Introduction

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Dopamine (DA) receptors are classified into two subfamilies according to their pharmacological profiles and sequence homologies: the D1-like receptor subtypes (D1, D5) and the D2-like subtypes (D2, D3, D4) (Sibley and Monsma, 1992). The density of DA receptors in the mammalian central nervous system can be regulated by a variety of pharmacological manipulations that alter dopaminergic neurotransmission. Several studies have demonstrated that D1-like and D2-like receptors can be regulated independently by such treatments. Until recently, a lack of specific ligands has hindered study of the regulatory properties of brain DA receptors within each subfamily (e.g., D2 and D3 receptors).

The D2 receptor is expressed abundantly and widely in all major dopaminoceptive brain areas (Boyson et al., 1986; Bouthenet et al., 1987). Chronic administration of D2 receptor antagonists increases the density of D2 receptors (Burt et al., 1977; McGonigle et al., 1989). This effect has been interpreted to be an adaptive increase in postsynaptic receptor sensitivity to DA in response to its decreased availability. Long-term depletion of monoamines with reserpine also produces increases in D2-like receptors (Norman et al., 1987; Neisewander et al., 1991). In contrast, treatment with D2-like receptor agonists decreases D2 receptor expression (Winkler and Weiss, 1989; Subramaniam et al., 1992).

The D3 receptor is expressed at fairly high levels in the nucleus accumbens (NA), the islands of Calleja, the ventral pallidum (VP), and in lobules 9 and 10 of the cerebellum (Levesque et al., 1992; Diaz et al., 1995; Stanwood et al., 1997). Lower levels of D3 expression are also detected in dopaminergic cell bodies in the substantia nigra (SN). Chronic treatment with antipsychotics has been reported to increase D3 mRNA in some studies (Buckland et al., 1991; Wang et al., 1996), but other studies have found no effect on D3 receptor mRNA or binding sites (Kung et al., 1995; Levesque et al., 1995; Tarazi et al., 1997). The ability of agonist treatments or DA depletion to regulate D3 receptor expression in the brain has not been investigated.

Activation or blockade of D2 and/or D3 receptors modulates neurochemical and behavioral activities associated with drugs of abuse and reward. Recent findings have suggested that D2 and D3 receptor activation may lead to different and perhaps opposing functional responses. 7-OH-DPAT and other agonists that are somewhat selective for the D3 subtype suppress locomotor activity at low (putative D3-selective) doses, but increase it at higher doses (Levesque, 1996; Levant, 1997). The interpretation of such studies is difficult, however, due to limitations in the in vivo pharmacological profiles of the available drugs. Nevertheless, support for this interpretation has come from the observation that D3 receptor mutant mice are spontaneously hyperactive (Accili et al., 1996), whereas selective knockout of D2 receptor expression leads to hypomotility (Balk et al., 1995). Additionally, blockade of D2 and D3 receptors leads to increases in neurotensin gene expression in regions of the NA primarily expressing the D2 receptor, but greatly decreases neurotensin mRNA in D3-rich regions (Diaz et al., 1994). Antisense oligonucleotides directed against D3 receptor mRNA also decreases neurotensin expression in the NA (Tremblay et al., 1997).

In this study, we have systematically evaluated the effects of pharmacological manipulations designed to either increase or decrease dopaminergic neurotransmission to establish and compare the regulatory properties of D2 and D3 receptors. We have capitalized on the sensitivity and in vitro selectivity of radiolabeled and unlabeled 7-OH-PIPAT to specifically measure D2 and D3 receptors in multiple brain regions. The opposing changes in receptor regulation observed after these treatments is consistent with opposing functional roles that have been postulated for these two DA receptors.

