Abstract
S18327 displayed modest affinity for human (h)D2 and hD3 receptors and high affinity for hD4receptors. At each, S18327 antagonized stimulation of [35S]guanosine-5′-O-(3-thio)triphosphate binding by dopamine (DA). It also blocked activation of mitogen-activated protein kinase at hD3 receptors. The affinity of S18327 at hD1 and hD5 sites was modest. S18327 showed pronounced affinity for human serotonin (h5-HT)2A receptors and human α1A-adrenergic receptors (hARs), at which it antagonized increases in intracellular Ca2+ concentration levels elicited by 5-HT and norepinephrine (NE), respectively. S18327 presented significant affinity for hα2A-ARs and antagonized NE-induced[35S]guanosine-5′-O-(3-thio)triphosphate binding both at these sites and at α2-ARs in rat amygdala. Reflecting blockade of α2-autoreceptors, S18327 enhanced firing of adrenergic neurons in locus ceruleus, accelerated hippocampal synthesis of NE, and increased dialysate levels of NE in hippocampus, accumbens, and frontal cortex. S18327 abolished inhibition of ventrotegmental area-localized dopaminergic neurons by apomorphine. However, S18327 alone did not affect their activity and only modestly enhanced cerebral turnover of DA and dialysate levels of DA in striatum and accumbens. In contrast, S18327 markedly increased dialysate levels of DA in frontal cortex, an action abolished by the selective α2-AR agonist, S18616. Finally, S18327 reduced synthesis and dialysate levels of 5-HT in striatum and suppressed firing of dorsal raphe-localized serotonergic neurons, an action attenuated by the α1-AR agonist cirazoline. In conclusion, S18327 possesses marked antagonist activity at α1-ARs and D4 and 5-HT2A receptors and less potent antagonist activity at α2-ARs and D1 and D2 receptors. Antagonism by S18327 of α2-ARs enhances adrenergic transmission and reinforces frontocortical dopaminergic transmission, whereas blockade of α1-ARs inhibits dorsal raphe-derived serotonergic pathways. As further described in the accompanying paper, this profile of activity may contribute to the potential antipsychotic properties of S18327.
Although neuroleptics such as haloperidol moderate the positive symptoms of schizophrenia via blockade of mesolimbic D2receptors, they are not effective in all patients and poorly alleviate negative-cognitive symptoms (Kinon and Lieberman, 1996). Furthermore, concomitant blockade of striatal and hypophyseal populations of D2 sites provokes motor and endocrine extrapyramidal side effects, respectively (Cunningham-Owens, 1996). In the design of improved antipsychotic agents (Meltzer, 1995), many recent studies have focused on the following conceptual developments of this classic, “mesolimbic dopaminergic/D2hyperactivity” theory of schizophrenia (Brunello et al., 1995; Kinon and Lieberman, 1996). First, a reduced activity of dopaminergic pathways innervating frontal cortex (FCX) may contribute to largely intractable, negative and cognitive symptoms (Knable and Weinberger, 1997; Parellada et al., 1998). Second, D3 and D4 receptors, which are closely related to their D2 counterparts, may be involved in the pathogenesis and management of schizophrenia (Levant, 1997; Wilson et al., 1998). Third, in addition to dopaminergic transmission, a dysfunction of serotonergic and adrenergic networks may be implicated in psychotic states and their management (Baldessarini et al., 1992;Nutt, 1994; Roth and Meltzer, 1995).
Within this framework, the dibenzodiazepine clozapine has attracted considerable interest because it preferentially reinforces mesocortical versus subcortical dopaminergic transmission, possesses marked affinity for D4 receptors, and interacts with a diversity of serotonergic and adrenergic receptors (ARs; Brunello et al., 1995;Meltzer, 1995; Josselin et al., 1997; Millan et al., 1998b; Wilson et al., 1998). These distinctive actions of clozapine likely underlie its superior (“atypical”) clinical profile (Brunello et al., 1995). Clozapine is, thus, efficacious in a proportion of patients refractory to neuroleptics, exerts antipsychotic activity in the absence of extrapyramidal side effects, and may control negative-cognitive symptoms, although its efficacy against primary negative symptoms is still debated (Brunello et al., 1995). Apart from the potential importance of non-D2 dopaminergic receptors, several serotonergic and AR subtypes have been implicated in the actions of clozapine. For example, the pronounced antagonist actions of clozapine at 5-hydroxytryptamine (5-HT)2Areceptors may permit antipsychotic efficacy in the relative absence of extrapyramidal side effects (Roth and Meltzer, 1995; Schmidt et al., 1995).
