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Involvement of dopaminergic system in the nucleus accumbens in the discriminative stimulus effects of phencyclidine
Neuropharmacology 42 (2002) 764–771 www.elsevier.com/locate/neuropharm
Involvement of dopaminergic system in the nucleus accumbens in the discriminative stimulus effects of phencyclidine Akitomo Mori a, Yukihiro Noda a, Taku Nagai a, Takayoshi Mamiya a, Hiroshi Furukawa b, Toshitaka Nabeshima a,∗ a
Department of Neuropsychopharmacology and Hospital Pharmacy, Nagoya University Graduate School of Medicine, Nagoya 466-8560, Japan b Department of Medicinal Chemistry, Faculty of Pharmacy, Meijo University, Nagoya 468-8503, Japan Received 31 August 2001; received in revised form 27 February 2002; accepted 28 February 2002
Abstract The effects of microinjection of phencyclidine (PCP) and dizocilpine, non-competitive NMDA receptor antagonists, and dopamine into the nucleus accumbens were examined in rats trained to discriminate PCP (1.5 mg/kg i.p.) from saline under a two-lever fixed ratio 20 schedule of food reinforcement. Microinjection of PCP (2–40 µg) and dizocilpine (2–12 µg) into the bilateral nucleus accumbens produced a dose-dependent increase in PCP-appropriate responding and fully substituted for systemically administered PCP, whereas microinjection of dopamine (1–4 µg) did not produce PCP-like discriminative stimulus effects. The performance of PCP discrimination was assessed after bilateral destruction of the dopaminergic nerve neurons in the nucleus accumbens with dopaminergic neurotoxin, 6-hydroxydopamine (6-OHDA, 4 µg/1 µl/side). The destruction of dopaminergic nerve neurons in the nucleus accumbens failed to prevent the performance of PCP discrimination. There was no difference in the average percentages of PCP-appropriate responding between vehicle and 6-OHDA-treated rats in the dose–response tests. These results suggest that the dopaminergic system in the nucleus accumbens does not play a critical role in the discriminative stimulus effects of PCP. 2002 Elsevier Science Ltd. All rights reserved. Keywords: Phencyclidine; Drug discrimination; NMDA receptor; Dopamine; Nucleus accumbens; Rat
1. Introduction Phencyclidine (PCP), a non-competitive N-methyl-daspartate (NMDA) receptor antagonist, has been demonstrated to produce psychotomimetic effects in humans and be a widely abused drug (Petersen and Stillman, 1978). In animals, several studies have shown that PCP produces the discriminative stimulus (Holtzman, 1980), self-administration (Balster and Woolverton, 1980) and conditioned place preference (Kitaichi et al., 1996; Noda et al., 1998). Thus, although it has been well known that PCP induces dependence in humans and animals, the mechanisms of PCP-induced dependence remain unclear. Drug discrimination procedures have proven a valu-
Corresponding author. Tel.: +81-52-744-2674; fax: +81-52-7442979. E-mail address: [email protected] (T. Nabeshima). ∗
able means of obtaining information relevant to the subjective effects of drugs (Colpaert, 1978). The results of drug discrimination studies have suggested that the discriminative stimulus induced through antagonism of the NMDA receptor is highly dependent on the nature of the antagonism (Jackson and Sanger, 1988; Koek et al., 1990; Witkin et al., 1996). Namely, the discriminative stimulus effects of PCP are mediated predominantly via PCP binding sites on the NMDA receptor-ion channel complex (Mori et al., 2001). Microinjection of PCP into a lateral ventricle fully substituted for systemic administration of PCP in the rats trained to discriminate PCP from saline (Slifer and Balster, 1985). These findings demonstrate that the discriminative stimulus effects of PCP are centrally mediated. However, the mechanism behind the discriminative stimulus effects of PCP is not well elucidated. The mesolimbic–mesocortical dopaminergic pathways have been implicated as important substrates in the rewarding qualities of abuse drugs (Wise and Bozarth,
0028-3908/02/$ - see front matter 2002 Elsevier Science Ltd. All rights reserved. PII: S 0 0 2 8 - 3 9 0 8 ( 0 2 ) 0 0 0 3 7 - 0
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1987). The fact that PCP produces psychotomimetic effects and it is widely abused in humans seems to suggest an involvement with mesolimbic–mesocortical dopaminergic pathways. It has been reported that the discriminative stimulus effects of cocaine and amphetamine, abuse drugs as well as PCP, can be attributed to their ability to elevate extracellular concentrations of dopamine (DA) in the nucleus accumbens (Nielsen and Scheel-Kruger, 1986; Wood and Emmett-Oglesby, 1989; Callahan et al., 1997). PCP is self-administrated into the nucleus accumbens (Carlezon et al., 1995; Carlezon and Wise, 1996) and elevates extracellular concentrations of DA in the nucleus accumbens (Carboni et al., 1989). Therefore, there is a possibility that the dopaminergic system in the nucleus accumbens is involved in the discriminative stimulus effects of PCP. The present study was conducted to elucidate further the role of the mesolimbic dopaminergic system of the nucleus accumbens in PCP-induced discriminative stimulus. For this purpose, in the first experiment we investigated the generalized effects of local bilateral microinjections of PCP, dizocilpine, non-competitive NMDA receptor antagonists and of DA into the nucleus accumbens in rats trained to discriminate PCP from saline. In the second experiment, the influence of the destruction of the dopaminergic nerve terminals in the nucleus accumbens after local bilateral microinjections of the neurotoxin, 6-hydroxydopamine (6-OHDA) was tested in PCP-induced discriminative stimulus.
ventilated and sound-attenuated cubicles. The chambers were equipped with two response levers, spaced 16 cm apart with a food pellet trough mounted midway between the levers. A houselight was located over the trough. Reinforcement consisted of a 45-mg food pellet (Bio Serv Inc., Frenchtown, NJ). Scheduling of reinforcement contingencies, reinforcement delivery and data recording were controlled by computer systems (Neuroscience Co.).
2. Materials and methods
Rats were initially trained to press each of the two levers under a fixed ratio (FR) 1 schedule of food reinforcement. The FR response requirement for food delivery was gradually increased from 1 to 20. After responses under the FR 20 schedule of food reinforcement had been stabilized, drug discrimination training was started. Training sessions were conducted daily. Rats were injected 10 min before the session with either saline or PCP (1.5 mg/kg i.p.) according to a previous report (Mori et al., 2001). In drug discrimination training sessions, PCP or saline was administered randomly to ensure that no olfactory cues associated with the two levers (Extance and Goudie, 1981; Mori et al., 2001), which would bias the discrimination. After administration of PCP, 20 consecutive responses (FR 20) on one lever produced a food pellet, whereas after that of saline, 20 consecutive responses on the other lever produced a food pellet. Responding to the incorrect lever reset the FR requirement for the correct lever. Each session ended after 20 food pellets were delivered or 20 min was elapsed. The criteria for learning the discrimination was five consecutive sessions with: (1) more than 85% correct-lever responding before the first reinforcement; (2) more than 90% correct-lever responding throughout the session.
