Introduction

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There is increasing evidence that drug-induced changes in glutamatergic neurotransmission might play a role in the development of alcoholism (Tsai et al., 1995; Tsai and Coyle, 1998). Acutely, alcohol exerts antagonistic effects on N-methyl-D-aspartate (NMDA) receptor function, and chronic alcohol consumption leads to an increase in NMDA receptor-mediated neurotransmission, which is presumably not due to a simple increase in NMDA receptor density, but to differential up-regulation of different NMDA receptor subunits that could result in changes in the composition and functioning of NMDA receptor complexes (Tabakoff and Hoffman, 1996; Rudolph et al., 1997; Darstein et al., 1998; Faingold et al., 1998). Therefore, modulators of the glutamatergic/NMDA receptor system are now considered in the search for pharmacotherapeutic agents that may be useful in the treatment of alcohol dependence (Parsons et al., 1998).

The functional NMDA receptor antagonist acamprosate was effective in a series of preclinical studies (Spanagel et al., 1996; Hölter et al., 1997; Heyser et al., 1998) and clinical trials (Sass et al., 1996; Whitworth et al., 1996) in reducing alcohol consumption and relapse (for review, see Spanagel and Zieglgänsberger, 1997). Competitive NMDA receptor antagonists attenuated operant responding for ethanol without affecting baseline levels of water self-administration (Rassnick et al., 1992). However, the selectivity of such an effect was questioned by demonstrating that the competitive NMDA receptor antagonist CPPene decreased both ethanol and saccharin self-administration (Shelton and Balster, 1997). Uncompetitive NMDA receptor antagonists such as phencyclidine and memantine also reduced alcohol intake (Shelton and Balster, 1997; Piasecki et al., 1998) and prevented relapse (Hölter et al., 1996).

Encouraged by these findings, new compounds based on a cyclohexan structure with similar characteristics to memantine were developed, MRZ 2/579 (1-amino-1,3,3,5,5-pentamethyl-cyclohexane hydrochloride) being one of the most promising of these agents. MRZ 2/579 possesses rapid blocking/unblocking kinetics and a strong voltage dependence, neuroprotective properties in vitro and in vivo and antiparkinsonian-like activity, whereas it lacks psychotomimetic potential (Parsons et al., 1995, 1999; Danysz et al., 1997; Wenk et al., 1998). This profile suggests that MRZ 2/579 could prove to be useful in a wide range of central nervous system disorders that involve disturbances of glutamatergic neurotransmission because such biophysical properties may allow therapeutically relevant concentrations to block chronic, low-level pathological activation of NMDA receptors whereas leaving their synaptic activation intact (Parsons et al., 1998).

A long-term free-choice ethanol self-administration paradigm in Wistar rats is a promising animal model for the search for pharmacotherapeutic agents in relapse prevention. After several months of voluntary ethanol consumption, the drug-taking behavior following a deprivation phase is characterized by increased ethanol intake and preference and by changes in intake patterns, resulting in an immediate increase in consumption of highly concentrated ethanol solutions both under operant and nonoperant conditions (Spanagel et al., 1996; Hölter et al., 1997, 1998). The phenomenon of a transient increase in ethanol consumption and preference after a period of imposed ethanol abstinence has been termed the alcohol deprivation effect (ADE) and has been observed in several species, including humans (Sinclair and Senter, 1967; Sinclair, 1971; Burish et al., 1981). Because the ADE in a long-term ethanol self-administration model is characterized by an increased motivation to obtain ethanol, outlasts long abstinence phases, and is hardly modified by external stimuli such as taste adulteration or social factors, it can be regarded as an animal model of relapse and possibly of craving (Wolffgramm and Heyne, 1991; Spanagel et al., 1996; Spanagel and Hölter, 1999). Moreover, it has been shown that the ADE can be suppressed by acamprosate (Spanagel et al., 1996; Hölter et al., 1997; Heyser et al., 1998) and naltrexone (Kornet et al., 1991; Hölter and Spanagel, 1999), compounds that are also effective in reducing relapse in abstinent alcoholics (O'Malley et al., 1992; Volpicelli et al., 1992; Sass et al., 1996; Whitworth et al., 1996).

The aim of the present study was to investigate the effects of MRZ 2/579 on ethanol-drinking behavior in long-term ethanol-experienced rats. Thus, we tested the effects of chronic MRZ 2/579 on the ADE in a home cage-drinking paradigm, and the effects of acute MRZ 2/579 in an operant ethanol self-administration paradigm because this allows for detailed analysis of the time course of the acute drug effects without disturbing the animals. In an additional experiment, MRZ 2/579 was tested for generalization to the ethanol cue in a drug discrimination test because previous studies have shown that uncompetitive NMDA-receptor antagonists dose dependently substitute for the ethanol cue (Colombo and Grant, 1992; Shelton and Balster, 1994; Hundt et al., 1998).

