morphine-sensitized rats As shown in Fig. 2, amnesia induced by post-training intra-VTA morphine (7.5 g/rat) was significantly decreased in rats which had previously received once daily injections of morphine (10 and 20 mg/kg, s.c.) for 3 days, compared with rats pretreated with saline (1 l/rat, intra-VTA) [Oneway ANOVA, F(3,28)) = 22.9, P < 0.0001]. Further analysis showed that a maximum response was obtained with 20 mg/kg of opioid. 3.3. Inhibition of morphine sensitization by naloxone administration and effects of intra-VTA administration of morphine on memory retention Fig. 3 shows the inhibition of morphine (7.5 g/rat, intraVTA)-induced amnesia in animals that had previously received the 3-day morphine (20 mg/kg, s.c.) treatment suppressed by once daily injections of the opioid receptor antagonist, naloxone (0.5, 1 and 2 mg/kg, s.c.) 30 min prior to s.c. injections of morphine (20 mg/kg/day × 3 days) [Oneway ANOVA, F(3,28) = 13.1, P < 0.001]. A maximum response was obtained with 2 mg/kg of naloxone (Tukey test, P < 0.001). 3.4. Inhibition of morphine sensitization by SCH 23390 or sulpiride administration and effects of intra-VTA administration of morphine on memory retention Fig. 4 shows that the inhibition of morphine (7.5 g/rat, intra-VTA)-induced amnesia in animals that had previously received the 3-day morphine (20 mg/kg, s.c.) treatment was dose-dependently decreased by once daily injections of the dopamine D1 receptor antagonist, SCH 23390 (0.025, 0.05 and 0.1 mg/kg, s.c.) 60 min prior to s.c. injections of morphine (20 mg/kg/day × 3 days) [One-way ANOVA, F(3,28) = 18.6, P < 0.001]. Fig. 5 also shows that the inhibition of morphine (7.5 g/rat, intra-VTA)-induced amnesia in animals that had previously received the 3-day morphine (20 mg/kg, s.c.) treatment was dose-dependently reduced by once daily injections of the dopamine D2 receptor antagonist, sulpiride (25, 50 and 100 mg/kg, s.c.) 90 min prior to s.c. injections of morphine (20 mg/kg/day × 3 days) [One-way ANOVA, F(3,28) = 7.5, P < 0.001].
4. Discussion In the present experiments, the involvement of VTA in morphine effects on memory retention of a one-trial passive
avoidance task in the morphine-sensitized rats was investigated. Post-training s.c. administration of different doses of morphine, dose-dependently, decreased learning on a onetrial passive avoidance task. Thus, in agreement with previous reports moderate doses of morphine causes impairment of memory retention [3,5]. In order to show the role of the VTA in the morphine-induced impairment of memory retention, the opioid has been injected in this site. The bilateral intraVTA injections of morphine impaired memory retention. This result revealed that the VTA may be involved in morphineinduced impairment of memory retention in rats. The VTA is an important site for synaptic modifications involved in learning and memory to associate morphine exposure with a specific environment [6]. It has also been suggested that the VTA is an important area for reward-related learning [34]. It has become apparent that repeated and intermittent morphine treatment produces an enhancement of motor activity associated with increases in mesolimbic dopaminergic cell firing [1,8,9,24]. Our previous results have shown that repeated administration of morphine (20 mg/kg), once daily for 3 days, followed by “a period of drug-free treatment” increased locomotion, indicating behavioural sensitization [38,36]. In the present study, morphine-sensitization has been achieved by administration of 3 days of morphine (5–20 mg/kg, s.c.) followed by 5 days of free drug treatment. Impairment of memory retention induced by post-training injection of morphine (7.5 g/rat) into the VTA region was significantly reduced in morphine (10 and 20 mg/kg)-sensitized rats. The data may indicate that similar to the amnesia induced by systemic administration of morphine in mice [36], amnesia induced by injection of morphine into the VTA can be altered in the morphine-sensitized rats. Thus, the repeated administration of morphine can result in the enhancement of memory retention, and this may imply that the sensitization induced by the opioid affected on memory processes of the VTA. There are several experiments that enhanced dopamine transmission in the VTA is associated with behavioral sensitization to morphine [9,29]. It has been reported that microinjections of D1 antagonists [26,30] into the VTA can disrupt the development of