2.1. Animals Male Fischer rats (Charles River Japan, Atsugi, Japan), weighing 230–250 g at the beginning of the experiments, were used according to a previous report (Mori et al., 2001; Shannon, 1982). Each animal was housed individually in regulated conditions (23±1°C, 50±5% humidity) under a 12h/12h light-dark cycle (lights on at 09:00 h). Animals were deprived of food to 85% of free feeding weight. Water was available ad libitum in each home cage. All experiments were performed in accordance with the Guidelines for Animal Experiments of the Nagoya University Graduate School of Medicine. The procedures involving animals and their care conformed to the international guidelines “Principles of Laboratory Animal Care” (NIH publication no. 85-23, revised 1985). 2.2. Apparatus Experiments were conducted in operant-conditioning chambers (Neuroscience Co., Tokyo, Japan) located in
2.3. Drugs Phencyclidine hydrochloride [1-(1-phenylcyclohexyl) piperidine; PCP] was synthesized by the authors according to the method of Maddox et al. (1965) and it was checked for purity. (+)-5-Methyl-10,11-dihydro-5Hdibenzo[a,d]cyclohepten-5,10-imine hydrogen maleate salt (dizocilpine) and nialamide were purchased from Research Biochemicals (Natick, MA, USA). Dopamine hydrochloride (DA), 6-hydroxydopamine hydrochloride (6-OHDA) and desipramine hydrochloride were purchased from Sigma Chemical Co. (St. Louis, MO, USA). PCP, dizocilpine and desipramine were dissolved in 0.9% NaCl solution, DA and 6-OHDA in saline containing 0.1% ascorbic acid and nialmide in 0.1N hydrochloride. The dose of each drug refers to the drug form listed above. 2.4. PCP discrimination procedure
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2.5. Surgical procedure When the rats fulfilled the criteria for learning the discrimination, implantation of the cannula was carried out. Each rat was anesthetized with pentobarbital (35 mg/kg i.p.) and mounted in a stereotaxic frame. The skulls were exposed and holes were drilled. Each rat was implanted with bilateral 22-gauge guide cannulae that terminated 1.1 mm above the nucleus accumbens (according to the brain atlas of Pellegrino et al., 1979: with the incisor bar elevated to 5 mm above the interaural line, the cannula entry was 3.5 mm anterior to bregma, 2.4 mm bilateral to the midline suture; to avoid the bilateral ventricle, the cannulae were lowered at 10° angle toward the midline to an angled depth of 5.9 mm below the dura). The guide cannula was cemented in place with dental acrylic and the skin resutured around the base of the cannula. A stainless steel dummy cannula (28-gauge), extending 1.1 mm beyond the tip of the guide cannula, was inserted and remained in place when the animals were not injected. 2.6. Drug testing procedure After the surgery, the rats were given a recovery period of at least 1 week before returning to discrimination training. When the rats reliably discriminated PCP from saline again, the dose–response and the substitution tests were initiated. Test sessions were conducted once a week using rats that fulfilled the criteria during three consecutive training sessions. Test sessions were identical to training sessions except that 20 consecutive responses on either lever resulted in delivery of a food pellet. In the dose–response tests, lever selection was examined after the intraperitoneal administration of various doses of PCP, which was injected 10 min before the session. In the substitution tests, lever selection was examined after the administration of various doses of PCP or novel compounds into the nucleus accumbens. Injections were administered via two injection cannulae which were connected to Hamilton syringes by polyethylene tubes over a period of 120 s. Nialamide (100 mg/kg i.p.), a monoamine oxidase inhibitor, was administered 22 h before DA test sessions. Immediately after the drug administration, the rats were placed into the chambers and test sessions were begun. 2.7. 6-OHDA microinjections into the nucleus accumbens After the drug test, half of the animals received 6OHDA (4 µg/1 µl/side) and half of them received the 6-OHDA-vehicle into the bilateral nucleus accumbens via two injection cannulae over a period of 3 min. All rats were pretreated with desipramine (25 mg/kg i.p.) to prevent lesions of noradrenergic neurons and nialamide
(100 mg/kg i.p.). After 6-OHDA microinjections, the rats were given a recovery period of at least 1 week before returning to discrimination training and dose– response test. 2.8. Determination of monoamine contents Immediately after the retraining sessions and subsequent dose–response tests, rats were sacrificed. Brains were removed rapidly, and the striatum and nucleus accumbens 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. The contents of monoamines were determined using a HPLC system with an electrochemical detector (Eicom, Kyoto, Japan) as described (Noda et al., 1997, 1998). Briefly, each frozen tissue sample was weighed, then homogenized with an ultrasonic processor (475 W, Model XL2020, Heat Systems Inc., NY, USA) in 350 µl of 0.2 M perchloric acid containing isoproterenol (internal standard). The homogenate was placed in ice for 30 min and then centrifuged at 20,000×g for 15 min at 4°C. The supernatant was mixed with 1 M sodium acetate to adjust the pH to 3.0 and then injected into a liquid chromatography system equipped with a reversedphase ODS-column [4.6×150 mm, Eicompak MA-5 ODS (diameter of stationary phase grains; 5 µm), Eicom] and an electrochemical detector (Model ECD100, Eicom). The column temperature was maintained at 25°C and the detector potential was set at +750 mV. The mobile phase was 0.1 M citric acid and 0.1 M sodium acetate, pH 3.9, containing 14 % methanol, 160 mg/l sodium-l-octanesulfonate and 5 mg/l ethylenediamine tetraacetic acid: the flow rate was 1 ml/min. 2.9. Data analysis The percentages of correct responding during the first trial and entire session (correct responding/responses on both levers) and response rate (responses on both levers/s to complete session) were calculated for each training session. In the test sessions, the percentage of PCP responding during entire session (PCP-lever responding/responses on both levers) and response rate were calculated. Drugs were considered to have generalized to the discriminative stimulus properties of PCP if more than 80% of the responses were on the drugappropriate lever. The non-paired Student’s t-test was used to compare the percentage of correct responding during the first trial and entire session, or response rate in the retraining sessions and monoamine levels between vehicle and 6-OHDA-treated rats. Repeated measure analysis of variance (ANOVA) was used to compare the average percentages of PCP-appropriate responding between vehicle and 6-OHDA-treated rats in the dose–
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response tests. P values less than 0.05 were taken to indicate statistically significant differences.
3. Results 3.1. Performance of PCP discrimination and the dose–response tests The PCP discrimination required an average of 42 training sessions (range 36–48 sessions). Once rats attained the criterion, drug–saline discrimination stabilized and was maintained with a high degree of accuracy (⬎93%) for the remainder of the investigation (Fig. 1). In the dose–response tests, PCP (0.1–3 mg/kg) produced a dose-related increase in PCP-appropriate responding and the average percentages at 0.1, 0.3, 1, 1.5 and 3 mg/kg were 1.8, 43.4, 76.7, 98.3 and 98.8%, respectively. There was full substitution in all subjects after administration of doses that were equal to or greater than the training dose.