	Materials and Methods

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Drug Treatments and Tissue Preparation. Male Sprague-Dawley rats (200-225 g, Charles River, Wilmington, MA) were housed two to three per cage and treated as follows: Quinpirole (1 mg/kg/day), 7-OH-DPAT (1 mg/kg/day), and cocaine (20 mg/kg/day) were administered for 14 days by osmotic minipumps (Alza, Palo Alto, CA). Reserpine (1 mg/kg/every other day) and baclofen (30 mg/kg/day) were administered by subcutaneous injection. Rats receiving baclofen were given 15 mg/kg on day 1 of treatment, 22.5 mg/kg on day 2, and 30 mg/kg for the remaining 12 days. Some rats receiving reserpine initially experienced severe weight loss and were given 6 to 8 ml of a liquid diet consisting of 200 ml of tap water, 100 ml of sweetened condensed milk, one package of chocolate-flavored instant breakfast mix, and 22.5 ml of kaolin/pectin (Kaopectate; McNeil Consumer Products, Fort Washington, PA) via intubation. All rats in this group were given ground rat chow moistened with the liquid diet to minimize weight loss and dehydration. N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ) (10 mg/kg) was administered by subcutaneous injection and animals were sacrificed 6 h later. The dose and duration of treatment for the EEDQ experiment were based on preliminary experiments designed to maximize the extent of D2 receptor inactivation (P. McGonigle, unpublished observations). Animals were decapitated on day 15 of each chronic treatment and brains were removed and frozen in isopentane (20°C). Brains were sectioned (20 µm) on a cryostat, thaw-mounted on gelatin-coated slides, desiccated under vacuum at 4°C for 2 to 3 h, and stored at 70°C. The quinpirole treatment was run in two separate squads of animals. There was no difference in the levels of receptor binding across the vehicle-injected control groups, so these data were pooled for statistical analysis. All procedures were approved by the University of Pennsylvania Animal Care and Use Committee (Assurance no. 3079-01).

Autoradiographic Procedures. Before incubation with radioligand, tissue sections were thawed and preincubated for 30 min at 30°C in incubation buffer containing 50 mM Tris, 40 mM NaCl, and 300 µM GTP to promote dissociation of endogenous DA from the receptors. D2 receptors were then labeled with [¹²⁵I]NCQ 298 (0.05 nM) in incubation buffer containing 30 nM 7-OH-PIPAT to prevent labeling of D3 receptors. Incubations were carried out for 2 h at room temperature and nonspecific binding (NSB) was defined with 2 µM (+)-butaclamol. D3 receptors were labeled in adjacent sections with [¹²⁵I]7-OH-PIPAT (0.2 nM) in incubation buffer containing 5 µM 1,3-di(2-tolyl)guanindine to prevent labeling of sigma sites. Sections were incubated for 90 min at room temperature and NSB was defined with 5 µM 7-OH-DPAT. [¹²⁵I]NCQ 298 labelings were conducted at pH 7.4 and [¹²⁵I]7-OH-PIPAT assays were performed at pH 7.0. In some experiments sections were labeled with 0.04 to 1.2 nM [¹²⁵I]7-OH-PIPAT to perform saturation analysis of D3 receptor binding. D1 receptors were labeled with [³H]SCH 23390 (4 nM) in 50 mM Tris (pH 7.4), 10 mM MgSO_4, 2 mM EDTA, 154 mM NaCl, 10 mg/l BSA. Incubations were carried out for 75 min at 37°C and NSB was defined with 2 µM (+)-butaclamol. After all incubations, slides were rinsed in buffer at 4°C for 20, 45, or 90 min for [³H]SCH 23390, [¹²⁵I]NCQ 298, and [¹²⁵I]7-OH-PIPAT labelings, respectively. Slides were then dipped in ice-cold double distilled H₂O and dried with a stream of warm air.

Data Analysis. Labeled sections were apposed to Amersham Hyperfilm-³H in lightproof X-ray cassettes and exposed at room temperature for 4 to 72 h, depending on the radioligand and the receptor density. A plastic tritium standard calibrated with tissue sections containing ¹²⁵I or ³H was included in each cassette as previously described (Artymyshyn et al., 1990). Films were developed in Kodak GBX developer (3 min), rinsed in water (20 s), fixed in Kodak GBX fixer (6 min), and rinsed in cool water for 15 to 20 min. The autoradiograms were analyzed using a Macintosh-based image processing system using NIH Image 1.47 software. Optical density was converted to femtomoles per milligram of protein based on calibration curves generated from the tritium-containing standards. Additional sections were stained with cresyl violet for anatomical analysis. Maximum receptor density and K_d values were determined by Scatchard transformation of saturation-specific binding data using unweighted linear regression analysis. Comparison between treatment groups was performed by unpaired Student's t tests.

Neurochemical Analysis. A separate group of animals was treated with reserpine or vehicle and tissue DA content was analyzed by HPLC using electrochemical detection. Striata were homogenized in 2 ml 0.1 N perchloric acid, 100 µM ascorbate. Samples were centrifuged at 20,000g for 5 min at 0°C. The supernatant and tissue pellet were stored separately at 70°C. Aliquots of the supernatant were filtered and injected into an HPLC separation system (BAS, West Lafayette, IN). The mobile phase consisted of 12.4 mM citric acid, 39.9 mM monobasic sodium phosphate, 0.25 mM EDTA, 0.74 mM 1-decanesulfonic acid, 10.0 mM NaCl, 0.2% triethylamine, and 16% methanol (pH 4.3). The mobile phase was filtered and degassed. Known amounts of DA were assayed to construct standard curves of the relationship between DA concentration and peak height deflection. Pellets were assayed for protein content.