Adrenergic mechanisms, via actions in the FCX and other corticolimbic loci, play an important role in the control of mood and cognition (Arnsten, 1997; Coull et al., 1997). Furthermore, a perturbation of adrenergic transmission has been related to positive crises, intensification of negative symptoms, and the risk of relapse after the discontinuation of treatment (Maes et al., 1993). Notably, clozapine displays antagonist properties at α1- and α2-ARs (Baldessarini et al., 1992; Nutt, 1994;Blake et al., 1998; Elman et al., 1999). In this respect, the following points deserve emphasis. First, psychostimulants, such as cocaine and amphetamine, increase extracellular levels of norepinephrine (NE); correspondingly, activation of α1-ARs in both mesolimbic structures and the FCX may be involved in their excitatory actions (Baldessarini et al., 1992; Darracq et al., 1998). Second, blockade of α1-ARs preferentially suppresses mesolimbic versus nigrostriatal dopaminergic transmission (Lane et al., 1990; Svensson et al., 1995). Third, antagonism of α1-ARs facilitates thalamic gating of sensory input to the cortex, a process compromised in psychotic patients (McCormick and Pape, 1995; Bakshi and Geyer, 1997). Fourth, blockade of inhibitory α2-AR heteroceptors on terminals of dopaminergic fibers in FCX enhances mesocortical DA release (Gobert et al., 1998). Fifth, combined treatment with the α2-AR antagonist idazoxan and the neuroleptic fluphenazine affords a “clozapine-like” profile of antipsychotic activity (Litman et al., 1996), and α2-AR antagonist actions are implicated in the functional actions of clozapine in humans (Nutt, 1994; Elman et al., 1999). Finally, α2-AR antagonist properties limit extrapyramidal side effects (Kalkman et al., 1998) and contribute to an improvement in mood (Nutt, 1994).
From these considerations, the potential interest of antipsychotics sharing the adrenergic receptorial profile of clozapine appears evident. However, the potent histaminic and muscarinic actions of clozapine are accompanied by autonomic/cardiovascular side effects, and its conversion to a “netrenium” metabolite can provoke agranulocytosis (Cunningham-Owens, 1996). In this light, the present (and accompanying) papers describe a novel, potential antipsychotic, S18327 (1-{2-[4-(6-Fluoro-1,2-benzisoxazol-3-yl)piperid-1-yl]ethyl}3-phenyl imidazolin-2-one), which is chemically distinct from clozapine. Thus, the latter is a tricyclic, dibenzodiazepine derivative, whereas S18327 (Fig. 1) is a benzoisoxazolepiperidine coupled to a phenylimidazoline via an ethylene linker. In contrast to clozapine, S18327 possesses only weak affinity for muscarinic and histaminic receptors. Nevertheless, S18327 mimics both the monoaminergic profile of clozapine and its distinctive pattern of antipsychotic versus extrapyramidal activity in vivo, actions to which its marked α1- and α2-AR antagonist properties make an important contribution.
Materials and Methods
Affinity of S18327 for Multiple Monoaminergic Receptors.
The affinities of S18327 for multiple, native and cloned, human dopaminergic, adrenergic, and serotonergic receptors were determined using standard protocols described in detail previously (Millan et al., 1998b) and summarized (Tables1-3). Nonlinear, regression analysis of isotherms was performed by use of the program Prism (GraphPAD Software, San Diego, CA), which yielded IC values. These were transformed into Kivalues according to the Cheng-Prusoff equation:Ki = IC50/(1 + L/Kd), where L corresponds to the radioligand concentration and Kd to its dissociation constant (Kenakin, 1997).
Antagonist Properties of S18327 at Specific Monoaminergic Receptors: [35S]GTPγS binding.
