Introduction

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Drug discrimination procedures have been used successfully to characterize the pharmacological properties of many psychotropic drugs including opiates, psychomotor stimulants, anxiolytics and compounds acting through different neurotransmitter receptor subtypes. Through the use of appropriate tests of substitution for, and antagonism of, a training drug, it is possible, using drug discrimination, to make precise comparisons between different drugs and to investigate their mechanism of action.

The discriminative stimulus effects of directly (Colpaert et al., 1975; Appel et al., 1988; Sanger et al., 1997a) and indirectly (Colpaert et al., 1976a; Young and Glennon, 1986) acting dopamine agonists have been investigated in some detail. In contrast, there are only a few reports dealing with the stimulus effects of dopamine antagonists (Nielsen, 1993). Stewart (1962) trained rats to discriminate 4 mg/kg of chlorpromazine in a shock-escape procedure although Overton (1966) was unsuccessful in attempting to train rats to discriminate a slightly higher dose of this drug (5 mg/kg) in a T-maze. In lever pressing operant procedures three studies (Barry et al., 1974; Colpaert et al., 1976b; Goas and Boston, 1978) have found chlorpromazine to serve effectively as a discriminative stimulus and Goas and Boston (1978) also reported that chlorpromazine-induced stimulus control generalized to haloperidol. More recently, McElroy et al., (1989) trained rats to discriminate a dose of 0.05 mg/kg of haloperidol. The stimulus produced by this drug was dose-dependent and generalized fully to chlorpromazine.

These reports indicate that, under appropriate conditions, dopamine antagonists serve effectively as discriminative stimuli in rats. Our study was therefore carried out to investigate the discriminative stimulus effects of a dopamine antagonist in detail. The substance chosen as the training drug was the benzamide derivative tiapride, which is used clinically in the treatment of geriatric agitation, alcohol dependence and some forms of dyskinetic movement (Steele et al., 1993; Peters and Faulds, 1994). Unlike most dopamine antagonists that have affinities for other central neurotransmitter receptors, tiapride binds selectively to D₂ and D₃ dopamine receptors in vitro and in vivo (Jenner et al., 1978; Bischoff et al., 1982; Chivers et al., 1988). Tiapride blocks the behavioral effects of dopamine agonists (Jenner et al., 1978; Puech et al., 1978; Steele et al., 1993). It has not been reported to induce catalepsy, produces limited sedation and there is some evidence that it may act selectively on limbic structures (Jenner et al., 1978; Bischoff et al., 1982; Sanger et al., 1997b). Because of this pharmacological profile (i.e., receptor selectivity, limited sedation) tiapride was considered an appropriate compound for use in a drug discrimination procedure. In our study, rats were first trained to discriminate between tiapride and vehicle and then drug substitution experiments were conducted to compare various dopamine receptor antagonists and other reference compounds for their capacity to reproduce the tiapride cue. Additional studies were then conducted to evaluate the extent to which the stimulus effects of tiapride could be attenuated by pretreatment with direct and indirect dopamine agonists.

	Methods

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Subjects. Twenty-one male Wistar rats obtained from IFFA CREDO (L'Arbresle, France) were used. They weighed 180 to 200 g when obtained from the suppliers and were allowed to gain weight during the experiment so that by the end they weighed between 400 and 500 g. Animals were restricted to the food obtained during sessions and a daily ration of 15 to 20 g of standard laboratory food given at the end of each day and over the weekend. Housing was in individual cages under standard laboratory conditions with lights on between 7:00 A.M. and 7:00 P.M.. Animals were housed and tested in accordance with current French legislation on animal experimentation.

Discrimination training. The method used for drug discrimination training was essentially that developed by Colpaert and co-workers (e.g., Colpaert et al., 1975). The animals were trained to press both levers in standard two-lever operant test chambers (MED-Associates, Inc. Georgia, VT) to obtain 45-mg food pellets (Noyes, formula P, Lancaster, NH). Only one lever was operational on any particular session. Initially, daily sessions were 30 min in duration and every lever press produced a pellet. The schedule requirement was then gradually increased until 10 lever presses were required for each pellet (fixed ratio 10: FR10). At this stage the daily session duration was reduced to 15 min and injections were started. Rats were given injections of either tiapride or physiological saline, 30 min before sessions, in the daily sequence SDDSSDSSDD (D = drug, S = saline). In some of the rats, responding on the right lever after drug injection and the left lever after saline injection was reinforced with food. For the other animals this relationship was reversed.