Fig. 1. Acquisition of PCP discrimination during the first 60 sessions in rats trained to discriminate PCP (1.5 mg/kg i.p.) from saline under a two-lever fixed ratio 20 schedule of food reinforcement. PCP and saline were injected 10 min before the session. Each point represents the mean±S.E.M. in 16 rats.
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3.2. Substitution tests The effects of microinjection of PCP, dizocilpine and DA into the bilateral nucleus accumbens in rats trained to discriminate PCP from saline are shown in Fig. 2. Control microinjections of saline into the bilateral nucleus accumbens resulted in less than 10% PCPappropriate responding and had no effects on the response rates. These results were nearly identical to those obtained from intraperitoneally administered saline. PCP (2–40 µg) and dizocilpine (2–12 µg), noncompetitive NMDA receptor antagonists, produced a dose-related increase in PCP-appropriate responding and fully substituted for PCP in all subjects. The average maximum percentage of PCP-appropriate responding was 92.2 and 97.3% after 40 µg PCP and 12 µg dizocilp-
Fig. 2. Effects of microinjection of PCP, dizocilpine and DA into the bilateral nucleus accumbens in rats trained to discriminate PCP (1.5 mg/kg i.p.) from saline. They were administered into the bilateral nucleus accumbens just before the session. Nialamide (100 mg/kg i.p.) was administered 22 h before dopamine test sessions. Each point represents the mean±S.E.M. in six to 16 rats. Points above PCP and SAL show control responses to i.p. administration of PCP (1.5 mg/kg) and saline, respectively. A dose of 0 µg shows microinjection of saline into the bilateral nucleus accumbens.
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ine, respectively. The response rate decreased at the highest dose of PCP and dizocilpine. DA (1–4 µg), however, did not engender PCP-appropriate responding and fully substituted for PCP in only one out of six subjects at a dose of 2 µg. The response rate decreased at the highest dose of DA. 3.3. Effects of 6-OHDA on the performance of PCP discrimination and the dose–response tests As shown in Fig. 3, 6-OHDA affected neither the discriminative effects of PCP during the first trial (A) and entire session (B) nor the response rates (C) in the retraining sessions. In Fig. 3B, the average percentage of PCP-appropriate responding during entire session in the retraining sessions was 90–99%. In both groups, the rats reliably discriminated PCP from saline and response rates were nearly identical. In the dose–response tests after the application of 6OHDA, there was no difference in the average percentages of PCP-appropriate responding between vehicle and 6-OHDA-treated rats (Fig. 4). 3.4. Monoamine levels in the nucleus accumbens and striatum The contents of DA in the nucleus accumbens of 6OHDA-treated rats were significantly decreased compared with those in the vehicle-treated rats (Table 1). In contrast, the contents of noradrenaline (NA) and serotonin (5-HT) in the nucleus accumbens and those and DA in striatum remained unaffected (Table 1).
4. Discussion A non-competitive NMDA receptor antagonist PCP (1.5 mg/kg, i.p.) was used as a discriminative stimulus in rats. The results of the present study showed that the discriminative stimulus effects of intraperitoneally administered PCP were similar to those of bilateral microinjections of PCP and dizocilpine, non-competitive NMDA receptor antagonists, into the nucleus accumbens. Several discriminative studies indicate that systemically administered non-competitive NMDA receptor antagonists substitute for PCP and the order of potency to produce PCP-like discriminative stimulus effects correlates significantly with relative affinity for the PCP binding sites on the NMDA receptor-ion channel complex (Tricklebank et al., 1987; Balster and Willetts, 1988; Koek et al., 1990). In the present study, the order of potency of producing PCP-like discriminative stimulus effects, dizocilpine ⬎PCP, was consistent with relative affinity for the PCP binding sites (Quirion et al., 1988). These results suggest that the discriminative stimulus effects of PCP are mediated via PCP binding
Fig. 3. Effects of 6-OHDA on the performance of PCP discrimination during the retraining sessions in rats trained to discriminate PCP (1.5 mg/kg i.p.) from saline under a two-lever fixed ratio 20 schedule of food reinforcement. (A) correct responding during the first trial (%), (B) correct responding during entire session (%), (C) response rate. PCP and saline were injected 10 min before the session. Each point represents the mean±S.E.M. in seven–nine rats.
sites on the NMDA receptor-ion channel complex in the nucleus accumbens. The nucleus accumbens has been shown to be involved in the mediation of the discriminative stimulus effects of cocaine (Wood and Emmett-Oglesby, 1989; Callahan et al., 1997) and amphetamine (Nielsen and Scheel-Kruger, 1986). These results demonstrate the importance of the nucleus accumbens in modulating the
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Fig. 4. Dose–response in the performance of PCP discrimination in 6-OHDA-treated rats. PCP was injected 10 min before the session. Each point represents the mean±S.E.M. in seven–nine rats. Table 1 Content of monoamines in the brain of rat treated with 6-OHDAa Treatment Striatum Vehicle 6-OHDA Nucleus accumbens Vehicle 6-OHDA a b
N
NA
DA
5-HT
7 9
8.5±1.4 9.8±1.4
3739.3±705.2 3919.3±313.6
21.5±2.3 23.2±3.3
7 9
44.0±17.7 71.9±17.3
2260.7±633.2 561.8±154.7b
24.9±3.4 20.1±3.8
Values are expressed as the mean±S.E.M. (ng/g wet tissue). P⬍0.05 vs. vehicle control group.