As described in detail previously (Newman-Tancredi et al., 1997, 1999; Millan et al., 1998b), and summarized (Table 4), the binding of [35S]guanosine-5′-O-(3-thio)triphosphate (GTPγS; 1000 Ci/mmol; NEN, Les Ulis, France) at human (h)D2, hD3, hD4, hα2A-AR, h5-HT1A, h5-HT1B, and h5-HT1D receptors was used as a measure of the efficacy with which S18327 interacts at these sites. S18327 was evaluated both alone (agonist activity) and, where appropriate, in the presence of a fixed concentration of dopamine (DA), NE, or 5-HT (antagonist activity). Agonist efficacy was expressed relative to that of a maximally effective concentration of DA, NE, or 5-HT (defined as 100%). For the antagonist studies, concentration-response curves of the blocking properties of S18327 were analyzed as described inNewman-Tancredi et al. (1999) to yieldKb values.
Antagonist Properties of S18327 at hα1A-ARs and h5-HT2A Receptors: Intracellular Ca2+Concentration ([Ca2+]i).
Chinese hamster ovary (CHO) cells expressing h5-HT2A receptors or hα1A-ARs were harvested and incubated for 60 min at room temperature with 5 μM Fura-2 acetoxymethyl ester. The cells were pelleted by centrifugation (1500g, 10 min, 10°C), resuspended in Hanks' medium, and incubated for 15 min at 37°C to allow hydrolysis of Fura-2 acetoxymethyl ester to Fura-2. The cells were then recentrifuged as above and resuspended in Hanks' medium with 5-HT or NE and/or S18327. After incubation for 12 min, assays were terminated by the addition of Triton X-100 (0.1%) and EGTA (10 mM). [Ca2+]ilevels were calculated from the fluorescence detected at a wavelength of 510 nm after excitation at wavelengths of 340 and 380 nm. The latter correspond to the absorbance peaks of Fura-2 in its Ca2+-complexed and noncomplexed forms, respectively.
Antagonist Properties of S18327 at hD3 Receptors: Mitogen-Activated Protein (MAP) Kinase.
CHO cells expressing hD3 receptors were grown in 6-well plates until 90% confluent. The cells were then washed with serum-free medium and incubated overnight in this medium. Drugs were diluted in serum-free medium and added to cells to obtain the appropriate, final concentration. Cells were preincubated for 5 min with antagonists and then stimulated with DA (100 nM) for 5 min. At the end of the incubation period, 0.25 ml/well of Laemmi's sample buffer containing 200 mM dithiotreitol was added. Whole-cell lysates were boiled for 3 min at 95°C. Then, 14 μl of cell extract was loaded onto 15-well, 10% polyacrylamide gels and “fully” activated MAP kinase was revealed using a monoclonal antibody specifically raised against the phosphorylated pp42mapk (ERK 2) and pp44mapk (ERK 1) forms on both threonine and tyrosine residues (NanoTools, Denzlingen, Germany), followed by enhanced chemiluminescence detection with horseradish peroxidase as a secondary antibody (Amersham, Les Ulis, France).
Studies In Vivo.
All studies in vivo used male Wistar rats weighing 220 to 240 g obtained from Iffa Credo (L'Arbresle, France). They were maintained in sawdust-lined cages with unlimited access to water and food. Laboratory humidity was 60 ± 5%, and temperature was 21 ± 1°C. Lights were on from 7:30 AM to 7:30 PM. All animals were adapted for at least 1 week to laboratory conditions before use.
Antagonist Properties of S18327 at Cerebral Populations of α2-ARs: Autoradiographic Evaluation of [35S]GTPγS Binding In Situ.