Drug testing. The training procedure was continued until the following criterion was met for a period of 10 successive days: the total number of responses on both levers before the first reinforcement was less than 15. When the criterion for successful discriminative control was reached, substitution and antagonism tests were carried out. During these tests the animal was placed in the test chamber at the appropriate time after injection and was reinforced after the first ratio of 10 responses had been completed on either lever. For the remainder of the 15-min session, responding on the lever on which the first 10 responses had occurred continued to be reinforced according to the FR10 schedule. Substitution tests were first carried out in each rat with several doses of tiapride before other drugs were tested. The tiapride dose-response function was also re-established approximately 18 mo later to investigate the possibility that tolerance or sensitization to the training drug had occurred.

Data analysis. The lever chosen and the total number of lever presses were recorded during each 15-min session. During tests of substitution and antagonism the results were expressed as the percentage of rats choosing the lever associated with the training dose of tiapride and the rate of responding expressed as a percentage of the rate on the preceding saline sessions. Drug effects on rates of lever pressing were analyzed statistically using Friedman analyses of variance followed by Wilcoxon matched-pairs signed-ranks tests. ED₅₀ values for the potencies of different drugs to substitute for tiapride (i.e., doses at which 50% of rats tested chose the tiapride associated-lever) or to decrease rates of responding (i.e., doses that reduced response rates to 50% of control values) were calculated using log-probit analysis.

Drugs. The drugs used were tiapride hydrochloride, amisulpride, sulpiride, sultopride, metoclopramide hydrochloride, raclopride, clebopride maleate, remoxipride hydrochloride, haloperidol, clozapine, olanzapine, risperidone, chlorpromazine, thioridazine hydrochloride, pimozide, d-amphetamine sulfate, apomorphine hydrochloride, 7-OH-DPAT hydrobromide, bromocriptine mesylate, quinpirole hydrochloride, SKF 38393 hydrochloride, nomifensine maleate, ethanol, cocaine hydrochloride, morphine sulfate, diazepam, lorazepam and dizocilpine maleate. Raclopride was donated by Astra (Sodertalije, Sweden), risperidone by Janssen (Beerse, Belgium) and olanzapine by Lilly (Indianapolis, IN). All other drugs were obtained from commercial sources or synthesized at Synthelabo Recherche. All doses are expressed as the bases and injection volume was 1 or 2 ml/kg except for ethanol (13 ml/kg). Drugs were injected as solutions or suspensions in saline containing two drops of Tween 80. All injections were given i.p. 30 min before the start of the sessions except for amisulpride, sulpiride, sultopride and pimozide (i.p., 60 min), ethanol (i.p., 10 min), cocaine (i.p., 15 min), apomorphine and morphine (s.c., 30 min).

	Results

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Effects of tiapride. For the 12 rats that began discrimination training at 10 mg/kg of tiapride, the discrimination was not acquired after 17 training sessions. The dose was then increased to 20 mg/kg and after a further 34 sessions to 30 mg/kg. Nine of these rats achieved the accuracy criterion after a further 18 to 64 training sessions. The other three rats were dropped from the experiment when it became apparent that they were not acquiring a discrimination. For the nine rats that began discrimination training at 30 mg/kg of tiapride, the mean number of sessions to criterion was 76 (range 42-149).

View larger version (15K): [in this window] [in a new window]   Fig. 1.   Effects of tiapride after initial training and 18 mo later (same rats). Data represent the percentage of rats selecting the tiapride-associated lever and the rates of responding expressed as a percentage of control rates obtained from the sessions preceding the test sessions. Points are means ± S.E.M. based on 13 rats. **P < .01 compared to saline control values.

Effects of dopamine antagonists. Several dopamine antagonists were evaluated for their capacity to reproduce the discriminative stimulus of tiapride. Figure 2 shows results for benzamide drugs, results for other dopamine antagonists are shown in figure 3. ED₅₀ values for the dose-response curves are presented in table 1.

View larger version (30K): [in this window] [in a new window]   Fig. 2.   Effects of benzamide dopamine antagonists in rats trained to discriminate tiapride (30 mg/kg) from saline. Data represent the percentage of rats selecting the tiapride-associated lever and the rates of responding expressed as a percentage of control rates obtained from the sessions preceding the test sessions. Points are means ± S.E.M. based on 6 to 8 rats except for tiapride where n = 18. *P < .05; **P < .01 compared to saline control values.

