NMDA receptor antagonists inhibit opiate antinociceptive tolerance ...

Psychopharmacology (2008) 196:497–509 DOI 10.1007/s00213-007-0984-8

ORIGINAL INVESTIGATION

NMDA receptor antagonists inhibit opiate antinociceptive tolerance and locomotor sensitization in rats Ian A. Mendez & Keith A. Trujillo

Received: 11 April 2007 / Accepted: 12 October 2007 / Published online: 10 November 2007 # Springer-Verlag 2007

Abstract Rationale N-Methyl-D-aspartate (NMDA) receptors have an important role in different forms of behavioral and neural plasticity. Evidence suggests that these receptors may also be involved in plasticity arising from long-term treatment with different drugs of abuse, including tolerance, sensitization, and physical dependence. There is abundant evidence demonstrating that NMDA receptors are involved in tolerance to opiate-induced antinociception; however, the role of these receptors in sensitization to the locomotor effects of opiates is more controversial. Objective The ability of NMDA receptor antagonists to modify the development of sensitization to the locomotor stimulant effect of three different opiates was examined. In selected studies, the ability of the antagonists to modify tolerance to the antinociceptive effects of the opiates was also examined. Materials and methods Adult male Sprague–Dawley rats were used to assess the effects of NMDA receptor antagonists (MK-801, memantine or LY235959) on tolerance and sensitization to three opiates: morphine, methadone, or buprenorphine. It was predicted that low, selective doses of

I. A. Mendez : K. A. Trujillo (*) Department of Psychology, California State University, 333 S. Twin Oaks Valley Road, San Marcos, CA 92096-0001, USA e-mail: [email protected] Present address: I. A. Mendez Department of Psychology, Texas A & M University, TAMU 4235, College Station, TX 77843, USA e-mail: [email protected]

the antagonists would inhibit the development of opiate tolerance and sensitization. Results Consistent with our predictions, the noncompetitive NMDA receptor antagonists MK-801 and memantine and the competitive NMDA receptor antagonist LY235959 inhibited the development of sensitization to the locomotor stimulant effect of morphine. Additionally, MK-801 inhibited the development of tolerance and sensitization to methadone and buprenorphine in a similar manner. Conclusions The results, together with previous research, suggest that NMDA receptors are broadly involved in opiateinduced plasticity, including the development of opiate tolerance and sensitization. Keywords Opiate . NMDA receptor . Tolerance . Sensitization . Analgesia . Locomotion Evidence suggests that N-methyl-D-aspartate (NMDA) glutamate receptors may be involved in the plasticity that arises from long-term administration of morphine (Herman et al. 1995; Inturrisi 1997; Mao 1999; Trujillo 1995, 2000, 2002). Initial evidence supporting this idea was provided by Trujillo and Akil (1991) who reported that the NMDA receptor antagonist MK-801 inhibited the development of tolerance to the antinociceptive effect of morphine and morphine physical dependence without affecting acute morphine antinociception. After this discovery, numerous laboratories replicated these findings, demonstrating that a variety of NMDA receptor antagonists have the ability to inhibit the development of opiate tolerance and dependence (for review, see Bisaga and Popik 2000; Herman et al. 1995; Inturrisi 1997; Mao 1999; Mayer et al. 1999; Trujillo 2000, 2002; and Trujillo and Akil 1995). Wolf and Jeziorski (1993) provided the first evidence that NMDA receptor antagonists inhibit sensitization to the

