Introduction

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There is substantial evidence for regional differences between the striatum and nucleus accumbens with regard to dopamine metabolism and/or the activity of dopamine neurons induced by dopamine receptor antagonists (Anden and Stock, 1973; Weisel and Sedvall, 1975; Zivkovic et al., 1975; Chiodo and Bunney, 1983; Yamada et al., 1995). However, the mechanism underlying the regional difference in dopamine receptor antagonist-induced changes in dopamine turnover remains unclear. Sokoloff et al. (1990) found that the mRNA encoding the dopamine D₃ receptor exhibited a unique distribution in the brain; with the highest density in the islands of Calleja, olfactory tubercules, and nucleus accumbens; but with very little in the striatum.

The physiological role of dopamine D₃ receptor remains unclear. The speculated role of the receptor was that of an autoreceptor because dopamine D₃ receptor mRNA was found in the 6-hydroxydopamine-sensitive neurons in the substantia nigra and ventral tegmental area (Sokoloff et al., 1990). Stimulation of D₃ receptors expressed in neuronal mesencephalic MN9D cells resulted in a dose-dependent inhibition of dopamine release (Tang et al., 1994). Furthermore, the D₃-preferring agonist 7-hydroxy-N,N,-di-propyl-2-aminotetralin produces a decrease in dopamine release in vivo as well as in accumbal slices (Damsma et al., 1993; Rivet et al., 1994; Yamada et al., 1994; Devoto et al., 1995; Gilbert et al., 1995; Gaintdinov et al., 1996). However, recent studies failed to detect D₃ receptor mRNA in the rat midbrain (Landwehrmer et al., 1993; Meador-Woodruff et al., 1994; Richtand et al., 1995; Healy and Meador-Woodruff, 1996). In this regard, recent studies have shown a lack of changes in dopamine autoreceptor function in dopamine D₃ receptor mutant mice (Koeltzow et al., 1998). These findings indicate that dopamine D₃ receptors may be present in postsynaptic sites but not in presynaptic sites. We postulated that the regional differences in dopamine release in response to dopamine receptor agonists and/or antagonists could be due to the different distribution of dopamine D₂ or D₃ receptors (Yamada et al., 1994, 1995).

The aim of the present study was to investigate the contribution of dopamine D₃ receptor to the regional difference in dopamine antagonist-induced increase in dopamine release from the nucleus accumbens and striatum. We also investigated the role of the cholinergic system in the above-mentioned regional differences. We used the in vitro slice superfusion method (Yamada et al., 1993) to examine the effect of a preferential dopamine D₂ antagonist, haloperidol, and the preferential dopamine D₃ antagonists 5,6-dimethoxy-2-(di-n-propylamine)indan (U-99194A) and cis-(+)-(1S,2R)-5-methoxy-1-methyl-2-(di-n-propylamino)tetralin HCl [(+)-UH232] with or without pirenzepine, on the electrically evoked dopamine release from the striatum and nucleus accumbens.

	Materials and Methods

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Slice Preparation. Adult male Wistar rats were used in the present experiments. The experimental protocol was approved by the Ethics Review Committee for Animal Experimentation of Kurume University School of Medicine. The rats were sacrificed by decapitation at 9:00 to 10:00 AM. The brain was immediately removed and immersed in an ice-cold Krebs' solution, and bubbled with a gas mixture of 95% O₂/5% CO₂. Four slices, 0.3 mm in thickness, were prepared with a Microslicer (Dohan EM Co., Kyoto, Japan) at coordinates A 9760 to A 7630, according to the atlas of Konig and Klippel (1963). One slice was used for control experiments (perfusion without drugs), whereas the other three slices were used for perfusion studies. The striatum and nucleus accumbens in the slices were punched out with a metal tube (2 mm i.d.). One slice of the striatum or the nucleus accumbens was placed in a chamber constructed from a Teflon tube, in which platinum electrodes were mounted to stimulate the slice. Eight slices were used in the stimulation studies, including two control (no drug)-superfused slices.