View larger version (24K): [in this window] [in a new window]   Fig. 3.   Effects of nonbenzamide dopamine antagonists in rats trained to discriminate tiapride (30 mg/kg) from saline. Data represent the percentage of rats selecting the tiapride-associated lever and the rates of responding expressed as a percentage of control rates obtained from the sessions preceding the test sessions. Points are means ± S.E.M. based on six to eight rats. *P < .05; **P < .01 compared to saline control values.

Effects of other reference compounds. Several reference compounds, including an opioid agonist (morphine), a dopamine agonist (apomorphine), a dopamine reuptake inhibitor (cocaine), a NMDA antagonist (dizocilpine), two benzodiazepines (diazepam and lorazepam) and ethanol were evaluated for tiapride-like discriminative stimulus effects (table 2). Ethanol was the only drug tested to produce more than 50% selection of the tiapride-associated lever.

Effects of pretreatment with dopamine agonists. Several direct and indirect dopamine agonists were evaluated for their ability to attenuate the stimulus effects of tiapride. As shown in figure 4, pretreatment with the indirect dopamine agonists, d-amphetamine and nomifensine, dose-dependently antagonized the stimulus effect of the training dose of tiapride. The dopamine agonists, quinpirole, 7-OH-DPAT, bromocriptine and apomorphine, partially blocked the stimulus effect of the training dose of tiapride whereas the D₁ dopamine agonist, SKF 38393, did not antagonize the discriminative stimulus effects of tiapride (fig. 4, top). The rate-decreasing effects of the dopamine agonists in combination with tiapride prevented testing of higher doses (fig. 4, bottom).

View larger version (20K): [in this window] [in a new window]   Fig. 4.   Effects of the training dose of tiapride after pretreatment with several direct and indirect dopamine agonists in rats trained to discriminate tiapride from saline. Data represent the percentage of rats selecting the tiapride-associated lever and the rates of responding expressed as a percentage of control rates obtained from the sessions preceding the test sessions. Points are means ± S.E.M. based on seven to eight rats. *P < .05; **P < .01 compared to tiapride (30 mg/kg) control values (T) obtained from the tiapride sessions preceding the test sessions.

View larger version (16K): [in this window] [in a new window]   Fig. 5.   Effects of tiapride after pretreatment with d-amphetamine in rats trained to discriminate tiapride from saline. Data represent the percentage of rats selecting the tiapride-associated lever and the rates of responding expressed as a percentage of control rates obtained from the sessions preceding the test sessions. Points are means ± S.E.M. based on 8 rats. *P < .05; **P < .01 compared to saline control values.

	Discussion

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Several previous studies reported that training animals to discriminate dopamine antagonists, such as haloperidol and chlorpromazine, was difficult, and it was hypothesized that the general depressant effects of the drugs and/or the long duration of action of haloperidol may have impaired the acquisition of the discrimination (Stewart, 1962; Barry et al., 1974; Colpaert et al., 1976b; Goas and Boston, 1978; McElroy et al., 1989). As tiapride has been reported to have dopamine antagonist activity with relatively limited depressant effects (Steele et al., 1993), it was chosen as the training drug in our study, in the hope that it would provide a more discriminable stimulus than other dopamine antagonists. The results of our study show that tiapride produces a discriminative stimulus that can reliably control responding in rats. Similar to results with other discriminable drugs, tiapride engendered dose-dependent increases in the percentage of rats selecting the drug-associated lever. Further, it retained this ability over the 18-mo period covered by the study with no differences between the initial and last dose-effect curves. However, as previously observed with haloperidol and chlorpromazine, tiapride required a relatively lengthy training period (mean of 76 sessions) to function as a discriminative stimulus.

Evidence from previous research has showed that chlorpromazine and haloperidol cross-generalized and that the indirect dopamine agonists amphetamine and cocaine blocked the haloperidol discriminative stimulus, suggesting that the discriminative stimulus effects of haloperidol are mediated by dopamine receptors (Goas and Boston, 1978; McElroy et al., 1989); however, only a limited number of dopaminergic and nondopaminergic drugs were tested in these studies. In our study, we investigated a number of reference compounds for their capacity to reproduce or to antagonize the discriminative stimulus of tiapride. Results indicate that D₂/D₃ dopamine receptors mediate the discriminative stimulus effects of tiapride. First, several dopamine antagonists substituted for the discriminative stimulus effect of tiapride. Second, several nondopaminergic drugs produced responding predominantly on the saline-associated lever. Finally, direct D₂/D₃ but not D₁ dopamine agonists reduced and indirect dopamine agonists blocked the discriminative stimulus effects of tiapride.