sensitization, suggesting that DA systems within the midbrain mediate the neuroadaptations that are responsible [33]. Our data show that impairment of memory retention induced by post-training morphine (7.5 g/rat, intra-VTA) is significantly reduced in the animals that previously received for 3 days either a dopamine D1 receptor antagonist, SCH 23390 or a dopamine D2 receptor antagonist, sulpiride, before morphine injection. These results once again support the theory that the dopaminergic system is involved in morphine-induced sensitization. Behavioral evidence suggests that changes in dopaminergic and/or opioid neurotransmission may be involved in the behavioral sensitization to morphine [13,28]. The present data also indicated that the inhibition of morphine-induced impairment of memory re-
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tention in morphine-sensitized rats was suppressed by 3 days injections of the opioid receptor antagonist naloxone, before morphine administration. Since morphine enhances both dopamine synthesis and release in the dopaminergic system [27] via activation of -opioid receptors in the VTA, It is likely that sensitization induced morphine was by a similar mechanism. Our results are in agreement with those of previous studies [31,35] and confirm that -opioid receptors play an important role in modulating morphine-induced sensitization. In summary, the results of the present study demonstrate that the VTA may play an important role in morphineinduced amnesia and morphine sensitization affects this process through opioid and dopamine receptors.
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Research report
Involvement of the ventral tegmental area (VTA) in morphine-induced memory retention in morphine-sensitized rats Mohammad-Reza Zarrindast a,b,c,∗ , Zahra Farajzadeh d , Parvin Rostami d , Ameneh Rezayof e , Parvaneh Nourjah d a
Department of Pharmacology and Iranian National Center for Addiction Studies, Tehran University of Medical Sciences, P.O. Box 13145-784, Tehran, Iran b School of Cognitive Science, Institute for Studies in Theoretical Physics and Mathematics, Tehran, Iran c Institute for Cognitive Science Studies, Tehran, Iran d Department of Biology, Teacher Training University, Tehran, Iran e Department of Biology, Faculty of Science, Tehran University, Tehran, Iran Received 8 March 2005; received in revised form 18 April 2005; accepted 18 April 2005 Available online 23 May 2005
Abstract In the present study, the effects of intra-ventral tegmental area (VTA) injections of morphine on memory retention of a one-trial passive avoidance task have been investigated in morphine-sensitized rats. Retrieval was examined 24 h after training and used as memory retention. Sensitization was obtained by subcutaneous (s.c.) injections of morphine, once daily for 3 and 5 days free of the opioid before training. Post-training administration of the both systemic (2.5, 5 and 7.5 mg/kg, s.c.) and intra-VTA (5 and 7.5 g/rat) of morphine, dose-dependently decreased memory retention. The response induced by post-training administration of intra-VTA morphine (7.5 g/rat) was significantly reversed in morphine-sensitized rats. The inhibition of morphine-induced amnesia in morphine-sensitized rats was decreased by once daily injections of naloxone (0.5, 1 and 2 mg/kg, s.c.), SCH 23390 (0.025, 0.05 and 0.1 mg/kg, s.c.) or sulpiride (25, 50 and 100 mg/kg, s.c.), during the sensitization. The results suggest that VTA has an important role in morphine-induced amnesia and morphine sensitization affects this process through opioid and dopamine receptors. © 2005 Elsevier B.V. All rights reserved. Keywords: Morphine; Ventral tegmental area; Sensitization; Naloxone; Dopamine receptor antagonists; Passive avoidance learning; Rat
1. Introduction A large body of evidence indicates that the ventral tegmental area (VTA) is the major source of dopamine (DA) neurons projecting to cortical and limbic regions involved in cognitive and motivational aspects of addiction [4]. Drugs of abuse influence neuronal plasticity in brain regions related to motivation and reward [12]. Other reports have shown that NMDA receptor-dependent long-term potentiation, which is widely believed to be a cellular basis of learning and memory, occurs at excitatory synapses in the VTA [2,15,18]. Recently it was ∗