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locomotor stimulant effect of morphine in a manner similar to their effect on tolerance and dependence, a finding that has been supported by others (Jeziorski et al. 1994; Kosten and Bombace 2000). Although these results suggest a role for NMDA receptors in opiate sensitization, there is controversy regarding this idea (Carlezon et al. 2000; Iijima et al. 1996; Jeziorski et al. 1994; Ranaldi et al. 2000; Tzschentke and Schmidt 1996, 1998). Some research has failed to replicate an ability of NMDA receptor antagonists to inhibit the development of sensitization, whereas other research has found inhibition but only at high doses of NMDA receptor antagonists that produce adverse side effects (Iijima et al. 1996; Tzschentke and Schmidt 1996, 1998; Tzschentke and Schmidt 2000; Vanderschuren et al. 1997). An additional controversy is over the selection of NMDA receptor antagonists used to explore the role of NMDA receptors in sensitization to the stimulant effect of morphine. Most studies on this topic have used the potent and selective antagonist MK-801 as the drug of choice. However, MK-801 has been criticized as problematic because of its behavioral effects alone and in combination with morphine (Carlezon et al. 2000; Ranaldi et al. 2000; Tzschentke and Schmidt 1998; Tzschentke and Schmidt 2000). There is, therefore, a need to further explore the potential role of NMDA receptors in opiate sensitization using alternative NMDA receptor antagonists. Another issue of importance is whether or not NMDA receptor antagonists have the ability to affect tolerance or sensitization to different opiates. Although these antagonists can inhibit the development of tolerance resulting from chronic morphine administration, inhibition of tolerance resulting from other opiates that act at mu opioid receptors has not been consistently reported. For example, Bilsky et al. (1996) reported that NMDA receptor antagonists do not inhibit the development of antinociceptive tolerance to mu opioid agonists other than morphine. On the other hand, Mao et al. (1998) and Allen and Dykstra (2000a) have reported that NMDA receptor antagonists can indeed inhibit tolerance to different mu opioids. Because of these conflicting findings and a relative lack of research, there is a need to further explore the effects of NMDA receptor antagonists on the development of tolerance and sensitization to opiates other than morphine. The purpose of the current studies is to address some of the controversies regarding the role of NMDA receptors in tolerance and sensitization to opiates. To date, the majority of studies that have investigated sensitization to the locomotor stimulant effect of opiates have used MK-801 as the NMDA receptor antagonist and morphine as the opioid. The present studies replicated earlier research with MK-801 but extended this work to other NMDA receptor antagonists, including memantine and LY235959. Memantine is a clinically available noncompetitive antagonist, whereas LY235959 is a

Psychopharmacology (2008) 196:497–509

competitive antagonist, and each produces a behavioral profile distinct from MK-801 (Bubser et al. 1992; Danysz et al. 1994; Danysz and Parsons 1998; Geter-Douglass and Witkin 1997, 1999; Parsons et al. 1999). In addition to examining different NMDA receptor antagonists, the present experiments utilized three different opiates: morphine, methadone, and buprenorphine. Each of these opiates is used clinically; however, they differ in affinity, selectivity, and efficacy for mu opioid receptors. The present studies are the first to examine the ability of memantine or LY235959 to affect sensitization to the stimulant effects of opiates and the first to examine the effects of NMDA receptor antagonists on tolerance and sensitization to methadone or buprenorphine. The general working hypothesis of this research is that NMDA receptors are involved in opiate-induced neural and behavioral plasticity. Given this hypothesis, it is predicted that low selective doses of the NMDA receptor antagonists will inhibit the development of tolerance and sensitization to morphine, methadone, and buprenorphine. If similar effects are obtained across the different combinations of NMDA receptor antagonists and opioids, this will support the idea that NMDA receptors are involved in both tolerance to the antinociceptive effects and sensitization to the stimulant effects of opiates. Six different experiments were performed in three sets (Table 1). The first set of experiments examined the effects of MK-801 or memantine on sensitization to the stimulant effect of morphine. These experiments replicated the ability of MK-801 to inhibit morphine sensitization and extended these findings to memantine. The second set of experiments examined the effects of MK-801 or LY235959 on both tolerance and sensitization to morphine in the same experimental animals. Similar findings across these two independent behaviors in the same experimental animals have the opportunity to strengthen the idea that the effects are caused by the inhibition of plasticity rather than a pharmacological interaction unique to a particular behavior. The third set of experiments examined the effects of MK-801 on both tolerance and sensitization to methadone and buprenorphine, thereby, extending the research to different mu opioids. Together, the findings support the idea that NMDA receptors are involved in opiate tolerance and sensitization.

Materials and methods Animals and groups One hundred thirty-one experimentally naive adult male Sprague–Dawley rats (200–225 g at time of purchase; Harlan, San Diego, CA, USA) were used in these experiments. The animals were housed in plastic cages, three rats per cage on a 12-h light/dark cycle, with


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Table 1 Summary of experiments Experiment

NMDA antagonist

Opiate

Test Sensitization Sensitization Tolerance and sensitization in same animals Tolerance and sensitization in same animals Tolerance and sensitization in same animals Tolerance and sensitization in different animals

Effects of MK-801 and memantine on sensitization to morphine Effects of MK-801 and LY235959 on tolerance and sensitization to morphine in the same experimental animals

