Introduction

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A place conditioning paradigm is used widely for determining both rewarding and aversive properties of drugs in animals (see review, Schechter and Calcagnetti, 1993). In this paradigm, many drugs of abuse such as amphetamines, cocaine and benzodiazepines produce a place preference in animals (Acquas et al., 1989; Schechter and Calcagnetti, 1993; Cervo and Samanin, 1995).

PCP has been a common drug of abuse in the United States during the past two decades, because it produces both physical and psychological dependence in humans (Petersen and Stillman, 1978). Like many other abused drugs, PCP reinforces drug self-administration behavior (Marquis and Moreton, 1987). In the place conditioning paradigm, PCP produces place aversion in naive animals (Barr et al., 1985; Kitaichi et al., 1996; Nabeshima et al., 1996), whereas it produces place preference in rats pretreated with PCP repeatedly (Kitaichi et al., 1996; Nabeshima et al., 1996). These phenomena observed in rats are similar to those in humans; although a single use of PCP produces aversive effects, long-term use of it causes abuse in humans (Isaacs et al., 1986). We previously found that the PCP-induced place aversion on the CPP in rats is attributed to interaction with the serotonergic system, particularly, the postsynaptic 5-HT₂ receptors (Nabeshima et al., 1996). However, few reports have addressed the pharmacological mechanisms of PCP-induced place preference.

PCP interacts with the dopaminergic system directly through an inhibition of dopamine reuptake (Smith et al., 1977) or, to a lesser extent, through a stimulation of DA release (Bowyer et al., 1984) in vitro. Several in vivo microdialysis studies have confirmed the ability of PCP to increase DA efflux in the nucleus accumbens (Bristow et al., 1993) and medial prefrontal cortex (Rao et al., 1989; Hondo et al., 1994). Further, long-term treatment with PCP produces the development of sensitization to PCP-induced hyperlocomotion and rearing (Nabeshima et al., 1987; Noda et al., 1996) and the increased turnover of DA in the brain (Nabeshima et al., 1987). These effects are antagonized by DA antagonists, which suggests that the dopaminergic system is involved in behavioral sensitization.

Several lines of evidence suggest that the dopaminergic system plays an important role in the rewarding and abuse properties of drugs in the place conditioning paradigm. Blockade of DA receptors reduces or abolishes the place preference induced by amphetamine and opiates (Leone and Di Chiara, 1987). A recent study has shown that cocaine administered i.p. increases extracellular concentrations of DA in the nucleus accumbens and induces CPP (Hemby et al., 1994), which suggests that the mesolimbic DA neurons mediate the rewarding properties of various drugs of abuse (Di Chiara and Imperato, 1988). These findings, together with the pharmacological properties of PCP, suggest that the dopaminergic system is involved in PCP-induced place preference in rats pretreated with PCP repeatedly.

Accordingly, in the present study we investigated the pharmacological characterization of PCP-induced place preference in the CPP test. First, we investigated whether PCP-induced place preference in mice was modified by hypofunction or overfunction of the dopaminergic system. Second, selective DA receptor antagonists then were tested to delineate the role of D₁ vs. D₂ receptors in the mediation of the PCP-induced place preference. Finally, we examined the effect of a 5-HT₂ receptor antagonist, ritanserin, on PCP-induced place preference, because ritanserin antagonizes the PCP-induced place aversion (Nabeshima et al., 1996).

	Methods

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Animals. Male mice of the ddY strain (Japan SLC Inc., Shizuoka, Japan), weighing 25 to 27 g at the beginning of the experiments, were used. The animals were housed in plastic cages and were kept in a regulated environment (23 ± 1°C, 50 ± 5% humidity), with a 12/12 hr light-dark cycle (light on at 8:00 A.M.). Food (CE2, Clea Japan Inc. Tokyo, Japan) and tap water were available ad libitum.

