Introduction

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In situ hybridization studies show that in a variety of species, including humans, dopamine (DA) D₃ receptor mRNA is enriched in limbic brain areas, such as ventral striatum, nucleus accumbens, dentate gyrus, and islands of Calleja (Sokoloff et al., 1990; Bouthenet et al., 1991; Herroelen et al., 1994; Levant, 1998; Suzuki et al., 1998; Gurevich and Joyce, 1999). Receptor autoradiographic studies with a variety of radioligands broadly confirm this distribution (Herroelen et al., 1994; Gurevich and Joyce, 1999), although D₃ receptor distribution in human brain appears to be more widespread and there are differences between human and rat in the distribution reported in motor striatum and ventral tegmental area (VTA) (Herroelen et al., 1994; Suzuki et al., 1998; Gurevich and Joyce, 1999).

The distribution of D₃ receptors suggests potential roles for the receptor in processes related to cognitive and emotional behavior (Herroelen et al., 1994; Suzuki et al., 1998; Gurevich and Joyce, 1999) and reinforcement and drug abuse (Pilla et al., 1999). Furthermore, the limbic structures in which dopamine D₃ receptors are located are thought to be involved in the pathogenesis of schizophrenia (Matthysse, 1973) and are considered important targets for antipsychotic agents (Sokoloff et al., 1990, 1992). Indeed, clinically effective antipsychotics have high affinity for dopamine D₃ as well as D₂ receptors and these drugs are thought to occupy both receptor subtypes at clinically active doses (Schwartz et al., 1993). Interestingly, postmortem data indicate that D₃ receptor number is elevated in the ventral striatum and striatopallidal targets in schizophrenia and this is reduced by concurrent antipsychotic treatment (Gurevich et al., 1997).

Although a number of putatively selective D₃ receptor antagonists such as (+)-UH232, (+)-AJ76 (Sokoloff et al., 1992; Sonesson et al., 1993), U 99194A (Waters et al., 1993), and nafadotride (Sautel et al., 1995) have been described, their selectivity is only 5- to 25-fold higher for D₃ versus D₂ receptors in vitro. The compounds (+)-S-14297 (Millan et al., 1994; Audinot et al., 1998) and PD 152255 are reported to have greater selectivity in vitro (Corbin et al., 1998), although Flietstra and Levant (1998) have reported that the selectivity of PD 152255 for D₃ versus D₂ receptors was only 6-fold. The compounds GR 103691 and PD 58491 (Whetzel et al., 1997) have D₃ versus D₂ selectivities of 132- and 120-fold in vitro (Audinot et al., 1998), respectively; however, GR 103691 has significant affinity for 5-hydroxytryptamine_1A and ₁-adrenoceptors (Audinot et al., 1998) and does not penetrate well into the brain (Audinot et al., 1998). Therefore, the pharmacological properties of these compounds make it difficult to attribute their effects solely to interactions with D₃ receptors. Indeed, the locomotor-stimulating effects of both l-nafadotride and U 99194A also are observed in DA D₃ receptor knockout mice, clearly indicating that the stimulant properties of both compounds are unrelated to their D₃ receptor-blocking activities (Xu et al., 1999). In addition, a recent study has shown that the differences in behaviors elicited by U 99194A, GR 103691, and nafadotride are not related to their relative selectivities for D₃ receptors in rats (Clifford and Waddington, 1998). A recent study has reported the synthesis of a novel benzopyrano[3,4-c] pyrole derivative, S 33084, which has a >100-fold greater affinity for D₃ versus D₂ receptors (Dubuffet et al., 1999), although its pharmacological profile still remains largely uncharacterized.

The compound SmithKline Beecham (SB)-277011-A {trans-N-[4-[2-(6-cyano-1,2,3,4-tetrahydroisoquinolin-2-yl) ethyl]-cyclohexyl]-4-quinolininecarboxamide} is a brain penetrant, DA D₃ receptor antagonist with high affinity for the human D₃ (pK_i = 7.97) and rat D₃ receptors and has a 100-fold selectivity over the human D₂ receptor and 66 other receptors, enzymes, and ion channels (Reavill et al., 2000). The compound is a competitive antagonist at the human D₃ receptor in a functional assay (pK_b = 8.3). In vivo microdialysis studies demonstrate that SB-277011-A reverses the quinelorane (D₂/D₃ receptor agonist)-induced reduction of dopamine efflux in the nucleus accumbens, an area with a high density of D₃ receptors (Reavill et al., 2000).