	Materials and Methods

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Subjects. Fourty-two male Wistar rats (Max Planck Institute of Biochemistry, Martinsried, Germany), weighing 220 to 250 g at the beginning of the experiment, were used in this study. All animals were housed individually in standard hanging rodent cages with tap water ad libitum. Animals that were used for the long-term voluntary ethanol self-administration and for pharmacokinetic experiments received food ad libitum. The weight of animals used for the drug discimination study was maintained at ~80% of the weight under free-feeding conditions by restricting their daily food consumption. Artificial light was provided daily from 7:00 AM until 7:00 PM. and room temperature and humidity were kept constant (temperature: 23 ± 1°C; humidity: 60 ± 5%). The experiments were approved by the Committee on Animal Care and Use of the relevant local governmental body and carried out following the German Law on the Protection of Animals.

Long-Term Ethanol Self-Administration. After 1 week of habituation to the animal room, all rats were given continuous access to tap water, and 5, 10, and 20% (v/v) ethanol solutions in their home cages. Spillage and evaporation were minimized by the use of bottle caps with ball bearings (Ehret, Emmendingen, Germany). With this procedure the ethanol concentration in any of the solutions stayed constant for at least 1 week (Hölter et al., 1998). All drinking solutions were renewed weekly and at that time the positions of the four bottles were changed to avoid location preferences. After 8 weeks of continuous access, ethanol solutions were repeatedly withdrawn for 3 days (deprivation phase) every 4 weeks.

ADE Measurement in Nonoperant Self-Administration Paradigm. In the first experiment, we determined the effects of chronic MRZ 2/579 treatment on the alcohol deprivation effect in rats with 11 months of ethanol experience in the long-term paradigm described above.

Operant Chambers. Eight operant chambers (Med Associates Inc., East Fairfield, VT) situated in sound attenuating cubicles with background noise provided by a fan were used for the second experiment. Each chamber was equipped with a house light, two levers (one on each side of the chamber), two liquid dispensers adjacent to the levers, and a food rack at the back wall. The pressing of one lever resulted in the delivery of a drop of tap water and pressing of the other lever resulted in the delivery of a drop of 20% (v/v) ethanol (FR1FR1). The volume per drop was 25 to 30 µl. The chambers were controlled and data automatically recorded by a personal computer.

Training and Testing Procedure in Operant Self-Administration Paradigm. In this experiment, we determined the effects of acute MRZ 2/579 treatment on the alcohol deprivation effect in rats with at least 16 months of ethanol experience. After 5 months of ethanol experience in the long-term, four-bottle paradigm described above, the operant chamber sessions began. All sessions were started at 10:00 AM and lasted 23 h. During all sessions water and 20% ethanol were concurrently available on an FR1FR1 schedule and food was available ad libitum from the food rack. The house light in the chambers was turned off at 7:00 PM and on at 7:00 AM to keep the animals in their regular light/dark cycle. The animals were tested in the chambers once a week.

Drug Discrimination. Standard operant chambers (Coulburn Instruments, Lehigh Valley, PA) were used for drug discrimination training. Each chamber was equipped with two levers, one on either side and equidistant from a food cup. The chambers were contained in ventilated, sound-attenuated cubicles equipped with a house light. The experiments were controlled by a computer (Med Associates Inc.) with a modified version of the software package described by Spencer and Emmett-Oglesby (1985).

Determination of MRZ 2/579 Concentrations. To determine brain and serum levels of MRZ 2/579 after chronic infusion, four age-matched ethanol-naive control rats with 11 months of individual housing were briefly anaesthetized with halothane and mini-osmotic pumps (model 2 ML1, pumping rate 10 µl/h for 1 week; Alzet) filled with MRZ 2/579 (40 mg/ml, resulting in a dose of 9.6 mg/day) were implanted s.c. Six days after surgery, the animals were sacrificed by decapitation after halothane anesthesia, a blood sample was taken, and the brain was removed.

Drugs. Ethanol-drinking solutions were made up from 96% pure ethanol diluted with tap water to the different concentrations. For injections, 96% pure ethanol was diluted with 0.9% saline to a 12% (v/v) solution. MRZ 2/579 (Merz) was dissolved in water ad iniectabilia (Braun, Melsungen, Germany). Drug doses refer to the weight of the salt.