subjective effects of cocaine and amphetamine, especially because direct administration of these stimulants into other brain regions, such as the prefrontal cortex (Wood and Emmett-Oglesby, 1989), amygdara (Bryan et al., 1993) or dorso- and ventrolateral areas of the caudate putamen (Nielsen and Scheel-Kruger, 1986; Wood and Emmett-Oglesby, 1989), fails to engender full substitution for systemic administration of psychomotor stimulants. In the present study, the nucleus accumbens was involved in the mediation of the discriminative stimulus effects of PCP. The discriminative stimulus effects of microinjection of PCP into the nucleus accumbens, however, were only about nine fold potent, on a mg/kg basis, than the training dose administered systemically. This difference in potency is much less than that reported for the discriminative stimulus effects of microinjection of cocaine and amphetamine into the nucleus accumbens (Nielsen and Scheel-Kruger, 1986; Callahan et al., 1997). The potency of PCP, indeed, was increased by no more than about seven-fold from intraperitoneally to intraventricular administration (Slifer and Balster, 1985). One possible explanation for this differ-
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ence in potency is that a systemically administered dose of PCP may induce the discriminative stimulus effects more effectively in other parts of the brain than the nucleus accumbens. This remains to be tested by injections of PCP into different areas of the brain to localize the specific sites that mediate the discriminative stimulus effects of PCP. The ventral tegmental area is likely to be a candidate, since from perikarya in the ventral tegmental area DA pathways innervate numerous limbic (e.g., nucleus accumbens, amygdala) and cortical structures (e.g., prefrontal cortex). Another possibility is that it may be caused by the high lipophilicity of PCP (Kamenka and Geneste, 1981), which would facilitate its rapid diffusion into the cerebrospinal fluid, not leaving a high enough concentration of PCP in the nucleus accumbens. PCP has at least two pharmacological properties that may contribute to its ability to elevate DA concentrations directly through an inhibition of DA re-uptake (Gerhardt et al., 1987), and it increases dopaminergic cell firing through its ability to block NMDA receptors (French and Ceci, 1990). Several microdialysis studies have confirmed the ability of PCP to increase DA efflux in the nucleus accumbens (Bristow et al., 1993). In the drug discrimination paradigms, a monoamine re-uptake inhibitor, cocaine produced partial substitution in rats trained with PCP (Mori et al., 2001), whereas PCP fully substituted in rats trained with cocaine (Kantak et al., 1995). It is suggested that the discriminative stimulus effects of cocaine are mediated predominantly via DA D1 receptors in the nucleus accumbens, since microinjections of DA D1 receptor antagonists into the nucleus accumbens and 6-OHDA lesions of DA terminals in the nucleus accumbens disrupted the discriminative stimulus effects of cocaine (Callahan et al., 1997). Therefore, a partial cross substitution between PCP and cocaine suggests that the discriminative stimulus effects of PCP are mediated, at least in part, via the dopaminergic system in the nucleus accumbens. In the present results, microinjection of DA into the nucleus accumbens did not substitute for systemically administered PCP. The same finding has been reported by Ando et al. (1994); microinjection of DA into the nucleus accumbens does not substitute for subcutaneous methamphetamine. The reason for this lack of the substitution of DA is not certain. However, it is considered that rates of distribution and transformation of DA may be different from those of endogenous DA released by PCP. We have investigated the performance of PCP discrimination and the dose–response tests after the application of 6-OHDA. The destruction of dopaminergic nerve neurons in the nucleus accumbens with 6-OHDA failed to prevent the performance of PCP discrimination and there was no difference in the average percentages of PCP-appropriate responding between vehicle and 6-OHDA-treated rats in the dose–response tests. A possible explanation is that
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PCP may induce the discriminative stimulus effects at doses below those needed to block DA uptake, since PCP blocks NMDA receptors at concentrations lower than those needed to block DA uptake (Chaudieu et al., 1989; Ohmori et al., 1992). This hypothesis is supported by the present results. Microinjection of dizocilpine, which shares the ability to block NMDA receptors with PCP, but not to block DA uptake into the nucleus accumbens, reproduced the discriminative stimulus effects of systemic administration of PCP. Consequently, it seems to be reasonable to conclude that the discriminative stimulus effects of PCP are mediated via PCP binding sites on the NMDA receptor-ion channel complex, not via DA receptors in the nucleus accumbens. Several recent studies investigated the effects of intraaccumbal application of 6-OHDA on other effects of PCP, such as locomotor activity, and it has been shown that lesions somewhat attenuated PCP-induced increases in some behavioral measures (Nabeshima et al., 1983; Steinpreis and Salamone, 1993; Millan et al., 1999). These results suggest that the locomotor effects of PCP are mediated, at least in part, via DA receptors in the nucleus accumbens. However, there is evidence that some behavioral effects of PCP are not involved in dopamine-dependent mechanisms in the nucleus accumbens. For example, the effects of PCP on social behavior, stereotypy and ataxia were not altered by depletion of DA in the nucleus accumbens (Steinpreis and Salamone, 1993). In the self-administration paradigms, self-administration of PCP directly into the nucleus accumbens was not altered by co-infusion of a dose of the DA antagonist sulpiride that effectively blocked intracranial self-administration of the DA uptake inhibitor nomifensine (Carlezon and Wise, 1996). This result supported the present result that the dopaminergic system in the nucleus accumbens does not play a critical role in the discriminative stimulus effects of PCP. However, the present result does not entirely rule out the possible involvement of the dopaminergic system in other parts of the brain or neurotransmitters (Nabeshima et al., 1983, 1984; Millan et al., 1999) other than DA. Further studies are needed to clarify the involvement of dopamine and other neurotransmitters in the discriminative stimulus effects of PCP. In conclusion, the present study is the first report analyzing involvement of the nucleus accumbens in the discriminative stimulus effects of PCP. Microinjections of PCP and dizocilpine into the nucleus accumbens reproduced the discriminative stimulus effects of systemically administered PCP, whereas that of DA did not. The destruction of dopaminergic nerve neurons in the nucleus accumbens using 6-OHDA failed to prevent the performance of PCP discrimination and there was no difference in the average percentages of PCP-appropriate responding between vehicle and 6-OHDA-treated rats in the dose–response tests. These data suggest that the
discriminative stimulus effects of PCP are mediated via PCP binding sites on the NMDA receptor-ion channel complex in the nucleus accumbens, and the dopaminergic system in the nucleus accumbens does not play a critical role in the discriminative stimulus effects of PCP.
Acknowledgements This work was supported, in part, by a Grant-in-Aid for COE Research and Scientific Research (10044260) from the Ministry of Education, Science, Sports and Culture of Japan, by Special Coordination Funds for Promoting Science and Technology, the Target-oriented Brain Science Research Program, from the Ministry of Science and Technology of Japan and by Health Scientific Research Grants for Research on Pharmaceutical and Medical Safety from the Ministry of Health and Welfare of Japan.
