Enhancement of antipsychoticlike properties of raclopride in rats using the selective serotonin2A receptor antagonist MDL 100,907

Enhancement of Antipsychoticlike Properties of Raclopride in Rats Using the Selective Serotonin2A Receptor Antagonist MDL 100,907 Marie-Louise G. Wadenberg, Paul B. Hicks, J. Travis Richter, and Keith A. Young Background: Selective suppression of conditioned avoidance response (CAR) is a standard animal screening test for predicting antipsychotic effect. Ability to suppress CAR is presumed to be due to antagonism at dopamine receptors, a property shared by all known antipsychotics. Methods: Using CAR behavior, in a conventional shuttlebox paradigm, as an index for antipsychotic efficacy, the effects of the selective serotonin2A receptor antagonist MDL 100,907 alone, and in combination with the dopamine D2 receptor antagonist raclopride, were studied in adult male Sprague-Dawley rats. Nonparametric procedures were employed for statistical evaluation. Results: MDL 100,907 (0.1–1.5 mg/kg, SC) alone did not suppress CAR in a manner predictive of antipsychotic activity; however, in the presence of an ED50 (0.14 mg/kg, SC) dose of raclopride, MDL 100,907 enhanced and prolonged the suppression of CAR. In the presence of a subthreshold (0.05 mg/kg, SC) dose of raclopride, MDL 100,907 induced a suppression of CAR. Conclusions: The results suggest that treatment with a selective serotonin2A receptor antagonist alone may not produce a robust antipsychotic effect; however, a selective serotonin2A receptor antagonist in the presence of a minimal dopamine D2 receptor blocking action could potentially be an adjunctive therapy resulting in improved antipsychotic efficacy and fewer extrapyramidal symptoms. Biol Psychiatry 1998;44:508 –515 © 1998 Society of Biological Psychiatry Key Words: Dopamine D2 receptor, serotonin2A receptor, conditioned avoidance response behavior, atypical antipsychotic drugs, rat

From the Department of Psychiatry, Scott & White Clinic and Memorial Hospital, Scott Sherwood and Brindley Foundation, Temple, Texas (MLGW, PBH, JTR); Neuroscience Laboratory, Central Texas Veterans Health Care System, Waco, Texas (KAY); and Texas A & M University Health Science Center College of Medicine, College Station, Texas (MLGW). Address reprint requests to Dr. M.-L. G. Wadenberg, Department of Physiology & Pharmacology, Karolinska Institute, S-171 77 Stockholm, Sweden. Received August 16, 1996; revised July 26, 1997; accepted August 8, 1997.

© 1998 Society of Biological Psychiatry

Introduction

T

raditionally, the suppression of conditioned avoidance response (CAR) has been used as an animal screening model to predict antipsychotic activity in drugs. All antipsychotics, both typical and atypical, are known to suppress CAR (Arnt 1982; Courvoisier 1956; Janssen et al 1966; Ponsluns 1962; Reynolds and Czudek 1995; Seeman 1992; Wadenberg et al 1993). The ability to suppress CAR is presumed to be due to antagonism at dopamine (DA) receptors, a property shared by all known antipsychotic medications (Arnt 1982). Drugs that act entirely through nondopaminergic mechanisms have not yet been shown to be antipsychotic. The functional mechanisms and site(s) of action in the mediation of CAR, however, are less well understood. Suppression of CAR has been viewed as reflecting an antipsychotic property of a drug rather than its extrapyramidal side effect (EPS) liability (Courvoisier et al 1957; Morpurgo 1965; Reynolds and Czudek 1995). Antipsychotic activity is thought to be mediated via mesocorticolimbic and not nigrostriatal DA pathways (Baldessarini 1996; Stevens 1973). A primary involvement of DA mesocorticolimbic pathways in the mediation of CAR is indicated by suppression of CAR after local application of the DA D2 receptor antagonist (2)sulpiride into the ventral striatum (nucleus accumbensshell), but not the dorsal striatum of the rat (Wadenberg et al 1990). The fact that suppression of CAR has been so accurate a predictor of antipsychotic response with typical antipsychotics and clozapine suggests that the use of CAR may provide some clues to which novel drugs would be expected to be antipsychotic. The most effective antipsychotic currently available is the atypical antipsychotic clozapine. The mechanism of action of clozapine is quite complex because of its high affinity interactions with a variety of brain neurotransmitter receptor sites. The favorable affinity ratio between the serotonin2A (5-HT2A) and the DA D2 receptor has been implicated as an important factor in the antipsychotic profile observed with clozapine (Meltzer 1989, 1995a, 1995b). Increasing the affinity for the 5-HT2A receptor in the presence of some affinity for the DA D2 receptor is 0006-3223/98/$19.00 PII S0006-3223(97)00424-1