The potential activity of S18327 at cerebral populations of α2-ARs, visualized by [35S]GTPγS in the amygdala, was determined as follows. Slides with three or four brain sections were incubated for 60 min in 50 mM HEPES, pH 7.5, 150 mM NaCl, 0.2 mM EGTA, 0.2 mM dithiothreitol, 2.5 mM GDP, 10 mM MgCl2, and 0.05 nM [35S]GTPγS plus NE and/or S18327. After incubation, sections were washed with ice-cold buffer and then dipped into ice-cold, deionized distilled water. The slides were dessicated under air flow and placed in x-ray cassettes apposed to35S-sensitive film for 4.5 days at −20°C. Binding densities were measured by computerized densitometry using a Leica Q600 image analyzer (Leica, Paris, France) and14C-standard microscales. Data are expressed as a percentage increase in [35S]GTPγS binding induced by drug treatment relative to that observed under basal (non-drug-treated) conditions (defined as 0%). The amygdala was selected for this study inasmuch as this central nervous system region shows a robust [35S]GTPγS response to NE, which is fully blocked by selective α2-AR antagonists (Newman-Tancredi et al., in press).
Influence of S18327 on Hypothermia Elicited by Preferential D3 versus D2 Agonist, PD128,907.
Core (rectal) temperature was measured in lightly restrained rats by the use of a digital thermisoprobe as described previously (Millan et al., 1995). Basal core temperature was determined; 30 min later, vehicle or S18327 was administered, and then 30 min later, vehicle or PD128,907 (0.63 mg/kg s.c.) was injected. Again, 30 min later, core temperature was redetermined, and the difference from pretreatment values calculated. Data were analyzed by ANOVA, and the ID50 plus 95% confidence limit (CL) values were calculated.
Influence of S18327 on Rotation in Unilateral, Substantia Nigra-Lesioned Rats.
The procedure used has been described previously (Millan et al., 1998b). Separate groups of rats with a unilateral 6-hydroxydopamine (8 μg/4 μl) lesion of the left or right substantia nigra, pars compacta, were trained with either the D1 agonist SKF38393 (0.63 mg/kg s.c.) or the D2 agonist quinpirole (0.02 mg/kg s.c.). The induction of rotation was measured 45 to 60 min and 20 to 50 min after the administration of quinpirole and SKF38393, respectively. Rotation was evaluated automatically using a harness coupled to a Rotacount 8 (Columbus Instruments, Columbus, OH) apparatus. In alternate sessions, rats were treated 25 min before SKF38393 or quinpirole with either S18327 or vehicle. This permitted expression of the action of S18327 as a function of the mean of the preceding and subsequent vehicle sessions (defined as 100%). Data were analyzed by a paired Student's t test, and the ID50 (95% CL) was calculated.
Influence of S18327 on 5-HT2A Receptor-Mediated Secretion of Corticosterone (CS).
The antagonist properties of S18327 at 5-HT2A receptors in vivo were evaluated by its ability to inhibit the increase in plasma levels of CS elicited by the 5-HT2A agonist (±)-1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI). Vehicle or S18327 and vehicle or DOI (2.5 mg/kg s.c.) were administered, respectively, 60 and 30 min before the determination of CS levels, performed with a CS radioimmunoassay (RPA548; Amersham). Cross-reactivity with other steroids was <0.1%. Levels are expressed in ng/ml of plasma. Data were analyzed by two-way ANOVA followed by Dunnett's test.
Modulation by S18327 of Electrical Activity of Dopaminergic, Adrenergic, and Serotonergic Neurons in Anesthetized Rats.