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kawa, H., Kameyama, T., 1983. Effect of lesions in the stratum, nucleus accumbens and medial raphe on phencyclidine-induced stereotyped behaviors and hyperactivity in rats. European Journal of Pharmacology 91, 455–462. Nabeshima, T., Yamaguchi, K., Hiramatsu, M., Amano, M., Furukawa, H., Kameyama, T., 1984. Serotonergic involvement in phencyclidine-induced behaviors. Pharmacology, Biochemistry and Behavior 21, 401–408. Nielsen, E.B., Scheel-Kruger, J., 1986. Cueing effects of amphetamine and LSD: elicitation by direct microinjection of the drugs into the nucleus accumbens. European Journal of Pharmacology 125, 85– 92. Noda, Y., Mamiya, T., Furukawa, H., Nabeshima, T., 1997. Effects of antidepressants on phencyclidine-induced enhancement of immobility in a forced swimming test in mice. European Journal of Pharmacology 324, 135–140. Noda, Y., Miyamoto, Y., Mamiya, T., Kamei, H., Furukawa, H., Nabeshima, T., 1998. Involvement of dopaminergic system in phencyclidine-induced place preference in mice pretreated with phencyclidine. Journal of Pharmacology and Experimental therapeutics 286, 44–51. Ohmori, T., Koyama, T., Nakamura, F., Wang, P., Yamashita, I., 1992. Effect of phencyclidine on spontaneous and N-methyl-d-aspartate (NMDA)-induced efflux of dopamine from superfused slices of rat striatum. Neuropharmacology 31, 461–467. Pellegrino, L.J., Pellegrino, A.S., Cushman, A.J., 1979. A Stereotaxic Atlas of the Rat Brain, Second ed. Plenum Press, New York. Petersen, R.C., Stillman, R.C., 1978. Phencyclidine abuse: An appraisal, in National Institute of Drug Abuse Research Monograp, 21. Department of Health, Education and Welfare, Washington, DC pp. 1–17. Quirion, R., Chicheportiche, R., Contreras, P.C., Johnson, K.M., Lodge, D., Tam, S.W., Woods, J.H., Zukin, S.R., 1988. Classification and nomenclature of phencyclidine and σ-receptor sites. Trends in Neuroscience 10, 444–446. Shannon, H.E., 1982. Phencyclidine-like discriminative stimuli of (+)and (⫺)-N-allylnormetazocine in rats. European Journal of Pharmacology 84, 225–228. Slifer, B.L., Balster, R.L., 1985. A comparison of the discriminative stimulus properties of phencyclidine, given intraperitoneally or intraventricularly in rats. Neuropharmacology 24, 1175–1179. Steinpreis, R.E., Salamone, J.D., 1993. The role of nucleus accumbens dopamine in the neurochemical and behavioral effects of phencyclidine: a microdialysis and behavioral study. Brain Research 612, 263–270. Tricklebank, M.D., Singh, L., Oles, R.J., Wong, E.H.F., Iversen, S.D., 1987. A role for receptors of N-methyl-d-aspartic acid in the discriminative stimulus properties of phencyclidine. European Journal of Pharmacology 141, 497–501. Wise, R.A., Bozarth, M.A., 1987. Psychomotor stimulant theory of addiction. Psychological Review 94, 469–492. Witkin, J.M., Steele, T.D., Sharpe, L.G., 1996. Effects of strychnineinsensitive glycine receptor ligands in rats discriminating dizocilpine or phencyclidine from saline. Journal of Pharmacological and Experimental therapeutics 280, 46–52. Wood, D.M., Emmett-Oglesby, M.W., 1989. Mediation in the nucleus accumbens of the discriminative stimulus produced by cocaine. Pharmacology, Biochemistry and Behavior 33, 453–457.
Involvement of dopaminergic system in the nucleus accumbens in the discriminative stimulus effects of phencyclidine Akitomo Mori a, Yukihiro Noda a, Taku Nagai a, Takayoshi Mamiya a, Hiroshi Furukawa b, Toshitaka Nabeshima a,∗ a
Department of Neuropsychopharmacology and Hospital Pharmacy, Nagoya University Graduate School of Medicine, Nagoya 466-8560, Japan b Department of Medicinal Chemistry, Faculty of Pharmacy, Meijo University, Nagoya 468-8503, Japan Received 31 August 2001; received in revised form 27 February 2002; accepted 28 February 2002
Abstract The effects of microinjection of phencyclidine (PCP) and dizocilpine, non-competitive NMDA receptor antagonists, and dopamine into the nucleus accumbens were examined in rats trained to discriminate PCP (1.5 mg/kg i.p.) from saline under a two-lever fixed ratio 20 schedule of food reinforcement. Microinjection of PCP (2–40 µg) and dizocilpine (2–12 µg) into the bilateral nucleus accumbens produced a dose-dependent increase in PCP-appropriate responding and fully substituted for systemically administered PCP, whereas microinjection of dopamine (1–4 µg) did not produce PCP-like discriminative stimulus effects. The performance of PCP discrimination was assessed after bilateral destruction of the dopaminergic nerve neurons in the nucleus accumbens with dopaminergic neurotoxin, 6-hydroxydopamine (6-OHDA, 4 µg/1 µl/side). The destruction of dopaminergic nerve neurons in the nucleus accumbens failed to prevent the performance of PCP discrimination. There was no difference in the average percentages of PCP-appropriate responding between vehicle and 6-OHDA-treated rats in the dose–response tests. These results suggest that the dopaminergic system in the nucleus accumbens does not play a critical role in the discriminative stimulus effects of PCP. 2002 Elsevier Science Ltd. All rights reserved. Keywords: Phencyclidine; Drug discrimination; NMDA receptor; Dopamine; Nucleus accumbens; Rat
1. Introduction Phencyclidine (PCP), a non-competitive N-methyl-daspartate (NMDA) receptor antagonist, has been demonstrated to produce psychotomimetic effects in humans and be a widely abused drug (Petersen and Stillman, 1978). In animals, several studies have shown that PCP produces the discriminative stimulus (Holtzman, 1980), self-administration (Balster and Woolverton, 1980) and conditioned place preference (Kitaichi et al., 1996; Noda et al., 1998). Thus, although it has been well known that PCP induces dependence in humans and animals, the mechanisms of PCP-induced dependence remain unclear. Drug discrimination procedures have proven a valu-
Corresponding author. Tel.: +81-52-744-2674; fax: +81-52-7442979. E-mail address: [email protected] (T. Nabeshima). ∗
able means of obtaining information relevant to the subjective effects of drugs (Colpaert, 1978). The results of drug discrimination studies have suggested that the discriminative stimulus induced through antagonism of the NMDA receptor is highly dependent on the nature of the antagonism (Jackson and Sanger, 1988; Koek et al., 1990; Witkin et al., 1996). Namely, the discriminative stimulus effects of PCP are mediated predominantly via PCP binding sites on the NMDA receptor-ion channel complex (Mori et al., 2001). Microinjection of PCP into a lateral ventricle fully substituted for systemic administration of PCP in the rats trained to discriminate PCP from saline (Slifer and Balster, 1985). These findings demonstrate that the discriminative stimulus effects of PCP are centrally mediated. However, the mechanism behind the discriminative stimulus effects of PCP is not well elucidated. The mesolimbic–mesocortical dopaminergic pathways have been implicated as important substrates in the rewarding qualities of abuse drugs (Wise and Bozarth,
0028-3908/02/$ - see front matter 2002 Elsevier Science Ltd. All rights reserved. PII: S 0 0 2 8 - 3 9 0 8 ( 0 2 ) 0 0 0 3 7 - 0
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1987). The fact that PCP produces psychotomimetic effects and it is widely abused in humans seems to suggest an involvement with mesolimbic–mesocortical dopaminergic pathways. It has been reported that the discriminative stimulus effects of cocaine and amphetamine, abuse drugs as well as PCP, can be attributed to their ability to elevate extracellular concentrations of dopamine (DA) in the nucleus accumbens (Nielsen and Scheel-Kruger, 1986; Wood and Emmett-Oglesby, 1989; Callahan et al., 1997). PCP is self-administrated into the nucleus accumbens (Carlezon et al., 1995; Carlezon and Wise, 1996) and elevates extracellular concentrations of DA in the nucleus accumbens (Carboni et al., 1989). Therefore, there is a possibility that the dopaminergic system in the nucleus accumbens is involved in the discriminative stimulus effects of PCP. The present study was conducted to elucidate further the role of the mesolimbic dopaminergic system of the nucleus accumbens in PCP-induced discriminative stimulus. For this purpose, in the first experiment we investigated the generalized effects of local bilateral microinjections of PCP, dizocilpine, non-competitive NMDA receptor antagonists and of DA into the nucleus accumbens in rats trained to discriminate PCP from saline. In the second experiment, the influence of the destruction of the dopaminergic nerve terminals in the nucleus accumbens after local bilateral microinjections of the neurotoxin, 6-hydroxydopamine (6-OHDA) was tested in PCP-induced discriminative stimulus.
ventilated and sound-attenuated cubicles. The chambers were equipped with two response levers, spaced 16 cm apart with a food pellet trough mounted midway between the levers. A houselight was located over the trough. Reinforcement consisted of a 45-mg food pellet (Bio Serv Inc., Frenchtown, NJ). Scheduling of reinforcement contingencies, reinforcement delivery and data recording were controlled by computer systems (Neuroscience Co.).