5-HT2A and Antipsychoticlike Action

expected to result in antipsychotic activity with low potential for EPS, and an increased efficacy against so-called negative symptoms of schizophrenia. According to this concept a new generation of potentially antipsychotic drugs with combined DA D2/5-HT2 receptor antagonist properties (ie., risperidone, olanzapine, and seroquel) has been developed (O’Neill and Palacios 1994). Recently, MDL 100,907, a potent 5-HT2A receptor antagonist with a 300-fold selectivity for the 5-HT2A site over the DA D2, a1, and 5-HT2C receptors (Dudley et al 1990; So¨rensen et al 1993), was proposed as potentially antipsychotic. In a variety of animal (rat) models, MDL 100,907 mimics the atypical antipsychotic clozapine. For example, MDL 100,907 selectively reduces the number of active neurons in the dopamine mesolimbic A10 area after chronic administration (So¨rensen et al 1993), reverses amphetamine-induced hyperlocomotion (Moser et al 1996), and causes a greater increase in DA release in the medial prefrontal cortex than typical neuroleptics (Schmidt et al 1992; So¨rensen et al 1993). Furthermore, MDL 100,907 has been shown to be an effective antagonist of two animal models of drug-induced psychosis. Thus, MDL 100,907 diminishes the dopamine-releasing effect in the striatum induced by the amphetamine structural analogue 3,4methylenedioxymethamphetamine (MDMA), and reverses the disruption of prepulse inhibition (PPI) induced by the 5-HT2A/2C receptor agonist DOI (Schmidt et al 1993; Sipes and Geyer 1995). Despite the performance in those preclinical studies suggesting antipsychotic activity, results from clinical trials are not currently available. Using the conditioned avoidance response behavior as an index for antipsychotic efficacy, the present studies were performed to evaluate the potential of MDL 100,907 to be an antipsychotic agent. In addition, MDL 100,907 was tested as an adjunctive treatment with the specific DA D2 receptor antagonist raclopride (Ko¨hler et al 1985) to determine if MDL 100,907 would potentiate the suppressive effects of raclopride on the conditioned avoidance responding.

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Conditioned Avoidance Response Behavior Rats were trained and tested in a set of four computer-assisted (LabView, National Instruments, Austin, TX) shuttleboxes made of Plexiglas, equipped with a tilting grid floor with micro switch detection, and connected to a high-resistance power supply (Department of Biomedical Engineering, Scott and White Memorial Hospital, Temple, TX). The boxes were divided into two parts of equal size by a partition with one opening. Each box was placed in a sound-attenuated, ventilated enclosure provided with a dim background light. Upon presentation of the 80-dB white noise conditioned stimulus (CS) (White Noise Generator, Lafayette Instruments, Lafayette, IN), the animals had 10 sec to move into the other compartment of the shuttlebox. If the rat remained in the same compartment for more than 10 sec, the unconditioned stimulus (UCS) was presented as an intermittent electric shock in the tilting grid floor (four shocks, approximately 0.6 mA, in 10 sec; duration 0.5 sec; intershock interval 2.5 sec) until an escape response was performed. If the animal did not respond within 60 sec, the trial was terminated (escape failure). The intertrial interval (end of trial to start of new trial) varied at random between 20 and 40 sec. The following behavioral variables were recorded: avoidance (response to CS within 10 sec); escape (response to CS 1 UCS); escape failures; and intertrial crosses. The animals were trained for 5 consecutive days. Each training session consisted of approximately 20 trials randomly distributed over 15 min, with the limitation that any two trials were separated by at least 20 sec. Experimental manipulations were preceded by a pretest. All pretest and experimental sessions were run for 10 min. Within each group the same animals were tested repeatedly according to a crossover design (Li 1964). The number of trials in which an avoidance response occurred was divided by the total number of trials per session to determine the percent avoidance response. Comparisons between groups were based on the median percent avoidance scores.