Drugs. Phencyclidine HCl (PCP) was synthesized by the authors according to the method of Maddox et al. (1965) and was checked for purity. AMPT, 6-OHDA and desipramine HCl were purchased from Sigma Chemical Co. (St. Louis, MO). Methamphetamine HCl (Philopone) was purchased from Dainippon Pharmaceutical Co. Ltd.(Osaka, Japan). (+) SCH-23390 HCl, () sulpiride and DSP-4 HCl were purchased from Funakoshi (Tokyo, Japan). Ritanserin was kindly provided from Janssen Kyowa Co. Ltd. (Tokyo, Japan). Other agents were obtained by standard commercial sources.

Apparatus. The apparatus used for the place conditioning task consisted of two compartments: a black Plexiglas box and a transparent Plexiglas box (both 15 × 15 × 15 cm high) with metal gird floor. To enable the mice to distinguish easily the transparent box from the black one, the floor of the transparent and black boxes were covered with white plastic mesh and with black frosting Plexiglas, respectively. Each box could be divided by a sliding door (10 × 15 cm high).

Preconditioning test. The place conditioning paradigm was performed according to the method of Kitaichi et al. (1996), with a minor modification. In the preconditioning test, the sliding door was opened and the mouse was allowed to move freely between both boxes for 15 min once a day for 3 days. On the third day of the preconditioning test, we measured the time that the mouse spent in the black and transparent boxes with use of Scanet SV-10 LD (Toyo Sangyo Co. Ltd., Toyama, Japan). The box in which the mouse spent the most time was referred to as the "preferred side," and the other box the "nonpreferred side."

Conditioning. Conditioning was performed during 6 successive days. Mice were given drugs or vehicle in the apparatus with the sliding door closed. That is, a mouse was given PCP and put in its preferred (for investigating the PCP-induced place aversion) or nonpreferred (for the PCP-induced place preference) side for 20 min. The next day, the mouse was given saline, and placed opposite the drug conditioning site for 20 min. These treatments were repeated for three cycles (6 days).

Postconditioning test. In the postconditioning test, the sliding door was opened, and we measured the time that the mice spent in the black and transparent boxes for 15 min with use of Scanet SV-10 LD.

Data analysis. Place conditioning behaviors were expressed by Post  Pre, which was calculated as: [(postvalue)  (prevalue)], where post- and prevalues were the difference in time spent in the drug conditioning and the saline conditioning sites in the postconditioning and preconditioning tests, respectively.

Drug administration. PCP (2-8 mg/kg s.c.) was injected immediately before the conditioning. Ritanserin (0.3 and 1 mg/kg i.p.), AMPT (50 and 100 mg/kg i.p.), (+) SCH-23390 (0.1 and 0.5 mg/kg s.c.) and () sulpiride (50 and 100 mg/kg i.p.) were administered 60, 180, 15 and 60 min, respectively, before every treatment with PCP (8 mg/kg). These drugs were administered for 3 alternating days in the 6-day conditioning period, and corresponding vehicles were administered for the other 3 days. 6-OHDA (100 µg/mouse i.c.v.) and DSP-4 (30 mg/kg i.p.) were administered 7 and 3 days, respectively, before the preconditioning test. To prevent the destruction of noradrenergic neurons, mice were administered desipramine (25 mg/kg i.p.) 30 min before 6-OHDA treatment.

Determination of monoamine contents. Immediately after the postconditioning test, the 6-OHDA-, AMPT- and DSP-4-treated and control mice were sacrificed. Brains were removed rapidly, and the prefrontal cortex and striatum were dissected out on an ice-cold plate according to the method of Glowinski and Iversen (1966). Each tissue sample was frozen quickly and stored in a deep freezer at 80°C until assayed.

Statistics. All data were expressed as means ± S.E. Statistical differences among values for individual groups were determined with the Student-Newmann-Keuls multiple comparisons test.

	Results

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Effect of PCP on the performance in the place conditioning paradigm. We have confirmed our previous results that PCP has both aversion and preference in the place conditioning paradigm depending on the treatment schedule. In the naive mice, PCP (2-8 mg/kg) showed place aversion in a dose-dependent manner (fig. 1A). In contrast to this finding, in mice pretreated with PCP (10 mg/kg/day s.c.) for 28 days, PCP (2-8 mg/kg) produced place preference in a dose-dependent manner (fig. 1B).

