Chapter 3 Excitatory amino acid receptor antagonists and naloxoneprecipitated withdrawal syndrome in morphine-dependent mice

Abstract - The effects of excitatory amino acid (EAA) receptor antagonists MK801 (non-competitive NMDA receptor antagonist), DNQX (competitive nonNMDA receptor antagonist) and 5,7-DCKA (antagonist of glycine site of NMDA receptor) have been examined on the naloxone (4 mg/kg, i.p.)-precipitated withdrawal jumping behaviour in morphine-dependent mice. The results indicate that withdrawal jumping behaviour in morphine-dependent mice was attenuated by all three EAA receptor antagonists, MK-801, DNQX and 5,7-DCKA. However, MK-801, DNQX and 5,7 -DCKA inhibited the jumping behaviour in a relatively narrow dose range Published in European Neuropsychopharmacology 3: 111-116, 1993.

Central excitatory amino acids (EAAs) with corresponding receptors have been the focus of much attention in order to clarify neuronal development, long-term potentiation, kindling, epilepsy, learning or memory (Cotman and Iversen, 1987). In addition, there is evidence of the involvement of EAA in drug dependence phenomena. It has been shown that MK-801 blocks alcohol withdrawal seizures in the rat (Morrisett et al., 1990). It seems that chronic alcohol treatment uregulates the number of N-methyl-D-aspartate (NMDA) receptors in the hippocampus (Grant et al., 1990), which might explain both the seizures in alcohol withdrawal and anticonvulsant activity of NMDA receptor antagonists.

Furthermore, recent data indicate that the non-selective antagonist of EAA receptors, kynurenic acid (Krystal et al., 1990) attenuated naloxone-precipitated withdrawal in rats. Similarly, the non-competitive (MK-801) and competitive (LY274614) NMDA antagonists suppressed the behavioural signs of withdrawal in morphine-dependent rats (Koyuncuoglu et al., 1992; Rasmussen et al., 1991; Trujillo and Akil, 1991). Evidently, the functional activity of NMDA receptors may have a modulatory effect on drug dependence phenomena in the rat. The role of other EAA receptors, besides the NMDA receptor, in morphine dependence remains unclarified.

The aim of this study is to examine the role of various EAA receptor antagonists in naloxone-precipitated withdrawal in morphine-dependent mice. We used the non-competitive NMDA receptor antagonist MK-801 (Wong et al., 1988), the non-NMDA receptor antagonist DNQX (Honor6 et al., 1988) and 5,7-DCKA, a selective antagonist of NMDA receptor-associated strychnine-insensitive glycine binding site (Baron et al., 1991). An additional reason for using 5,7-DCKA was that glycine receptor antagonism may produce motor effects different from the competitive NMDA receptor antagonists (Koek and Colpaert, 1990). Materials and Methods

Animals

Male Swiss mice weighing 25-35 g were used in all experiments. The animals were housed singly in polyethylene cages with food and water ad libitum. Artificial light was supplied in a 12-h light-dark cycle.

Morphine dependence

In general, the experimental model for the opioid withdrawal in mice was followed (Way et al., 1969; Kosersky et al., 1974). Chronic morphine dependence in mice was induced by morphine pellets (25 mg morphine base/mouse, s.c.) implanted on the back of the animal under ether anaesthesia. The morphine withdrawal was precipitated by administration of naloxone (4 mg/kg, i.p.), 72 h after the implantation of the pellet. The withdrawal severity was quantified by counting the frequency of jumping from a circular platform (30 cm high, 12 cm diameter). The general behaviour of drug-treated naive and morphine-dependent mice was observed and registered.

The animal was pretreated with vehicle or one EAA receptor antagonist, 30 min prior to naloxone. The pretreated animal was placed on the platform and observed for the following 30 min (in time intervals of 5 min). At the end of the 30-min period, the animal was given naloxone and placed again on the platform in order to be observed in a similar way, for the following 30 min.

Experimental protocol

Morphine-dependent mice were divided into five groups, pretreated intraperitoneally with vehicle (saline, 0.5 ml, n=5; DMSO, 0.5 ml, n=5), MK-801 (1-80 Vg/kg, n=78), DNQX (0.6310 mg/kg, n=40) or 5,7-DCKA (5-160 mg/kg, n=35). In all these animals, the withdrawal jumping behaviour was precipitated by administration of naloxone (4 mg/kg, i.p.), 30 min after drug pretreatment. In order to observe the behavioural effect of EAA antagonist in morphine-dependent mice, naloxone was replaced by saline. These three additional groups (n=14-36) were pretreated (30 min before saline) with various doses of EAA receptor antagonists. Each animal was used only once.

