Introduction

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Classical neuroleptics, such as haloperidol, control the positive symptoms of schizophrenia (hallucinations, delusions, etc.) via the blockade of limbic D₂ receptors targeted by hyperactive mesolimbic dopaminergic pathways (Holcomb et al., 1996; Kahn and Davis, 1995). However, neuroleptics are poorly effective against negative-cognitive symptoms, such as mutism and blunted affect. These symptoms reflect a disruption in the activity of mesocortical dopaminergic pathways and, more generally, a perturbation in the function of the prefrontal cortex and FCX, commonly termed "hypofrontality" (Jentsch et al., 1997; Knable and Weinberger, 1997). Indeed, neuroleptics may exacerbate negative symptoms by blocking FCX-localized D₂ receptors and provoking an extrapyramidal syndrome. An additional disadvantage of neuroleptics is that a substantial population of patients do not respond satisfactorily to their administration (Kane and Freeman, 1994). Further, neuroleptics induce a pronounced hyperprolactinemia and associated endocrinological disorders by antagonism of tonically active D₂ receptors on hypophyseal lactotrophs, and a marked extrapyramidal motor syndrome due to blockade of D₂ receptors in the basal ganglia (Cunningham-Owens, 1996). Finally, long-term treatment with neuroleptics may ultimately result in the emergence of tardive dyskinesias, an irreversible motor problem likely related to striatal D₂ receptor blockade, although its precise origin remains uncertain (Cunningham-Owens, 1996).

The above observations suggest that the improved treatment of schizophrenia requires drugs with characteristics different to those of typical neuroleptics and targeting sites other than, or in addition to, D₂ receptors. In this respect, the dibenzodiazepine, clozapine, has attracted enormous interest inasmuch as this "atypical" antipsychotic manifests only modest affinity for D₂ receptors yet is effective in a subpopulation of neuroleptic-resistant patients, improves negative symptomology, presents a benign extrapyramidal potential and does not elicit tardive dyskynesia (Kane and Freeman, 1994; Meltzer, 1995). An ongoing challenge is to identify the key receptorial interactions underlying the superior clinical profile of clozapine and, in this regard, numerous hypotheses have been formulated (Brunello et al., 1995; Kinon and Lieberman, 1996). These include: equilibrated antagonist activity at D₁ and D₂ receptors (Gerlach and Hansen, 1992); preferential antagonist activity at D₄ vs. D₂ receptors (Seeman et al., 1997) and pronounced antagonist activity at adrenergic (AR) receptors (Baldessarini et al., 1992). In addition, a convincing body of evidence points to the importance of 5-HT_2A and, possibly, 5-HT_2C receptors: these are concentrated in corticolimbic regions and the basal ganglia, are involved in the modulation of mood and motor behavior and modulate the activity of dopaminergic pathways (Brunello et al., 1995; Casey, 1993; Kelland and Chiodo, 1996; Kennett et al., 1997; Roth and Meltzer, 1995; Schmidt and Fadayel, 1995). Thus, clozapine has marked affinity for 5-HT_2C receptors, blockade of which facilitates mesocortical dopaminergic transmission (Gobert et al., 1998 and unpublished observations; Kennett et al., 1997; Pessia et al., 1994). Further, a preferential blockade of 5-HT_2A vs. D₂ receptors by antipsychotic drugs, such as clozapine, has been convincingly correlated with a low propensity to elicit an extrapyramidal motor syndrome (Meltzer, 1995; Roth and Meltzer, 1995; Wadenberg, 1996) and changes in the levels of 5-HT_2A receptors have been documented in the FCX of schizophrenic patients (Burnet et al., 1996; Gurevich and Joyce, 1997). Alterations in 5-HT_1A receptor levels have also been documented in schizophrenia and, more recently, a potential significance of actions at 5-HT_1A receptors in the treatment of schizophrenia has been evoked (Burnet et al., 1996; Simpson et al., 1996) (see "Discussion").

Notwithstanding the improved antipsychotic profile of clozapine, it cannot be considered as an ideal antipsychotic agent. First, there remains a population of patients irresponsive to clozapine, and its impact upon primary negative symptoms may be limited (Kane and Freeman, 1994; Meltzer, 1995). Second, clozapine provokes autonomic and cardiovascular side-effects via actions at nonmonoaminergic receptors, notably histaminic and muscarinic sites. In addition, a minority (~5%) of patients display seizures, probably due to interference with central GABAergic and glutamatergic transmission (Cunningham-Owens, 1996). Third, the chemical structure of clozapine is associated with a potentially fatal agranulocytosis in 1 to 2% of patients treated (Liu and Uetrecht, 1995).

In the light of the above observations, it would clearly be of interest to develop antipsychotic agents possessing the beneficial properties of clozapine yet lacking its disadvantages. In our efforts to identify such antipsychotic drugs, we have characterized a novel benzodiozopyrrolidine, S 16924 (fig. 1). Herein, the receptorial profile of S 16924 is characterized, together with its modulation of dopaminergic, serotoninergic and adrenergic transmission in cortical, limbic and striatal regions. In the following article, the putative antipsychotic as compared to extrapyramidal properties of S 16924 are documented.

View larger version (8K): [in this window] [in a new window]   Fig. 1.   Chemical structure of S 16924.

	Methods

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Binding. Competition binding studies were performed at multiple dopaminergic, serotonergic and AR receptor types, as well as at DA, 5-HT and NAD reuptake sites. Assay conditions are summarized in tables 1, 2 and 3 (see also Millan et al., 1995). Isotherms were analyzed by nonlinear regression, using the program "PRISM" (Graphpad Software Inc., San Diego, CA) to yield Inhibitory Concentration (IC)₅₀ values. K_is were derived from IC₅₀ values according to the Cheng-Prusoff equation: K_i = IC₅₀/(1 + L/K_d) where L is the concentration of radioligand and K_d is the dissociation constant of the radioligand.

Measurement of agonist efficacy and antagonist potency at hD₂, hD₃, hD₄ and h5-HT_1A receptors. Receptor-linked G-protein activation at hD₂, hD₃, hD₄ and h5-HT_1A receptors was determined by measuring the stimulation of [³⁵S]-GTPS (1000 Ci/mmol; NEN, Les Ulis, France) binding as described in Newman-Tancredi et al. (1997). Briefly, CHO membranes (50 µg protein) expressing the respective receptors were incubated (20 min, 22°C) with agonists and/or antagonists in a buffer containing HEPES 20 mM (pH 7.4), GDP (3 µM), MgCl₂ (3 mM for hD₃ and hD₄, 10 mM for hD₂), NaCl (100 mM for hD₃ and hD₄, 150 mM for hD₂) and [³⁵S]-GTPS (0.1 nM for hD₂ and hD₄, 1 nM for hD₃). Nonspecific binding was defined with GTPS (10 µM). Agonist efficacy was expressed relative to that of DA or 5-HT (=100%) which were tested at maximally effective concentrations in each experiment. For antagonist studies, membranes were preincubated with antagonist and a single concentration of agonist for 30 min before the addition of [³⁵S]-GTPS. For concentration-response curves of the inhibition of DA-stimulated [³⁵S]-GTPS binding, K_b values were calculated as described in Newman-Tancredi et al. (1997). Experiments were terminated by rapid filtration through Whatman GF/B filters using a Brandel cell harvester. Radioactivity retained on the filters was determined by liquid scintillation counting. Protein concentration was determined colorimetrically using a bicinchoninic acid assay kit (Sigma Chimie, St Quentin-Fallavier, France).