Drugs Raclopride tartrate (Astra, So¨derta¨lje, Sweden) was dissolved in physiological saline. R-(1)-a(2,3-dimethoxyphenyl)-1-[2(4-fluorophenethyl)]-4-piperidinemethanol, MDL 100,907 (Hoechst Marion Roussel, Inc., Cincinnati, OH), was dissolved in a minimal amount of glacial acetic acid and made up to volume with isotonic glucose. Drugs were given subcutaneously in a volume of 2 mL/kg body weight.

Methods and Materials Animals Adult male Sprague-Dawley rats (280 –300 g) were purchased from Sasco King Inc. (Omaha, NE), and housed 2 per cage for a minimum of 3 days prior to CAR training. After behavioral training was initiated, rats were housed singly, maintained on a 12-hour on/off light cycle (lights off 09:00 AM). Rats were kept on a calorie-restricted diet throughout the study. Water was available ad libitum. Room temperature was maintained at 21 6 1°C. Relative humidity averaged 50%, but varied with ambient conditions. Experiments were performed between the 4th and 10th hour of the daylight cycle.

Statistics Statistical evaluation was performed by means of the Friedman two-way analysis of variance (ANOVA) by ranks, followed by the Wilcoxon matched-pairs signed-ranks test for appropriate post hoc comparisons (Siegel and Castellan 1988).

Results No escape failures were recorded under any treatment condition. The decrease in avoidance responding was always accompanied by a corresponding increase in es-

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Table 1. Effects of MDL 100,907 on the Performance of a Conditioned Avoidance Response Behavior in Rats Time after MDL 100,907 (min) 21

Dose (mg/kg )

Baseline

20

60

120

240

x24

0 0.1 0.5 1.0 1.5 x24

100 6 3 100 6 3a 100 6 1a 100 6 1a 100 6 0a 3.90a

100 6 0a 95 6 19a 100 6 3a 98 6 6a 100 6 0a 8.44a

100 6 3a 92 6 12b,c 95 6 8b,c 95 6 30b,c 100 6 0a 12.86b

100 6 2a 95 6 7a 98 6 3a 98 6 12a 100 6 1a 9.38a

100 6 0a 95 6 9a 100 6 1a 100 6 3a 100 6 3a 9.06a

3.06a 4.59a 9.83b 2.95a 6.61a

The selective 5-HT2A receptor antagonist MDL 100,907 (0.1–1.5 mg/kg, SC) or vehicle was administered 20 min before the first observation time. The medians (percent avoidance) 6 semi-interquartile range are shown based on repeated observations of the same 12 animals serving as their own controls in a crossover design (Li 1964). Statistical evaluation was performed by means of the Friedman two-way ANOVA followed by the Wilcoxon matched-pairs signed-ranks test for appropriate post hoc comparisons (Siegel and Castellan 1988). a p . .05. b p , .05. c Compared to controls at 60 min.

capes. Latencies to escape did not exceed the criterion time for escape at any occasion. There were no significant differences in latencies to escape between treatment groups.

Effects of MDL 100,907 on the Performance of Conditioned Avoidance Response in Rats The selective 5-HT2A receptor antagonist MDL 100,907 (0.1, 0.5, or 1.0 mg/kg, SC) produced a slight, but statistically significant (p , .05), suppression of the conditioned avoidance response at 60 min after administration when compared to control conditions. A higher dose (1.5 mg/kg) had no effect. No effects were found at any other time interval (Table 1).

Effects of MDL 100,907 on the Number of Intertrial Crosses in Conditioned Avoidance Response Performance in Rats The number of intertrial crosses decreased from the baseline testing period in all testing conditions. In the

vehicle controls, the decrease in intertrial crosses occurred after 20 min. There were fewer intertrial crosses by all rats treated with MDL 100,907 at 20 min after administration when compared to controls at the same time interval (p , .01). There were no significant differences of MDL 100,907 compared to controls on intertrial crosses at any other time interval (Table 2).