Drugs. [¹²⁵I]R-(+)-trans-7-Hydroxy-2-(N-n-propyl-N-3'-iodo-2'-propenyl)aminotetralin ([¹²⁵I]7-OH-PIPAT), and [¹²⁵I]NCQ 298 were obtained as a gift from Drs. Hank Kung and Mei-Ping Kung (Department of Radiology, University of Pennsylvania). [³H]SCH 23390 was purchased from DuPont NEN (Boston, MA). (±)-7-OH-DPAT, cocaine HCl, quinpirole, reserpine, (±)-baclofen, 1,3-di(2-tolyl)guanidine, EEDQ and (+)-butaclamol were obtained from Research Biochemicals International (Natick, MA). All other reagents were purchased from Sigma (St. Louis, MO). Reserpine and baclofen were dissolved in glacial acetic acid, and then diluted to the correct concentration in distilled water. Controls for these groups were injected with solutions with the same concentration of dilute acid. EEDQ was administered in 50% ethanol/50% 0.9% NaCl solution. All other drugs were administered in a 0.9% NaCl vehicle.

	Results

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Autoradiographic Labeling of D2 and D3 Receptors. We have presented a detailed characterization of [¹²⁵I]7-OH-PIPAT labeling of D3 receptors in Stanwood et al. (2000). D2 receptors, defined by [¹²⁵I]NCQ 298 binding, are present throughout the caudate-putamen, NA, and olfactory tubercle. D3 receptors labeled with [¹²⁵I]7-OH-PIPAT, on the other hand, are expressed at lower levels and primarily in the shell of the NA and the islands of Calleja within the olfactory tubercle. Both D2 and D3 receptors are expressed in ventral pallidum. At the level of the substantia nigra, D2 receptors are expressed in all regions containing dopaminergic cell bodies, whereas D3 receptor binding is expressed primarily in the medial substantia nigra.

Reserpine Treatment. Chronic treatment with the monoamine depleting agent reserpine for 14 days decreased striatal DA content by 92% (Table 1). The effects of this treatment on D2 and D3 receptor expression were examined by measuring the binding of [¹²⁵I]NCQ 298 and [¹²⁵I]7-OH-PIPAT, respectively. D2 receptor binding was significantly increased in the NA (+20%), VP (+11%), and SN (+16%) (Fig. 1A). In contrast, [¹²⁵I]7-OH-PIPAT binding to D3 receptors was significantly decreased after chronic treatment with reserpine (Fig. 1B). D3 levels were reduced by 19, 11, and 29% in the NA, VP, and SN, respectively (Fig. 1B). Saturation experiments in the medial shell of the NA confirmed that this decrease in D3 receptor binding was due to a decrease in B_max (46 versus 64 fmol/mg in the NA) rather than a change in the dissociation constant of the radioligand (Fig. 2).

View larger version (58K): [in this window] [in a new window]   Fig. 1.   Effect of chronic reserpine treatment on [125I]NCQ 298 binding to D2 receptors (A) and [125I]7-OH-PIPAT binding to D3 receptors (B) (n = 10-12 animals/group). Data shown are percentage of control binding values ± S.E.M. *P < .05, **P < .01.

View larger version (19K): [in this window] [in a new window]   Fig. 2.   Scatchard analysis of [125I]7-OH-PIPAT binding to D3 receptors in the NA after chronic reserpine treatment (n = 8 animals/group). Vehicle: Kd = 0.25 ± 0.02 nM, Bmax = 64.3 ± 5.55 fmol/mg; reserpine: Kd = 0.22 ± 0.02 nM, Bmax = 46.3 ± 3.87 fmol/mg.

Quinpirole, 7-OH-DPAT Treatments. Chronic treatment with the direct D2-like agonist quinpirole for 14 days produced moderate, but significant, decreases in D2 receptor expression in all regions (approximately 20-25%) (Fig. 3A), and fairly large increases in D3 expression in the VP (+27%) and SN (+52%) (Fig. 3B). D3 receptor binding was unchanged in the NA. Treatment with an additional D2-like agonist, 7-OH-DPAT, produced the same pattern of effects on D2 and D3 receptors (Fig. 4, A and B). 7-OH-DPAT decreased D2 levels by about 20% in each region, and increased D3 expression in the VP and SN by 20 and 36%, respectively (Fig. 4B). As was the case after reserpine treatment, Scatchard analyses demonstrated that these changes in D3 receptor expression were due to increases in B_max rather than changes in K_d in both the VP (Fig. 5A) and SN (Fig. 5B).

