Elucidation of D3 receptor function in vivo: do D3 receptors mediate inhibition of dopamine neuronal activity?

A number of studies have recently suggested that the D3 receptor [ 11 is located presynaptically and functions as an autoreceptor [2-41. These studies have demonstrated that 7-hydroxy-di-npropyl-2-aminotetralin [ ( + )-7-OH-DPAT], a ligand with significant D3 receptor affinity, decreases dopamine release in limbic and striatal regions. However, the selectivity of (+)-7-OHDPAT for the D3 receptor is based on binding data [ 1,2]. When characterized functionally, there is little difference (fourto seven-fold) in the efficacy of (+)-7-OH-DPAT at the D3 and D2 receptor [5,6]. This calls into question the claims that certain in vivo responses, which have been characterized using ( +)-7-OH-DPAT, are mediated via D3 receptors. It also suggests that (+)-7-OH-DPAT may not be the ligand of choice for elucidating D3 receptor function in vivo. The discrepancy between binding and functional values for D3 receptor ligands may, however, be a consequence of using recombinant systems; it remains to be determined if this difference is extrapolated from cloned human receptors expressed in artificial cell lines to native animal tissue. Whether using functional or binding data, one of the most selective D3 ligands to date is quinelorane (LY163502). This compound has at least 100-fold greater affinity for the D3 than for the D2 receptor. Functionally (stimulation of mitogenesis), this compound is over 20-fold more selective for the D3 receptor [6]. In the present study we have characterized the profile for quinelorane plus a number of other D3 and DJD2 receptor ligands on dopamine release and synthesis in the rat nucleus accumbens (n. accumbens) and striatum (the n. accumbens has a higher DJD2 receptor density than the striatum) in attempts to characterize further D3 receptor function in vivo. For synthesis studies, male CDl mice (Charles River) were administered varying concentrations of apomorphine, 7-OH-DPAT, ( +) PHNO [ ( +)-4-propyl-9-hydroxynaphthoxazine-HCl], quinelorane (LY-163502) and (-)quinpirole (LY-171555) followed 5 min later by administration of y-hydroxybutyrolactone (GBL, 750 mg/kg intraperitoneally) or vehicle (0.9% NaCl, 0.1 mVkg intraperitoneally). After an additional 5 min, all mice received the dopa decarboxylase inhibitor NSD 10 15 (100 mg/kg intraperitoneally) and were then killed 30 min later. The striatum and n. accumbens were removed and frozen for analysis of L-dopa using high-performance liquid chromatography with electrochemical detection (HPLC-ECD) as previously described [ 71. Significant differences were identified using two-way analysis of variance (ANOVA) followed by the Tukey-Kramer test; P < 0.05 was regarded as significant. For microdialysis studies, male SpragueDawley rats (260-320 g; Charles River) were anaesthetized and their heads secured in a stereotaxic frame. A microdialysis probe was implanted into either the n. accumbens or striatum and secured in position (tip co-ordinates: n. accumbens, P+2.7, L + / 1.6, V-7.4; striatum, P 0.0, L+/-2.8, V-6.0; reference point Bregma according to [S]). Animals were allowed to recover for 24 h, after which probes were perfused with artificial cerebrospinal fluid (aCSF: NaCl 125mM, KCl 2.5mM, MgC12 1.18mM, CaC12 1.26 mM, pH 7.4) at a flow rate of 2 pV min, and 20-min dialysis samples were collected. After a 2-h stabilization period, three stable baseline control samples were taken. Agonist or vehicle was then administered subcutaneously and samples were collected for a further 3 h. For antagonist studies, three baseline control samples were taken followed by administration of antagonist or vehicle. Thirty minutes later agonist or vehicle was administered and samples collected for a further 3 h. The samples were assayed for dopamine by HPLC-ECD as previously described [7]. Data were analysed by twoway ANOVA with repeated measures followed by post-hoc testing using the Tukey-Kramer test; P < 0.05 was regarded as significant. All compounds significantly (P < 0.05) decreased dopamine synthesis in the n. accumbens compared with vehicle controls with the following order of potency (ED5", pg/kg S.C. in parentheses): (+) PHNO (0.55) >quinelorane (1.8) >apomorphine (21) = 7-OH-DPAT (23) = quinpirole (33). Dopamine synthesis in the striatum was decreased with a similar order of