One model that can be used to screen for compounds with potential antipsychotic activity is the in vivo electrophysiological model measuring the number of spontaneously active midbrain DA neurons in anesthetized rats. Previous studies have shown that the repeated administration of typical antipsychotic drugs, such as haloperidol, produces a significant decrease in the number of spontaneously active DA neurons in the VTA (or A10) and substantia nigra pars compacta (SNC or A9; Bunney and Grace, 1978; Chiodo and Bunney, 1983; White and Wang, 1983; Minabe et al., 1991, 1992). In contrast, the repeated administration of atypical antipsychotic drugs, such as clozapine, which have a lower propensity for inducing neurological side effects, significantly decreases the number of spontaneously active VTA DA neurons (Chiodo and Bunney, 1983; White and Wang, 1983; Minabe et al., 1991; Ashby and Wang, 1996). The repeated administration of compounds that lack antipsychotic action does not significantly decrease the number of midbrain DA neurons (Chiodo and Bunney, 1983; White and Wang, 1983; Chiodo, 1988). It has been postulated that the decrease in the number of spontaneously active VTA and SNC DA neurons produced by the repeated administration of compounds may be correlated with their therapeutic and neurological side effects, respectively (Chiodo and Bunney, 1983; White and Wang, 1983).

In this study, we examined the effect of the acute and repeated administration of SB-277011-A on the number, as well as the firing pattern, of spontaneously active SNC and VTA DA neurons in anesthetized male rats. This was accomplished with the electrophysiological technique of in vivo extracellular recording. The effect of the i.v. administration of SB-277011-A on the firing pattern of spontaneously active VTA and SNC DA neurons also was determined.

	Materials and Methods

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Animals and Surgery

Male albino Sprague-Dawley rats (200-225 g for acute studies and 300-350 g at the end of the chronic regimen; Taconic Farms, Inc., Germantown, NY) were anesthetized with chloral hydrate (400 mg/kg i.p.) and mounted in a stereotaxic instrument. A lateral tail vein was cannulated with a 25-gauge needle for the administration of additional anesthetic or drug solution. The animals were placed on a heating pad to maintain a constant body temperature of 37-38°C. A hole was drilled over the A9 (anterior 3.0-3.5 mm to the lambda, lateral 1.8-2.5 mm to the midline, and 6.0-8.5 mm ventral to the cortical surface) and A10 (anterior 3.0-3.5 mm, lateral 0.5-1.0 mm, and ventral 6.0-8.5 mm; according to the atlas of Paxinos and Watson, 1986) regions and the dura retracted.

Extracellular Single-Unit Recording Procedure

Single barrel microelectrodes were used for recording single-cell activity. Glass micropipettes, which were pulled with an electrode puller (Narishige PE-2) and the tip broken back under a light microscope, were filled with a solution of 2 M NaCl saturated with 1% fast green dye. The impedance of the electrodes was usually 0.8 to 1.2 M measured at 135 Hz in vitro and 1.5 to 2.0 m in vivo.

Identification of DA Neurons

During the recording sessions, a neuron encountered in the A9-A10 area was considered dopaminergic if it possessed the following characteristics: 1) a wide action potential (>2.5 ms), with a distinct initial segment and late positive component; 2) a characteristic low-pitch sound when monitored through an audioamplifier; 3) a slow, regular, or bursting firing pattern; and 4) a spontaneous firing rate of 2 to 9 Hz (Bunney et al., 1973; Wang, 1981).