The dopamine antagonists examined in substitution tests included benzamide (tiapride, amisulpride, sulpiride, sultopride, clebopride, raclopride, metoclopramide and remoxipride) and nonbenzamide (clozapine, olanzapine, pimozide, haloperidol, thioridazine, chlorpromazine, risperidone) derivatives. All dopamine antagonists, except clozapine, fully or partially (averaging a maximum substitution of about 70%) produced dose-related increases in the percentage of rats selecting the tiapride-associated lever. The lack of substitution by clozapine is consistent with previous findings that clozapine itself produces discriminative stimulus effects that are not mediated by dopaminergic mechanisms but are related to cholinergic muscarinic antagonism and 5HT₂ receptor blockade (Goas and Boston, 1978; Browne and Koe, 1982; Nielsen, 1988; Hoenicke et al., 1992, Wiley and Porter, 1992). It is of interest that olanzapine, which structurally and pharmacologically resembles clozapine and has been found to substitute for clozapine (Moore et al., 1992), produced partial substitution for tiapride. Although both clozapine and olanzapine show affinities for muscarinic, adrenergic, histamine and serotonin receptors, olanzapine exhibits higher affinity for D₂ dopamine receptors than clozapine (Ashby and Wang, 1996; Bymaster et al., 1996).

Tiapride-like stimulus effects of dopamine antagonists were observed at doses that markedly reduced rates of responding. The question thus arises as to whether drugs substitute for tiapride on the basis of their rate-decreasing effects. To examine further the pharmacological specificity of tiapride discrimination, several reference compounds were evaluated for tiapride-like discriminative stimulus effects (table 2). The finding that drugs such as morphine and lorazepam did not substitute for tiapride at doses that decreased rate of responding indicates that the discriminative stimulus effects of tiapride are not mediated by nonspecific depressant effects. The pharmacological specificity of tiapride discrimination is further supported by the finding that ethanol was the only drug tested to produce more than 50% selection of the tiapride-associated lever. In addition to its action at GABA_A and NMDA receptors, ethanol also interacts with other transmitters including dopamine (Deitrich et al., 1989; Grant, 1994). Specifically, ethanol stimulates dopamine release in terminal dopaminergic areas (Di Chiara and Imperato, 1988). However, morphine and cocaine which also increase extracellular dopamine concentrations (Di Chiara and Imperato, 1988) did not substitute for tiapride in our study. The mechanism of the partial substitution obtained with ethanol is, thus, not clear, although it is tempting to speculate that it may be related to the therapeutic efficacy of tiapride in the management of alcohol abuse (Steele et al., 1993; Peters and Faulds, 1994; Shaw et al., 1994).

Studies were conducted to evaluate the ability of several direct and indirect dopamine agonists to attenuate the stimulus effects of tiapride. The nonselective dopamine agonist, apomorphine, and the D₂/D₃ dopamine agonists, quinpirole, 7-OH-DPAT and bromocriptine (Sokoloff et al., 1990; Levesque et al., 1992), partially blocked the stimulus effect of the training dose of tiapride. In contrast, the D₁ dopamine agonist, SKF 38393 (Arnt et al., 1992), did not affect the discrimination. These findings provide support for a D₂/D₃ dopamine receptor mediation of tiapride discrimination. Although none of these drugs produced full antagonism, their depressant effects on rate of responding prevented testing of higher doses.

The indirect dopamine agonists, d-amphetamine and nomifensine, dose-dependently antagonized the discriminative stimulus effects of tiapride. The doses required to antagonize the discriminative stimulus effects of tiapride were, however, much lower than those necessary to produce direct behavioral effects in rats (Colpaert et al., 1979). The reasons for this sensitivity of the tiapride discrimination to antagonism by the indirect dopamine agonists are not clear. It is possible that chronic treatment with tiapride had produced supersensitivity of dopamine receptors although the dose-response curves established to tiapride itself showed no evidence that either sensitization or tolerance had developed to the effects of tiapride.