Effects of MDL 100,907 on Raclopride-Induced Suppression of Conditioned Avoidance Response in Rats The selective dopamine D2 receptor antagonist raclopride (0.14 mg/kg, SC; ED50 dose) alone suppressed the conditioned avoidance response at 30 min after administration (p , .05). This effect was completely gone at later observation time intervals. Pretreatment with MDL 100,907 (0.5–1.5 mg/kg, SC, administered 60 min before the first observation time) obviously enhanced and prolonged the raclopride-induced suppression of the conditioned avoidance response at 30, 90, and 240 min (MDL 100,907 0.5 mg/kg only) after raclopride administration

Table 2. Effects of MDL 100,907 on the Number of Intertrial Crosses in a Conditioned Avoidance Response Performance in Rats Time after MDL 100,907 (min) 21

Dose (mg/kg )

Baseline

20

60

120

240

0 0.1 0.5 1.0 1.5 x24

10 6 1 7 6 3a 9 6 5a 5.5 6 3a 9 6 5a 2.17a

11 6 4a 2 6 1c,d,e 5 6 2c,d,e 3 6 3d,e 3 6 3c,d,e 9.88b

4 6 3b,c 3 6 1b,c 4 6 2c,d 2.5 6 4a 3.5 6 3c,d 1.21a

4 6 3c,d 2 6 2c,d 2 6 2c,d 3 6 2a 3 6 3c,d 4.18a

3 6 2c,d 4 6 1a 4 6 3b,c 5 6 2a 3 6 3b,c 4.35a

x24 16.75d 11.91b 9.92b 4.50a 9.67b

The selective 5-HT2A receptor antagonist MDL 100,907 (0.1–1.5 mg/kg, SC) or vehicle was administered 20 min before the first observation time. The medians (number of intertrial crosses) 6 semi-interquartile range are shown based on repeated observations of the same 12 animals serving as their own controls in a crossover design (Li 1964). Statistical evaluation was performed as in Table 1. a p . .05. b p , .05. c Compared to baseline. d p , .01. e Compared to controls at 20 min.

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Figure 1. Effects of MDL 100,907 on raclopride-induced suppression of a conditioned avoidance response behavior in rats. The selective dopamine D2 receptor antagonist raclopride (racl) (0.14 mg/kg, SC; ED50 dose) and the selective 5-HT2A receptor antagonist MDL 100,907 (0.5–1.5 mg/kg, SC) were administered 30 and 60 min, respectively, before the first observation time. The medians 6 semi-interquartile range are shown based on repeated observations (30, 90, 240 min and 24 hours after raclopride) of the same 6 animals (top: avoidance percent; bottom: number of intertrial crosses) serving as their own controls in a crossover design (Li 1964). Statistical evaluation was performed by means of the Friedman two-way ANOVA followed by the Wilcoxon matched-pairs signed-ranks test for appropriate post hoc comparisons (Siegel and Castellan 1988). nsp . .05; *p , .05; **p , .01. veh, vehicle.

(p , .05; p , .01). The effect lasted up to 4 hours (Figure 1, top). The number of intertrial crosses was not altered by raclopride alone. Cotreatment with MDL 100,907 and raclopride, however, produced a decrease in the number of intertrial crosses at 30 and 90 min after raclopride administration (p , .05; p , .01), paralleling the time course of action observed for the suppression of the conditioned avoidance response under the same treatment conditions (Figure 1, bottom). The estimated ED50 dose for raclopride, according to the procedure of Litchfield and Wilcoxon (1949), was based on a dose–response study using a subset of animals

from the same batch as those participating in the present study.

Effects of MDL 100,907 Combined with a Subthreshold Dose of Raclopride on Conditioned Avoidance Response Behavior in Rats Raclopride given alone at a dose of 0.05 mg/kg, SC, had no effect on the conditioned avoidance response performance; however, cotreatment with MDL 100,907 (1 mg/ kg, SC, administered 60 min before the first observation time) and raclopride (0.05 mg/kg, SC, administered 30 min before the first observation time) produced a dramatic