In vivo studies. Male Wistar rats (Iffa Credo, L'arbresle, France, 220-240 g body weight) were housed in sawdust-lined cages with free access to food and water. Laboratory temperature was 21 ± 1.0°C and humidity 60 ± 5%. There was a 12 hr/12 hr light-dark cycle with lights on at 7:30.

Influence upon striatal DA and 5-HT turnover. As described in detail previously (Gobert et al., 1995a), the influence of drugs upon striatal DA and 5-HT turnover in rats was evaluated by measuring the levels of the DA precursor, DOPA, and of the 5-HT precursor, 5-HTP in the striatum 60 min after s.c. injection of drugs and 30 min after injection of the decarboxylase inhibitor, NSD 1015 (100 mg/kg, s.c.). Tissues were homogenized in 500 µl of 0.1 M HClO₄ containing 0.5% Na₂S₂O₅ and 0.5% EDTA and centrifuged at 15,000 × g for 15 min at 4°C. Supernatants were diluted in the mobile phase. HPLC analysis followed by electrochemical detection was used for determination of tissue levels of DOPA and 5-HTP. The column characteristics and elution phases were as follows: column, Hypersil ODS (5 µm), C18, 150 × 4.6 mm maintained at 25°C; mobile phase, KH₂PO₄ (100 mM), EDTA (0.1 mM), sodium octylsulfonate (0.5 mM), methanol (5%) adjusted to pH 3.15 with PO₄H₃. The flow rate was 1 ml/min. Electrochemical detection was performed using a Waters M460 detector with a working potential of 850 mV against an Ag/AgCl reference. Levels of DOPA and 5-HTP were expressed relative to control (vehicle) values (=0%). Data were analyzed by ANOVA followed by Dunnett's test.

Influence on cerebral DA turnover. As described in detail previously (Millan et al., 1995), the ratio of DOPAC to DA levels was determined in various cerebral tissues 30 min after s.c. administration of drugs. Levels were determined by HPLC/electrochemical detection as described above. DOPAC/DA ratios were expressed relative to control (vehicle) values (=0%). Data were analyzed by ANOVA followed by Dunnett's test.

Influence upon the electrical activity of dopaminergic neurones. As previously described (Lejeune et al., 1997), rats were anesthetized with chloral hydrate (400 mg/kg, i.p.), the femoral vein was catheterized and rats were placed in a stereotaxic apparatus. A tungsten micro-electrode was lowered into the VTA. Dopaminergic neurones were identified as previously and baseline recording performed over 5 min. Drugs were dissolved in sterile water and injected i.v. in a volume of 0.5 ml/kg, followed by a 0.1 ml saline flush. Compounds were administered cumulatively i.v. at intervals of 2 to 5 min. In antagonist studies, they were administered (single dose) 2 min after the injection of apomorphine (0.031 mg/kg, i.v.). Data acquisition and analysis were performed using Spike 2 software (C.E.D., Cambridge, England) and results are expressed as firing rate (60-sec bins at time of peak drug action) as a percentage of baseline pre-injection values (=0%).

Influence on the electrical activity of serotoninergic neurones. For evaluation of the influence of drugs upon the activity of serotoninergic neurones of the DRN, an identical protocol was used as described above for dopaminergic neurones (Lejeune et al., 1994, 1997). Drugs were administered in cumulative doses i.v. at intervals of 2 to 5 min. In antagonist studies, drugs were administered at a single dose followed, 2 min later, by a single injection of WAY 100,635 (0.031 mg/kg, i.v.) or () tertatotol (2.0 mg/kg, i.v.).

Determination of extracellular levels of DA, 5-HT and NAD in the FCX, accumbens and striatum. The procedure used has been described in detail elsewhere (Gobert et al., 1995b, 1998). Under pentobarbital anesthesia (60 mg/kg, i.p.), rats were placed in a stereotaxic apparatus and a guide cannula implanted in the FCX or in both the accumbens and the contralateral striatum. Five days later, a Cuprophan CMA/11 probe of 4 mm length (FCX and striatum) or of 2 mm length (accumbens) and 0.24 mm o.d. was lowered into position and perfused at 1 µl/min with a phosphate-buffered Ringer solution (147.2 mM NaCl, 4 mM KCl and 2.3 mM CaCl₂, pH 7.3). Two hours later, dialysis was commenced and samples taken every 20 min. Three basal samples were taken, then the drug was injected. Samples were then taken for another 3 hr. For interaction studies, WAY 100,635 (0.16 mg/kg, s.c.) was injected followed, 20 min later, by either S 16924 (2.5) or clozapine (2.5). Levels of DA, 5-HT and NAD were simultaneously quantified in individual samples using HPLC and coulometric detection with the following conditions: 20-µl dialysate samples were diluted with 20 µl of mobile phase (NaH₂PO₄: 75 mM, EDTA: 20 µM, sodium decanesulphonate: 1 mM, methanol: 17.5%, triethylamine 0.01%, pH 5.70) and 33-µl samples were analyzed by HPLC with a column (hypersil ODS 5 µm, C18, 150 × 4.6 mm) maintained at 43°C for separation and a coulometric detector (ESA 5014, Coulochem II) for quantification. The first electrode of the detector was set at 70 mV (reduction) and the second at +280 mV (oxidation). The mobile phase was delivered at a flow rate of 2 ml/min. The assay sensitivity was between 0.1 and 0.2 pg/sample for DA, NAD and 5-HT. Drug effects were expressed as a percentage of basal values (=0%). Data were analyzed by ANOVA with sampling time as the repeated within-subject factor.

Drugs. All drugs were dissolved in sterile water, if necessary with a few drops of lactic acid. The pH was adjusted to as close to neutrality as possible (>5.0). Drugs were injected s.c. unless otherwise specified. Drug sources and salts were as follows. Clozapine and apomorphine HCl (Research Biochemicals International, Natick, MA). S 16924 HCl, WAY 100,635 HCl and haloperidol were synthetized by O. Muller and G. Lavielle (Servier).

	Results

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Patterns of displacement. At each of the receptors presented in tables 1 to 3, S 16924, clozapine and haloperidol presented monophasic isotherms for displacement of the respective radioligands (slope factors not significantly differing from unity) (not shown). In figure 2, the overall receptor profiles of S 16924, haloperidol and clozapine at several key receptor types are depicted. It may be seen that the profiles of S 16924 and clozapine corresponded closely, whereas that of haloperidol was markedly different.