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reported that many abused drugs, including morphine and cocaine, induce long-term potentiation at glutamatergic receptors on VTA dopaminergic neurons [22]. Opiates are known to interact with DA systems by suppressing GABA inhibitory input to DA neurons in the VTA, thereby augmenting DA release [6,7]. It has also been shown that morphine and other opioidergic agents modulate learning and memory processes [14]. Morphine exerts amnestic effects in different models of memory [16,17,20]. In the step-down passive avoidance task, morphine impairs retention of memory, dose- and timedependently, when administered before or post-training [37]. Intermittent repeated exposure to opiates in various animal species can result in an enhancement of their behavioral effects [24,25]. This phenomenon, referred to
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as sensitization, is suggested to play an effective role in the psychopathology of drug abuse [21,23]. This enhanced response appears to be related to changes in the mesolimbic DA pathway, which arises in the VTA and projects mainly to the nucleus accumbens [10,11,32]. Behavioral sensitization has been shown to be involved in the locomotor stimulant, rewarding and discriminative effects of morphine [13]. Recently the importance of this process in learning and memory has also been realized [36]. Our previous study showed that amnesia induced by pretraining morphine was significantly inhibited in morphine-sensitized mice [36]. Thus, regarding the functional involvement of the VTA in cognitive and motivational aspects of addiction, and considering the influence of morphine sensitization on morphine-induced amnesia, it was postulated that the VTA may be involved in memorymodulating effects of morphine in morphine-sensitized rats. Accordingly, we examined the possible role of the VTA in the effect of morphine on memory retention using a stepthrough passive avoidance task in morphine-sensitized rats. If our hypothesis is correct, we also intend to investigate the possible role of dopamine receptor subtypes in this model.
2. Materials and methods 2.1. Animals Male Wistar rats (Pasteur Institute; Tehran, Iran) weighing 250–300 g at the time of surgery were used. The animals were kept in an animal house with a 12-h light/12-h dark cycle and controlled temperature (22 ± 2 ◦ C). They had ad libitum access to food and water. All animals were allowed to adapt to the laboratory conditions for at least 1 week before surgery and were handled for 5 min/day during this adaptation period. Each animal was used once only. Eight animals were used in each experimental group. All procedures were carried out in accordance with institutional guidelines for animal care and use. 2.2. Surgical and infusion procedures All surgical procedures were conducted under ketamine– xylazine (100 mg/kg ketamine–5 mg/kg xylazine) anaesthesia. Stainless steel, 22-gauge guide cannulas were implanted (bilaterally) 1.5 mm above the intended site of injection according to the atlas of Paxinos and Watson [19]. Stereotaxic coordinates for VTA were incisor bar (−3.3 mm), −5.8 mm posterior to the bregma, ±0.9 mm lateral to the sagittal suture and 8 mm from the top of the skull. Cannulae were secured to anchor jewellers screws with dental acrylic. To prevent clogging, stainless steel stylets (27 gauge) were placed in the guide cannulae until the animals were given the VTA injection. All animals were allowed 1 week to recover from surgery and clear anesthetic. For drug infusion, the animals were gently restrained by hand; the stylets were removed from the guide cannulae and replaced by 27-gauge injection needles (1.5 mm below the tip of the guide cannulae). Each injection unit was connected by polyethylene tubing to 1 l Hamilton syringe. The left and right VTA were infused with a 0.5 l solution on each side (1 l/rat) over a 60-s period. The injection needles were left in place additional