View larger version (57K): [in this window] [in a new window]   Fig. 3.   Effect of chronic quinpirole (1 mg/kg/day, 14 days) treatment on [125I]NCQ 298 binding to D2 receptors (A) and [125I]7-OH-PIPAT binding to D3 receptors (B) (n = 14-16 animals/group). Data shown are percentage of control binding values ± S.E.M. *P < .05, **P < .01.

View larger version (54K): [in this window] [in a new window]   Fig. 4.   Effect of chronic 7-OH-DPAT (1 mg/kg/day, 14 days) treatment on [125I]NCQ 298 binding to D2 receptors (A) and [125I]7-OH-PIPAT binding to D3 receptors (B) (n = 10 animals/group). Data shown are percentage of control binding values ± S.E.M. *P < .05, **P < .01.

View larger version (21K): [in this window] [in a new window]   Fig. 5.   Scatchard analysis of [125I]7-OH-PIPAT binding to D3 receptors in the VP (A) and SN (B) after chronic quinpirole and 7-OH-DPAT treatment (n = 8-9 animals/group). VP: vehicle: Kd = 0.26 ± 0.02 nM, Bmax = 39.5 1.83 ± fmol/mg; 7-OH-DPAT: Kd = 0.26 ± 0.02 nM, Bmax = 48.2 ± 2.01 fmol/mg; quinpirole: Kd = 0.27 ± 0.01 nM, Bmax = 53.7 ± 3.91 fmol/mg. SN: vehicle: Kd = 0.30 ± 0.03 nM, Bmax = 11.6 ± 0.72 fmol/mg; 7-OH-DPAT Kd = 0.29 ± 0.03 nM, Bmax = 15.5 ± 1.09 fmol/mg; quinpirole: Kd = 0.29 ± 0.03 nM, Bmax = 18.4 ± 1.61 fmol/mg.

Cocaine Treatment. We also examined whether D2 and D3 expression would be altered by administration of the indirect DA agonist and psychomotor stimulant cocaine. Continuous administration of cocaine for 14 days via an osmotic minipump produced a significant decrease in D1-like receptor expression in the striatum (20%; data not shown). However, this treatment did not affect either D2 or D3 receptor expression in any region examined (Fig. 6).

View larger version (58K): [in this window] [in a new window]   Fig. 6.   Effect of chronic cocaine (20 mg/kg/day, 14 days) treatment on [125I]NCQ 298 binding to D2 receptors (A) and [125I]7-OH-PIPAT binding to D3 receptors (B) (n = 7 animals/group). Data shown are percentage of control binding values ± S.E.M.

Baclofen Treatment. The GABA_b agonist baclofen has been reported to acutely inhibit DA cell firing (Grace and Bunney, 1980), decrease extracellular DA (Westerink et al., 1996), and induce decreases in D3 mRNA expression when given subchronically (Levesque et al., 1995). Administration of baclofen for 14 days, however, did not alter D2 or D3 receptor binding in the NA, VP, or SN (data not shown).

EEDQ Treatment. Acute administration of the irreversible antagonist EEDQ reduces binding to many DA, serotonin, and other neurotransmitter receptor subtypes (Norman et al., 1987; Levant, 1995; Raghupathi et al., 1996). Previous reports have suggested that D3 receptors in the NA are resistant to inactivation by EEDQ in vivo, presumably because they are normally bound by endogenous DA (McGonigle et al., 1994; Levant, 1995). We tested the ability of systemically administered EEDQ to inactivate D2 and D3 receptors in multiple brain regions. [¹²⁵I]NCQ 298 binding to D2 receptors was decreased by >75% in all regions 6 h after EEDQ administration (Fig. 7A), which is consistent with previous reports (Norman et al., 1987; Levant, 1995). [¹²⁵I]7-OH-PIPAT binding to D3 receptors was unaffected in the NA (Fig. 7B). However, [¹²⁵I]7-OH-PIPAT binding was significantly decreased both in the VP (30%) and the SN (75%).