Procedures described in detail previously (Aghajanian et al., 1977;Lejeune et al., 1997) were used for evaluation of the influence of S18327 on the electrical activity of dopaminergic, adrenergic, and serotonergic perikarya localized in the ventrotegmental area (VTA), locus ceruleus (LC), and dorsal raphe nucleus (DRN), respectively. Separate groups were used for each nucleus. Rats were anesthetized with chloral hydrate (400 mg/kg i.p.) and placed in a stereotaxic apparatus, after which a tungsten microelectrode was lowered into the VTA, LC, or DRN. Coordinates were as follows: VTA, AP = 5.5 from bregma, L = 0.7, and H = −7/8.5 from dura; LC, AP = −1.1 from 0, L = 1.2, and H = −5.5/6.5; and DRN, AP = −7/−8 from bregma, L = 0.0, and H = −5/−6.5. Specific populations of dopaminergic, adrenergic, and serotonergic neurons were identified according to their waveform as described previously (above citations) and baseline recording performed during 5 min. The influence of S18327 (dissolved in sterile water and injected i.v. in a volume of 0.5 ml/kg) on firing rate was evaluated after its administration in cumulative doses at intervals of 2 to 3 min. For examination of the antagonist properties of S18327 on dopaminergic neurons of the VTA, it was administered 3 min after a single dose of the dopaminergic agonist apomorphine (0.031 mg/kg i.v.). To examine mechanisms underlying the inhibitory influence of S18327 on serotonergic neurons of the DRN, a single dose of S18327 was injected (0.25 mg/kg i.v.) followed, 3 min later, by an injection of the 5-HT1A antagonist WAY100,635 (0.031 mg/kg i.v.). In an additional experiment, as detailed in Lejeune et al. (1994), a single dose of the α1-AR agonist cirazoline (0.005 mg/kg i.v.) was injected 3 min before a single dose of S18327 (0.25 mg/kg i.v.). All drug effects were determined during 60 s at the time of peak drug action. Data acquisition and analysis were performed with Spike 2 software (CED, Cambridge, England). Data are percent change from preinjection, basal values (defined as 0%). Data were analyzed by ANOVA followed by Newman-Keuls test.
Influence of S18327 on Cerebral Synthesis of DA, NE, and 5-HT.
The modulation by S18327 of cerebral turnover of DA, NE, and 5-HT was evaluated as described previously (Millan et al., 1998b). DA and 5-HT turnover was determined in the striatum (rich in DA, but not NE), and NE and 5-HT turnover was evaluated in the hippocampus (rich in NE, but not DA). The actions of S18327 were measured 60 min after its administration and 30 min after injection of the decarboxylase inhibitor NSD1015 (100 mg/kg s.c.). HPLC analysis followed by electrochemical detection was used for determination of tissue levels of l-dihydroxyphenylalanine (l-DOPA) and 5-hydroxytryptophan (5-HTP) as described previously (Millan et al., 1998b). Levels of l-DOPA and 5-HTP were expressed relative to those of vehicle values (defined as 0%). Data were analyzed by ANOVA followed by Dunnett's test.
Influence of S18327 on Cerebral DA Turnover.
As detailed previously (Millan et al., 1998b), the ratio of levels of the DA metabolite dihydroxyphenylalaninecarboxylic acid (DOPAC) to those of DA itself were determined in projection regions of the nigrostriatal pathway (striatum), the mesolimbic pathway (nucleus accumbens and olfactory tubercles), and the mesocortical pathway (FCX) 30 min after the administration of S18327. Levels of DOPAC and DA were determined by HPLC and electrochemical detection. DOPAC/DA ratios were expressed relative to those of vehicle values (defined as 0%). Data were analyzed by ANOVA followed by Dunnett's test.
Influence of S18327 on Dialysate Levels of DA, NE, and 5-HT in FCX, Nucleus Accumbens, Striatum, and Hippocampus of Freely Moving Rats.
The procedures used for quantification of DA, NE, and 5-HT levels in single dialysate samples of the FCX, accumbens and striatum of freely moving rats have been extensively documented elsewhere (Gobert et al., 1998; Millan et al., 1998b). Using essentially identical procedures, we also examined levels of NE and 5-HT in single dialysates of the hippocampus of freely moving rats with a guide cannula (CMA 11) implanted under pentobarbital anesthesia (60.0 mg/kg i.p.) at the coordinates AP = −3.6, L = ±1.2, and DV = −2.3. A cuprophane CMA/11 probe, 2 mm length, 0.24 mm diameter, was used for this structure. Experiments were performed 5 days after the placement of guide cannulas. Samples were taken every 20 min, and after three basal samples, S18327 or vehicle was injected s.c. and sampling continued for an additional 3 h. The influence of S18327 and vehicle was expressed relative to basal values (defined as 0%). In the interaction experiments performed in the FCX, WAY100,635 (0.16 mg/kg s.c.) or S18616 (0.00063 mg/kg s.c.) was injected 20 min before S18327 (1.25 mg/kg s.c.) or vehicle. The assay sensitivity was ≈0.1 to 0.2 pg/sample for DA, NE, and 5-HT in each case. Data were analyzed by ANOVA with sampling time as the repeated within-subject factor.