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60 s to allow diffusion and then the stylets were reinserted into the guide cannulae. 2.3. Passive avoidance apparatus A learning box consisted of two compartments, one light (white opaque resin, 20 cm × 20 cm × 30 cm) and the other dark (black opaque resin, 20 cm × 20 cm × 30 cm). A guillotine door opening (7 cm × 9 cm) was made on the floor in the center of the partition between the two compartments. Stainless steel grids (2.5 mm in diameter) were placed at 1-cm intervals (distance between the centers of grids) on the floor of the dark compartment to produce foot shock. Intermittent electric shocks (50 Hz, 1.5 s, 2.5 mA intensity) were delivered to the grid floor of the dark compartment by an insulated stimulator. 2.4. Training All animals were allowed to habituate in the experimental compartments for 30 min prior to testing. All training and testing was done between 08.00 and 14.00 h. All experimental groups were first habituated to the apparatus. Each animal was gently placed in the light compartment for 20 s, after which the guillotine door was lifted and the latency with which the animal crossed to the dark (shock) compartment was timed. Animals that waited more than 100 s to cross to the other side were eliminated from the experiment. Once the animal crossed with all four paws to the next compartment, the door was closed and the rat was taken from the dark compartment into the home cage. The habituation trial was repeated after 30 min and was followed after the same interval by the acquisition trial during which the guillotine door was closed and a foot shock (50 Hz, 1.5 s, 2.5 mA) was delivered immediately after the rat had entered the dark compartment. After 20 s, the rat was removed from the apparatus and placed temporarily into the home cage. Two minutes later, the rat was retested in the same way as before; if the rat did not enter the dark compartment during 120 s, successful acquisition of passive avoidance response was recorded. Otherwise, when the rat entered the dark compartment a second time, the door was closed and the rat received the same shock as above. After retesting, if the rat acquired acquisition of passive avoidance successfully, it was removed from the apparatus and injected with morphine subcutaneously or intra-VTA via the guide cannula. 2.5. Retention test Twenty-four hours after training, a retention test was performed to determine long-term memory. Each animal was placed in the light compartment for 20 s, the door was opened, and the step-through latency was measured for entering into the dark compartment. The test session ended when the animal entered the dark compartment or remained in the light compartment for 300 s (criterion for retention). During these sessions, no electric shock was applied. 2.6. Drugs The drugs used in the study were morphine sulfate (Temad Co., Teharan, Iran), naloxone hydrochloride (Tolid-Daru, Tehran, Iran), SCH 23390 and sulpiride (Sigma, St. Louis, CA, USA). All drugs were dissolved in sterile 0.9% saline, just before the experiment, except for sulpiride that was dissolved in one drop of glacial acetic
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acid with a Hamilton micro-syringe and made up to a volume of 5 ml with sterile 0.9% saline and was then diluted to the required volume. Control animals received either saline or vehicle. 2.7. Experimental procedure Eight animals were used in each experimental group. In experiments where animals received two or three injections, the control groups also received two or three saline or drug vehicle injections. The intervals of drug administration were based on previous studies in order to obtain a maximum response. 2.7.1. Experiment 1: effects of morphine on memory retention The experiment examined the effects of subcutaneous (s.c.) or intra-VTA administration of different doses of morphine on memory retention in rat. Four groups of animal received post-training administration of saline (1 ml/kg, s.c.) or morphine (2.5, 5 and 7.5 mg/kg, s.c.) (Fig. 1A). Another four groups of animals received intra-VTA injections of saline (1 l/rat) or morphine (2.5, 5 and 7.5 g/rat) just after training (Fig. 1B).
Fig. 2. The effect of bilateral intra-VTA injection of morphine on memory retention in the morphine-sensitized rats. In order to induce sensitization to morphine, the animals received morphine (5, 10 and 20 mg/kg, s.c.), once daily for 3 days, in the colony room. After 5 days, they received post-training morphine (7.5 g/rat, intra-VTA), immediately after the training session. Two control groups of rats received once daily injection of saline for 3 days, in the colony room. Five days later, they received saline (1 l/rat) or morphine (7.5 g/rat, intra-VTA), immediately after the training session. The animals were tested 24 h after training. Data are expressed as mean ± S.E.M. of eight animals per group. *** P < 0.001 different from the saline/saline group. + P < 0.05, +++ P < 0.001 different from saline/morphine group.