View larger version (56K): [in this window] [in a new window]   Fig. 7.   Effect of acute EEDQ (10 mg/kg) administration on [125I]NCQ 298 binding to D2 receptors (A) and [125I]7-OH-PIPAT binding to D3 receptors (B) (n = 6 animals/group). Animals were sacrificed 6 h after receiving EEDQ or vehicle control. Data shown are percentage of control binding values ± S.E.M. *P < .05, **P < .01.

	Discussion

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Previous studies have examined the regulation of D2-like binding after chronic drug treatments but the radioligands used could not distinguish between D2 and D3 receptor subtypes. Based on the significantly lower level of expression of D3 receptors, it has been assumed that these earlier studies accurately represented the responses of D2 receptors to such treatments. The current study used the antagonist ligand [¹²⁵I]NCQ 298, which has high affinity for D2 and D3, but not D4, receptors in the presence of cold 7-OH-PIPAT to restrict labeling to D2 receptors. Chronic treatment with the DA-depleting agent reserpine produced significant increases in D2 receptors, consistent with the results described in previous studies (Norman et al., 1987; Neisewander et al., 1991).

Somewhat surprisingly, long-term depletion of DA with reserpine produced significant decreases in D3 receptor expression measured with [¹²⁵I]7-OH-PIPAT, suggesting that attenuation of dopaminergic neurotransmission results in a down-regulation of D3 receptors. This result would seem to complement the functional data suggesting opposing roles for D2 and D3 receptors. It is also consistent with our previous observation that 6-hydroxydopamine lesion of dopaminergic neurons produces a decrease in D3 receptor expression (Stanwood et al., 2000), although the loss of D3 receptors after lesion is more likely due to a loss of presynaptic receptors on degenerating axon terminals. The observed decrease in [¹²⁵I]7-OH-PIPAT binding to D3 receptors after 14 days of reserpine treatment is in contrast to the lack of changes in D3 mRNA expression in the NA after 5 days of reserpine exposure reported by Levesque et al. (1995). This difference may be due to the different durations of treatment used or could indicate that the alteration in D3 receptor binding observed in the current study is due to regulation of translational or post-translational events. Because the absolute decrease in D3 receptors is relatively small compared with the total number of D2 and D3 receptors, this decrease would not have resulted in a substantial underestimate of D2 receptor up-regulation measured in previous studies using less specific radioligands.

Chronic treatment with direct D2-like agonists decreases [³H]spiperone binding and produces receptor subsensitivity (Winkler and Weiss, 1989; Subramaniam et al., 1992), whereas chronic antagonist treatment increases D2-like receptor binding (Burt et al., 1977; McGonigle et al., 1989). In the current study, continuous treatment with the D2-like receptor agonists quinpirole or 7-OH-DPAT for 14 days produced decreases in D2 expression in all regions examined and increases in D3 expression in the SN and VP. D3 receptor expression in the NA was unchanged. These data suggest that increases in DA receptor stimulation can produce up-regulation of D3 receptors in certain regions and complement functional data suggesting opposing roles for D2 and D3 receptors. These data also highlight the presence of regional heterogeneities in the regulation of D3 receptors.

Similar to the effects we observed in brain tissues, treatment of D3 receptor-transfected C₆ glioma cells with saturating concentrations of quinpirole or other DA agonists can produce 5-fold increases in D3 receptor expression (Cox et al., 1995). The up-regulation of D3 receptor binding in C₆ cells is blocked by cyclohexamide, suggesting that the synthesis of new receptors is involved. However, this increase in protein is not accompanied by an induction of D3 mRNA, suggesting that the increase in receptor synthesis may be due to translational or post-translational mechanisms.

Long-term administration of the psychomotor stimulant cocaine produces many alterations in receptor binding and functional responses (Inada et al., 1992; Neisewander et al., 1994, 1996). Moreover, compounds possessing a high affinity at D3 receptors may modulate the reinforcing effects of cocaine (Caine and Koob, 1993). D3 receptor mRNA and binding are also increased in the ventral striatum of human cocaine fatalities (Staley and Mash, 1996; Segal et al., 1997). This observation is consistent with the premise that a sustained increase in dopaminergic neurotransmission produces an up-regulation of D3 receptors. No significant changes in D2 or D3 receptor expression were observed after continuous administration of cocaine for 2 weeks in the current study. This treatment did decrease D1-like receptors, however, consistent with a previous report from our laboratory (Neisewander et al., 1994). Despite the absence of changes in D2-like receptor binding, animals treated continuously with cocaine for 2 weeks develop a decreased sensitivity to quinpirole-induced locomotion (Neisewander et al., 1996). We had speculated that these changes in D2-like receptor-mediated functions might be due to altered expression of D3 receptors, but our results do not support this hypothesis.