Firing Pattern Analysis

The DA neuronal spikes obtained after the acute and chronic administration of vehicle, haloperidol, or SB-277011-A were fed into an SX 486 computer via an interface Rate/ISI Histogram program for PC; Symbolic Logic, Inc., Grapevine, TX). The data regarding the firing pattern of the discriminated DA neurons were analyzed off-line and the following parameters were calculated or determined: spikes per burst, percentage of events in bursts, mean interspike interval (interval between successive spikes), coefficient of variation (ratio of standard deviation and mean interspike interval × 100), and percentage of cells exhibiting burst firing (defined as DA neurons that showed two or more bursts of at least three spikes of a series of 500 spikes). These values were determined over a period of 500 intervals between successive DA neuron spikes. The onset of a burst was defined by an interval less than 80 ms and the termination of a burst as an interval exceeding 160 ms. These parameters were programmed into the Rate/ISI program.

Determination of Number of Spontaneously Active A9 and A10 DA Neurons

The number of spontaneously active DA neurons was determined in 10 stereotaxic electrode descents or tracks as previously described (White and Wang, 1983; Chiodo, 1988). Briefly, 10 electrode tracks (separated from each other by 200 µm), whose sequence was constant from animal to animal, were made in the A9 and A10 areas. Each electrode descent was made in a slow (1-3 µm/s), uniform speed with a hydraulic microdrive. Only those cells whose electrophysiological profile matched those previously established for midbrain DA cells were counted.

In addition, after counting the number of spontaneously active A9 and A10 DA cells per track in the animals treated for 21 days with 1, 3, or 10 mg/kg p.o. SB-277011-A, 50 µg/kg i.v. (+)-apomorphine was given and the number of spontaneously active A9 and A10 DA cells recounted in five different stereotaxic descents. The 50-µg/kg i.v. dose of (+)-apomorphine was chosen because it has been previously shown that this dose will effectively reverse the depolarization inactivation of A9 and A10 DA cells produced by the chronic administration of antipsychotic drugs (Chiodo and Bunney, 1983; White and Wang, 1983).

Drug Treatment Regimens

Effect of i.v. SB-277011-A on Basal Firing Rate and Pattern of DA Neurons. In a series of experiments, the effect of i.v.-administered SB-277011-A (0.01-1.28 mg/kg) on the basal firing rate of spontaneously active A9 and A10 DA cells was examined. In each rat, either an SNC or VTA DA cell was isolated and a stable baseline firing rate was recorded for 3 to 5 min. Subsequently, SB-277011-A was administered, beginning with a dose of 10 µg/kg and each consecutive dose was double that of the preceding one until a cumulative dose of 1.28 mg/kg was reached. The time between each injection was 1 min. In these experiments, only one cell was studied in each animal.

Determination of Number of Spontaneously Active A9 and A10 Cells per Track. In the experiments examining the effect of SB-277011-A on the number of spontaneously active SNC and VTA DA cells, rats were randomly allocated to one of following treatment groups: SB-277011-A (1, 3, or 10 mg/kg p.o.), haloperidol (0.5 mg/kg p.o.), or vehicle (1 ml/kg p.o. 2% methylcellulose solution). All drug solutions were made up fresh daily in 2% methylcellulose. The various solutions were given with a 1-ml syringe (VWR Scientific, Inc., Bridgeport, NJ) to which a 3-inch, curved, stainless steel gavage needle (Henry Schein Co., Long Island, NY) was attached. Rats were given only one administration (acute preparation) or one administration daily for 21 consecutive days (chronic preparation) of either SB-277011-A or vehicle. All animals were then prepared for the recording of SNC and VTA DA cells 2 h after the last treatment. We chose the 2-h postadministration time point based on data indicating the antagonistic action of SB-277011-A appears about 1 h after its administration and that this effect can be maintained for a period of 5 h (Reavill et al., 2000). The experimenter was "blind" as to the treatment of each animal. In addition, for half the rats in each group, the order of recording was SNC-VTA and the order was reversed for the other rats. The treatments were administered with a Latin square design.

Histology

At the end of each experiment, a 25-µA cathodal current was passed through the electrode, a procedure that leads to the deposition of a discrete spot of fast green dye in the brain. The animals were overdosed with chloral hydrate and perfused transcardially with 10% buffered formalin for 10 min. The brains were removed and serial coronal sections were cut at 50-µm intervals, stained with cresyl violet, and counterstained with neutral red. The dye spot was viewed under a light microscope and served as a reference point for the location of each cell.