2.7.2. Experiment 2: effects of intra-VTA injection of morphine on memory retention in morphine-sensitized rats The effect of bilateral intra-VTA injection of morphine on memory retention in the morphine-sensitized rats has been examined. In order to induce sensitization to morphine, the animals received either saline or morphine (5, 10 and 20 mg/kg, s.c.), once daily for 3 days (days 1–3) in the colony room. After 5 days (no drug treatment), all groups of animals received intra-VTA morphine (7.5 g/rat), just after training (Fig. 2). 2.7.3. Experiment 3: inhibition of morphine sensitization by naloxone administration and effect of intra-VTA administration of morphine on memory retention The effects of bilateral intra-VTA injection of morphine on memory retention in animals that had previously received a 3-day morphine treatment regimen in combination with naloxone have been examined. Four groups of animals received once daily injections of saline or the opioid receptor antagonist naloxone (0.5, 1 and 2 mg/kg, s.c.), 30 min prior to s.c. injections of morphine (20 mg/kg/day × 3 days). After 5 days (no drug treatment), all groups of animals received intra-VTA morphine (7.5 g/rat), just after training (Fig. 3).
Fig. 1. The effects of post-training administration of morphine on memory retention. In panel A, animals received post-training systemic administration of saline (1 ml/kg, s.c.) or morphine (2.5, 5 and 7.5 mg/kg, s.c.). In panel B, animals received intra-VTA injections of saline (1 l/rat) or morphine (2.5, 5 and 7.5 g/rat) just after training. Animals were tested 24 h after training. Data are expressed as mean ± S.E.M. of eight animals per group. ** P < 0.01, *** P < 0.001 different from the control group.
2.7.4. Experiment 4: inhibition of morphine sensitization by SCH 23390 or sulpiride administration and effects of intra-VTA administration of morphine on memory retention The effect of bilateral intra-VTA injection of morphine on memory retention in animals that had previously received a 3-day morphine treatment regimen in combination with SCH 23390 or sulpiride have been examined. Four groups of animals received once daily injections of saline or the dopamine D1 receptor antagonist, SCH 23390 (0.025, 0.05 and 0.1 mg/kg, s.c.) 30 min prior to s.c. injections of morphine (20 mg/kg/day × 3 days; Fig. 4). Another four
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Fig. 3. The effect of bilateral intra-VTA injection of morphine on memory retention in morphine-sensitized rats in the presence or absence of naloxone. The animals received once daily injections of saline or the opioid receptor antagonist, naloxone (0.5, 1 and 2 mg/kg, s.c.), 30 min prior to s.c. injections of morphine (20 mg/kg/day × 3 days). After 5 days, all groups received morphine (7.5 g/rat, intra-VTA) immediately after training and tested 24 h after training. Data are expressed as mean ± S.E.M. of eight animals per group. ** P < 0.01, *** P < 0.001 different from the control group.
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Fig. 5. The effect of bilateral intra-VTA injection of morphine on memory retention in morphine-sensitized rats in the presence or absence of SCH 23390. The animals received once daily injections of saline or the dopamine D2 receptor antagonist, sulpiride (25, 50 and 100 mg/kg, s.c.), 90 min prior to s.c. injections of morphine (20 mg/kg/day × 3 days). After 5 days, all groups received morphine (7.5 g/rat, intra-VTA) immediately after training and tested 24 h after training. Data are expressed as mean ± S.E.M. of eight animals per group. ∗ P < 0.05, *** P < 0.001 different from the control group.