Drugs.
S18327 and other drugs were dissolved in sterile water, if required with the addition of a few drops of lactic acid. In this case, the pH was readjusted to as close to neutrality as possible (>5.0). Unless otherwise indicated, drugs were administered s.c. Cirazoline HCl was obtained from Synthélabo (Bagneux, France). Apomorphine HCl was obtained from Sigma (Chesnes, France). SKF38393 [(±)-1-phenyl-2,3,4,5-tetrahydro-(1H)-3-benzazepine-7,8-diol HCl], quinpirole HCl, DOI (1-[2,5-dimethoxy-4-iodophenyl]2-aminopropane HCl), and PD128,907 [(+)-(4aR,10bR)-3,4,4a,10b-tetrahydro-4-propyl-2H,5H-[1]benzopyrano-[4,3-b]1,4-oxazin-9-ol) HCl] were obtained from Research Biochemicals (Natick, MA). S18327, WAY100,635 [(N-{2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl}-N-(2-pyridinyl)cyclo-hexanecarboxamide fumarate], and S18616 ([7,8](2-chlorobenzo)-2-amino-1-aza-3-oxa-[4,5]spirodeca-1,7-diene HCl) were synthetized by Servier chemists (J.-L. Peglion and A. Cordi).
Results
Binding Profile of S18327 at Multiple Dopaminergic Receptors.
S18327 displayed affinities of about 70–90 nM for native and cloned rat and human D2 receptors (Table 1). Its affinity for cloned rat and human D3 receptors was similar. In contrast, the affinity of S18327 for hD4 receptors was 10-fold greater than that at hD2 sites. At native rat D1, cloned hD1, and cloned hD5 receptors, S18327 manifested affinities of ≈60 to 160 nM. S18327 had negligible affinity for DA reuptake sites (>1000 nM).
Binding Profile of S18327 at Multiple ARs.
S18327 showed high affinities (≈1.0 nM) for rat frontocortical α1-ARs as well as for native rat α1A- and α1B-ARs (Table2). Similarly, the affinities of S18327 for cloned hα1A-, hα1B-, and hα1D-ARs were high (≈1.0 nM). At cortical α2-ARs and cloned human hα2A-, hα2B-, and hα2C-ARs, the affinities of S18327 were ≈40 to 200 nM. The affinity of S18327 at β-ARs and NE reuptake sites was negligible (>1000 nM).
Binding Profile of S18327 at Multiple Serotonergic Receptors.
S18327 displayed affinities of 63 and 45 nM for native rat 5-HT1A and cloned h5-HT1Areceptors, respectively, yet low (>1000 nM) affinity for native and cloned 5-HT1B receptors (Table 3). Its affinity for h5-HT1D receptors was 374 nM. The affinity of S18327 for both native and cloned 5-HT2Areceptors was high (≈4.0 nM). On the other hand, it showed comparatively low affinities of ≈50 to 500 nM for cloned h5-HT2B receptors and for native 5-HT2C and cloned h5-HT2Csites. S18327 manifested negligible (≥1000 nM) affinity for 5-HT3, 5-HT4, 5-HT5A, and 5-HT6 receptors and 5-HT reuptake sites. However, the affinity of S18327 at 5-HT7 receptors was 15 nM.
Binding Profile of S18327 for Other, Nonmonoaminergic Receptors and Enzymes.
S18327 displayed affinity of 80 nM for native, rat ς1 receptors. Its affinity for native, rat ς2 sites was 530 nM. S18327 had negligible affinity (>10 μM) for all other receptor types examined: nicotinic, acetylcholine uptake, α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid, N-methyl-d-aspartate, glycine, γ-aminobutyric acidA, γ-aminobutyric acidB, benzodiazepine, adenosine1, adenosine2, imidazoline1, imidazoline2, μ-opioid, cannabinoid1, neuropeptide Y, neurokinin1, neurokinin2, Ca2+ channels, site 2 Na+channels, K+ channels, monoamine oxidase A, thromboxane2, estrogen, progesterone, testosterone, bradykinin2, endothelinA, endothelinB, nitric oxide synthase, phospholipase A2, phosphodiesterase, 5-lipoxygenase, acetylcholinesterase, and cyclooxygenase. The interaction of S18327 at histaminic and muscarinic sites is described in detail in the accompanying paper; however, it should be noted here that its affinity was 17.5 nM at native, histamine1 receptors and 600 nM at cloned, human muscarine1 receptors.