View larger version (12K): [in this window] [in a new window]   Fig. 2.   "Radar" representation of competition binding profiles of S 16924, clozapine and haloperidol at dopaminergic, serotonergic and adrenergic receptor types potentially implicated in the actions of clozapine and other antipsychotic agents: h, cloned, human; r, native rat and rc, cloned, rat. The distance from the center of the radar is proportional to the affinity (pKi) of the compound at the receptor. pKi (log Ki) values were derived from results shown in tables 1, 2 and 3.

Affinities of S 16924, clozapine and haloperidol at multiple dopaminergic receptors (table 1). Whereas haloperidol displayed potent affinity at native rat and cloned hD₂ receptors, S 16924 mimicked the modest affinity of clozapine at these sites. Similarly, the affinity of S 16924 and clozapine at cloned rat and hD₃ receptors was modest in contrast to the marked affinity of haloperidol for these sites. Haloperidol manifested about 5-fold lower affinity for hD₄ (the hD_4.4 isoform) as compared to hD₂ receptors whereas clozapine displayed a mild preference (about 2-fold) for hD₄ sites. This preferential affinity for hD₄ vs. hD₂ sites was more pronounced for S 16924 (about 5-fold selectivity). Compared with D₂ receptors, the affinity of haloperidol was markedly lower at both native D₁ and cloned, hD₁ receptors. In distinction, both S 16924 and clozapine presented comparable and modest affinity for native D₁ and cloned hD₁ receptors as compared with D₂ receptors. Haloperidol, S 16924 and clozapine all showed similar affinities for cloned hD₅ receptors as compared to hD₁ receptors. The affinity of S 16924 at DA reuptake sites was negligible.

Affinities of S 16924, clozapine and haloperidol at multiple serotoninergic receptors (table 2). Haloperidol showed negligible affinity at native rat 5-HT_1A and cloned h5-HT_1A receptors although the modest affinity of clozapine at these sites was similar to its affinity at D₂ receptors (table 1). In contrast, S 16924 showed pronounced affinity at both rat 5-HT_1A and h5-HT_1A receptors that was 20-fold superior to its affinity at D₂ sites. Haloperidol showed negligible affinity for 5-HT_1B sites, at which clozapine displayed very weak and S 16924 only low affinity. The affinity of haloperidol at native 5-HT_2A and cloned h5-HT_2A receptors was weak vs. its affinity at D₂ sites, and haloperidol displayed negligible affinity for native 5-HT_2C and cloned h5-HT_2C sites as well as for h5-HT_2B sites. In distinction, clozapine and S 16924 both showed markedly higher affinity at native, 5-HT_2A and cloned h5-HT_2A vs. D₂ receptors. Similarly, in contrast to haloperidol, both S 16924 and clozapine manifested marked affinity for native 5-HT_2C and cloned h5-HT_2C receptors. Interestingly, for all ligands, affinities were higher at cloned, h5HT_2C vs. native, porcine 5-HT_2C sites, and this difference was significant (P < .05) for S 16924 and clozapine. Whether this observation reflects a species difference, or a difference between native, tissue vs. cloned, transfected receptors, remains to be elucidated. S 16924 and haloperidol also showed pronounced affinity for h5-HT_2B sites. At 5-HT₃ receptors, clozapine displayed modest affinity whereas neither haloperidol nor S 16924 displayed significant affinity. The affinity of all drugs for 5-HT₄ and 5-HT_5A sites was low. However, both S 16924 and clozapine, in contrast to haloperidol, showed significant affinity at 5-HT₆ and 5-HT₇ sites. S 16924 did not manifest significant affinity for 5-HT reuptake sites.

Influence of S 16924, clozapine and haloperidol at multiple adrenergic receptors (table 3). S 16924, clozapine and haloperidol all shared potent affinity for native ₁-AR as well as _1A- and _1B-AR receptors. However, when expressed relative to their affinity at D₂ receptors, S 16924 and clozapine, but not haloperidol, revealed a marked preference for ₁-, _1A- and _1B-AR sites in each case. S 16924 and clozapine showed modest affinity at both native _2A- and cloned h_2A-AR receptors. Further, they also showed modest affinity for cloned h_2B- and h_2C-AR receptors. The affinity of haloperidol for each of these ₂-AR receptor types was negligible. S 16924 did not show significant affinity for ₁- and ₂-AR receptors or for NAD reuptake sites.

Influence of S 16924 as compared to clozapine and haloperidol upon [³⁵S]-GTPS binding at hD₂, hD₃ and hD₄ receptors. Dopamine elicited a concentration-dependent increase in [³⁵S]-GTPS binding to cloned hD₂, hD₃ and hD₄ receptors with Effective Concentration (EC)₅₀ values of 353 ± 52, 15.6 ± 3.9 and 109 ± 15 nM, respectively. In contrast, neither S 16924, clozapine nor haloperidol stimulated binding at these receptors (fig. 3 and not shown). Indeed, they all behaved as antagonists at hD₂, hD₃ and hD₄ receptors, concentration-dependently inhibiting the stimulation of [³⁵S]-GTPS binding induced by DA (3, 1 and 1 µM respectively) (fig. 3 and not shown). K_b values calculated for S 16924 were: hD₂, 34.2 ± 3.7 nM; hD₃, 79.8 ± 7.2 nM and hD₄, 5.0 ± 1.8 nM. K_b values calculated for clozapine were: hD₂, 71.7 ± 11.1 nM; hD₃, 251 ± 80 and hD₄, 48.4 ± 3.7 nM. K_b values calculated for haloperidol were: hD₂, 0.58 ± 0.10; hD₃, 28.8 ± 11.3 and hD₄, 1.37 ± 0.18 nM.

View larger version (16K): [in this window] [in a new window]   Fig. 3.   Effect of S 16924 on receptor-mediated stimulation of [35S]-GTPS binding at hD2 (upper panel), hD3 (middle panel) and hD4 (lower panel) receptors expressed in mammalian (CHO) cells. [35S]-GTPS binding was determined in the presence of S 16924 alone or with a fixed concentration of dopamine (3 µM at hD2 and 1 µM at hD3 and hD4 receptors). Points shown are means of triplicate determinations from representative experiments repeated on at least three occasions.

Influence of S 16924 as compared to clozapine and haloperidol upon [³⁵S]-GTPS binding at h5-HT_1A receptors. Serotonin concentration-dependently increased [³⁵S]-GTPS binding at h5-HT_1A receptors with an EC₅₀ of 16.8 ± 3.9 (fig. 4). Even at a very high concentration (10 µM), haloperidol failed to stimulate [³⁵S]-GTPS binding (not shown). However, clozapine (EC₅₀ of 1740 ± 736 nM) stimulated binding to 43.8 ± 3.1% of levels attained with 5-HT (defined as 100%) (not shown). S 16924 stimulated [³⁵S]-GTPS binding by 54.1 ± 11.3%, but with considerably greater potency than clozapine: the EC₅₀ for S 16924 was 11.3 ± 0.4 nM (fig. 4). S 16924-stimulated [³⁵S]-GTPS binding was inhibited by the selective 5-HT_1A antagonist, WAY 100,635, with an IC₅₀ of 3.18 ± 0.53 (fig. 4). WAY 100,635 was inactive alone (not shown).