2.8. Histology groups of animals received once daily injections of vehicle or the dopamine D2 receptor antagonist, sulpiride (25, 50 and 100 mg/kg, s.c.) 90 min prior to s.c. injections of morphine (20 mg/kg/day × 3 days; Fig. 5). After 5 days (no drug treatment), all groups of animals received intra-VTA morphine (7.5 g/rat), just after training.
After completion of behavioral testing, each animal was killed with an overdose of chloroform. Animals received a 0.5 l/side injection of ink (1% aquatic methylene blue solution) into the VTA. The brains were then removed and fixed in a 10% formalin solution for 10 days before sectioning. Sections were examined to determine location of the cannulae aimed at the VTA. The cannulae placements were verified using the atlas of Paxinos and Watson [19]. Data from animals with injections sites located outside the VTA region were not used in the analysis. 2.9. Statistics Comparisons between groups were made with one-way analysis of variance (ANOVA) following Tukey test. A difference with P < 0.05 between experimental groups was considered statistically significant. Calculations were performed using the SPSS statistical package.
3. Results 3.1. Effects of morphine on memory retention
Fig. 4. The effect of bilateral intra-VTA injection of morphine on memory retention in morphine-sensitized rats in the presence or absence of SCH 23390. The animals received once daily injections of saline or the dopamine D1 receptor antagonist, SCH 23390 (0.025, 0.05 and 0.1 mg/kg, s.c.), 30 min prior to s.c. injections of morphine (20 mg/kg/day × 3 days). After 5 days, all groups received morphine (7.5 g/rat, intra-VTA) immediately after training and tested 24 h after training. Data are expressed as mean ± S.E.M. of eight animals per group. ** P < 0.01, *** P < 0.001 different from the control group.
Fig. 1A shows the effects of post-training systemic administration of morphine on step-through latency. One-way ANOVA revealed that post-training systemic morphine (2.5, 5 and 7.5 mg/kg, s.c.) dose dependently reduced the step-through latency in the one-trial passive avoidance task [F(3,28) = 34.7, P < 0.001]. Fig. 1B also shows that post-training intra-VTA injections of morphine (5 and 7.5 g/rat) decreased step-through latency [F(3,28) = 20.39,
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P < 0.001]. Both systemic and intra-VTA injections of morphine attenuated memory retention and induced amnesia. 3.2. Effects of intra-VTA administration of morphine on memory retention in morphine-sensitized rats As shown in Fig. 2, amnesia induced by post-training intra-VTA morphine (7.5 g/rat) was significantly decreased in rats which had previously received once daily injections of morphine (10 and 20 mg/kg, s.c.) for 3 days, compared with rats pretreated with saline (1 l/rat, intra-VTA) [Oneway ANOVA, F(3,28)) = 22.9, P < 0.0001]. Further analysis showed that a maximum response was obtained with 20 mg/kg of opioid. 3.3. Inhibition of morphine sensitization by naloxone administration and effects of intra-VTA administration of morphine on memory retention Fig. 3 shows the inhibition of morphine (7.5 g/rat, intraVTA)-induced amnesia in animals that had previously received the 3-day morphine (20 mg/kg, s.c.) treatment suppressed by once daily injections of the opioid receptor antagonist, naloxone (0.5, 1 and 2 mg/kg, s.c.) 30 min prior to s.c. injections of morphine (20 mg/kg/day × 3 days) [Oneway ANOVA, F(3,28) = 13.1, P < 0.001]. A maximum response was obtained with 2 mg/kg of naloxone (Tukey test, P < 0.001). 3.4. Inhibition of morphine sensitization by SCH 23390 or sulpiride administration and effects of intra-VTA administration of morphine on memory retention Fig. 4 shows that the inhibition of morphine (7.5 g/rat, intra-VTA)-induced amnesia in animals that had previously received the 3-day morphine (20 mg/kg, s.c.) treatment was dose-dependently decreased by once daily injections of the dopamine D1 receptor antagonist, SCH 23390 (0.025, 0.05 and 0.1 mg/kg, s.c.) 60 min prior to s.c. injections of morphine (20 mg/kg/day × 3 days) [One-way ANOVA, F(3,28) = 18.6, P < 0.001]. Fig. 5 also shows that the inhibition of morphine (7.5 g/rat, intra-VTA)-induced amnesia in animals that had previously received the 3-day morphine (20 mg/kg, s.c.) treatment was dose-dependently reduced by once daily injections of the dopamine D2 receptor antagonist, sulpiride (25, 50 and 100 mg/kg, s.c.) 90 min prior to s.c. injections of morphine (20 mg/kg/day × 3 days) [One-way ANOVA, F(3,28) = 7.5, P < 0.001].