Antagonist Properties of S18327 at hD2, hD3, and hD4 Receptors: Blockade of DA-Stimulated [35S]GTPγS Binding and MAP Kinase.
As described previously (Newman-Tancredi et al., 1997, 1999), DA markedly enhanced [35S]GTPγS binding at hD2, hD3, and hD4 receptors with EC50values of 364.0 ± 42.0, 13.2 ± 2.0, and 102.0 ± 13.0 nM, respectively (Fig. 2, A–C). These actions of DA (defined as 100%) were concentration-dependently inhibited by S18327, which did not itself modify [35S]GTPγS binding.Kb values for S18327 at hD2, hD3, and hD4 sites were 34.0 ± 7.4 (Fig. 2A), 119.0 ± 28.0 (Fig. 2B), and 12.5 ± 3.2 nM (Fig. 2C), respectively. DA also markedly stimulated the activity of MAP kinase in CHO cells transfected with hD3 receptors (Fig.2D); this action was similarly abolished by S18327, which did not itself influence MAP kinase activity.
Antagonist Properties of S18327 at D3/D2Receptors In Vivo: Inhibition of Hypothermia Elicited by PD128,907.
The preferential D3 versus D2 agonist, PD128,907 (0.63 mg/kg s.c.), elicited hypothermia in rats (PD128,907, −2.0 ± 0.2°C; vehicle, +0.5 ± 0.1°C; P < .05. Its action was dose-dependently inhibited by S18327 with an ID50(95% CL) value of 6.1 (2.4–15.1) mg/kg s.c.
Antagonist Actions of S18327 at D1 and D2Receptors: Inhibition of SKF38393- and Quinpirole-Elicited Rotation in Rats.
The preferential agonists at D1 and D2 receptors, SKF38393, and quinpirole, respectively, each provoked contralateral rotation in rats sustaining unilateral lesions of the substantia nigra, pars compacta. S18327 dose-dependently and fully blocked their actions with ID50 (95% CL) values of 1.4 (0.4–3.6) and 0.5 (0.2–1.2) mg/kg s.c., respectively.
Antagonist Actions of S18327 at hα1A-ARs: Inhibition of Increases in [Ca2+]i Levels Elicited by NE.
At cloned hα1A-ARs, NE elicited a pronounced elevation in [Ca2+]i levels with an EC50 value of 95.0 ± 32.0 nM and a maximal effect of 1119 ± 37 nM (Fig. 3). Typical basal levels were 140 to 200 nM (Fig. 3A). In analogy to the prototypical α1-AR antagonist prazosin, which blocked the action of NE with a Kbvalue of 0.4 ± 0.1 nM, S18327 concentration-dependently abolished the action of NE with a Kb value of 3.6 ± 1.3 nM (Fig. 3B). Applied alone, at concentrations up to 10 μM, S18327 was inactive.
Antagonist Actions of S18327 at hα2A-ARs: Inhibition of Activation of [35S]GTPγS Binding by NE.
At cloned hα2A-ARs transfected into CHO cells, NE enhanced [35S]GTPγS binding with an EC50 value of 230.0 ± 26.0 nM and a maximal effect of 2.7 ± 0.4-fold stimulation relative to basal values (Fig. 4A). This effect was abolished by the prototypical α2-AR antagonist yohimbine with aKb value of 2.1 ± 0.5 nM. S18327 also concentration-dependently abolished the action of NE with aKb value of 162.0 ± 46.0 nM (Fig. 4B). Moreover, in the presence of fixed concentrations of S18327, the concentration-response curve for NE was progressively displaced in parallel to the right without any loss of maximal effect (Fig. 4C). The pA2 acquired from Schild analysis (Fig. 4D), 6.9, corresponded well to its pKi at these sites of 7.0. Furthermore, the slope of 1.00 ± 0.07 (r = 0.99) is consistent with competitive antagonist properties of S18327 at hα2-ARs. Alone, S18327 (10 μM) did not affect [35S]GTPγS binding.