View larger version (16K): [in this window] [in a new window]   Fig. 4.   Effect of S 16924 on receptor-mediated stimulation of [35S]-GTPS binding at h5-HT1A receptors expressed in mammalian (CHO) cells. A, Stimulation of [35S]-GTPS binding by S 16924 as compared of 5-HT. B, Inhibition of S 16924 (100 nM)-stimulated [35S]-GTPS binding by the selective 5-HT1A receptor antagonist, WAY 100,635. Points shown are means of triplicate determinations from representative experiments repeated on at least three occasions.

Influence of S 16924 as compared to clozapine and haloperidol upon cerebral turnover of DA and 5-HT. As determined by the ratio of tissue levels of DA to those of its metabolite, DOPAC, haloperidol potently and markedly enhanced DA turnover in projection areas of mesocortical (FCX), mesolimbic (olfactory tubercles and nucleus accumbens) and nigrostriatal (striatum) pathways (fig. 5). In contrast, S 16924 and clozapine only weakly and less markedly increased DA synthesis in each of these regions (fig. 5). Similarly, on determination of levels of the DA precursor, DOPA, after pretreatment with the decarboxylase inhibitor, NSD 1015 (100 mg/kg, s.c.), haloperidol elicited a potent and pronounced induction in striatal DA synthesis whereas S 16924 and clozapine were only weakly active (fig. 5). Haloperidol failed to modify striatal levels of the 5-HT precursor, 5-HTP, an index of 5-HT synthesis, whereas striatal levels of 5-HTP were potently and markedly decreased by S 16924 and slightly depressed by clozapine (fig. 5).

View larger version (27K): [in this window] [in a new window]   Fig. 5.   Influence of S 16924 as compared to clozapine and haloperidol upon cerebral turnover of dopamine and serotonin. A to D depict ratios of tissue levels of DA as compared to those of its metabolite, DOPAC and E and F represent the accumulation of the DA precursor, DOPA and the 5-HT precursor, 5-HTP, in rats pretreated with the decarboxylase inhibitor, NSD 1015 (100 mg/kg, s.c.). Data are means ± S.E.M. N > 5 per value. ANOVA as follows. A, S 16924, F(4,17) = 52.8, P < .001; clozapine, F(3,46) = 0.9, P > .05 and haloperidol, F(10,62) = 29.1, P < .001. B, S 16924, F(4,17) = 32.7, P < .001; clozapine, F(3,40) = 2.4, P > .05 and haloperidol, F(10,54) = 97.0, P < .001. C, S 16924, F(4,17) = 81.2, P < .001; clozapine, F(3,56) = 7.4, P < .001 and haloperidol, F(10,62) = 98.7, P < .001. D, S 16924, F(4,17) = 62.8, P < .001; clozapine, F(3,56) = 7.2, P < .001 and haloperidol, F(10,62) = 131.9, P < .001. E, S 16924, F(4,30) = 0.8, P > .05; clozapine, F(5,42) = 2.9, P < .05 and haloperidol, F(6,29) = 150.0, P < .001. F, S 16924, F(4,30) = 19.0, P < .001; clozapine, F(5,42) = 5.1, P < .01 and haloperidol, F(6,29) = 3.2, P < .05. Asterisks indicated significance of differences to vehicle in Dunnett's test following ANOVA. * P < .05.

Influence of S 16924 as compared to clozapine and haloperidol upon the electrical activity of VTA-localized dopaminergic neurones. The dopaminergic agonist, apomorphine (0.031 mg/kg, i.v.), markedly reduced the firing rate of dopaminergic neurones in the VTA (fig. 6). This action was dose-dependently inhibited by haloperidol and, less potently, by S 16924 and clozapine (fig. 6). ID₅₀s (95% CLs) were as follows: 0.004 (0.002-0.006), 0.18 (0.12-0.19) and 0.22 (0.15-0.34), respectively. Administered alone, haloperidol and, less potently, clozapine and S 16924 slightly but dose-dependently and significantly increased firing rate (fig. 6).

View larger version (18K): [in this window] [in a new window]   Fig. 6.   Influence of S 16924 as compared to clozapine and haloperidol upon the electrical activity of ventrotegmental dopaminergic neurones. A, Dose-response curves for antagonism of the inhibitory action of apomorphine (0.031 mg/kg, i.v.). B, Dose-dependent modulation of firing rate upon administration of drugs alone. Data are means ± S.E.M. N  4 per value. ANOVA as follows. A, S 16924, F(4,16) = 76.0, P < .001; clozapine, F(4,16) = 30.8, P < .001 and haloperidol, F(3,12) = 71.0, P < .001. B, S 16924, F(5,25) = 8.6, P < .001; clozapine, F(5,25) = 13.1, P < .001 and haloperidol, F(5,25) = 3.9, P < .01. Asterisks indicate significance of differences to vehicle in Newman-Keuls test (paired data) after ANOVA. * P < .05.

Influence of S 16924 as compared to clozapine and haloperidol upon the electrical activity of DRN-localized serotoninergic neurones. S 16924 potently and dose-dependently inhibited the firing of DRN-localized serotoninergic neurones with an ID₅₀ (95% CLs) of 0.02 (0.01-0.03) (fig. 7). Clozapine also inhibited DRN firing over a higher dose-range: ID₅₀ (95% CLs) = 0.08 (0.02-0.3). Haloperidol was also effective, although only at high doses: ID₅₀ (95% CLs) = 0.4 (0.2-0.9) (fig. 7). The inhibitory influence of S 16924 was blocked by WAY 100,635 and a further 5-HT_1A antagonist, ()-tertatolol, neither of which significantly modified firing rate upon administration alone (fig. 7). The ac- tions of clozapine and haloperidol were not affected by WAY 100,635 or ()-tertatotol (fig. 7).

View larger version (20K): [in this window] [in a new window]   Fig. 7.   Influence of S 16924 as compared to clozapine and haloperidol on the electrical activity of dorsal raphe serotoninergic neurones. A, Dose-dependent inhibition of firing rate on administration of drugs alone. B, Influence on the actions of S 16924, clozapine and haloperidol of the 5-HT1A antagonists, WAY 100,635 (0.031 mg/kg, i.v.) and ()tertatolol (2.0 mg/kg, i.v.). Data are means ± S.E.M. N  4 per value. ANOVA as follows. A, S 16924, F(5,35) = 93.9, P < .001; clozapine, F(4,16) = 30.6, P < .001 and haloperidol, F(4,12) = 9.6, P < .001. Asterisks indicate significance of differences (P < .05) to vehicle in Newman-Keuls test after ANOVA. Panel B: S 16924 (0.125 mg/kg, i.v.), F(2,11) = 27.8, P < .001; clozapine (1.0 mg/kg, i.v.), F(2,16) = 1.1, P > .05 and haloperidol (1.0 mg/kg), F(2,12) = 0.7, P > .05. Asterisks indicate significance of differences of WAY 100,635/drug or ()-tertatolol/drug to vehicle/drug values in Dunnett's test after ANOVA. * P < .05.