Superfusion Experiments. The slice was perfused with Krebs' solution, and bubbled with a gas mixture of 95% O₂/5% CO₂ at a flow rate of 1 ml/min at 37°C. The composition of the Krebs' solution was as follows: 118.0 mM NaCl, 4.9 mM KCl, 25.0 mM NaHCO₃, 1.25 mM NaH₂PO₄, 1.25 mM CaCl₂, 1.18 mM MgCl₂, and 11.0 mM glucose with added nomifensine (3 µM), a dopamine reuptake inhibitor to prevent the reuptake of released dopamine. After a steady state of 50 min, electric field stimulation was performed with the spiral platinum electrodes set up at the end of the chamber. Electric stimulation consisted of 20 mA rectangular pulses of 2-ms duration at a frequency of 1 Hz for 2 min, which was applied 50 min (S1) and 72 min (S2) after commencement of superfusion. Haloperidol (0.1, 0.3, or 0.6 µM; Research Biochemicals Inc., Natick, MA), a preferential D₂ receptor antagonist, and U-99194A (0.1, 1, or 10 µM; Research Biochemicals Inc.) or (+)-UH232 (0.1, 0.3, or 1 µM; Upjohn Company, Kalamazoo, MI) (preferential D₃ receptor antagonists) were added to the perfusate at various concentrations, 15 min before the S2 stimulus with or without pirenzepine (0.1-2 µM; Sigma Chemical Co., St. Louis, MO), a muscarinic M₁ receptor antagonist, or the dopamine D₂/₃ receptor agonist quinpirole (1 µM; Research Biochemicals Inc.). The superfusate was collected in tubes as the 7-min fraction. Six animals were used for the superfusion experiment observing the effect of halopridol or (+)-UH232 and 12 animals were used for U-99194A.

Dopamine Assay. Dopamine released into the superfusate was adsorbed on alumina, eluted with 300 µl of 0.1 M acetic acid, and quantitated by HPLC with electrochemical detection (ECD-100, EICOM Co., Kyoto, Japan). Dopamine release evoked by each electric stimulus was estimated from the total amount minus spontaneous dopamine release during stimulation, expressed as S1 or S2. Spontaneous release was estimated as the amount released for 7 min immediately before electric stimulation. The method used for HPLC with electrochemical detection has been described previously (Yamada et al., 1993).

Data Analysis. Data were expressed as means ± S.E. in nanograms of dopamine per milligram protein per 7-min perfusate fraction, as the S2/S1 ratio or as a percentage of control S2/S1 ratio. Differences between control and drug-induced level of evoked dopamine release from the striatum or nucleus accumbens were examined for statistical significance by one-way ANOVA with Fisher post hoc comparisons. In addition, a two-way ANOVA was used to assess regional differences and effects of pirenzepine in drug-induced changes in dopamine release. A p value < .05 denoted the presence of statistically significant difference.

	Results

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Effects of Drugs on Evoked Dopamine Release from the Striatum and Nucleus Accumbens. The evoked dopamine release from striatal slices (2.15 ± 0.16 ng/mg protein/7-min fraction, n = 21) was higher than that from the accumbens slices (0.91 ± 0.09 ng/mg protein/7-min fraction, p < .05). Under control conditions, the S2/S1 ratio for the striatum was almost similar to that of the nucleus accumbens. Both D₂ and D₃ antagonists [up to a concentration of 0.6 µM for haloperidol, 1 µM for (+)-UH232, or 10 µM U-99194A] had no effect on the spontaneous release of dopamine (data not shown). Haloperidol resulted in a significant increase in electrically evoked dopamine release from the striatum as well as nucleus accumbens (one-way ANOVA, p = .0004 for the striatum and p < .0001 for the nucleus accumbens), but there was no regional difference in the drug-induced dopamine release (two-way ANOVA, p = .93) (Table 1). (+)-UH232 also increased the release of dopamine from both regions (one-way ANOVA, p < .0001 for the striatum and p = .0006 for the nucleus accumbens). (+)-UH232-induced dopamine released from the nucleus accumbens was significantly higher than that from the striatum (two-way ANOVA, p = .001) (Table 1). U-99194A (1-10 µM) enhanced the evoked dopamine release from the nucleus accumbens (one-way ANOVA, p = .036) but not from the striatum (p = .81, regional difference; two-way ANOVA, p = .003) (Table 1).

Effects of U-99194A on Quinpirole-Induced Reduction of Evoked Dopamine Release. As shown in Fig. 1, the addition of the D₂/D₃ receptor agonist quinpirole (1 µM) caused a greater reduction in the evoked dopamine release from the nucleus accumbens (55%) than from the striatum (68%), in agreement with our previous report (Yamada et al., 1994). U-99194A dose dependently attenuated the effect of quinpirole on evoked dopamine release from the nucleus accumbens but not from the striatum (two-way ANOVA, interaction of region × U-99194A, p = .015) (Fig. 1).