2. Materials and methods
Rats were initially trained to press each of the two levers under a fixed ratio (FR) 1 schedule of food reinforcement. The FR response requirement for food delivery was gradually increased from 1 to 20. After responses under the FR 20 schedule of food reinforcement had been stabilized, drug discrimination training was started. Training sessions were conducted daily. Rats were injected 10 min before the session with either saline or PCP (1.5 mg/kg i.p.) according to a previous report (Mori et al., 2001). In drug discrimination training sessions, PCP or saline was administered randomly to ensure that no olfactory cues associated with the two levers (Extance and Goudie, 1981; Mori et al., 2001), which would bias the discrimination. After administration of PCP, 20 consecutive responses (FR 20) on one lever produced a food pellet, whereas after that of saline, 20 consecutive responses on the other lever produced a food pellet. Responding to the incorrect lever reset the FR requirement for the correct lever. Each session ended after 20 food pellets were delivered or 20 min was elapsed. The criteria for learning the discrimination was five consecutive sessions with: (1) more than 85% correct-lever responding before the first reinforcement; (2) more than 90% correct-lever responding throughout the session.
2.1. Animals Male Fischer rats (Charles River Japan, Atsugi, Japan), weighing 230–250 g at the beginning of the experiments, were used according to a previous report (Mori et al., 2001; Shannon, 1982). Each animal was housed individually in regulated conditions (23±1°C, 50±5% humidity) under a 12h/12h light-dark cycle (lights on at 09:00 h). Animals were deprived of food to 85% of free feeding weight. Water was available ad libitum in each home cage. All experiments were performed in accordance with the Guidelines for Animal Experiments of the Nagoya University Graduate School of Medicine. The procedures involving animals and their care conformed to the international guidelines “Principles of Laboratory Animal Care” (NIH publication no. 85-23, revised 1985). 2.2. Apparatus Experiments were conducted in operant-conditioning chambers (Neuroscience Co., Tokyo, Japan) located in
2.3. Drugs Phencyclidine hydrochloride [1-(1-phenylcyclohexyl) piperidine; PCP] was synthesized by the authors according to the method of Maddox et al. (1965) and it was checked for purity. (+)-5-Methyl-10,11-dihydro-5Hdibenzo[a,d]cyclohepten-5,10-imine hydrogen maleate salt (dizocilpine) and nialamide were purchased from Research Biochemicals (Natick, MA, USA). Dopamine hydrochloride (DA), 6-hydroxydopamine hydrochloride (6-OHDA) and desipramine hydrochloride were purchased from Sigma Chemical Co. (St. Louis, MO, USA). PCP, dizocilpine and desipramine were dissolved in 0.9% NaCl solution, DA and 6-OHDA in saline containing 0.1% ascorbic acid and nialmide in 0.1N hydrochloride. The dose of each drug refers to the drug form listed above. 2.4. PCP discrimination procedure
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2.5. Surgical procedure When the rats fulfilled the criteria for learning the discrimination, implantation of the cannula was carried out. Each rat was anesthetized with pentobarbital (35 mg/kg i.p.) and mounted in a stereotaxic frame. The skulls were exposed and holes were drilled. Each rat was implanted with bilateral 22-gauge guide cannulae that terminated 1.1 mm above the nucleus accumbens (according to the brain atlas of Pellegrino et al., 1979: with the incisor bar elevated to 5 mm above the interaural line, the cannula entry was 3.5 mm anterior to bregma, 2.4 mm bilateral to the midline suture; to avoid the bilateral ventricle, the cannulae were lowered at 10° angle toward the midline to an angled depth of 5.9 mm below the dura). The guide cannula was cemented in place with dental acrylic and the skin resutured around the base of the cannula. A stainless steel dummy cannula (28-gauge), extending 1.1 mm beyond the tip of the guide cannula, was inserted and remained in place when the animals were not injected. 2.6. Drug testing procedure After the surgery, the rats were given a recovery period of at least 1 week before returning to discrimination training. When the rats reliably discriminated PCP from saline again, the dose–response and the substitution tests were initiated. Test sessions were conducted once a week using rats that fulfilled the criteria during three consecutive training sessions. Test sessions were identical to training sessions except that 20 consecutive responses on either lever resulted in delivery of a food pellet. In the dose–response tests, lever selection was examined after the intraperitoneal administration of various doses of PCP, which was injected 10 min before the session. In the substitution tests, lever selection was examined after the administration of various doses of PCP or novel compounds into the nucleus accumbens. Injections were administered via two injection cannulae which were connected to Hamilton syringes by polyethylene tubes over a period of 120 s. Nialamide (100 mg/kg i.p.), a monoamine oxidase inhibitor, was administered 22 h before DA test sessions. Immediately after the drug administration, the rats were placed into the chambers and test sessions were begun. 2.7. 6-OHDA microinjections into the nucleus accumbens After the drug test, half of the animals received 6OHDA (4 µg/1 µl/side) and half of them received the 6-OHDA-vehicle into the bilateral nucleus accumbens via two injection cannulae over a period of 3 min. All rats were pretreated with desipramine (25 mg/kg i.p.) to prevent lesions of noradrenergic neurons and nialamide
(100 mg/kg i.p.). After 6-OHDA microinjections, the rats were given a recovery period of at least 1 week before returning to discrimination training and dose– response test. 2.8. Determination of monoamine contents Immediately after the retraining sessions and subsequent dose–response tests, rats were sacrificed. Brains were removed rapidly, and the striatum and nucleus accumbens 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. The contents of monoamines were determined using a HPLC system with an electrochemical detector (Eicom, Kyoto, Japan) as described (Noda et al., 1997, 1998). Briefly, each frozen tissue sample was weighed, then homogenized with an ultrasonic processor (475 W, Model XL2020, Heat Systems Inc., NY, USA) in 350 µl of 0.2 M perchloric acid containing isoproterenol (internal standard). The homogenate was placed in ice for 30 min and then centrifuged at 20,000×g for 15 min at 4°C. The supernatant was mixed with 1 M sodium acetate to adjust the pH to 3.0 and then injected into a liquid chromatography system equipped with a reversedphase ODS-column [4.6×150 mm, Eicompak MA-5 ODS (diameter of stationary phase grains; 5 µm), Eicom] and an electrochemical detector (Model ECD100, Eicom). The column temperature was maintained at 25°C and the detector potential was set at +750 mV. The mobile phase was 0.1 M citric acid and 0.1 M sodium acetate, pH 3.9, containing 14 % methanol, 160 mg/l sodium-l-octanesulfonate and 5 mg/l ethylenediamine tetraacetic acid: the flow rate was 1 ml/min. 2.9. Data analysis The percentages of correct responding during the first trial and entire session (correct responding/responses on both levers) and response rate (responses on both levers/s to complete session) were calculated for each training session. In the test sessions, the percentage of PCP responding during entire session (PCP-lever responding/responses on both levers) and response rate were calculated. Drugs were considered to have generalized to the discriminative stimulus properties of PCP if more than 80% of the responses were on the drugappropriate lever. The non-paired Student’s t-test was used to compare the percentage of correct responding during the first trial and entire session, or response rate in the retraining sessions and monoamine levels between vehicle and 6-OHDA-treated rats. Repeated measure analysis of variance (ANOVA) was used to compare the average percentages of PCP-appropriate responding between vehicle and 6-OHDA-treated rats in the dose–
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response tests. P values less than 0.05 were taken to indicate statistically significant differences.