Influence of S 16924 as compared to clozapine and haloperidol upon extracellular levels of DA, 5-HT and NAD in the FCX, nucleus accumbens and striatum of freely-moving rats. Haloperidol (0.63) elicited a modest increase in dialysate levels of DA in the FCX and a similar, though more sustained, elevation in extracellular levels of DA levels in the nucleus accumbens and striatum (fig. 8). In contrast S 16924 (2.5) and clozapine (2.5) increased dialysate levels of DA in the FCX without markedly modifying those of DA in either the accumbens or striatum (fig. 8). This facilitatory influence of S 16924 on FCX levels of DA was expressed dose-dependently and, in parallel, it dose-dependently increased and decreased FCX levels of NAD and 5-HT, respectively (fig. 9). S 16924 also markedly decreased 5-HT levels in both accumbens and striatum (fig. 10). Clozapine also increased dialysate levels of NAD in the FCX (fig. 11) without significantly affecting levels of 5-HT either in this structure or in the accumbens, although it significantly decreased 5-HT levels in striatum (fig. 10). Haloperidol similarly provoked a modest increase in levels of NAD in FCX (fig. 11) without modifying levels of 5-HT in the FCX, accumbens or striatum (fig. 10). The influence of S 16924 upon FCX levels of DA and 5-HT was significantly inhibited by WAY 100,635 (fig. 12). In contrast, WAY 100,635 did not significantly modify the influence of clozapine upon FCX levels of DA, 5-HT or NAD (fig. 13).

View larger version (23K): [in this window] [in a new window]   Fig. 8.   Influence of S 16924 as compared to clozapine and haloperidol upon extracellular levels of DA in the frontal cortex as compared to the nucleus accumbens and striatum of freely-moving rats. Data are means ± S.E.M. N = 5 to 12 per value. They are expressed as a percentage of basal, preinjection values which were defined as 0%. These were 1.2 ± 0.09, 8.4 ± 1.3 and 12.9 ± 1.6 pg/20 min dialysate for DA in the frontal cortex, nucleus accumbens and striatum, respectively, in vehicle-treated animals. For comparison of individual values with the vehicle-treated group (open circles), ANOVA was performed over 40 to 180 min. Influence of S 16924: frontal cortex, F(1,16) = 56.5, P < .01; nucleus accumbens, F(1,18) = 0.2, P > .05 and striatum, F(1,16) = 9.9, P < .01. Influence of clozapine: frontal cortex, F(1,17) = 27.4, P < .01; nucleus accumbens, F(1,18) = 2.0, P > .05 and striatum, F(1,17) = 0.2, P > .05. Influence of haloperidol: frontal cortex, F(1,19) = 25.3, P < .01; nucleus accumbens, F(1,16) = 39.2, P < .01 and striatum, F(1,16) = 154.1, P < .01. Asterisks indicate significance of drug-treated groups to the vehicle-treated group. * P < .05.

View larger version (24K): [in this window] [in a new window]   Fig. 9.   Dose-dependent modulation by S 16924 of extracellular levels of 5-HT, DA and NAD in the frontal cortex of freely-moving rats. Data are means ± S.E.M. N = 5 to 13 per value. They are expressed as a percentage of basal, pre-injection values which were defined as 0%. These were 0.80 ± 0.07, 1.20 ± 0.09 and 1.46 ± 0.21 pg/20 min dialysate for 5-HT, DA and NAD, respectively, in vehicle-treated animals. For comparison of individual values with the vehicle-treated group, ANOVA with dose as between factor and time as within factor, was performed over 40 to 180 min. S 16924 (0.16): 5-HT, F(1,9) = 1.7, P > .05; DA, F(1,16) = 5.7, P < .05 and NAD, F(1,14) = 0.3, P > .05. S 16924 (0.63): 5-HT, F(1,10) = 13.0, P < .01; DA, F(1,17) = 33.7, P < .01 and NAD, F(1,15) = 19.4, P < .01. S 16924 (2.5): 5-HT, F(1,9) = 31.3, P < .01; DA, F(1,16) = 56.5, P < .01 and NAD, F(1,14) = 37.2, P < .01. S 16924 (10.0): 5-HT, F(1,11) = 76.5, P < .01; DA, F(1,18) = 78.6, P < .01 and NAD, F(1,16) = 4.7, P < .05. Asterisks indicate significance of drug-treated groups to vehicle-treated group. * P < .05.

View larger version (25K): [in this window] [in a new window]   Fig. 10.   Influence of S 16924 as compared to clozapine and haloperidol upon extracellular levels of 5-HT in the frontal cortex as compared to the nucleus accumbens and striatum of freely-moving rats. Data are means ± S.E.M. N = 6 to 11 per value. They are expressed as a percentage of basal, pre-injection values which were defined as 0%. These were 0.80 ± 0.07, 0.65 ± 0.09 and 0.55 ± 0.07 pg/20 min dialysate for 5-HT in the frontal cortex, nucleus accumbens and striatum, respectively, in vehicle-treated animals. For comparison of individual values with the vehicle-treated group (open circles), ANOVA was performed over 40 to 180 min. Influence of S 16924, frontal cortex, F(1,9) = 31.3, P < .01; nucleus accumbens, F(1,14) = 43.5, P < .01 and striatum, F(1,11) = 29.6, P < .01. Influence of clozapine, frontal cortex, F(1,10) = 2.4, P > .05; nucleus accumbens, F(1,14) = 0.1, P > .05 and striatum, F(1,11) = 6.2, P < .05. Influence of haloperidol, frontal cortex, F(1,11) = 2.1, P > .05; nucleus accumbens, F(1,14) = 0.3, P > .05 and striatum, F(1,9) = 1.3, P > .05. Asterisks indicate the significance of drug-treated groups to vehicle-treated group. * P < .05.

View larger version (22K): [in this window] [in a new window]   Fig. 11.   Influence of clozapine and haloperidol on extracellular levels of NAD in the frontal cortex of freely-moving rats. Data are means ± S.E.M. N = 6 to 11 per value. They are expressed as a percentage of basal, preinjection values that were defined as 0% (see legend to fig. 10). For comparison of individual values with the vehicle-treated group, ANOVA was performed over 40 to 180 min. Influence of clozapine, F(1,15) = 30.9, P < .01 and influence of haloperidol, F(1,17) = 16.7, P < .01. Asterisks indicate significance of drug-treated groups to vehicle-treated group. * P < .05.