Data Analysis

The software used to conduct the statistical analysis was GB-STAT, version 6.5 (Dynamics Microsystems, Inc., Silver Spring, MD). The data obtained from the experiments examining the effects of the various treatments on the number of spontaneously active A9 and A10 DA cells were analyzed with a one-way ANOVA and post hoc analyses were conducted with the Student-Newman-Keul's test. The percentage of events as bursts and number of bursts data were analyzed with the Kruskal-Wallis ANOVA (or H test) because these data were not normally distributed. The mean interspike interval, spikes per burst, and coefficient of variation data were analyzed with a random, repeated ANOVA. The data for the percentage of neurons exhibiting a burst firing pattern were analyzed with the ² test.

Drug Sources

Chloral hydrate and methylcellulose were purchased from Sigma Chemical Co. (St. Louis, MO). Haloperidol was obtained from Research Biochemicals International (Natick, MA), and SB-277011-A was obtained from SmithKline Beecham Pharmaceuticals (Harlow, UK).

	Results

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Effect of i.v. Administration of SB-277011-A on Firing Rate and Pattern of Spontaneously Active A9 and A10 DA Neurons

The i.v. administration of SB-277011-A (0.01-1.28 mg/kg) did not significantly alter the firing rate or pattern of spontaneously active DA neurons in either the A9 or A10 regions of the rat brain (Tables 1 and 2).

Effect of Acute p.o. Administration of SB-277011-A on Number of Spontaneously Active A9 and A10 DA Neurons

The values obtained for the number of spontaneously active A9 and A10 DA neurons after vehicle treatment are similar to those previously obtained from this laboratory (Minabe et al., 1998). As previously reported (Minabe et al., 1998), a single p.o. administration of 0.5 mg/kg haloperidol produced a significant increase in the number of spontaneously active SNC (A9) and VTA (A10) DA neurons compared with vehicle (Table 3). A single p.o. administration of 1 mg/kg SB-277011-A did not significantly alter the number of spontaneously active A9 DA neurons, whereas the 3- and 10-mg/kg doses produced a significant increase compared with vehicle-treated animals (Table 3). Neither the 1- nor 3-mg/kg doses of SB-277011-A significantly altered the number of spontaneously active A10 DA neurons compared with vehicle-treated animals (Table 3). In contrast, the 10-mg/kg dose of SB-277011-A produced a significant increase in the number of spontaneously active A10 DA neurons compared with vehicle-treated animals (Table 3).

Effect of Acute p.o. Administration of SB-277011-A 277011A on Firing Pattern of All (Bursting and Nonbursting Neurons) and Burst Firing Spontaneously Active A9 and A10 DA Neurons

Comparison of SB-277011-A 277011A to Vehicle-Treated Animals (Control): All DA Neurons. A9 DA neurons. The acute administration of 1 mg/kg SB-277011-A 277011A produced a significant increase in the spikes per burst and percentage of neurons exhibiting a bursting pattern in all spontaneously active A9 DA neurons compared with vehicle-treated animals (Table 4). However, neither the 3- nor 10-mg/kg doses significantly altered the firing pattern of A9 DA neurons compared with control (Table 4).

A10 DA neurons. The acute administration of 3 mg/kg SB-277011-A 277011A produced a significant decrease in the following parameters in all A10 DA neurons compared with vehicle-treated animals: spikes per burst, firing rate (note: 1/interspike interval = firing rate; thus, a higher interspike interval = decreased firing rate), number of bursts, and percentage of neurons bursting (Table 4). The acute administration of 10 mg/kg SB-277011-A 277011A produced a significant decrease in spikes per burst and firing rate of all spontaneously active A10 DA neurons compared with vehicle-treated animals (Table 4).

Comparison of SB-277011-A and Burst Firing DA Neurons to Vehicle-Treated Animals. A9 DA neurons. The 1-mg/kg dose of SB-277011-A produced a significant increase in the percentage of events as bursts in A9 DA neurons exhibiting a bursting pattern compared with vehicle-treated animals (Table 5). Neither the 3- nor 10-mg/kg dose of SB-277011-A significantly altered the firing pattern of A9 DA neurons firing in a bursting pattern compared with vehicle-treated animals (Table 5).