View larger version (25K): [in this window] [in a new window]   Fig. 1.   Effect of U-99194A on quinpirole-induced reduction of evoked dopamine release from the nucleus accumbens (closed columns) and striatum (open columns). U-99194A attenuated the quinpirole-induced reduction of dopamine release from the nucleus accumbens but not from the striatum (two-way ANOVA, interaction of region × U-99194A, p = .015). Data are means ± S.E.

Effects of Pirenzepine on Haloperidol, U-99194A, or (+)-UH232-Induced Increase in Evoked Dopamine Release. Pirenzepine itself had no effect on the evoked dopamine release from the slices of either region, even at 2 µM (data not shown). However, U-99194A-induced increase in dopamine release from the nucleus accumbens was abolished by superfusion with 0.3 µM pirenzepine (two-way ANOVA, treatment, p < .003) (Fig. 2A). (+)-UH232-induced increase in evoked dopamine release from the nucleus accumbens was reduced by superfusion with 0.3 µM pirenzepine (two-way ANOVA, treatment, p = .0024). In contrast, (+)-UH232-induced increase in dopamine release from the striatum was unchanged by pirenzepine (Fig. 2B). The regional difference in U-99194A- and (+)-UH232-induced increase in dopamine release from the striatum and nucleus accumbens was abolished when the slices were superfused with 0.3 µM pirenzepine (Fig. 2, A and B). Haloperidol- induced increase in dopamine release from the nucleus accumbens and striatum was attenuated by superfusion with pirenzepine (one-way ANOVA, p = .014 for the striatum and p = .043 for the nucleus accumbens) (Fig. 2C). There were no regional differences in haloperidol-induced increase in dopamine release irrespective of the presence or absence of pirenzepine (two-way ANOVA, treatment, p = .93 in the absence of pirenzepine; p = .39 in the presence of pirenzepine).

View larger version (49K): [in this window] [in a new window]   Fig. 2.   Effects of pirenzepine on U-99194A- (A), (+)-UH-232- (B), or haloperidol (C)-induced increase in evoked dopamine release from slices of nucleus accumbens (closed bars) or striatum (open bars). Pirenzepine (0.3 µM) reduced the U-99194A- or (+)-UH232-induced increase in the evoked dopamine release from the nucleus accumbens (gray bars) but not from the striatum (hatched bars) and abolished the regional difference in evoked dopamine release (two-way ANOVA, treatment, p = .0024 for (+)-UH-232 in the nucleus accumbens; p = .003 for U-99194A in the nucleus accumbens). There was no regional difference in haloperidol-induced dopamine release from the striatum and nucleus accumbens (two-way ANOVA, treatment, p = .93). Pirenzepine reduced haloperidol-induced increase in the evoked dopamine release from both region (two-way ANOVA, treatment, p = .014 for the striatum; p = .043 for the nucleus accumbens). Data are means ± S.E. of percentage of control (no drugs) S2/S1 ratio.

	Discussion

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The major finding of the present study was that (+)-UH232 and U-99194A but not haloperidol caused a preferential increase in the electrically evoked dopamine release from the nucleus accumbens relative to that from the striatum. In particular, U-99194A, a specific dopamine D₃ receptor antagonist, had no effect on dopamine release from the striatum (Table 1). It is thought that enhancement of dopamine release by superfusion with dopamine D₂ or D₃ receptor antagonists is probably mediated by blockade of autoreceptors on dopamine nerve terminals (Dismukes and Mulder, 1977; Starke et al., 1978; Nowak et al., 1983; Dwoskin and Zahniger, 1986; Yamada et al., 1993; Gaintdinov et al., 1994; Gobert et al., 1996). However, (+)-UH232 is thought to be a partial agonist of the dopamine D₃ receptor with an intrinsic activity of 0.2 to 0.4 (Griffon et al., 1995). Becasue the presence of nomifensine in the superfusate increases dopamine levels in the synaptic gap, it is likely that (+)-UH232 acted as a dopamine D₃ receptor antagonist in the present experimental conditions.

