Introduction SHORT COMMUNICATION. Irma V. Belozertseva Anton Yu. Bespalov

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1 Naunyn-Schmiedeberg s Arch Pharmacol (1998) 358: Springer-Verlag 1998 SHORT COMMUNICATION Irma V. Belozertseva Anton Yu. Bespalov Effects of NMDA receptor channel blockers, dizocilpine and memantine, on the development of opiate analgesic tolerance induced by repeated morphine exposures or social defeats in mice Received: 13 December 1997 / Accepted: 15 April 1998 A.Y. Bespalov ( ) I.V. Belozertseva Laboratory of Behavioral Pharmacology, Institute of Pharmacology, I.P. Pavlov State Medical University, 6/8 Lev Tolstoy St., St. Petersburg , Russia Abstract Development of tolerance to opiates involves various neurochemically and pharmacologically distinct processes. For instance, the diversity of opiate tolerance has been suggested by experiments comparing the establishment of diminished response to different effects of opiate agonists. Antagonists acting at N-methyl-D-aspartate (NMDA) receptors has become a very useful tool for studying opiate tolerance mechanisms since these drugs have been shown to retard the development of tolerance to analgesic properties of opiates. The present study compared the ability of two NMDA receptor channel blockers, dizocilpine and memantine, to affect the development of tolerance to morphine analgesia induced by repeated social defeat or by repeated morphine administrations. Male BALB/c mice were assessed for the tail-flick response before and after the defeat in five social confrontations, or before and after repeated morphine injections (20 mg/kg, s.c., once daily for 8 days). Repeated morphine injections were explicitly paired with environmental cues. Socially-defeated as well as morphine-treated mice developed significant tolerance to morphine analgesia. Separate groups of mice were exposed to repeated social confrontations or injections of morphine with each defeat or morphine injection followed by administration of either dizocilpine ( mg/kg, i.p.) or low-affinity channel blocker memantine (3 30 mg/kg, i.p.). Both dizocilpine and memantine were effective in preventing the development of repeated morphine-induced tolerance to acute morphine analgesia. Treatments with NMDA receptor antagonists that retarded development of non-associative tolerance also suppressed the establishment of associative tolerance significantly. Social defeat-induced tolerance was prevented by dizocilpine but not by memantine. Our results suggest some degree of similarity in the mechanisms of morphine analgesic tolerance induced by pharmacological, contextual and social stimuli. Key words Morphine Tolerance Associative learning Social defeat NMDA receptor antagonist Tail-flick test Mice Introduction Considerable evidence accumulated over the last several years indicates that N-methyl-D-aspartate (NMDA) antagonists prevent development of tolerance to morphine (Herman et al. 1995), diazepam (File and Fernandes 1994), nicotine (Shoaib et al. 1994), ethanol (Szabo et al., 1994), 9 - tetrahydrocannabinol (Thorat and Bhargava 1994), sensitization to cocaine, morphine, amphetamines (Wolf and Jeziorski 1993), nicotine (Shoaib and Stolerman 1992), as well as attenuate establishment and/or expression of morphine (Herman et al. 1995), ethanol (Liljequist 1991), barbiturate (Rabbani et al. 1994) and benzodiazepine dependence (Steppuhn and Turski 1993). This suggests that certain common mechanisms may underlie long-term adaptation to neuroactive drugs. However, at least for opiates, it is quite likely that development of tolerance involves various neurochemically and pharmacologically distinct processes. First, the diversity of opiate tolerance has been suggested by experiments comparing the establishment of diminished response to different effects of opiate agonists. Fernandes and colleagues (1982) have shown differences in the threshold doses for induction of tolerance to analgesic, motor and thermoregulatory effects of opiates. There is also evidence for differential disappearance of tolerance to thermal, hormonal and locomotor effects of opiates (Rauhala et al. 1995). Rats trained to discriminate an opiate from vehicle showed no decrease in the stimulus control of behavior by the training drug after several months of continued drug exposure, despite of the development of tolerance to analgesic effects (Colpaert et al., 1976). Finally, defeats in social confrontations have pro-