Data Analysis. The effects of chronic MRZ 2/579 treatment on the occurrence of an ADE were analyzed by three-way ANOVA with repeated measures (treatment × ADE × days). Given that there were no significant group differences in baseline levels, treatment effects on postabstinence days were assessed by two-way ANOVA with repeated measures (treatment × days). Drug effects on food and fluid consumption were analyzed by two-way ANOVA with repeated measures (treatment × days). Concerning operant chamber data, dose-response effects of drug treatment on ethanol intake, ethanol preference, and the total number of lever presses were assessed individually for each group by one-way ANOVA with repeated measures. Dose-response effects on accumulated first-hour data were analyzed by two-way ANOVA with randomized blocks (dose × time interval). Lever latency data of drug discrimination testing was analyzed by a one-way ANOVA. The chosen level of significance was P  .05. Fisher's least-significant difference (Protected-t) test was applied for post hoc comparisons when appropriate.

	Results

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Effects of Chronic MRZ 2/579 on ADE. After 2 weeks of abstinence, a significant ADE occurred in the vehicle group (Fig. 1), characterized by a transient increase in ethanol intake (factor ADE: F_1,42 = 12.59, P < .01) and a transient increase in ethanol preference (factor ADE: F_1,42 = 7.49, P < .05). Chronic MRZ 2/579 treatment completely suppressed the occurrence of an ADE in terms of the increased ethanol intake (interaction treatment × ADE: F_1,42 = 12.48, P < .01), but did not affect ethanol preference (interaction treatment × ADE: F_1,42 = 0.05, NS). After a second alcohol deprivation phase of 2 weeks, again an ADE occurred in the vehicle group, but the suppressant effect on ethanol intake in the MRZ 2/579 group was no longer present in the absence of the drug (Fig. 1). Water and food intake were not affected by chronic MRZ 2/579 treatment (Fig. 2). After 6 days of chronic infusion, serum levels of MRZ 2/579 were 0.52 ± 0.046 µM and its concentration in brain tissue was 87.8 ± 7.79 µmol/kg tissue (mean ± S.E., n = 4).

View larger version (18K): [in this window] [in a new window]   Fig. 1.   Effects of chronic MRZ 2/579 treatment on the ADE. Top, effect of treatment on ethanol intake during the ADE; bottom, effect of treatment on ethanol preference during the ADE. Note that preabstinence data represent baseline levels of each group before drug treatment. AD, alcohol deprivation for 2 weeks. Data are presented as means + S.E. (n = 8). **P < .01, significant difference versus vehicle.

View larger version (17K): [in this window] [in a new window]   Fig. 2.   Effects of chronic MRZ 2/579 treatment on water (top) and food intake (bottom) during the ADE. Note that preabstinence data represent baseline levels of each group before drug treatment. AD, alcohol deprivation for 2 weeks. Data are presented as means + S.E. (n = 8).

Effects of Acute MRZ 2/579 on ADE. Previous experiments in this paradigm have shown that drug effects are most pronounced at the beginning of the session (Hölter et al., 1997). Furthermore, at lower doses drug effects can wear off before the end of the session. Thus, data were analyzed for both the first hour and the cumulative total for the 23-h test session.

View larger version (27K): [in this window] [in a new window]   Fig. 3.   Effects of acute MRZ 2/579 treatment on basal drinking and the ADE in an operant 23-h session. Left, effects during the first hour; right, effects during the whole 23-h session. Top, effects on ethanol intake; middle, effects on ethanol preference; and bottom, effects on total lever-pressing activity. Data are presented as means + S.E. (n = 6-8). *P < .05, **P < .01, significant difference versus vehicle.

View larger version (24K): [in this window] [in a new window]   Fig. 4.   Effects of acute MRZ 2/579 treatment on basal drinking (right) and the ADE (left) during the first hour of the operant 23-h session. Top, effects on lever pressing for ethanol; bottom, effects on lever pressing for water. Data are presented as means + S.E. (n = 6-8). *P < .05, **P < .01, significant difference versus vehicle.

Effects of MRZ 2/579 on Drug Discrimination. As shown in Fig. 5, MRZ 2/579 dose dependently generalized to the ethanol cue in rats trained to discriminate ethanol from saline. Drug treatment also affected lever response latency (F_4,34 = 3.99, P < .05), but only at the highest dose tested (4 mg/kg) (Fig. 5, bottom). At this dose, only four of eight animals completed the test session.

View larger version (13K): [in this window] [in a new window]   Fig. 5.   Results of discrimination testing with different doses of MRZ 2/579 in rats discriminating ethanol from saline. The mean percentage of responses on the ethanol lever was used as a measure of ethanol-appropriate responding (top). Drug effects on response latency (bottom). Data are presented as means + S.E. (n = 4-8). *P < .05, significant difference versus vehicle.