Drugs

The following drugs were used: MK-801 [(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d] cyclohepten 5,10-imine maleate, Research Biochemicals Inc. USA], DNQX (6,7dinitroquinoxaline 2,3-dione, Tocris Neuramin) and 5,7-DCKA (5,7-dichlorokynurenic acid, Brunschwig Chemie). Two compounds, MK-801 and DNQX, were dissolved in distilled water. The pH of MK-801 was adjusted to 7-8, while the pH of DNQX was adjusted to 9 in order to solubilise the compound completely. 5,7-DCKA was dissolved in 10% DMSO (dimethylsulfoxide) and adjusted to pH 7-8. In the control experiments the vehicle solutions were adjusted to the corresponding pH value of drugs. All drug solutions were administered i.p. and given in an equal volume (0.5 ml/injection).

Statistics

Data are expressed as medians. The effects of drug and vehicle treatment were evaluated statistically using the non-parametric Kruskal-Wallis one-way analysis of variance, followed by the Mann-Whitney U-test, with a level of P<0.05 being considered significant (Glantz, 1989).

Results

MK-801

In our preliminary experiments we observed that administration of MK-801, in a dose range of 0.1-10 mg/kg (i.p.), induced a pronounced locomotor dysfunction in both naive and morphine-dependent mice, consisting of wild running (hyperlocomotion), jumping, ataxia and convulsion. The incidence of these locomotor disturbances was dose related, while the higher doses of MK-801 (1-10 mg/kg) induced mainly ataxia and convulsions. However, concentrations of MK-801 below 0.1 mg/kg did not affect a normal behaviour of naive or morphinedependent mice.

In order to avoid the influence of disturbed locomotion on the withdrawal jumping in mice, we used MK-801 in a dose range of 1-80 pg/kg (i.p.) which did not affect locomotion in naive or morphine-dependent mice. MK-801 significantly attenuated the naloxone precipitated jumping behaviour in a dose range of 5-20 pg/kg, i.p. (Fig. 1). The higher doses of MK-801 (40-80 pg/kg, i.p.) were without significant effect on withdrawal jumping behaviour of mice

Fig. 1. Effect of MK-801 (pg/kg, i.p., 30 min before naloxone) on jumping behaviour precipitated by naloxone (4 mg/kg, i.p.) in morphine-dependent mice. Histograms represent medians and dots indicate individual animal scores (n=9-14 in MK-801-treated groups, control group = 36). * Significant difference (P<0.05) from the value in vehicle (saline)-pretreated animals.

DNQX

DNQX (20-80 mg/kg, i.p.) caused convulsions (observation period 60 min after drug administration) and/or death (observation period 72 h after drug administration) in morphinedependent mice. However, in naive mice neither convulsions nor death has been observed (Table 1). Therefore, in our further experiments we selected lower concentrations of DNQX (0.63-10 mg/kg, i.p.), which did not induce convulsions in morphine-dependent or naive mice. DNQX reduced naloxone-precipitated withdrawal jumping in mice in a dose range of 1.25-5 mg/kg, i.p. Similarly to MK-801, the dose-response curve of DNQX was also U-shaped (Fig. 2)

Table 1. Lethal (observation period 72 h after drug administration) and convulsant (observation period 60 min after drug administration) effect of DNQX (20-80 mg/kg, i.p.) on morphine-dependent and naive mice.

Fig. 2. Effect of DNQX (mg/kg, i.p., 30 min before naloxone) on jumping behaviour precipitated by naloxone (4 mg/kg, i.p.) in morphine-dependent mice. Histograms represent medians and dots indicate individual animal scores (n=6-8 in DNQX-treated groups, control group = 36).* Significant difference (P<0.05) from the value in the vehicle (saline)-pretreated animals

5, 7-DCKA
Administration of 5,7-DCKA in a relatively wide dose range (5-160 mg/kg, i.p.) did not affect the usual behavioral pattern of either naive or morphine-dependent mice. However, 5,7DCKA in a dose range of 20-40 mg/kg, i.p. significantly attenuated the naloxone-precipitated jumping behaviour in morphine-dependent mice (Fig. 3). The maximal effect was seen after treatment with 40 mg/kg 5,7-DCKA, while higher doses (80-160 mg/kg) were ineffective

Fig. 3. Effect of 5,7 DCKA (mg/kg, i.p., 30 min before naloxone) on jumping behaviour precipitated by naloxone (4 mg/kg, i.p.) in morphine-dependent mice. Histograms represent medians and dots indicate individual animal scores (n=5-7 in 5,7-DCKA-treated groups, control group = 16). * Significant difference (P<0.05) from the value in vehicle (DMSO)-pretreated animals.