View larger version (20K): [in this window] [in a new window]   Fig. 1.   Effect of PCP on the performance in the place conditioning paradigm in naive mice (A) and in mice pretreated with PCP (10 mg/kg/day) for 28 days (B). The experimental protocol is described in the text. Each column represents the mean ± S.E.M. Numbers in the parentheses are the number of animals used. *P < .05, **P < .01 vs. corresponding saline-treated group.

Effect of AMPT and 6-OHDA on the PCP-induced place preference. The contents of DA in the prefrontal cortex and striatum of AMPT-treated mice were decreased significantly to 40.6% and 63.5%, respectively, compared with those in the vehicle-treated mice (table 1). In contrast, the contents of NA and 5-HT in the prefrontal cortex and striatum remained unaffected (table 1). When PCP (8 mg/kg) was administered in combination with AMPT (100 mg/kg) during the conditioning phase, the PCP-induced place preference in mice pretreated with PCP repeatedly was attenuated significantly (fig. 2A). AMPT (100 mg/kg) itself did not produce either place preference or aversion (fig. 2A).

View larger version (21K): [in this window] [in a new window]   Fig. 2.   Effect of AMPT (A) and 6-OHDA (B) on the preference induced by PCP in mice. The experimental protocol is described in the text. Each column represents the mean ± S.E.M. Numbers in the parentheses are the number of animals used. *P < .05 vs. (vehicle + saline)-treated group. #P < .05 vs. (vehicle + PCP)-treated group.

Effects of repeated methamphetamine treatment on the motivational properties of PCP in mice. PCP (8 mg/kg) significantly produced place aversion in mice pretreated with saline repeatedly. In mice pretreated with methamphetamine (1 mg/kg/day) for 7 days, however, PCP (8 mg/kg) produced neither place aversion nor preference. On the other hand, PCP (8 mg/kg) significantly induced place preference in mice treated with methamphetamine (1 mg/kg/day) for 14 days (fig. 3).

View larger version (25K): [in this window] [in a new window]   Fig. 3.   Effects of repeated methamphetamine treatment on the motivational properties of PCP in mice. The experimental protocol is described in the text. Each column represents the mean ± S.E.M. Numbers in the parentheses are the number of animals used. *P < .05, **P < .01 vs. (saline + saline)-treated group. ##P < .01 vs. (saline + PCP)-treated group.

Effects of (+) SCH-23390 and () sulpiride on the PCP-induced place preference. As shown in figure 4, (+) SCH-23390 (0.5 mg/kg) significantly attenuated place preference produced by PCP in mice pretreated with PCP repeatedly. (+) SCH-23390 (0.5 mg/kg) itself did not produce either place preference or place aversion (fig. 4). However, () sulpiride (50 and 100 mg/kg) failed to modify the place preference produced by PCP.

View larger version (30K): [in this window] [in a new window]   Fig. 4.   Effects of (+) SCH-23390 and () sulpiride on the place preference induced by PCP in mice pretreated with PCP repeatedly. The experimental protocol is described in the text. Each column represents the mean ± S.E.M. Numbers in the parentheses are the number of animals used. *P < .05 vs. corresponding (vehicle + saline)-treated group. #P < .05 vs. corresponding (vehicle + PCP)-treated group.

Effect of ritanserin and DSP-4 on the PCP-induced place preference. In agreement with a previous report (Nabeshima et al., 1996), ritanserin (0.3 and 1 mg/kg) inhibited the place aversion produced by PCP in naive mice (fig. 5). However, it (1 mg/kg) failed to inhibit the place preference produced by PCP in mice pretreated with PCP repeatedly (fig. 6A). Ritanserin (1 mg/kg) itself did not produce either place preference or aversion (fig. 5).

View larger version (27K): [in this window] [in a new window]   Fig. 5.   Effect of ritanserin on the place aversion induced by PCP in mice. The experimental protocol is described in the text. Each column represents the mean ± S.E.M. Numbers in the parentheses are the number of animals used. *P < .05 vs. (vehicle + saline)-treated group. #P < .05 vs. (vehicle + PCP)-treated group.