View larger version (22K): [in this window] [in a new window]   Fig. 12.   Influence of WAY 100,635 on the modulation by S 16924 of extracellular levels of 5-HT, DA and NAD in the frontal cortex of freely-moving rats. Data are means ± S.E.M. N = 6 to 11 per value. They are expressed as a percentage of basal, preinjection values which were defined as 0%. These were 0.61 ± 0.12, 0.97 ± 0.10 and 1.2 ± 0.23 pg/20 min dialysate for 5-HT, DA and NAD, respectively, in vehicle-treated animals. ANOVA with drug as between factor and time as within factor was performed over 80 to 160 min. Upper panel, 5-HT, influence of WAY 100,635, F(1,13) = 30.0, P < .01; influence of time, F(4,52) = 0.6, P > .05 and interaction, F(4,52) = 1.2, P > .05. DA, influence of WAY 100,635, F(1,13) = 7.2, P < .05; influence of time, F(4,52) = 2.4, P > .05 and interaction, F(4,52) = 1.0, P > .05. NAD, influence of WAY 100,635, F(1,13) = 3.0, P > .05; influence of time, F(4,52) = 5.9, P < .01 and interaction, F(4,52) = 0.1, P > .05. Lower panel, 5-HT, influence of WAY 100,635, F(1,19) = 0.1, P > .05; influence of time, F(4,76) = 0.8, P > .05 and interaction, F(4,76) = 0.8, P > .05. DA, influence of WAY 100,635, F(1,19) = 0.2, P > .05; influence of time, F(4,76) = 0.7, P > .05 and interaction, F(4,76) = 1.6, P > .05. NAD, influence of WAY 100,635, F(1,14) = 0.6, P > .05; influence of time, F(4,56) = 0.2, P > .05 and interaction, F(4,56) = 0.3, P > .05. Asterisks indicate significance of the WAY 100,635-treated group to the vehicle-treated group. * P < .05.

View larger version (19K): [in this window] [in a new window]   Fig. 13.   Lack of influence of WAY 100,635 on the modulation by clozapine of extracellular levels of 5-HT, DA and NAD in the frontal cortex of freely-moving rats. Data are means ± S.E.M. N = 5 to 6 per value. They are expressed as a percentage of basal, preinjection values which were defined as 0% (see legend to fig. 12). ANOVA with drug as between factor and time as within factor was performed over 80 to 160 min. 5-HT, influence of WAY 100,635, F(1,9) = 2.3, P > .05; influence of time, F(4,36) = 1.4, P > .05 and interaction, F(4,36) = 1.0, P > .05. DA, influence of WAY 100,635, F(1,9) = 0.1, P > .05; influence of time, F(4,36) = 20.9, P < .01 and interaction, F(4,36) = 0.8, P > .05. NAD, influence of WAY 100,635, F(1,8) = 0.1, P > .05; influence of time, F(4,32) = 19.0, P < .01 and interaction, F(4,32) = 0.3, P > .05.

	Discussion

Top Abstract Introduction Methods Results Discussion References

S 16924 displays a profile of interaction at multiple dopaminergic, serotoninergic and adrenergic receptors similar to that of the "atypical" antipsychotic, clozapine and different to that of the neuroleptic, haloperidol. In particular, it shares the more marked activity of clozapine at hD₄, 5-HT_2A, 5-HT_2C and ₁-AR as compared to D₂ receptors. In addition, in contrast to both clozapine and haloperidol, S 16924 possesses potent, partial agonist properties at 5-HT_1A receptors. This distinctive binding profile of S 16924 is reflected in vivo by its ability, upon acute administration, to inhibit serotoninergic transmission and to preferentially reinforce frontocortical vs. subcortical dopaminergic transmission.

Antagonist properties at cloned hD₂, hD₃ and hD₄ receptors. S 16924 displayed, as with clozapine, only modest affinity at hD₂ and hD₃ receptors. In previous studies, clozapine has shown a mild, although variable and radioligand-dependent, preference for hD₄ vs. hD₂ receptors (Newman-Tancredi et al., 1997; Seeman et al., 1997), a finding confirmed herein, and S 16924 also displayed higher affinity at hD₄ than hD₂ receptors. Human D₂, hD₃ and hD₄ receptors all couple via G proteins to (specific isoforms) of adenylyl cyclase and other intracellular transduction systems (Levant, 1997; Newman-Tancredi et al., 1997). Thus, activation and blockade of hD₂, hD₃ and hD₄ sites can be evaluated by the binding of [³⁵S]-GTPS, which interacts with "activated" G proteins after their ligand-induced dissociation from the corresponding receptor. Using this approach, we recently demonstrated that clozapine and haloperidol behave as antagonists at hD₄ receptors (Newman-Tancredi et al., 1997) and S 16924 also behaved as a potent antagonist at hD₄ sites. Our findings also show that S 16924, haloperidol and clozapine behave as antagonists at hD₂ and hD₃ receptors. The antagonist properties of S 16924 at hD₂ receptors are of significance inasmuch as blockade of postsynaptic D₂ receptors in limbic structures, by counteracting the hyperactivity of mesolimbic dopaminergic pathways, may reduce the positive symptoms of schizophrenia (Kahn and Davis, 1995). Whether antagonism of postsynaptic D₃ receptors, which are enriched in limbic tissue, is of importance to the actions of antipsychotic drugs is still under debate (Levant, 1997). The more pronounced activity of S 16924 at hD₄ vs. hD₂ receptors is of interest inasmuch as such a preference has been proposed to account for the superior antipsychotic profile of clozapine vs. haloperidol (Seeman et al., 1997). This contention has, however, been challenged (Roth et al., 1995) and putative alterations in levels of mRNA encoding D₄ receptors in schizophrenics are controversial (Marzella et al., 1997; Mulcrone and Kerwin, 1996; Seeman et al., 1997). Further, the selective D₄ receptor antagonist, L 745,870, was not effective in controlling psychosis in a clinical study (Kramer et al., 1997). In addition, the preclinical profiles of L 745,870 and other selective D₄ antagonists provide little evidence that antagonism of D₄ receptors controls the positive symptoms of schizophrenia (Bristow et al., 1997). Nevertheless, D₄ receptor blockade may improve the cognitive-attentional symptoms of schizophrenia (Tallman, 1997; Millan MJ and Dekeyne A, unpublished observations).

Interaction with D₁ and D₅ receptors. The similar affinity of S 16924 and clozapine at D₁ vs. D₂ receptors contrasts to the preference of haloperidol for the latter. This observation is of interest inasmuch as 1) a dysequilibrium in the activity of striatal populations of D₁ and D₂ receptors may contribute to extrapyramidal, side-effects and 2) actions at D₁ receptors may contribute to antipsychotic properties (Gerlach and Hansen, 1992) (companion paper). D₅ receptors present marked similarities to D₁ receptors, and clozapine and haloperidol possess similar affinity at hD₅ vs. hD₁ sites (Sunahara et al., 1991). Similarly, the modest affinity of S 16924 at hD₅ receptors was comparable to its affinity at hD₁ receptors. Although D₅ receptors are differentially localized to D₁ receptors, their functional significance remains unknown and certain actions ascribed to D₁ sites may actually be mediated by D₅ receptors (Bergson et al., 1995; Meador-Woodruff et al., 1996).