3. Results 3.1. Performance of PCP discrimination and the dose–response tests The PCP discrimination required an average of 42 training sessions (range 36–48 sessions). Once rats attained the criterion, drug–saline discrimination stabilized and was maintained with a high degree of accuracy (⬎93%) for the remainder of the investigation (Fig. 1). In the dose–response tests, PCP (0.1–3 mg/kg) produced a dose-related increase in PCP-appropriate responding and the average percentages at 0.1, 0.3, 1, 1.5 and 3 mg/kg were 1.8, 43.4, 76.7, 98.3 and 98.8%, respectively. There was full substitution in all subjects after administration of doses that were equal to or greater than the training dose.
Fig. 1. Acquisition of PCP discrimination during the first 60 sessions in rats trained to discriminate PCP (1.5 mg/kg i.p.) from saline under a two-lever fixed ratio 20 schedule of food reinforcement. PCP and saline were injected 10 min before the session. Each point represents the mean±S.E.M. in 16 rats.
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3.2. Substitution tests The effects of microinjection of PCP, dizocilpine and DA into the bilateral nucleus accumbens in rats trained to discriminate PCP from saline are shown in Fig. 2. Control microinjections of saline into the bilateral nucleus accumbens resulted in less than 10% PCPappropriate responding and had no effects on the response rates. These results were nearly identical to those obtained from intraperitoneally administered saline. PCP (2–40 µg) and dizocilpine (2–12 µg), noncompetitive NMDA receptor antagonists, produced a dose-related increase in PCP-appropriate responding and fully substituted for PCP in all subjects. The average maximum percentage of PCP-appropriate responding was 92.2 and 97.3% after 40 µg PCP and 12 µg dizocilp-
Fig. 2. Effects of microinjection of PCP, dizocilpine and DA into the bilateral nucleus accumbens in rats trained to discriminate PCP (1.5 mg/kg i.p.) from saline. They were administered into the bilateral nucleus accumbens just before the session. Nialamide (100 mg/kg i.p.) was administered 22 h before dopamine test sessions. Each point represents the mean±S.E.M. in six to 16 rats. Points above PCP and SAL show control responses to i.p. administration of PCP (1.5 mg/kg) and saline, respectively. A dose of 0 µg shows microinjection of saline into the bilateral nucleus accumbens.
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ine, respectively. The response rate decreased at the highest dose of PCP and dizocilpine. DA (1–4 µg), however, did not engender PCP-appropriate responding and fully substituted for PCP in only one out of six subjects at a dose of 2 µg. The response rate decreased at the highest dose of DA. 3.3. Effects of 6-OHDA on the performance of PCP discrimination and the dose–response tests As shown in Fig. 3, 6-OHDA affected neither the discriminative effects of PCP during the first trial (A) and entire session (B) nor the response rates (C) in the retraining sessions. In Fig. 3B, the average percentage of PCP-appropriate responding during entire session in the retraining sessions was 90–99%. In both groups, the rats reliably discriminated PCP from saline and response rates were nearly identical. In the dose–response tests after the application of 6OHDA, there was no difference in the average percentages of PCP-appropriate responding between vehicle and 6-OHDA-treated rats (Fig. 4). 3.4. Monoamine levels in the nucleus accumbens and striatum The contents of DA in the nucleus accumbens of 6OHDA-treated rats were significantly decreased compared with those in the vehicle-treated rats (Table 1). In contrast, the contents of noradrenaline (NA) and serotonin (5-HT) in the nucleus accumbens and those and DA in striatum remained unaffected (Table 1).
4. Discussion A non-competitive NMDA receptor antagonist PCP (1.5 mg/kg, i.p.) was used as a discriminative stimulus in rats. The results of the present study showed that the discriminative stimulus effects of intraperitoneally administered PCP were similar to those of bilateral microinjections of PCP and dizocilpine, non-competitive NMDA receptor antagonists, into the nucleus accumbens. Several discriminative studies indicate that systemically administered non-competitive NMDA receptor antagonists substitute for PCP and the order of potency to produce PCP-like discriminative stimulus effects correlates significantly with relative affinity for the PCP binding sites on the NMDA receptor-ion channel complex (Tricklebank et al., 1987; Balster and Willetts, 1988; Koek et al., 1990). In the present study, the order of potency of producing PCP-like discriminative stimulus effects, dizocilpine ⬎PCP, was consistent with relative affinity for the PCP binding sites (Quirion et al., 1988). These results suggest that the discriminative stimulus effects of PCP are mediated via PCP binding
Fig. 3. Effects of 6-OHDA on the performance of PCP discrimination during the retraining sessions in rats trained to discriminate PCP (1.5 mg/kg i.p.) from saline under a two-lever fixed ratio 20 schedule of food reinforcement. (A) correct responding during the first trial (%), (B) correct responding during entire session (%), (C) response rate. PCP and saline were injected 10 min before the session. Each point represents the mean±S.E.M. in seven–nine rats.
sites on the NMDA receptor-ion channel complex in the nucleus accumbens. The nucleus accumbens has been shown to be involved in the mediation of the discriminative stimulus effects of cocaine (Wood and Emmett-Oglesby, 1989; Callahan et al., 1997) and amphetamine (Nielsen and Scheel-Kruger, 1986). These results demonstrate the importance of the nucleus accumbens in modulating the
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Fig. 4. Dose–response in the performance of PCP discrimination in 6-OHDA-treated rats. PCP was injected 10 min before the session. Each point represents the mean±S.E.M. in seven–nine rats. Table 1 Content of monoamines in the brain of rat treated with 6-OHDAa Treatment Striatum Vehicle 6-OHDA Nucleus accumbens Vehicle 6-OHDA a b
N
NA
DA
5-HT
7 9
8.5±1.4 9.8±1.4
3739.3±705.2 3919.3±313.6
21.5±2.3 23.2±3.3
7 9
44.0±17.7 71.9±17.3
2260.7±633.2 561.8±154.7b
24.9±3.4 20.1±3.8
Values are expressed as the mean±S.E.M. (ng/g wet tissue). P⬍0.05 vs. vehicle control group.