View larger version (30K): [in this window] [in a new window]   Fig. 6.   Effect of ritanserin (A) and DSP-4 (B) on the place preference induced by PCP in mice. The experimental protocol is described in the text. Each column represents the mean ± S.E.M. Numbers in the parentheses are the number of animals used. *P < .05, ** P < .01 vs. (vehicle + saline)-treated group.

	Discussion

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In the present study, PCP produced a dose-dependent place preference in mice pretreated with PCP for 28 days, as reported previously (Kitaichi et al., 1996; Nabeshima et al., 1996). This finding that PCP has rewarding properties is consistent with the results of several studies that PCP is self-administered by animals (Marquis and Moreton, 1987). Further, such a phenomenon also has been observed in humans; although single use of PCP produces aversive effects, long-term use of it causes abuse (Isaacs et al., 1986). Thus, these findings suggest that some functional changes induced by repeated PCP treatment play a critical role in PCP-induced place preference.

The dopaminergic system plays an important role in the rewarding and abuse properties of drugs in the place conditioning paradigm (Leone and Di Chiara, 1987). Several in vivo microdialysis studies have confirmed the ability of PCP to increase DA efflux in the nucleus accumbens (Bristow et al., 1993) and medial prefrontal cortex (Rao et al., 1989; Hondo et al., 1994). Repeated PCP treatment produces the behavioral sensitization (Nabeshima et al., 1987; Noda et al., 1996) and the increase of DA turnover in the striatum and nucleus accumbens (Nabeshima et al., 1987). Taken together, the dopaminergic system may be involved in PCP-induced place preference in rats pretreated with PCP repeatedly. The present study showed that PCP-induced place preference in mice pretreated with PCP for 28 days was blocked by coadministration of a tyrosine hydroxylase inhibitor, AMPT, and lesion of the dopaminergic system by 6-OHDA. Analysis of the neurochemical effects of 6-OHDA and AMPT treatment revealed a marked decrease of DA level and no reduction of NA and 5-HT levels in the brain. Although AMPT depletes both NA and DA contents in the brain, the reasons for which no depletion of NA was obtained by AMPT in the present biochemical study are unclear. A possible explanation is that the doses of AMPT used are lower than those of the depleted NA contents. DSP-4, a NA neurotoxin, at the dose of 30 mg/kg which caused significant depletion of NA, but not of DA and 5-HT, in the brain, failed to affect the PCP-induced place preference. Thus, it is suggested that the abolition of PCP-induced place preference results specifically from the destruction of DA neurons.

A 5-HT₂ receptor antagonist, ritanserin, attenuated the PCP-induced place aversion in agreement with a previous report (Nabeshima et al., 1996), whereas it had no affect on the PCP-induced place preference in mice pretreated with PCP repeatedly. These findings demonstrate that PCP-induced place preference, but not aversion, is not mediated via the postsynaptic 5-HT₂ receptors. A 5-HT_1A receptor agonist, 8-OH-DPAT, induces place preference in rodents (Fletcher et al., 1993). Because PCP has a 5-HT uptake inhibitory action, it is possible that 5-HT_1A receptors may be activated by the uptake inhibitory action of PCP. However, repeated PCP treatments produced the decrease of 5-HT turnover in the brain and behavioral tolerance; serotonergic system-mediated behaviors, including 5-HT_1A receptor-mediated behavior, are decreased (Nabeshima et al., 1987). Therefore, it can be hypothesized that the activity of DA, but not of NA and 5-HT, neurons may be necessary for the expression of the rewarding effects of PCP.

This hypothesis also is supported by the other present finding. Namely, in mice pretreated with methamphetamine repeatedly, PCP induced the place preference, but not aversion. Repeated treatment with amphetamine or methamphetamine enhances several behaviors (hyperlocomotion and stereotypy) and the DA efflux produced by these drugs in rats either in vitro and in vivo (Nabeshima et al., 1987; Yamada et al., 1988; Robinson et al., 1988; Kazahaya et al., 1989). These findings have demonstrated that hyperfunction of the dopaminergic system is produced by repeated methamphetamine treatment. Thus, in view of the repeated PCP-induced hyperfunction in the dopaminergic system, together with the critical importance of the dopaminergic system to the rewarding properties of drugs, it is suggested that the place preference induced by repeated administration of PCP for 28 days is caused by functional changes in the dopaminergic system.