2 271 duced selective tolerance to morphine-induced analgesia, but not to the decrement in operant performance or discriminative stimulus effects of morphine (Miczek and Winslow 1987; Miczek 1991). Second, there are data indicating that the NMDA receptor blockade by dizocilpine prevents development of tolerance to morphine analgesia (Herman et al. 1995) but not to morphine s discriminative stimulus effects (Bespalov et al. 1997; A.M. Young, personal communication). Tolerance to analgesic effects of fentanyl was not blocked by concurrent administration of NMDA receptor antagonist (Bilsky et al. 1996), while there is evidence for cross-tolerance between morphine and fentanyl with respect to their analgesic effects (Carter and Tiffany 1996). Moreover, neurochemical distinction between associative and non-associative components of morphine analgesic tolerance in the rat spinal cord has been demonstrated using blockade of NMDA and neurotensin receptors (Grisel et al. 1996). The present study compared the ability of two NMDA receptor channel blockers, dizocilpine and memantine, to affect the development of tolerance to morphine analgesia induced by repeated morphine administration (non-associative vs. associative components) or by repeated social defeat. Dizocilpine (MK-801) was selected as a representative, selective phencyclidine (PCP)-like noncompetitive antagonist (Wong et al. 1986) and memantine as a low-affinity channel blocker that was thought to be safer than high-affinity blockers, e.g. dizocilpine (Rogawski 1993; Parsons et al. 1995). Materials and methods Animals. Two hundred and eighty-five adult male drug- and experimentally na BALB/c mice (20 29 g; State Breeding Farm Rappolovo, St. Petersburg, Russia) were used. Animals were housed in groups (n=7 10), or individually with food and water available ad libitum. All experiments were conducted during the light period of a 12/12-h day-night cycle (09:00 21:00 h). Drugs. The following drugs were used: morphine hydrochloride (purchased from commercial sources), dizocilpine maleate (MK-801; RBI, Natick, Mass., USA), and memantine (gift from Merz, Germany). Morphine, dizocilpine and memantine were dissolved in phosphate-buffered saline (ph 7.2). Morphine and its vehicle were injected s.c. while dizocilpine, memantine and their vehicles were administered i.p. All injections delivered a solution in a volume of 10 ml/kg. Doses are based upon the forms of the drugs listed above. Tail-flick procedure. The tail of a mouse (about 1 cm from the base) was exposed to a focussed heat source (300 W white bulb). By withdrawing or removing the tail from the path of the stimulus and thereby exposing a photocell located in the apparatus ( Farmakolog, St. Petersburg, Russia) immediately below the tail, the mouse could terminate the noxious stimulation and the reaction time was then recorded. An animal that failed to respond before 10 s (cutoff time) was removed from the apparatus and assigned latency of 10 s. A mean (±SEM) baseline tail-flick latency for drug-na mice was 4.1±0.1 s (n=233). On each test day, tail-flick latencies were measured 3 times for each animal. Saline was injected immediately after initial measurement of baseline tail-flick latency. Some 30 min after the saline administration tail-flick latencies were re-assessed and immediately thereafter mice were treated with the test dose of morphine (5 mg/kg). The third tail-flick test was made 30 min after the morphine injection. Mice were returned to their home cages after each injection and/or tail-flick test. Experiment 1. On day 1 of the experiment, the effects of saline vehicle and morphine were assessed in group-housed experimental mice as described above. In the second phase of the experiment (days 2 9), tail-flick latencies were not measured but each experimental mouse received two s.c. injections and one i.p. injection daily. First, mice were given s.c. saline (between 9:00 and 10:00 a.m.) and then, 6 h later, s.c. injection of morphine (20 mg/kg) followed immediately by i.p. injections of dizocilpine (0.03, 0.1 or 0.3 mg/kg), memantine (3, 10 or 30 mg/kg) or saline. For each treatment group, half of the mice received morning injection with saline in the animal facility while evening injections (morphine plus NMDA receptor antagonist) were administered in the experimental room. For the other half, the order was reversed. When injections were performed in the experimental room, cages with mice remained there for 3 h before being returned to the animal facility. Additional control groups received second daily treatment with s.c. saline (instead of morphine) followed immediately