	Discussion

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The results of this study show that: 1) chronic MRZ 2/579 treatment abolished the increased ethanol intake during the ADE in long-term ethanol-experienced rats without affecting water and food intake; 2) this effect required the presence of the drug and did not affect a second ADE in the absence of the drug; 3) acute MRZ 2/579 treatment had a short-lasting, reductive effect on ethanol intake both during basal drinking and during the ADE without affecting water intake; and 4) in the dose range used for acute treatment, MRZ 2/579 generalized to the ethanol cue in a drug discrimination test.

The acute effects of MRZ 2/579 were only significant at the beginning of the session, probably due to the short half-life of this drug. Plasma and brain extracellular fluid t_1/2 values for a dose of 5 mg/kg i.p. are 1.63 and 1.95 h, respectively (Hesselink et al., 1999). The dose range used in the present study was even lower (1-4 mg/kg). With longer-acting drugs, reductions in lever pressing for ethanol also can be seen at later hours of the session (Hölter et al., 1997; Hölter and Spanagel, 1999).

At the doses used in this study, both acute and chronic administration of MRZ 2/579 specifically reduced ethanol intake without affecting water or food intake. Similar doses of acute MRZ 2/579 (2.5-7.5 mg/kg) also inhibited the reinforcing effects of morphine (Popik et al., 1998) and starting at 1 mg/kg, NMDA receptors were blocked sufficiently to provide neuroprotection from NMDA toxicity in the nucleus basalis magnocellularis (Wenk et al., 1998). Higher doses of acute MRZ 2/579 were not used because they were likely to induce unspecific reductions of lever-pressing activity due to an impairment of motor coordination (Danysz et al., 1997). However, chronic s.c. infusion of MRZ 2/579 at a dose leading to steady-state brain levels that are sufficient to inhibit NMDA receptors (Danysz et al., 1997) was well tolerated without visible side effects, which is in line with previous findings with memantine, another uncompetitive NMDA receptor antagonist with similar biophysical properties (Parsons et al., 1995; Hölter et al., 1996). Although MRZ 2/579 is strongly accumulated in brain tissue, free brain (extracellular fluid) levels are only 2-fold lower than free serum levels as found by microdialysis with in vivo recovery (Hesselink et al., 1999). Thus, considering the serum levels attained in this study after chronic infusion, free brain levels can be estimated to have reached 0.26 µM. Higher concentrations can be expected after acute injections (e.g., at 4 mg/kg) because injection of 5 mg/kg i.p. resulted in a free brain concentration of 0.70 µM (Hesselink et al., 1999). MRZ 2/579 selectively blocked NMDA receptor-mediated responses with an IC₅₀ value of 1.11 µM in cultured hippocampal neurons (Hesselink et al., 1999) and with an IC₅₀ value of 0.42 µM in Xenopus oocytes (Parsons et al., 1999). Hence, the doses used in the present study were most likely sufficient for selective NMDA receptor blockade. Therefore, we conclude that the observed behavioral effect of selective reduction of ethanol intake is due to selective NMDA receptor blockade.

Because NMDA receptors are involved in the mediation of learning and memory processes, it could have been possible that the suppression of the ADE by chronic MRZ 2/579 had long-lasting consequences leading also to a reduction of subsequent ADEs in the absence of the drug, but this was not the case. Thus, the suppression of one ADE by chronic MRZ 2/579 did not lead to extinction of the ADE.

The finding that MRZ 2/579 generalized to the ethanol cue in the drug discrimination test suggests that the reductive effects of this drug on ethanol intake might be due, at least in part, to a substitution of the stimulus properties of alcohol. This is not surprising, bearing in mind that alcohol also antagonizes NMDA receptor function, and it fits in with the lack of any effects in the absence of the drug (see above). These data are in line with previous drug discrimination studies that showed that a variety of uncompetitive NMDA receptor antagonists generalized to the ethanol cue (Colombo and Grant, 1992; Shelton and Balster, 1994; Hundt et al., 1998). Moreover, it has recently been demonstrated that ketamine produced dose-related ethanol-like subjective effects in detoxified alcoholics (Krystal et al., 1998), supporting the clinical importance of NMDA receptor antagonism among the mechanisms underlying the subjective effects of ethanol in humans.

In summary, in the treatment regimen used in this study, MRZ 2/579 specifically reduced ethanol intake without visible side effects independent of the strength of the motivation to consume ethanol. We conclude that this novel uncompetitive NMDA-receptor antagonist might exert its ethanol consumption-reducing effect by substituting for some of the subjective effects of ethanol. This does not necessarily reduce the potential therapeutic usefulness of this compound in the treatment of alcohol dependence because one of the main goals of treatment is the reduction of alcohol consumption to reduce the pathological consequences of chronic alcohol abuse. In contrast, the lack of obvious side effects of chronic treatment with MRZ 2/579 within the therapeutic dose range encourages hope that this drug might be therapeutically applicable. Further studies are necessary to clarify this issue.