A recent study showed that cocaine administered i.p. increases extracellular concentrations of DA in the nucleus accumbens and induces CPP (Hemby et al., 1994), which suggests that the mesolimbic DA neurons mediate the rewarding properties of various drugs of abuse (Di Chiara and Imperato, 1988). Repeated PCP treatment was demonstrated by an increase of DA turnover in the striatum and nucleus accumbens of rats treated with PCP repeatedly (Nabeshima et al., 1987). Thus, it is suggested that both PCP- and cocaine-induced place preferences are mediated via the mesolimbic DA neurons. However, we could not determine the changes of DA turnover in the nucleus accumbens of mice showing PCP-induced place preference, because it is difficult to dissect exactly only the nucleus accumbens in mice. Our recent in vivo microdialysis study in rats has found that DA turnover is increased in the nucleus accumbens of rats showing the place preference induced by PCP, compared with that in control rats, which suggests that the hyperfunction in the mesolimbic dopaminergic neurons is involved in the expression of PCP-induced place preference.

Another possibility is that the serotonergic system, in particular 5-HT₂ receptors, mediating the aversion may became desensitized by repeated PCP treatment because some biochemical studies have demonstrated an decrease of 5-HT turnover and 5-HT₂ receptor density in the brain of rats treated with PCP repeatedly (Nabeshima et al., 1985, 1987). Previously our finding showed that PCP-induced place aversion is diminished in rats pretreated with PCP for 14 days (Nabeshima et al., 1996), which indicates that PCP induces neither place aversion nor preference. In such rats, PCP-induced head-twitch behavior, which may be mediated by 5-HT₂ receptors, also was diminished (Nabeshima et al., 1996), which suggests that such a regimen produces the down-regulation of 5-HT₂ receptors. In the present study, we found that PCP induced place preference in mice pretreated with methamphetamine repeatedly. Although repeated methamphetamine treatment produces the hyperfunction of the dopaminergic system, there is little evidence that the serotonergic system is desensitized by methamphetamine. Thus, the present result, that PCP induces place preference in mice pretreated with PCP repeatedly, may not be caused only by desensitization of the serotonergic system by repeated PCP treatment. However, further studies are necessary to clarify the significance of desensitized serotonergic system.

Recent studies with selective D₁ and D₂ receptor antagonists have demonstrated that blockade of D₁, but not D₂ receptors prevents behavioral sensitization to amphetamine (Drew and Glick, 1990). An increase in D₁ receptor density in the brain has been found after daily treatment with methamphetamine (Ujike et al., 1991). Further, amphetamine regulates the expression of several genes, including c-fos, via D₁ receptors in the rat brain (Nguyen et al., 1992). Considering these findings and the present finding that PCP induced the place preference in rats pretreated with methamphetamine repeatedly, there is a possibility that the rewarding effect of PCP is mediated via D₁ receptors. The present results showed that the selective D₁ receptor antagonist (+) SCH-23390 blocked the PCP-induced place preference. In contrast, the selective D₂ receptor antagonist, () sulpiride failed to block it, although we used enough doses to block the central D₂ receptors (Ljungberg and Ungerstedt, 1978). The doses of (+) SCH-23390 used in this study were ineffective as a conditioning stimulus, and SCH-23390 has been shown to attenuate the drug-conditioned place preference as well as drug-conditioned place aversion (Acquas et al., 1989). Thus, the attenuation of PCP place conditioning cannot be attributed to an aversive action of the antagonist by itself. A similar effect of SCH-23390 has been observed in the cocaine-induced place preference in rats as well as the PCP-induced place preference. Namely, Cervo and Samanin (1995) demonstrated that SCH-23390 administered before cocaine during the conditioning phase significantly blocked the establishment of place conditioning, whereas () sulpiride had no effect. These findings suggest that D₁ receptors play a more important role in cocaine place conditioning as well as that of PCP. It is unlikely that the relatively high affinity of SCH-23390 for 5-HT₂ receptors (Bischoff et al., 1986) plays a role, because (+) SCH-23390 at the doses used in the present study does not affect the in vivo binding of [³H]spiperone in the rat prefrontal cortex (Bischoff et al., 1986). In addition, in the present study, ritanserin, a selective 5-HT₂ receptor antagonist, failed to affect the PCP-induced place preference in mice pretreated with PCP repeatedly. Taken together with these findings, the present results suggest that PCP can sensitize D₁ receptors to DA, enabling them to act independently from D₂ receptors, as observed in some cases of sensitization (Breese et al., 1985), and that D₁ receptors are involved in the conditioning of the rewarding effect of PCP.