A10 DA neurons. The acute p.o. administration of 1 mg/kg SB-277011-A did not significantly alter the firing pattern of A10 DA neurons firing in a bursting pattern compared with vehicle-treated animals (Table 5). In contrast, the acute p.o. administration of the 3-mg/kg dose produced a significant decrease in the coefficient of variation for burst firing A10 DA neurons compared with vehicle-treated animals (Table 5). The interspike interval in burst firing A10 DA neurons was significantly greater than that in vehicle-treated animals after the acute p.o. administration of 10 mg/kg SB-277011-A (Table 5). The coefficient of variation and interspike interval measured after the 10-mg/kg dose of SB-277011-A was greater than that of the 3-mg/kg dose (Table 5).

Effect of Repeated p.o. Administration of SB-277011-A or Haloperidol on Number of Spontaneously Active A9 and A10 DA Neurons

The repeated administration (1 p.o. administration per day for 21 consecutive days) of 0.5 mg/kg haloperidol, as previously reported, significantly decreased the number of spontaneously active A9 DA neurons compared with vehicle-treated animals (Table 6). In contrast, the number of spontaneously active A9 DA neurons was not significantly altered after the repeated p.o. administration of either 1, 3, or 10 mg/kg SB-277011-A compared with vehicle-treated animals (Table 6). The repeated p.o. administration of 1, 3, and 10 mg/kg SB-277011-A produced a significant decrease in the number of spontaneously active A10 DA neurons, with 1- and 3-mg/kg doses producing equipotent effects (Table 6). Interestingly, the number of spontaneously active A10 DA neurons increased after the 10-mg/kg dose of SB-277011-A compared with the 1- and 3-mg/kg doses (Table 6).

To determine the threshold dose of SB-277011-A, we examined the effect of the repeated administration of 0.1 and 0.3 mg/kg p.o. SB-277011-A on the number of spontaneously active A10 DA neurons. Overall, neither the 0.1- nor the 0.3-mg/kg dose produced a significant alteration in the number of spontaneously active A10 DA neurons compared with vehicle-treated animals (Table 7).

Previously, it has been shown that the decrease in the number of spontaneously active DA neurons produced by the repeated administration of antipsychotic drugs is related to an excessive depolarization of DA neurons (Bunney and Grace, 1978; Chiodo and Bunney, 1983; White and Wang, 1983; Grace and Bunney, 1986). This results in the inactivation of the spike-generating mechanism of the DA neurons (depolarization inactivation), which can reversed by experimental manipulations that produce membrane hyperpolarization (e.g., i.v. apomorphine, i.v. baclofen). Therefore, to gain insight into the mechanism by which SB-277011-A decreases the number of spontaneously active DA neurons, we administered 50 µg/kg i.v. (+)-apomorphine and determined the number of spontaneously active DA neurons in five additional stereotaxic descents. As can be seen in Table 8, i.v. apomorphine did not reverse the SB-277011-A-induced decrease in the number of spontaneously active A10 DA neurons; in fact, i.v. apomorphine produced a further decrease in the number of spontaneously active A10 DA neurons elicited by the 10-mg/kg dose of SB-277011-A.

Effect of Repeated p.o. Administration of SB-277011-A on Firing Pattern of All (Bursting and Nonbursting Neurons) and Bursting Spontaneously Active A9 and A10 DA Neurons

Comparison of SB-277011-A and Vehicle-Treated Animals (Control): All DA Neurons. A9 DA neurons. After repeated administration, none of the doses of SB-277011-A significantly altered the firing pattern of spontaneously active DA neurons in the A9 area compared with vehicle-treated animals (Table 9).

A10 DA neurons. The repeated administration of SB-277011-A did not significantly alter the firing pattern of spontaneously active DA neurons in the A9 area compared with vehicle-treated animals (Table 9).