by i.p. injections of dizocilpine (0.3 mg/kg), memantine (30 mg/kg) or saline. On days 10 and 11 of the experiment, the effects of saline vehicle and morphine were re-assessed in experimental mice as described above for day 1. For half of the mice, tests were conducted in the animal facility on day 10 and repeated in the experimental room on day 11. For the remaining half, this order was reversed. Each treatment group consisted of mice. Experiment 2. Three different pools of mice used for this experiment: experimental mice, resident mice, and intruder mice. Resident mice (n=22) were housed individually for at least 6 weeks prior to the experiment. Starting after the first 3 weeks of isolation, resident mice were repeatedly (twice weekly) allowed to attack for 4 min a grouphoused stimulus intruder mouse (randomly selected from a pool of 30 mice). Intruder mice responded to the residents bites and threat behavior by assuming defensive postures, attempting to escape, and eventually displaying submissive behavior (upright defensive posture, limp forelimbs, retracted ears, squealing to the approaching resident). Only the resident mice that consistently exhibited attack behavior towards intruders were used in the experiment. On day 1 of the experiment, the effects of saline vehicle and morphine were assessed in group-housed experimental mice as described above. In the second phase of the experiment (days 2 6), tail-flick latencies were not measured and each experimental mouse was subjected to resident-intruder confrontations. Specifically, experimental mice were placed as intruders into a cage of an aggressive resident mouse. Each resident-intruder confrontation lasted 4 min. There were attacks displayed by an intruder within one confrontation session. Immediately after a confrontation with the resident, experimental mice received i.p. injections of dizocilpine (0.03, 0.1 or 0.3 mg/kg), memantine (3, 10 or 30 mg/kg), or saline. On day 7 of the experiment, the effects of saline vehicle and morphine were re-assessed in experimental mice as described above for day 1. Each treatment group consisted of 9 19 mice. An additional control group (n=9) served to estimate nociceptive responding in tail-flick test after a single confrontation with an aggressive resident. Data analysis. Tail-flick latencies were converted to percentages of the maximal possible effect. The individual mouse values of the percent of analgesia were calculated according to the formula, [ ] ( T T ) 100 / 10 T ( ) EXP BL BL where T EXP is the test tail-flick latency and T BL the baseline latency. Since the control tests indicated no significant differences between the performances on days 10 and 11, the data were combined and subjected to statistical analysis separately for tests conducted in morphine-paired and saline-paired environments. Data were analysed using SAS-STAT software (6.11, SAS Institute, Cary, N.C., USA). Data were subjected to the distribution-free (1)

3 272 Fig. 1 Effects of dizocilpine (left panel) and memantine (right panel) on the development of morphine tolerance induced by repeated morphine exposures in mice. Tail-flick latencies were assessed following saline (SAL) and 5 mg/kg of morphine (MOR) injections before (PRE) and after (POST) repeated morphine (40 mg/kg, once daily, 8 days) or saline administration. Repeated morphine injections were given either in the laboratory or in the animal facility. During the tolerance induction period, dizocilpine and memantine were co-administered with morphine. Following the tolerance induction period, analgesic potency of acute morphine was tested in the environment that was either paired (+) or not paired ( ) with morphine injections. Data are expressed as mean ±SEM percentage of analgesia, n= *,# P<0.05 (Wilcoxon s test) vs MOR-pre and MOR-post ( ) measurements, respectively Fig. 2 Effects of repeated exposures to dizocilpine and memantine on the acute analgesic activity of morphine in mice. Tail-flick latencies were assessed following saline (SAL) and 5 mg/kg morphine (MOR) injections before (PRE) and after (POST) repeated administration of saline in combination with dizocilpine (0.3 mg/kg), memantine (30 mg/kg) or their vehicle (once daily, 8 days). Data are expressed as mean ± SEM percentage of analgesia, n=10 three-factorial analysis of variance (ANOVA) with repeated measures on one factor. Factors were as follows: 1, social confrontations (experiment 1) or repeated morphine exposures (experiment 2); 2, NMDA receptor antagonist dose; and 3, acute morphine treatment. Wilcoxon