The relative affinities of the dopamine D₂/D₃ receptor for haloperidol, (+)-UH-232, and U-99194A are 0.05, 4.3, and 20, respectively (Sokoloff et al., 1990; Waters et al., 1993). The preferential affinity to dopamine D₃ receptors of the compounds used in the present study appears to parallel their ability to preferentially affect dopamine release in the rat nucleus accumbens in vitro, in agreement with the conclusions of previous studies (Waters et al., 1994; Yamada et al., 1995) that compounds with a high K_I D₂/K_I D₃ ratio cause a marked increase in dopamine release from the nucleus accumbens compared with that from the striatum.

Our results also showed that quinpirole caused a greater reduction in the evoked dopamine release from nucleus accumbens than from the striatum, as previously reported (Yamada et al., 1994). Furthermore, U-99194A attenuated the quinpirole-induced reduction of evoked dopamine release from the nucleus accumbens but not from the striatum. Our results clearly showed for the first time that U-99194A modulated dopamine release from the nucleus accumbens but not from the striatum. Because quinpirole has a higher affinity for D₃ receptors than that for D₂ receptors, D₃ receptor blocking property of U-99194A would antagonize the effect of quinpirole in the nucleus accumbens.

Previous studies have shown that dopamine D₃ receptors are predominantly located in the limbic system and are somewhat less common in the striatum (Lavesque et al., 1992). Furthermore, recent studies failed to detect D₃ receptor mRNA in the rat midbrain (Landwehrmer et al., 1993; Meador-Woodruff et al., 1994; Richtand et al., 1995; Healy and Meador-Woodruff, 1996) and there is no change in dopamine autoreceptor function in dopamine D₃ receptor mutant mice (Koeltzow et al., 1998). Moreover, Bowery et al. (1996) reported that putative D₃ autoreceptor effects in rats are mediated by D₂ receptors. These findings indicate that D₃ receptor may lack autoreceptor function and that postsynaptic D₃ receptors may regulate dopamine release via a short-loop feedback mechanism in the nucleus accumbens. The most striking finding in the present study was that superfusion with the muscarinic M₁ receptor antagonist pirenzepine, which had no effect on dopamine release by itself, reduced (+)-UH232- and U-99194A-induced increase in the evoked dopamine release from the nucleus accumbens and abolished the regional differences in drug-induced increase in evoked dopamine release (Fig. 2, A and B). This is a first report suggesting that preferential dopamine D₃ antagonist-induced increase in evoked dopamine release from the nucleus accumbens could, at least partially, be mediated by activation of muscarinic M₁ receptors. There is substantial evidence that acetylcholine enhances dopamine release from dopamine nerve terminals via muscarinic M₁ receptors (Westfall, 1974; Giorguieff et al., 1977; Butcher and Talbot, 1978; Lehmann and Langer, 1982; de Belleroche and Gardiner, 1985; Xu et al., 1989; Gongoraalfaro et al., 1996). Admittedly, the concentration of pirenzepine used in our experiments was 0.3 µM, which was too high to be specific for muscarinic M₁ receptors. Further studies are necessary to examine the effect of nanomolar concentrations of pirenzepine on (+)-UH232 or U-99194A effects on evoked dopamine release to determine the exact muscarinic receptor subtype involved in the inhibitory effect of pirenzepine. Whether this enhancement is due to the activation of muscarinic M₁ or M₃ receptors located on dopamine nerve terminals or whether it is due to interaction with other neuronal elements is not clear at present. Previous studies have shown that postsynaptic dopamine D₂ or D₃ receptors on cholinergic neurons tonically inhibit the release of acetylcholine (Baud et al., 1985; Meltzer et al., 1994). Another study suggested the presence of postsynaptic muscarinic receptors (M₁ type) on glutamatergic terminals (Hersch et al., 1994). Combined, the above-mentioned results together with those of the present study indicate that the blocking of postsynaptic dopamine D₃ receptor could enhance the release of acetylcholine from intrinsic acetylcholine neurons in the nucleus accumbens, resulting in the enhancement of dopamine release from dopamine nerve terminals. In contrast, pirenzepine partially reduced the haloperidol-induced increase in dopamine release from the nucleus accumbens to the same extent as that in the striatum, which indicates that some part of the haloperidol-induced increase in evoked dopamine release could be caused by blocking of postsynaptic dopamine D₂ receptors in both regions. The regional differences in responsiveness to dopamine D₃ receptor antagonists may be mediated by the cholinergic system, which has a significant number of dopamine D₃ receptors in the nucleus accumbens, but fewer such receptors in the striatum.