Antagonist actions at D₂ and D₃ receptors in vivo: modulation of cerebral DA synthesis. The activity of dopaminergic pathways is tonically inhibited by D₂ (and D₃) receptors localized on their dendrites and terminals and, possibly, by postsynaptic populations of D₃ sites acting via a feedback loop (Gobert et al., 1995b; Koeltzow et al., 1998; Tepper et al., 1997). Correspondingly, S 16924, clozapine and, more potently, haloperidol elevated DA synthesis in regions innervated by mesocortical (FCX), mesolimbic (olfactory tubercles and accumbens) and nigrostriatal projections (striatum) (Gobert et al., 1995a and b; Kahn and Davis, 1995). Interestingly, the magnitude of the increases in DA turnover evoked by S 16924 and clozapine were less marked than for haloperidol. One possible explanation is that elevations in DA turnover reflect inverse agonist actions at D₂ autoreceptors rather than blockade of tonic DA activity (Nilsson et al., 1996). However, as with haloperidol, clozapine possesses negative efficacy at hD₂ sites (Hall and Strange, 1997). An alternative explanation is that the serotoninergic and/or adrenergic actions of S 16924 and clozapine (see below) may intervene to moderate their influence upon DA synthesis. Irrespective of the underlying mechanisms, the finding that DA synthesis was little perturbed by S 16924 in the striatum is of importance inasmuch as extrapyramidal motor effects are correlated with an elevation of striatal DA synthesis (Lucas et al., 1997) (companion paper).

Partial agonist actions at 5-HT_1A receptors in vivo. Whereas S 16924 preferentially interacted at 5-HT_1A vs. D₂ receptors, clozapine interacted with equivalent potency, and haloperidol interacted exclusively with D₂ vs. 5-HT_1A receptors. In a [³⁵S]GTPS binding model, S 16924 behaved as a partial agonist with an efficacy equivalent to that of clozapine (Newman-Tancredi et al., 1996). This cellular model of 5-HT_1A receptor stimulation possesses a sensitivity comparable to that of postsynaptic 5-HT_1A receptors (Newman-Tancredi et al., 1997; Lejeune et al., 1997), at which S 16924 behaves as a partial agonist in vivo (companion paper). Inhibitory 5-HT_1A autoreceptors on serotoninergic cell bodies are more sensitive than their postsynaptic counterparts (Meller et al., 1990; Newman-Tancredi et al., 1997). Correspondingly, S 16924 markedly reduced striatal and accumbens release of 5-HT and it reduced striatal turnover of 5-HT at doses substantially lower than those enhancing striatal DA turnover. These data, underpinned by the WAY 100,635-reversible inhibitory influence of S 16924 upon DRN firing rate and FCX dialysate levels of 5-HT, suggest that S 16924 acutely inhibits serotoninergic transmission via agonist actions at 5-HT_1A autoreceptors. This activity is of particular significance in several respects. First, stimulation of 5-HT_1A receptors facilitates the activity of mesocortical dopaminergic (and adrenergic) pathways (Lejeune et al., 1997; Millan et al., 1997) (see below). Second, an inhibition in 5-HT release is associated with a reduction in anxious states (Coplan et al., 1995; Meller et al., 1990) and S 16924 possesses anxiolytic properties (Dekeyne A, and Millan MJ, unpublished observations). Third, activation of 5-HT_1A autoreceptors may counter the induction of extrapyramidal motor symptoms due to striatal D₂ receptor blockade (Lucas et al., 1997) and S 16924 does not elicit catalepsy in rats (companion paper). In contrast to S 16924, clozapine only modestly inhibited striatal release and turnover of 5-HT and failed to modify dialysate levels of 5-HT in the accumbens or FCX. Further, the inhibitory influence of clozapine on DRN firing is mediated by its antagonist properties at ₁-AR receptors, a mechanism that may also intervene in the weak reduction of DRN firing by haloperidol (Lejeune et al., 1994).

Serotonin 5-HT_2A and 5-HT_2C receptors. A preferential blockade of 5-HT_2A vs. D₂ receptors has been associated with a reduced propensity to elicit extrapyramidal side-effects and, possibly, an improved efficacy in the control of resistant patients and negative-cognitive symptoms (Roth and Meltzer, 1995; Schmidt and Fadayel, 1995). Thus, it is of significance that, in analogy to clozapine (Canton et al., 1994; Roth and Meltzer, 1995), S 16924 showed more pronounced affinity at 5-HT_2A than D₂ receptors. Indeed, antagonism of 5-HT_2A receptors is an important, clozapine-like feature of the pharmacology of S 16924 (companion paper). The higher affinity of clozapine at 5-HT_2C vs. D₂ sites (Canton et al., 1994; Roth and Meltzer, 1995) was similarly mimicked by S 16924. Although the significance of 5-HT_2C receptor blockade has been questioned as regards a reduced propensity to elicit extrapyramidal symptoms (Roth and Meltzer, 1995), there are several further, potentially important consequences of 5-HT_2C receptor blockade. First, antagonism of 5-HT_2C receptors markedly facilitates mesocortical dopaminergic transmission (Gobert et al., 1998; Kelland and Chiodo, 1996; Pessia et al., 1994). Second, 5-HT_2C receptor antagonists display anxiolytic properties (Kennett et al., 1997). Third, based on studies of transgenic mice lacking 5-HT_2C receptors, it has been suggested that the weight gain provoked by antipsychotics reflects 5-HT_2C receptor blockade (Cunningham-Owens, 1996; Tecott et al., 1995). Nevertheless, certain antipsychotics, such as risperidone, elicit weight gain despite low affinity at 5-HT_2C receptors (Cunningham-Owens, 1996). Thus, other mechanisms, such as histamine₁ receptor blockade, may also be involved (Cunningham-Owens, 1996). An interesting question concerns the functional significance of the combined blockade of 5-HT_2A/2C receptors and activation of 5-HT_1A receptors, as shown by S 16924. Serotoninergic transmission is inhibited by 5-HT_1A autoreceptors, and postsynaptic 5-HT_1A vs. 5-HT_2A/2C receptors exert an opposite influence on cellular transduction mechanisms, resulting in neuronal hyperpolarization and excitation, respectively. Thus, these properties may, as suggested previously, act synergistically (Millan et al., 1992): for example, in enhancing mesocortical dopaminergic transmission (see below). It would be of interest to perform long-term studies of the antipsychotic and other actions of the parallel activation and blockade of 5-HT_1A and 5-HT_2A/2C receptors, respectively.