subjective effects of cocaine and amphetamine, especially because direct administration of these stimulants into other brain regions, such as the prefrontal cortex (Wood and Emmett-Oglesby, 1989), amygdara (Bryan et al., 1993) or dorso- and ventrolateral areas of the caudate putamen (Nielsen and Scheel-Kruger, 1986; Wood and Emmett-Oglesby, 1989), fails to engender full substitution for systemic administration of psychomotor stimulants. In the present study, the nucleus accumbens was involved in the mediation of the discriminative stimulus effects of PCP. The discriminative stimulus effects of microinjection of PCP into the nucleus accumbens, however, were only about nine fold potent, on a mg/kg basis, than the training dose administered systemically. This difference in potency is much less than that reported for the discriminative stimulus effects of microinjection of cocaine and amphetamine into the nucleus accumbens (Nielsen and Scheel-Kruger, 1986; Callahan et al., 1997). The potency of PCP, indeed, was increased by no more than about seven-fold from intraperitoneally to intraventricular administration (Slifer and Balster, 1985). One possible explanation for this differ-
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ence in potency is that a systemically administered dose of PCP may induce the discriminative stimulus effects more effectively in other parts of the brain than the nucleus accumbens. This remains to be tested by injections of PCP into different areas of the brain to localize the specific sites that mediate the discriminative stimulus effects of PCP. The ventral tegmental area is likely to be a candidate, since from perikarya in the ventral tegmental area DA pathways innervate numerous limbic (e.g., nucleus accumbens, amygdala) and cortical structures (e.g., prefrontal cortex). Another possibility is that it may be caused by the high lipophilicity of PCP (Kamenka and Geneste, 1981), which would facilitate its rapid diffusion into the cerebrospinal fluid, not leaving a high enough concentration of PCP in the nucleus accumbens. PCP has at least two pharmacological properties that may contribute to its ability to elevate DA concentrations directly through an inhibition of DA re-uptake (Gerhardt et al., 1987), and it increases dopaminergic cell firing through its ability to block NMDA receptors (French and Ceci, 1990). Several microdialysis studies have confirmed the ability of PCP to increase DA efflux in the nucleus accumbens (Bristow et al., 1993). In the drug discrimination paradigms, a monoamine re-uptake inhibitor, cocaine produced partial substitution in rats trained with PCP (Mori et al., 2001), whereas PCP fully substituted in rats trained with cocaine (Kantak et al., 1995). It is suggested that the discriminative stimulus effects of cocaine are mediated predominantly via DA D1 receptors in the nucleus accumbens, since microinjections of DA D1 receptor antagonists into the nucleus accumbens and 6-OHDA lesions of DA terminals in the nucleus accumbens disrupted the discriminative stimulus effects of cocaine (Callahan et al., 1997). Therefore, a partial cross substitution between PCP and cocaine suggests that the discriminative stimulus effects of PCP are mediated, at least in part, via the dopaminergic system in the nucleus accumbens. In the present results, microinjection of DA into the nucleus accumbens did not substitute for systemically administered PCP. The same finding has been reported by Ando et al. (1994); microinjection of DA into the nucleus accumbens does not substitute for subcutaneous methamphetamine. The reason for this lack of the substitution of DA is not certain. However, it is considered that rates of distribution and transformation of DA may be different from those of endogenous DA released by PCP. We have investigated the performance of PCP discrimination and the dose–response tests after the application of 6-OHDA. The destruction of dopaminergic nerve neurons in the nucleus accumbens with 6-OHDA failed to prevent the performance of PCP discrimination and there was no difference in the average percentages of PCP-appropriate responding between vehicle and 6-OHDA-treated rats in the dose–response tests. A possible explanation is that
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PCP may induce the discriminative stimulus effects at doses below those needed to block DA uptake, since PCP blocks NMDA receptors at concentrations lower than those needed to block DA uptake (Chaudieu et al., 1989; Ohmori et al., 1992). This hypothesis is supported by the present results. Microinjection of dizocilpine, which shares the ability to block NMDA receptors with PCP, but not to block DA uptake into the nucleus accumbens, reproduced the discriminative stimulus effects of systemic administration of PCP. Consequently, it seems to be reasonable to conclude that the discriminative stimulus effects of PCP are mediated via PCP binding sites on the NMDA receptor-ion channel complex, not via DA receptors in the nucleus accumbens. Several recent studies investigated the effects of intraaccumbal application of 6-OHDA on other effects of PCP, such as locomotor activity, and it has been shown that lesions somewhat attenuated PCP-induced increases in some behavioral measures (Nabeshima et al., 1983; Steinpreis and Salamone, 1993; Millan et al., 1999). These results suggest that the locomotor effects of PCP are mediated, at least in part, via DA receptors in the nucleus accumbens. However, there is evidence that some behavioral effects of PCP are not involved in dopamine-dependent mechanisms in the nucleus accumbens. For example, the effects of PCP on social behavior, stereotypy and ataxia were not altered by depletion of DA in the nucleus accumbens (Steinpreis and Salamone, 1993). In the self-administration paradigms, self-administration of PCP directly into the nucleus accumbens was not altered by co-infusion of a dose of the DA antagonist sulpiride that effectively blocked intracranial self-administration of the DA uptake inhibitor nomifensine (Carlezon and Wise, 1996). This result supported the present result that the dopaminergic system in the nucleus accumbens does not play a critical role in the discriminative stimulus effects of PCP. However, the present result does not entirely rule out the possible involvement of the dopaminergic system in other parts of the brain or neurotransmitters (Nabeshima et al., 1983, 1984; Millan et al., 1999) other than DA. Further studies are needed to clarify the involvement of dopamine and other neurotransmitters in the discriminative stimulus effects of PCP. In conclusion, the present study is the first report analyzing involvement of the nucleus accumbens in the discriminative stimulus effects of PCP. Microinjections of PCP and dizocilpine into the nucleus accumbens reproduced the discriminative stimulus effects of systemically administered PCP, whereas that of DA did not. The destruction of dopaminergic nerve neurons in the nucleus accumbens using 6-OHDA failed to prevent the performance of PCP discrimination and there was no difference in the average percentages of PCP-appropriate responding between vehicle and 6-OHDA-treated rats in the dose–response tests. These data suggest that the
discriminative stimulus effects of PCP are mediated via PCP binding sites on the NMDA receptor-ion channel complex in the nucleus accumbens, and the dopaminergic system in the nucleus accumbens does not play a critical role in the discriminative stimulus effects of PCP.
Acknowledgements This work was supported, in part, by a Grant-in-Aid for COE Research and Scientific Research (10044260) from the Ministry of Education, Science, Sports and Culture of Japan, by Special Coordination Funds for Promoting Science and Technology, the Target-oriented Brain Science Research Program, from the Ministry of Science and Technology of Japan and by Health Scientific Research Grants for Research on Pharmaceutical and Medical Safety from the Ministry of Health and Welfare of Japan.
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