4. Discussion In the present experiments, the involvement of VTA in morphine effects on memory retention of a one-trial passive
avoidance task in the morphine-sensitized rats was investigated. Post-training s.c. administration of different doses of morphine, dose-dependently, decreased learning on a onetrial passive avoidance task. Thus, in agreement with previous reports moderate doses of morphine causes impairment of memory retention [3,5]. In order to show the role of the VTA in the morphine-induced impairment of memory retention, the opioid has been injected in this site. The bilateral intraVTA injections of morphine impaired memory retention. This result revealed that the VTA may be involved in morphineinduced impairment of memory retention in rats. The VTA is an important site for synaptic modifications involved in learning and memory to associate morphine exposure with a specific environment [6]. It has also been suggested that the VTA is an important area for reward-related learning [34]. It has become apparent that repeated and intermittent morphine treatment produces an enhancement of motor activity associated with increases in mesolimbic dopaminergic cell firing [1,8,9,24]. Our previous results have shown that repeated administration of morphine (20 mg/kg), once daily for 3 days, followed by “a period of drug-free treatment” increased locomotion, indicating behavioural sensitization [38,36]. In the present study, morphine-sensitization has been achieved by administration of 3 days of morphine (5–20 mg/kg, s.c.) followed by 5 days of free drug treatment. Impairment of memory retention induced by post-training injection of morphine (7.5 g/rat) into the VTA region was significantly reduced in morphine (10 and 20 mg/kg)-sensitized rats. The data may indicate that similar to the amnesia induced by systemic administration of morphine in mice [36], amnesia induced by injection of morphine into the VTA can be altered in the morphine-sensitized rats. Thus, the repeated administration of morphine can result in the enhancement of memory retention, and this may imply that the sensitization induced by the opioid affected on memory processes of the VTA. There are several experiments that enhanced dopamine transmission in the VTA is associated with behavioral sensitization to morphine [9,29]. It has been reported that microinjections of D1 antagonists [26,30] into the VTA can disrupt the development of sensitization, suggesting that DA systems within the midbrain mediate the neuroadaptations that are responsible [33]. Our data show that impairment of memory retention induced by post-training morphine (7.5 g/rat, intra-VTA) is significantly reduced in the animals that previously received for 3 days either a dopamine D1 receptor antagonist, SCH 23390 or a dopamine D2 receptor antagonist, sulpiride, before morphine injection. These results once again support the theory that the dopaminergic system is involved in morphine-induced sensitization. Behavioral evidence suggests that changes in dopaminergic and/or opioid neurotransmission may be involved in the behavioral sensitization to morphine [13,28]. The present data also indicated that the inhibition of morphine-induced impairment of memory re-
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tention in morphine-sensitized rats was suppressed by 3 days injections of the opioid receptor antagonist naloxone, before morphine administration. Since morphine enhances both dopamine synthesis and release in the dopaminergic system [27] via activation of -opioid receptors in the VTA, It is likely that sensitization induced morphine was by a similar mechanism. Our results are in agreement with those of previous studies [31,35] and confirm that -opioid receptors play an important role in modulating morphine-induced sensitization. In summary, the results of the present study demonstrate that the VTA may play an important role in morphineinduced amnesia and morphine sensitization affects this process through opioid and dopamine receptors.
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