Abundant evidence exists that SCH-23390 impairs learning and memory in rodents. Thus, it is possible that the effect on place conditioning may be resulted from a generalized disruption of behavior rather than a specific motivational deficits. This possibility, however, is unlikely because SCH-23390 has failed to modify the place preference induced by [D-Ala2]deltorpine II, a selective delta-1 opioid receptor agonist (Suzuki et al., 1996). Therefore, (+) SCH-23390 may not disrupt learning, but directly affect a reward process in our present experiment.

(+) SCH-23390 blocks the PCP-induced place preference by the involvement of D₁ receptors in mediating the rewarding effects of PCP as assessed by expression of place preference. Kobayashi and Inoue (1993) reported that the systemic administration of MK-801, which is a noncompetitive NMDA receptor antagonist, as well as PCP, enhances the in vivo binding of [³H]SCH-23390 in the striatum. In addition, MK-801 enhances the stimulant effect of a D₁, but not a D₂, receptor agonist, in monoamine-depleted mice (Goodwin et al., 1992; Svensson et al., 1992). On the based of these results, we speculate that the functional changes in the dopaminergic system, particularly in D₁ receptors, were produced during repeated PCP treatment for 28 days, and then, the rewarding effect of PCP resulted from an increase in D₁ receptor activation, secondary to an increase of DA release. If such is the case, then the administration of (+) SCH-23390 before the conditioning phase would attenuate the motivational effect of PCP by masking PCP-induced activation of D₁ receptors. However, studies that could associate the neurochemical and behavioral actions of PCP should be performed to elucidate the mechanisms of PCP abuse.

The conditioned place preference consists of an acquisition or development phase during which the animals receive the drug in one distinctive environment, and a test or expression phase in which drug-free animals are tested for their preference of the environment previously paired with the drug. Blockade of D₁ receptors blocks the acquisition and expression of amphetamine-induced place preference (Hiroi and White, 1991), whereas it blocks only acquisition of cocaine-induced place preference (Cervo and Samanin, 1995). In the present study, (+) SCH-23390, administered before PCP during the conditioning, blocked the acquisition, whereas it is unclear whether (+) SCH-23390 blocks the expression of PCP-induced place preference. Thus, the significance of D₁ receptors and/or others in expression of PCP-induced place preference should be clarified by further study with use of D₁ receptors and/or other antagonists, because it has been suggested that different neurochemical mechanisms appear to mediate the acquisition and expression of this incentive learning (Hiroi and White, 1991).

In summary, the present results indicate that the repeated administration of PCP produces place preference, and that dopaminergic systems, but not serotonergic and/or noradrenergic systems, are involved in PCP-induced place preference and that some changes in dopaminergic systems induced by repeated PCP treatment play a critical role in the addiction of this drug. Further, the present study demonstrates an involvement of D₁ receptors in the conditioning of the rewarding effect of PCP. The previously documented (Leone and DiChiara, 1987; Shippenberg et al., 1993) effectiveness of systemically administered (+) SCH-23390 in attenuating the rewarding effects of opioids and other drugs of abuse suggests that D₁ receptor ligands may be useful therapeutic agents for the treatment of drug addiction.