1.1 1.2 2.1

MK-801 Memantine MK-801

Morphine Morphine Morphine

2.2

LY235959

Morphine

Effects of MK-801 on tolerance and sensitization to methadone and buprenorphine

3.1

MK-801

Methadone

3.2

MK-801

Buprenorphine

food and water available ad libitum, and allowed at least 1 week of acclimation before use in the experiments. Experimental protocols were approved by the California State University San Marcos Institutional Animal Care and Use Committee and are in compliance with the National Institutes of Health Guide for Care and Use of Laboratory Animals. Instruments An IITC model 33 tail-flick apparatus was used to measure antinociception. Methods were similar to those used previously (Redwine and Trujillo 2003; Trujillo and Akil 1991, 1994). Rats were gently restrained by hand for tail flick tests. Three tail flicks in a row, 10–20 s apart, were performed for each animal, with the average used for data analysis. The heat was focused between 2 and 8 cm from the tip of the tail, with the focus of the heat moved on each trial to avoid irritation caused by repeated testing at the same site. The amount of time an animal took to move its tail from the heat was determined. A heat setting that yielded a 2–4 s baseline response was used. A cutoff latency of 8 s was used to prevent tissue damage. Locomotor stimulation was automatically assessed using a Kinder Scientific open-field motor monitor system. This system consists of eight 16″×15″×16″ plexiglas enclosures with two 16″×16″ photocell arrays surrounding each chamber. The first array is placed 5 cm above the floor and is used to measure horizontal activity; the second is 12.5 cm above the floor and measures vertical (rearing) activity. An automated personal computer collects data on photocell beam interruptions from both arrays and calculates distance traveled, time at rest, number of rears, ambulations (interruptions of successive photocell beams resulting from displacement of the animal horizontally, typically associated with forward movement), and fine movements (interruptions of single photocell beams, typically associated with short repetitive movements).

Drugs Three NMDA receptor antagonists were used in these studies. The antagonists included the non-competitive antagonists MK-801 (0.1 mg/kg) and memantine (3.0 mg/kg) and the competitive antagonist LY235959 (1.0 mg/kg). Doses chosen for these studies were based on previous research demonstrating that they effectively block NMDA receptors but produce minimal behavioral side-effects (Allen and Dykstra 1999; Bilsky et al. 1996; Bubser et al. 1992; Carter 1994; Danysz et al. 1994; Popik and Kozela 1999; Popik et al. 2000; Redwine and Trujillo 2003; Trujillo et al. 2001a; Trujillo et al. 2000; Trujillo et al. 2001b). Memantine hydrochloride and MK-801 hydrogen maleate were purchased from Sigma, and LY235959 was purchased from Tocris. The opiates used were morphine (5.0 mg/kg), methadone (3.0 mg/kg), and buprenorphine (0.3 or 1.0 mg/kg). Morphine sulfate and buprenorphine hydrochloride were generous gifts from the National Institute on Drug Abuse Drug Supply Program, whereas methadone hydrochloride was purchased from Sigma. All drugs in this study were dissolved in 0.9% saline, except for buprenorphine, which was dissolved in 1% Tween-80; drugs were injected at a volume of 1.0 ml/kg Protocol Groups were assigned an initial injection of saline or an NMDA antagonist followed by a second injection of saline or an opiate. The experimental design for each study resulted in four treatment groups for each of six experiments (Table 2). Six animals were included in each group, except where otherwise noted. The comparison of interest is between the opiate group and the experimental group. The antagonist control group is also significant as a measure of any effects the antagonists alone had on the selected behavior. Three sets of experiments were performed. The first (experiments 1.1 and 1.2) examined the effects of MK-801 and memantine on sensitization to the locomotor stimulant

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Table 2 Treatment groups for experimental design Pretreatment