s test was used for post-hoc between-group pairwise comparisons. Results Tests conducted on day 1 of the experiments indicated that tail-flick latencies following the saline challenge (4.1±0.1 s) were not different from baseline levels (4.1±0.1 s; n=233). Treatment with morphine (5 mg/kg) produced significant analgesia (mean tail-flick latency: 9.6±0.0 s) and 79% of mice failed to respond before 10 s (cut-off time). Experiment 1 The results of experiment 1 are summarized in Fig. 1. Repeated administration of morphine in combination with saline (as a vehicle for NMDA receptor antagonist) resulted in the development of tolerance to morphine analgesia. Analgesic activity of the test dose of morphine on days 9 10 was 48±11% of the maximal possible effect when tested in the saline-unpaired environment and 19±10% when the mice were tested in morphine-paired environment. On day 1 of the experiment, the analgesic activity of morphine was 90±5% of maximal effect in the same mice (n=20). Dizocilpine and memantine treatment prevented development of tolerance to morphine. For both dizocilpine and memantine, the effects on morphine tolerance development were dose dependent (dizocilpine: F(3,85)=4.66, P=0.0045; memantine: F(3,88)=4.77, P=0.0040). Tests conducted in the saline-paired environment revealed that concurrent administration of 0.1 and 0.3 mg/kg of dizocilpine as well as 30 mg/kg of memantine blocked the development of morphine tolerance. Tests conducted in the morphine-paired environment showed that the same doses of dizocilpine and memantine were effective in retarding the development of morphine tolerance. Pairwise comparisons revealed no differences in morphine analgesic potency tested in morphine- vs. saline-environments in mice with prior repeated exposures to morphine combined with either dizocilpine (0.1 and 0.3 mg/kg) or memantine (10 and 30 mg/kg). Repeated administration of NMDA receptor antagonists (0.3 mg/kg of dizocilpine or 30 mg/kg of memantine) in combination with s.c. saline (instead of morphine) did not affect baseline pain thresholds or tail-flick latencies after saline and morphine challenge (dizocilpine: F(3,32)=1.12, P=0.1987; memantine: F(3,36)=0.58, P=0.3468; Fig. 2). Experiment 2 The results of experiment 2 are summarized in Fig. 3. When defeated for the first time, mice (n=10) showed a significant increase in the tail-flick latency (31±10%). The average

4 273 Fig. 3 Effects of dizocilpine (left panel) and memantine (right panel) on the development of morphine tolerance induced by repeated social defeats in mice. Tail-flick latencies were assessed following saline (SAL) and 5 mg/kg of morphine (MOR) injections before (PRE) and after (POST) repeated social confrontations period (4-min encounter with an aggressive conspecific once daily for 5 days). Dizocilpine and memantine were administered immediately after each social confrontation. Data are expressed as mean ±SEM percent of analgesia, n=9. P<0.05 (Wilcoxon s test) vs MOR-pre measurements baseline latency for this group of miee was 4.2±0.4 s, and after the defeat this latency increased to 6.0±0.5 s. After acute challenge with morphine on day 7 mice with repeated experiences of social defeats (n=19) exhibited shorter tail-flick latencies (6.6±0.3 s) compared with control levels (9.3±0.2 s). ANOVA revealed significant effects of exposures to social confrontations [F(1,18)=12.9, P=0.0021], as well as a significant interaction between test treatment (saline and morphine) and social confrontation factors [F(1,18)=23.53, P=0.0001]. Low doses of both dizocilpine (0.03 mg/kg) and memantine (3 and 10 mg/kg) caused non-significant increases in the degree of morphine analgesic tolerance while higher doses of dizocilpine (0.1 and 0.3 mg/kg), but not memantine, after each social confrontation prevented the development of morphine tolerance [dizocilpine: F(3,44)=7.44, P=0.0004; memantine: F(3,45)=1.24, P=0.3047]. Discussion The present results indicate firstly that explicit pairing of repeated morphine injections with environmental cues resulted in the development of associative tolerance to analgesia produced by acute morphine challenge. Secondly, concurrent administration of either dizocilpine or memantine prevented development of both non-associative and associative components of morphine analgesic tolerance. Thirdly, repeated social defeats led to diminished analgesic activity of the test dose of morphine and finally, administration of dizocilpine, but not memantine, after each social confrontation prevented toleranee to