An increase in D3 receptor expression occurs as a result of repeated intermittent administration of levodopa in 6-hydroxydopamine-lesioned rats (Bordet et al., 1997). Interestingly, the induction of D3 mRNA and binding sites parallels the induction of locomotor sensitization in these animals. Moreover, this behavioral sensitization is blocked by the putative D3-preferring antagonist nafadotride, suggesting that the D3 receptor may play a role in the development of sensitization to certain drug effects. Repeated administration of quinpirole or 7-OH-DPAT also leads to the development of behavioral sensitization (Szechtman et al., 1994; Khroyan et al., 1995; Mattingly et al., 1996). Although we did not measure behavioral sensitization in our studies, it is possible that the increases in D3 expression observed after chronic quinpirole or 7-OH-DPAT may contribute to the development of locomotor sensitization.

Chronic treatments with neuroleptics and other D2-like antagonists have been reported to increase the expression of D3 mRNA in some studies (Buckland et al., 1991; Wang et al., 1996), but have no effect on D3 mRNA or binding sites in other reports (Kung et al., 1995; Levesque et al., 1995; Tarazi et al., 1997). Interestingly, D3 receptor binding is elevated in schizophrenic patients not receiving antipsychotic drugs for at least a month before death, but D3 levels are normalized in patients who received antipsychotic drugs shortly before death (Gurevich et al., 1997). The elevation in D3 receptors in unmedicated schizophrenics is consistent with the hypotheses that schizophrenia results from hyperdopaminergic function and increased dopaminergic neurotransmission results in D3 receptor up-regulation. Moreover, the normalization of D3 levels after neuroleptic treatment is consistent with our observation that decreased DA receptor stimulation induces a down-regulation of D3 receptors.

The high affinity of the D3 receptor for DA may result in a high level of occupancy of D3 receptors in the NA by endogenous transmitter (McGonigle et al., 1994; Levant, 1995). The results of the current study using EEDQ suggest that although D3 receptors in the accumbens may be tonically bound by DA in vivo, D3 receptors in other brain regions are comparatively less occupied. Congruent with this finding, chronic administration of the DA agonists quinpirole and 7-OH-DPAT altered D3 receptor density in the VP and SN, but not in the NA. The lack of EEDQ inactivation of D3 receptor binding in the NA cannot be explained by a difference in affinity of EEDQ for D2 and D3 receptors because EEDQ is capable of inactivating D2 and D3 receptors with similar potency in vitro (Levant, 1995).

These findings are particularly interesting in the context of reports that have concluded that postsynaptic D2 receptors activate locomotor activity in the NA and D3 receptor stimulation may inhibit locomotion (Levesque, 1996; Levant, 1997). These studies must be interpreted with caution due to limitations in the selectivity of available D2 and D3 compounds. However, this hypothesis is supported by studies of D2 and D3 receptor knockout mice (Balk et al., 1995; Accili et al., 1996). Interestingly, specific decreases in D3 receptor expression in the NA accompany locomotor hyperactivity after neonatal lesion of the ventral hippocampus (Flores et al., 1996). Furthermore, Pilla et al. (1999) recently reported that a D3 partial agonist inhibits cue-controlled cocaine-seeking behavior in the absence of intrinsic reinforcing effects. Note, however, that the results of our current EEDQ experiment as well as that of Levant (1995) suggest that most of the D3 receptors in the NA are occupied by endogenous DA. It would therefore be surprising if exogenous administration of D2-like agonists could further stimulate D3 receptors in this region. It is possible that these effects are mediated by D3 receptors in the VP or SN or by a subpopulation of D3 receptors in the NA that are not labeled by [¹²⁵I]7-OH-PIPAT in our assays. In further support of this supposition, administration of putative "D3 receptor-selective" compounds to D3 receptor mutant and wild-type mice has near-identical effects (Xu et al., 1999).

In summary, we have observed regional and subtype-specific heterogeneities in the regulation of D2 and D3 receptors by chronic drug treatments. The expression of D2 receptors is modulated as expected by traditional models of receptor regulation. In contrast, D3 receptor density regulates in the opposite direction to D2 receptors in response to these challenges. The differential regulatory responses we have observed after chronic DA depletion and agonist treatments may represent cellular correlates of differences in the behavioral activities of D2 and D3 receptor subtypes. The direct testing of this hypothesis awaits the development of ligands with greater in vivo D2 and D3 receptor selectivity than those currently available.