Discussion
Our present findings demonstrate that antagonists of various glutamate receptors, such as MK-801 (non-competitive NMDA receptor antagonist), DNQX (competitive non-NMDA receptor antagonist) and 5,7-DCKA (antagonist of glycine site of NMDA receptors), attenuated the jumping withdrawal behaviour in morphine-dependent mice. These results are consistent with previous works showing that the non-selective EAA antagonist kynurenic acid (Krystal et al., 1990) and selective non-competitive and competitive NMDA receptor antagonists MK-801 and LY274614, respectively, suppressed the withdrawal signs in morphine-dependent rats (Koyuncuoglu et al., 1992; Rasmussen et al., 1991; Trujillo and Akil, 1991). However, in addition to NMDA receptors, this study indicates an involvement of the glycine site of NMDA receptors and non-NMDA receptors in drug withdrawal as well. Each of these three substances has a U-shaped dose-effect curve. Although unusual, a Ushaped dose-effect curve should not be considered as an exceptional phenomenon in the research of drug withdrawal. For example, buprenorphine in lower doses (0.01-0.5 mg/kg) precipitated abstention symptoms in morphine-dependent mice, while the higher doses (1-50 mg/kg) were less active or completely inactive (Lizasoain et al., 1991). The reason for the Ushaped dose effect curve is not known, but it could be assumed that a new mechanism(s) activated by higher drug concentrations may induce effects that are different from those produced by administration of lower doses. As regards the substances used in this study, it is known that higher doses of EAA receptor antagonists (in contrast to the lower doses), exerted a prominent excitatory/proconvulsant effect in naive animals (Jurson and Freed, 1990; Schoepp et al., 1990).

An additional relevant point is that morphine withdrawal in humans and animals is associated with an increase of neuronal and behavioral excitation (Wise and Bozarth, 1987). It seems that a further increase of central neuro-excitability induced by higher doses of drugs creates a new situation, which is presumably not favourable for an attenuation of morphine withdrawal. This might explain a failure of the higher doses of EAA receptor antagonists to attenuate morphine withdrawal and corresponding U-shaped doses-response curves. As a matter of fact, it should be expected that a further increase of neuro-excitability, due to elevated concentrations of EAA receptor antagonists, might even aggravate a withdrawal syndrome. Recent experiments support this idea, since administration of 0.1 mg/kg of MK-801 attenuated naloxone-precipitated withdrawal in rats, while a higher dose (0.3 mg/kg) increased the severity of the same abstention syndrome (Koyuncuoglu et al., 1992).

The mechanism of the suppressing effect of EAA antagonists on morphine withdrawal remains to be elucidated. There is evidence that the noradrenergic system plays an important role in opioid withdrawal. It has been reported that opioid withdrawal is associated with noradrenaline (NA) release (Laverty and Roth, 1980) and increased activity of noradrenergic cells in the locus coeruleus (Aghajanian, 1978; Valentino and Wehby, 1989). Several studies, in vitro and in vivo, indicate that NMDA antagonists may decrease NA release (Jones et al., 1987; Pittaluga and Raiteri, 1992) or activity (Burgard et al., 1989; Dahl and Sarvey, 1990; Loscher et al., 1991). In this respect, it is of importance that MK-801 significantly decreased the levels of NA and adrenaline in the amygdala of naive rats (Loscher et al., 1991). This might be of relevance, since the amygdala was implicated in emotion-related behaviour (Coulombe and White, 1978) and fear response (Hitchcock and Davis, 1986). An anxiolytic activity of NMDA antagonists, observed in naive mice (Trullas et al., 1989) and rats (Kehne et al., 1991), might play a role in the attenuation of withdrawal jumping in mice, since anxiety is a symptom of opioid withdrawal in humans and animals (Lal and Emmett-Oglesby, 1983).

An observation that relatively high doses of DNQX promote convulsions in morphinedependent mice, while the naive mice remained unaffected, deserves a comment. Recent data indicate that morphine dependence is associated with upregulation and/or supersensitivity of NMDA receptors (Marek et al., 1991). Other EAA receptors were not examined. A derangement of these, and possibly other EAA receptors, may explain a different reactivity to EAA receptor antagonists in morphine-dependent and naive mice.

In conclusion, the attenuating effect of EAA receptor antagonists on opioid withdrawal in mice might be due to complex changes in the activity of neurotransmitters and corresponding behavioral alterations in the addicted subjects. For further study the role of the NMDA glycine site is of particular interest, since drugs acting on this site are devoid of muscle-relaxant properties and possess significant anxiolytic effects.

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