morphine analgesia, while both drugs exhibited a tendency to increase the degree of morphine analgesic tolerance when administered at low doses. Both dizocilpine and memantine effectively retarded the development of morphine tolerance induced by repeated morphine exposures. To our knowledge, this is the first demonstration that memantine prevents development of opiate tolerance. Various NMDA receptor antagonists have been tested to date against the development of opiate tolerance and the list of antagonists employed includes noncompetitive antagonists, competitive antagonists and a partial agonist of the glycine site (Herman et al. 1995). Memantine is capable of blocking the maintenance of opiate dependence (Popik and Skolnick 1996) as well as of attenuating motivational components of opiate withdrawal syndrome (Popik and Danysz 1997). Taking into account the relatively safe profile of memantine compared with other NMDA receptor antagonists, it is tempting to suggest trying memantine as an adjunct for opiate-based management of chronic pain. In mice not treated with NMDA receptor antagonists, analgesic activity of the test dose of morphine was significantly higher in the saline-paired than in the morphinepaired environment. Concurrent administration of either dizocilpine or memantine eliminated the environment-related difference between morphine s acute effects. In other words, both dizocilpine and memantine were capable of preventing the development of associative tolerance to morphine analgesia. These data are in agreement with our earlier findings on prevention of associative tolerance to morphine analgesia by phencyclidine (Bespalov et al. 1994). However, contrasting results have been reported by Trujillo and Akil (1994) who concluded that dizocilpine did not interfere with associative tolerance. In fact, morphine-induced analgesia in dizocilpine-treated and saline-treated groups did not differ when both induction and testing of morphine tolerance took place in the same experimental room ( associative groups). Although procedual differences may be suggested as a possible source of the disagreement, we found no grounds for such speculations. In addition, although there is no clear evidence that mechanisms of morphine tolerance might somehow be different in rats and mice, it should be noted that the present study used mice while Trujillo and Akil (1994) reported results of the experiments conducted in rats. The present study also showed that dizocilpine, but not memantine, prevented establishment of morphine analgesic tolerance in socially defeated mice. There are two possible interpretations of these findings. First, the dizocilpine data

5 274 suggest that the development of morphine analgesic tolerance is functionally dependent upon NMDA receptors, irrespective of whether the tolerance is induced by pharmacological (i.e., repeated morphine injections) or social stimulation. Second, the memantine data suggest that the ability to block the development of morphine tolerance may be different amongst various NMDA receptor antagonists. Similarly, tolerance to discriminative stimulus effects of morphine is prevented by competitive and polyamine-site antagonists (Bespalov et al. 1997), but not non-competitive antagonist (Bespalov et al. 1997; A.M. Young, personal communication). In conclusion, the present report provides further evidence for NMDA receptors being involved in the development of morphine tolerance. Our results suggest some degree of similarity in the mechanisms of morphine analgesic tolerance induced by pharmacological, contextual and social stimuli. Differences between various site-selective NMDA receptor antagonists prompt for a search for the drug that would target selectively the development of tolerance to a specific effect of an opiate. Acknowledgements Supported in part by Fogarty International Research Collaboration Award #1R03TW Authors wish to thank Merz and Co. for the generous contribution of memantine. The experiments were performed in accordance with the recommendations and policies of the International Association for the Study of Pain and of the Ethics Committee of Pavlov Medical University. References Bespalov AY, Dumpis MA, Piotrovsky LB, Zvartau EE (1994) Tolerance to opiate analgesia: a complex effect of antagonists ot excitatory amino acid receptors. Biull Eksp Biol Med 117: Bespalov AY, Balster RL, Beardsley PM (1997) NMDA receptor antagonists attenuate tolerance to morphine s discriminative stimulus effects. 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