5-HT₆ and 5-HT₇ receptors. S 16924 displayed significant affinity at 5-HT₆ receptors and it has been proposed that an action of clozapine at these sites may contribute to its atypical profile (Monsma et al., 1993). Although Roth et al. (1994) suggested that relatively high affinity at 5-HT₆ vs. D₂ receptors may not be a distinguishing feature of "atypical" antipsychotics, the preferential corticolimbic localization of 5-HT₆ receptors is of pertinence regarding the negative and cognitive-attentional symptoms of schizophrenia (Sleight et al., 1997). S 16924 also mimicked the high affinity of clozapine at 5-HT₇ receptors. These are enriched in several limbic and cortical regions and have been implicated in depressive states, which can aggravate the negative symptoms of schizophrenia (Sleight et al., 1997; although see Gobbi et al., 1996). Moreover, sleep cycles are disrupted in schizophrenics and 5-HT₇ receptors are concentrated in the suprachiasmatic nucleus wherein they fulfill an important role in controlling circadian rhythms (Lovenberg et al., 1993).

Interaction at ₁-AR receptors. A perturbation of adrenergic transmission is related to positive crises in schizophrenic patients and to an intensification of negative symptoms and the risk of relapse after treatment withdrawal (Mass et al., 1993). S 16924 mimicked the pronounced affinity of clozapine at ₁-ARs (as well as _1A- and _1B-ARs) (table 3), which are enriched in the thalamus, hippocampus, FCX and other structures implicated in the control of mood and in the pathophysiology of psychiatric disorders (Baldessarini et al., 1992). Several lines of evidence suggest that ₁-AR blockade may afford advantages in the treatment of schizophrenia. First, blockade of (limbic or cortical) ₁-AR receptors inhibits the induction of locomotion by psychostimulants (Blanc et al., 1994; Prinssen et al., 1994; Svensson et al., 1995). Second, coadministration of ₁-AR antagonists with haloperidol results in a clozapine-like, preferential inhibition of the activity of mesolimbic vs. nigrostriatal dopaminergic pathways (Lane et al., 1988). Third, blockade of thalamic ₁-AR receptors may improve the gating of sensory information to the cortex, a process that is defective in psychotic patients (Goldberg and Gold, 1995). Although peripheral ₁-AR blockade is associated with orthostatic hypotension, drug titration circumvents this effect, to which tolerance may develop (Cunningham-Owens, 1996).

Modulation of the electrical activity of VTA dopaminergic neurones. S 16924, clozapine and, more potently, haloperidol blocked suppression of the firing of VTA-localized dopaminergic neurones by apomorphine, consistent with their antagonist properties at D₂ (and D₃) autoreceptors. In fact, they slightly enhanced firing rate when administered alone. This action of haloperidol may be attributed to the interruption of a tonic, inhibitory tone at D₂ receptors (Gobert et al., 1998), a mechanism that may similarly contribute to the actions of higher doses of S 16924 and clozapine. Further, the antagonist actions of S 16924 and clozapine at 5-HT_2C receptors, or their partial agonist actions at 5-HT_1A receptors, might also be involved in exciting dopaminergic cell bodies in the VTA (Gobert et al., 1998; Kelland and Chiodo, 1996; Pessia et al., 1994).

The influence of S 16924 on frontocortical dopaminergic and adrenergic transmission. A dysruption in cortical function ("hypofrontality") is involved in the negative- and cognitive-attentional symptoms of schizophrenia (Andreasen et al., 1992). In this regard, a perturbation of dopaminergic input to this region has been implicated (Jentsch et al., 1997; Knable and Weinberger, 1997). Correspondingly, a potentiation of mesocortical dopaminergic transmission may improve the negative-cognitive symptoms of schizophrenia. In line with previous work (Moghaddam and Bunney, 1990; see Meltzer, 1995), haloperidol elicited only a modest increase in dialysate levels of DA in the FCX, and a marked (and more prolonged) rise was seen in nucleus accumbens and striatum: these actions may reasonably be attributed to its antagonist properties at D₂ (and D₃) autoreceptors (Gobert et al., 1995b, 1998). This increase in limbic release of DA by haloperidol may contribute to its lack of antipsychotic efficacy in certain "refractory" patients. In contrast to haloperidol, both clozapine and S 16924 markedly increased dialysate levels of DA in the FCX at doses not markedly modifying DA levels in the accumbens or striatum. This suggests that they may correct FCX hypofrontality without perturbing DA levels in limbic or striatal regions. In view of this regional specificity, an action at D₂ (D₃) autoreceptors is not likely to be the principal mechanism involved in the influence of S 16924 on FCX levels of DA. Notably, dopaminergic neurones in the parabrachial subdivision of the VTA, which project primarily to the FCX, are subject to a more pronounced serotoninergic control than their paranigral counterparts projecting to limbic structures (Lejeune et al., 1997; Svensson et al., 1995) and selective 5-HT_1A receptor agonists increase dialysate levels of DA in the FCX (Kelland and Chiodo, 1996; Lejeune et al., 1997). In line with these observations, WAY 100,635 attenuated the increase in FCX levels of DA elicited by S 16924. WAY 100,635 did not, however, abolish the elevation in DA levels provoked by S 16924 and, as mentioned above, an action of S 16924 at 5-HT_2A or 5-HT_2C receptors controlling FCX release of DA may also underlie its enhancement of FCX levels of DA. Such mechanisms may also intervene in the elevation in FCX dialysate levels of DA elicited herein by clozapine inasmuch as its actions were insensitive to WAY 100,635 (but see Rollema et al., 1997). The activity of mesocortical adrenergic neurones is subject to an inhibitory _2A-AR autoreceptor mediated-tone as well as a complex pattern of modulatory serotoninergic influence involving (indirect) facilitatory and inhibitory effects mediated via 5-HT_1A and 5-HT_2A/5-HT_2C receptors, respectively (Gobert et al., 1998; Haddjeri et al., 1997; Millan et al., 1997). Inasmuch as the increase in FCX levels of NAD elicited by S 16924 and clozapine was not markedly attenuated by WAY 100,635, activation of 5-HT_1A receptors may play a less important role in these actions than blockade of 5-HT_2C (or ₂-AR) receptors. In any case, adrenergic pathways in the FCX fulfill an important role in mechanisms controlling vigilance and memory formation (Foote and Aston-Jones, 1995) suggesting that the potentiation in mesocortical adrenergic transmission by S 16924 may be of use in improving cognitive-attentional performance.

Conclusions. For antipsychotic agents, it is their global pattern of interaction at multiple monoaminergic receptor types, and the relationship between the affinity at specific receptor types to that at D₂ receptors, which determines their functional activity in vivo (see fig. 2). In this respect, S 16924 displays a profile of action that differs markedly to that of haloperidol and closely resembles that of clozapine, despite their chemical distinctiveness. In addition, the partial agonist properties of S 16924 at 5-HT_1A receptors are more pronounced than those of clozapine. This distinctive compon