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Figure 2. Effects of MDL 100,907 combined with a subthreshold dose of raclopride (racl) on a conditioned avoidance response behavior in rats. The selective dopamine D2 receptor antagonist raclopride (0.05 mg/kg, SC) and the selective 5-HT2A receptor antagonist MDL 100,907 (1 mg/kg, SC) were administered 30 and 60 min, respectively, before the first observation time. The medians 6 semi-interquartile range are shown based on repeated observations (30, 90, and 240 min after raclopride) of the same 10 animals (top: avoidance percent; bottom: number of intertrial crosses) serving as their own controls in a crossover design (Li 1964). Statistical evaluation was performed as in Figure 1. nsp . .05; *p , .05 compared to controls; **p , .01 compared to controls; † p , .05 compared to raclopride alone; †† p , .01 compared to raclopride alone. veh, vehicle.

suppression of the conditioned avoidance response compared to controls at 30, 90, and 240 min (p , .002, p , .02, p , .05, respectively), and compared to animals treated with raclopride alone at 30 and 240 min (p , .03) after raclopride (Figure 2, top). The number of intertrial crosses was not altered by raclopride alone at the 30- and 90-min time intervals, but was significantly higher than controls (p , .05) at the 240-min time interval. Cotreatment with MDL 100,907 and raclopride produced a decrease in the number of intertrial crosses compared to controls at 30 and 90 min after raclopride administration (p , .001, p , .004, respectively). The number of intertrial crosses in the combined treatment was also significantly lower compared to animals treated with raclopride alone at all time

intervals (p , .003, p , .002, p , .05, respectively) (Figure 2, bottom).

Discussion The conditioned avoidance response is an animal screening model with high reliability in predicting antipsychotic efficacy in drugs. In the present study, we have used the conditioned avoidance response model to determine the antipsychotic potential of the selective 5-HT2A receptor antagonist MDL 100,907. MDL 100,907 alone did not suppress CAR in rats in a manner that would predict sufficient antipsychotic activity; however, in the presence of an ED50 (0.14 mg/kg, SC) dose of raclopride, MDL 100,907 enhanced and prolonged the suppression of CAR.

5-HT2A and Antipsychoticlike Action

In the presence of a subthreshold (0.05 mg/kg, SC) dose of raclopride, MDL 100,907 induced the suppression of CAR. These effects lasted up to 4 hours. Using the raclopride and MDL 100,907 combination, the suppression of CAR was always paralleled by a corresponding decrease in intertrial crosses. The fact that MDL 100,907, alone, does not produce a robust suppression of CAR suggests a lack of sufficient antipsychotic property of this compound. In the presence of a subthreshold DA D2 receptor blockade, however, the addition of an effective 5-HT2A receptor blockade by MDL 100,907 produces a suppression of CAR identical to that observed with clinically effective antipsychotics. The effect is in all probability not due to pharmacokinetic interactions between the two compounds. A threefold range in doses of MDL 100,907 did not alter the duration of the effect of MDL 100,907. Furthermore, it was shown in a recent study that the 5-HT2A/2C receptor antagonist ritanserin (Leysen et al 1985), which is chemically different from MDL 100,907, also prolonged the suppression of CAR induced by raclopride (Wadenberg et al 1996). Finally, MDL 100,907 also potentiates suppression of CAR induced by the DA receptor antagonist haloperidol in a similar manner (Wadenberg et al 1997). The decrease in the number of intertrial crosses observed with the combined treatment of raclopride and MDL 100,907 is presumably an effect primarily mediated via DA mesolimbic pathways. It has previously been shown that exploratory locomotor activity is dependent on mesolimbic dopamine neural transmission (Fink and Smith 1980). Furthermore, local application of a DA D2 receptor antagonist into the nucleus accumbensshell, but not into the dorsolateral neostriatum, produces suppression of CAR with a concomitant decrease in intertrial crosses (Wadenberg et al 1990). It is known that clinically effective potent 5-HT2A receptor antagonists like clozapine that do suppress CAR (Arnt 1982; Davidson and Weidley 1976; Reynolds and Czudek 1995; Sanger 1984; Wadenberg et al 1993) also have measurable DA receptor antagonist activity. Positron emission tomography (PET) studies with schizophrenic patients show 40 – 65% DA D2 receptor occupancy for clozapine in clinically effective doses (Farde et al 1989). Clozapine is also claimed to have greater efficacy than typical neuroleptics, for both positive and negative symptoms of schizophrenia (Kane et al 1988). With clozapine as a prototype, a potent 5-HT2A/DA D2 receptor antagonist risperidone (Janssen et al 1988) has been introduced for clinical use. A recently published meta-analysis of the results of multiple double-blind trials indicates that risperidone is more effective than traditional antipsychotics at producing antipsychotic response. Specifically, a significant amelioration of negative symptoms has been reported