Comparison of SB-277011-A Vehicle-Treated Animals (Control): Burst Firing DA Neurons. Overall, there were no significant differences in any of the firing pattern parameters determined for burst firing A9 and A10 DA neurons between any dose of SB-277011-A and control (Table 10). However, the interspike intervals measured after the repeated administration of 3 and 10 mg/kg SB-277011-A were significantly lower than that for the 1-mg/kg dose (Table 10). The number of spikes per burst in burst firing A9 DA neurons after the repeated administration of 10 mg/kg SB-277011-A was significantly greater than that of the 1-mg/kg dose (Table 10). The number of bursts in burst firing A10 DA neurons after the repeated administration of 3 mg/kg SB-277011-A was significantly lower than that of either 1 or 10 mg/kg SB-277011-A (Table 10).

	Discussion

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One of the major findings of this study was that the administration of SB-277011-A altered the activity of midbrain DA neurons. The acute administration of 1 mg/kg SB-277011-A did not significantly alter the number of spontaneously active DA neurons compared with vehicle-treated animals. However, the 3-mg/kg dose only significantly increased the number of spontaneously active A9 DA neurons and the 10-mg/kg dose significantly increased the number of spontaneously active DA neuron in both the A9 and A10. It has been shown that the acute administration of typical antipsychotics significantly increases the number of spontaneously active DA neuron in the A9 and A10 (Bunney and Grace, 1978; Chiodo and Bunney, 1983; White and Wang, 1983; Chiodo, 1988; for review, see Arnt and Skarsfeldt, 1997). Thus, at least at the 10-mg/kg dose, SB-277011-A produces effects that resemble those of the typical antipsychotics after acute administration. However, SB-277011-A, at doses used in this study, does not produce catalepsy or inhibit spontaneous locomotor activity and does not block the locomotor effects of amphetamine (Reavill et al., 2000). This is in contrast to haloperidol that induces catalepsy, inhibits spontaneous locomotor activity, and blocks amphetamine-induced locomotor activity (Arnt and Skarsfeldt, 1997). Thus, if one compares the acute i.v. and p.o. effects of SB-277011-A, there is a dissociation: i.v. dosing produces no effect on firing, whereas p.o. alters the number of spontaneously active DA neurons. In contrast, i.v. haloperidol increases the firing rate of DA neurons (Bunney et al., 1973; Bunney and Aghajanian, 1975) and i.p. or p.o. administration (Bunney and Grace, 1978; Chiodo and Bunney, 1983; White and Wang, 1983; Chiodo, 1988) increases the number of spontaneously active DA neurons. The lack of a significant effect of i.v. SB-277011-A on the activity of midbrain neurons may be explained by the fact that perhaps a metabolite of SB-277011-A is required to alter DA neuronal activity. Thus, such an effect could occur during the time frame of the cells per track study experiments, which is 2 to 3 h, as opposed to the i.v. study, where only 15 to 20 min has elapsed, which may be an insufficient time period for metabolite formation. An alternative explanation is that although the brain/plasma ratio under steady-state i.v. dosing conditions for SB-277011A is 3.6 (Reavill et al., 2000), sufficient brain concentrations of the compound are not achieved within the 15- to 20-min time frame of the experiment to affect DA neuronal activity. Further studies would be required to distinguish between these alternatives.

The acute administration of SB-277011-A (3 and 10 mg/kg) appeared to have a greater effect on the firing pattern of spontaneously active A10, compared with A9 DA neurons, with the general effect being to decrease bursting activity (fewer spikes per burst) and decrease firing rate. Previously, it has been shown that DA neurons firing in a bursting pattern release greater concentrations of DA at their target areas compared with neurons firing in a regular, single-spike mode (i.e., nonbursting pattern; Gonon, 1988; Chergui et al., 1994). Therefore, one might postulate that SB-277011-A administration may produce a decrease in basal DA levels in brain areas innervated by A10 DA neurons (e.g., amygdala, nucleus accumbens, frontal cortex). Preliminary microdialysis studies of DA efflux in the rat nucleus accumbens after acute administration of SB-277011-A (Reavill et al., 2000) do not support this but further studies are required before a definitive conclusion can be reached.