Treatment

Purpose

Saline NMDA antagonist Saline NMDA antagonist

Saline Saline Opioid agonist Opioid agonist

Saline control NMDA antagonist control Opioid agonist group Experimental group

effects of morphine. The second set of studies (experiments 2.1 and 2.2) expanded on these findings, examining the effects of MK-801 and LY235959 on both tolerance to the antinociceptive effect and sensitization to the stimulant effect of morphine in the same experimental animals. Finally, to explore other opiates, the third set of studies (experiments 3.1 and 3.2) examined the effects of MK-801 on both tolerance to the antinociceptive effect and sensitization to the stimulant effect of methadone and buprenorphine (see Table 1). The experimental approach was similar to that used in earlier research (Peterson et al. 2002; Swadley-Lewellen and Trujillo 1998; Trujillo 1995, 2000). For all experiments, animals received, in their home environment, once daily injections of saline or an NMDA receptor antagonist followed 30 min later by an injection of saline or an opiate. On the opiate challenge day, for testing of sensitization, the animals were habituated to the locomotor testing room for 30 min. In the testing room, white noise was used to mask outside sounds, and overhead incandescent lights were dimmed. After habituation to the testing room, the animals were placed in the locomotor apparatus for 30 min, then removed from the enclosure and injected with an opiate in the absence of pretreatment. Upon injection, the animals were immediately placed back in the enclosure, and locomotor activity was recorded for an additional 3.5 h. This approach was used to minimize the opportunity for associative factors to contribute to sensitization. Because repeated injections of the opiate occurred in the home environment, whereas sensitization was assessed in the testing room under unique conditions, association of drug effects with environmental cues was not likely a key factor in the results. To examine the development of tolerance to the antinociceptive effects of the opiates, baseline tail-flick latencies were taken on the two consecutive days preceding the first day of treatment. Beginning on day 1 of treatment, tail-flick scores were determined 60 min after the second injection (vehicle or opiate) on odd numbered days. The tests during the course of chronic injections occurred in the home environment under the influence of the NMDA receptor antagonist. To further assess tolerance, on the final day of the experiment, during the test for sensitization, the animals were removed briefly 60 min after the opiate injection to obtain a tail-flick score (the time out of the enclosure for tail-flick testing was 2 min or less).

This final tail-flick test occurred in the absence of the NMDA receptor antagonist. In this manner (testing for tolerance in both the presence and the absence of the NMDA receptor antagonist), we were able to determine if the effects of the NMDA receptor antagonists resulted from state-dependence (see Trujillo 2000 for further discussion). The experimental treatment schedule for morphine experiments consisted of 10 days of injections, followed by an opiate challenge on day 11. Development of sensitization to the stimulant effects of methadone and buprenorphine, however, required a 16-day treatment schedule, with sensitization tested on day 17. For methadone, tolerance and sensitization were examined in the same experimental animals using this 17-day protocol. However, because of the different doses and time courses required for buprenorphine tolerance and sensitization, different experimental groups were used to assess tolerance and sensitization. Therefore, 1.0 mg/kg was used to assess the development of tolerance to the antinociceptive effect of buprenorphine over 9 days of treatment (similar to morphine), whereas 0.3 mg/kg was used to assess the development of sensitization to the stimulant effect over 17 days of treatment. Data analysis For analysis of tail-flick latencies, raw scores in seconds were determined and used as a measure of pain responsiveness. Effects over days were analyzed by two-way repeated measures analysis of variance (ANOVA; treatment group × day), followed by Fisher’s protected least significant difference (PLSD) post hoc analysis for comparison between individual groups. On test day, a one-way factorial ANOVA was used with Fisher’s PLSD for post hoc comparisons. For locomotor stimulation, activity was collected at 10-min intervals for a total of 210 min post-injection. Raw photo beam breaks (basic movements) were analyzed using a twoway repeated measures ANOVA (treatment group × time) to determine the overall effect of treatment, followed by Fisher’s PLSD post hoc analysis for individual group comparisons. In the present experiments, sensitization for morphine appeared most prominent during the first 60 min after injection, whereas sensitization for methadone and buprenorphine appeared most prominent during the first 120 min post-injection. Therefore, secondary statistical analyses focused on these respective time frames, with a one-way factorial ANOVA examining group differences in the total activity scores during the first 60 min (morphine) or the first 120 min (methadone and buprenorphine) post-injection.

Results 1. Effects of MK-801 and memantine on sensitization to morphine


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Experiment 1.1. Effects of the NMDA receptor antagonist MK-801 on sensitization to morphine-induced locomotor stimulation Animals that received morphine for the first time on day 11 (Sal/Sal and MK/Sal groups) showed a modest level of activity immediately after the morphine injection and a slowly emerging increase in activity that was evident late in the session. In contrast, animals that received morphine throughout the study (Sal/Mor group) showed a potent increase in activity that was evident within 10 min after injection, consistent with the development of sensitization. The group that received MK-801 and morphine (MK/Mor group) showed effects very similar to those that received morphine for the first time (Fig. 1a). Statistical analysis confirmed differences among the groups, showing a significant effect of treatment [F(3,14)=8.24, p