D₂/D₃ dopamine receptors are thought to serve as both postsynaptic receptors and presynaptic (auto)receptors that can control dopamine synthesis and release. In our study, all dopamine antagonists substituted for tiapride at doses that have been shown to antagonize apomorphine-induced effects (hyperactivity) mediated by postsynaptic receptors (Costall and Naylor 1975; Puech et al., 1978; Perrault et al., 1997). This is particularly true for the few dopamine antagonists (amisulpride, sulpiride) that discriminate between pre- and postsynaptic dopamine receptors (Costall et al., 1980; Puech et al., 1981; Perrault et al., 1997; Schoemaker et al., 1997). Drugs that increase extracellular dopamine concentrations, including morphine and cocaine, failed to engender tiapride-like discriminative effects and, in contrast, d-amphetamine and nomifensine blocked the tiapride cue, further suggesting that dopamine release induced by presynaptic activity of tiapride does not participate in its interoceptive cue. These findings suggest that presynaptic dopamine antagonism does not play an important role in the discriminative stimulus effects of tiapride and indicate that blockade of postsynaptic D₂/D₃ dopamine receptors is sufficient to create an interoceptive discriminative stimulus.

Based on the ED₅₀ values shown in table 1, dopamine antagonists can be classified according to their substitution to response-reduction dose ratio. Tiapride (0.054), amisulpride (0.17), sulpiride (0.24), sultopride (0.33), pimozide (0.63), clebopride (0.81) and thioridazine (0.83) substituted for the discriminative stimulus effects of tiapride at doses lower than those required to decrease response rate (ratio < 1). Olanzapine and chlorpromazine produced a tiapride-like discriminative stimulus at doses that reduced responding (ratio = 1). Raclopride (1.2), metoclopramide (1.3), risperidone (1.4), haloperidol (1.6) and remoxipride (1.8) generalized to tiapride only at doses that disrupted lever-pressing performance (ratio > 1).

It appears from our results, and previously published data, that drugs which displayed tiapride-like discriminative effects with an ED₅₀ about 3 to 20 times lower than the ED₅₀ for reducing response rate, i.e., tiapride, amisulpride, sulpiride and sultopride, display limbic selectivity as reflected by regional selectivity for D₂/D₃ dopamine receptors and a weak antagonism of apomorphine-induced stereotypies (Costall and Naylor, 1975; Jenner et al., 1978; Bischoff et al., 1982; Perrault et al., 1997, Schoemaker et al., 1997). In contrast, antagonism of apomorphine-induced stereotypies has been observed with the other dopamine antagonists at doses close to those producing generalization and decreases in response rate (Costall and Naylor, 1975; Jenner et al., 1978; Bischoff et al., 1982; Moore et al., 1992; Leysen et al., 1993; Perrault et al., 1997). These findings suggest that the discriminative stimulus and the rate-decreasing effects of tiapride may not be mediated by the same neuronal mechanism. Further, the observation that the drugs with the highest substitution to response-reduction dose ratios (risperidone, haloperidol and remoxipride) are those with the highest D₂:D₃ selectivity ratios (Sokoloff et al., 1990, 1992; Leysen et al., 1993) suggests that D₂ receptors may mediate decreases in rates of operant responding. However, other factors may have contributed to the rate decreasing effects of the drugs. Thus, tiapride, amisulpride, sulpiride and sultopride have selective affinities for D₂/D₃ dopamine receptors (Chivers et al., 1988; Leysen et al., 1993; Schoemaker et al., 1997) whereas thioridazine, olanzapine, chlorpromazine, risperidone and haloperidol display affinities for 5HT₂, ₁, H₁ and other neurotransmitter receptors (Leysen et al., 1993; Bymaster et al., 1996). H₁ histamine receptor and ₁ adrenergic antagonism is associated with sedative effects. In addition, clebopride, remoxipride and haloperidol have affinities for  sites (Largent et al., 1988; Leysen et al., 1993), which have been suggested to be important in mediating the motor side effects of neuroleptics (Walker et al., 1988). It is therefore possible that substitution to response-reduction dose ratios are based on the lack of specificity of the drugs rather than on limbic versus striatal or D₂ versus D₃ selectivity.

In conclusion, our study showing that tiapride produces a discriminative stimulus that can reliably control responding in rats supports previous findings that dopamine antagonists can serve as discriminative stimuli. Our results also suggest that D₂/D₃ dopamine receptors play a primary role in mediating the discriminative stimulus effects of tiapride.