The i.v. administration of SB-277011-A did not significantly alter the firing rate or pattern of spontaneously active DA neurons in either the A9 or A10. Previously, it has been reported that typical antipsychotics such as haloperidol and chlorpromazine produce an increase in the firing rate and degree of bursting of spontaneously active DA neurons in both the A10 and A9 (Bunney and Aghajanian, 1975; Bunney and Grace, 1978; Hand et al., 1987; for review, see Chiodo, 1988). In contrast, atypical antipsychotics such as clozapine appear to selectively increase the firing rate of A10 DA neurons (but see Bunney and Aghajanian, 1975; Souto et al., 1979; Hand et al., 1987). The findings after the acute administration of SB-277011-A show that acute antagonism of the D₃ receptor does not alter the firing rate or pattern of DA neurons. In addition, it suggests that antipsychotics such as haloperidol, chlorpromazine, and clozapine do not increase the firing rate of spontaneously active DA neurons via blockade of D₃ receptors. Our finding is consistent with a previous study demonstrating that the infusion of antisense deoxynucleotides against the mRNA for the D₃ receptor into the substantia nigra of rats did not significantly alter the firing rate or pattern of spontaneously active SNC DA neurons compared with controls (Tepper et al., 1997).

The repeated administration of 1, 3, and 10 mg/kg SB-277011-A produced a selective decrease in the number of spontaneously active A10 DA neurons. The explanation for the decreased effectiveness of the 10-mg/kg dose to decrease the number of A10 DA neurons compared with the other doses is unknown. It is unlikely to be related to an interaction with other receptors at this dose because SB-277011A has a low affinity for numerous other receptors (Reavill et al., 2000). Furthermore, the drug does not possess dopaminergic agonist properties (Reavill et al., 2000).

Previously, with the use of anesthetized, male Sprague-Dawley rats, it has been shown that the repeated administration of atypical antipsychotics, such as clozapine, produce a selective decrease in the number of spontaneously active A10 DA neurons (Chiodo and Bunney, 1983, White and Wang, 1983; Minabe et al., 1991; for review regarding clozapine, see Ashby and Wang, 1996). Thus, these results indicate that, after repeated administration, SB-277011-A has a profile similar to that of atypical antipsychotic drugs. It is unlikely that SB-277011-A is inactivating or decreasing the number of spontaneously active A10 DA neurons via depolarization inactivation because its action was not reversed by i.v. apomorphine. This is consistent with behavioral data indicating that the systemic administration of SB-277011-A does not block the apomorphine- or quinpirole-induced disruption of prepulse inhibition (Reavill et al., 2000). This tentatively suggests that the SB-277011-A-induced decrease in the number of spontaneously active VTA DA neurons is not the result of depolarization inactivation, although the appropriate intracellular recording studies must be conducted to verify this statement.

The mechanism by which the repeated administration of SB-277011-A decreases the number of spontaneously active VTA DA neurons is unknown. Although speculative, it is possible that the antagonism of D₃ receptors in brain areas that send feedback projections to the VTA may be of importance. The link between drug effects on the firing pattern of A9 and A10 DA neurons in the cells per track model and clinical efficacy is not clearly understood. Furthermore, the exact significance of the alteration in the number of cells per track after acute administration of drugs remains to be determined, particularly because it takes at least 3 to 6 weeks of treatment with antipsychotics to produce a therapeutic efficacy. However, after repeated drug administration, the electrophysiological model used in this study appears to have good predictive validity for antipsychotic efficacy, i.e., drugs that decrease the number of spontaneously active A9 and A10 DA cells per track have a typical antipsychotic drug profile, whereas those that selectively decrease the number of spontaneously active A10 DA neurons have an atypical profile. This predictive validity is especially good for drugs that have DA receptor antagonist action.

In conclusion, our results indicate that the administration of SB-277011-A significantly alters the activity of midbrain DA neurons in anesthetized, male Sprague-Dawley rats. One important finding is that the repeated administration of SB-277011-A produces a significant decrease in the number of spontaneously active A10 DA cells. However, the repeated administration of SB-277011-A did not significantly alter the number of spontaneously active A9 DA cells. Based on the interpretation of past results with this procedure the data are consistent with the hypothesis that that selective D₃ receptor blockade will be associated with therapeutic efficacy in schizophrenia without eliciting extrapyramidal side effects (Reavill et al., 2000). Confirmation of this hypothesis will require appropriate clinical trials of selective D₃ receptor antagonists in humans.