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(Carman et al 1995). Trazodone (selective 5-HT2A receptor antagonist) did not display sufficient overall antipsychotic efficacy in clinical trials; however, it was reported to have some efficacy against negative and depressionlike symptoms (Ayd and Settle 1982). Finally, a reduction of negative symptoms by ritanserin as an adjunctive therapy to a traditional neuroleptic has been reported (Duinkerke et al 1993; Reyntjens et al 1986; cf. also Kapur and Remington 1996). Taken together, these reports indicate that the 5-HT2 receptor blockade might be of importance in the amelioration of negative symptoms, and that the presence of some dopamine receptor antagonist activity, together with the 5-HT2 receptor blockade, is necessary for a complete (ie., therapeutic efficacy against both positive and negative symptoms) antipsychotic efficacy. 5-HT2A receptors are found in parts of the limbic system and basal ganglia, but have their highest density in brain neocortex (Hoyer et al 1994). Assuming that the CAR model involves primarily mesocorticolimbic DA pathways (see Introduction), which are thought to mediate cognitive and motivational functions, serotonin might well interact with the DA system in the suppression of CAR via 5-HT2A receptors on a neocortical or mesolimbic level. In support for this notion, electrophysiological and biochemical data have shown that the 5-HT2A/2C receptor antagonist ritanserin is able to confer an atypical (ie., producing similar effects as the atypical antipsychotic clozapine) profile on the DA D2 receptor antagonist raclopride. Thus, ritanserin has been shown to potentiate the stimulatory effects of the DA D2 receptor antagonist raclopride on neuronal activity and DA release selectively in the mesocorticolimbic dopaminergic system (Andersson et al 1995). Ritanserin also induced increased burst firing in mesocorticolimbic-projecting ventral tegmental DA neurons in rats treated with raclopride (Svensson et al 1993). Finally, it is interesting to note that the 5-HT2A/2C receptor antagonist ritanserin, although ineffective by itself, was recently shown to also enhance and prolong the suppression of CAR induced by the DA D2 receptor antagonist raclopride in rats; however, ritanserin was not able to induce suppression of CAR in the presence of a subthreshold dose of raclopride (Wadenberg et al 1996). The present data demonstrate that MDL 100,907, with a much greater selectivity for the 5-HT2A receptor than ritanserin, can induce suppression of CAR in the presence of a subthreshold dose of raclopride. This suggests that the 5-HT2A receptor subtype might be of special importance in the mediation of dopamine–serotonin interactions with respect to CAR behavior. Subsequently, the role of the 5-HT2A receptor in the suppression of CAR would predict a role for the 5-HT2A receptor in antipsychotic efficacy. Indeed, clozapine’s affinity for 5-HT2A receptors has

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specifically been implicated as responsible for its favorable clinical profile (O’Neill and Palacios 1994). In conclusion, the selective 5-HT2A receptor antagonist MDL 100,907 alone did not suppress CAR in rats in a manner that would predict antipsychotic activity; however, in the presence of an ED50 dose of raclopride, MDL 100,907 enhanced and prolonged the suppression of CAR. In the presence of a subthreshold dose of raclopride, MDL 100,907 induced a suppression of CAR. The results suggest that using a selective 5-HT2A receptor antagonist as adjunctive therapy to a minimal DA D2 receptor blocking activity may produce improved antipsychotic activity with potential for few extrapyramidal symptoms. The study was supported by grants from The Scott, Sherwood and Brindley Foundation (Grant #837), Temple, Texas; The Swedish Institute, SI (#303/40); The Swedish Medical Research Council, MRC (#B95-21R-11125); and The Swedish Research Council for Humanistic and Social Sciences, HSFR (#F1302/94); (Dr. M.-L. Wadenberg). MDL 100,907 and raclopride were generously provided by Hoechst Marion Roussel, Cincinnati, OH, USA and Astra, So¨derta¨lje, Sweden, respectively. Patricia Nun˜ez, LATG, RVT and her staff are greatly acknowledged for their professional animal care. For skillful preparation of the figures we want to thank Jeff Browning, MS.

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