Antagonist Actions of S18327 at hα2A-ARs: Inhibition of Activation of [35S]GTPγS Binding by NE at α2-ARs in Rat Amygdala In Situ.
In analogy to these studies of cloned hα2A-ARs, NE was shown to markedly activate [35S]GTPγS binding in rat amygdala (Fig. 5B). S18327 (10 μM), which did not itself modify [35S]GTPγS binding (Fig. 5C), abolished the action of NE (Fig. 5D).
Antagonist and “Inverse Agonist” Actions of S18327 at h5-HT1A, h5-HT1B, and h5-HT1DReceptors: Blockade of 5-HT-Stimulated [35S]GTPγS Binding.
5-HT markedly stimulated [35S]GTPγS binding at h5-HT1A, h5-HT1B, and h5-HT1D receptors with EC50values of 17.5 ± 2.0, 8.9 ± 1.4, and 1.3 ± 0.2 nM, respectively. At h5-HT1A sites, S18327 elicited a very mild stimulation, attaining 14.2 ± 0.8% of the maximal influence of 5-HT (100%) and with an EC50 value of 97.1 ± 30.1 nM. Furthermore, it exerted antagonist properties at these sites in concentration-dependently inhibiting the stimulation elicited by the 5-HT1A receptor agonist (±)-8-hydroxy-2-dipropylaminotetralin (100 nM), with aKb value of 129.0 ± 13.0 nM. At 5-HT1B and 5-HT1Dreceptors, S18327, applied alone, inhibited basal binding by 27.0 ± 6.1% and 51.2 ± 4.5%, respectively, suggesting “inverse agonist” properties. The inhibition was concentration dependent at both h5-HT1B and h5-HT1Dreceptors with IC50 values of 4074 ± 566 and 70 ± 18 nM, respectively. In view of the low affinity of S18327 at h5-HT1B and h5-HT1D sites and its inverse agonist actions, interaction studies with 5-HT were not undertaken.
Antagonist Properties of S18327 at h5-HT2A Receptors: Inhibition of Increases in [Ca2+]i Levels Elicited by 5-HT.
At cloned h5-HT2Areceptors, 5-HT evoked an increase in [Ca2+]i levels with an EC50 value of 124 ± 14 nM and a maximal effect of 728 ± 45 nM. Typical basal levels were 300 to 350 nM (Fig. 6A). This action of 5-HT was blocked by the prototypical antagonist mianserin with aKb value of 3.9 ± 1.9 nM. In analogy, S18327 concentration-dependently blocked the action of 5-HT with a Kb value of 1.4 ± 0.5 nM (Fig. 6B). S18327 was inactive alone at concentrations up to 10 μM.
Antagonist Properties of S18327 at Native 5-HT2AReceptors: Inhibition of DOI-Induced CS Secretion.
The 5-HT2A agonist DOI increased circulating levels of CS. This effect of DOI was dose-dependently and significantly inhibited by S18327, which, at the highest dose tested, tended itself (nonsignificantly) to elevate CS levels (Fig. 6C). [Notably, clozapine and certain other antipsychotics likewise elicit modest increases in CS levels, but the mechanisms underlying the intrinsic influence of S18327 and these agents on CS secretion remain to be clarified (Rivet et al., 1997, 1999)].
Influence of S18327 on Firing Rate of LC-Localized Adrenergic Neurons.
S18327 elicited a dose-dependent increase in the firing rate of adrenergic neurons localized in the LC with an AD50 (95% CL) value of 120 (70–200) μg/kg i.v. (Fig. 7A).
Influence of S18327 on Synthesis and Dialysate Levels of NE in Hippocampus and Accumbens.
In freely moving rats, at a dose (10.0 mg/kg s.c.) that increased hippocampal NE turnover, S18327 markedly increased extracellular levels of NE in the nucleus accumbens (Fig.7B). Similarly, a marked rise in dialysate levels of NE in the hippocampus was seen with S18327 (10.0 mg/kg; Fig. 7C). After suppression of decarboxylase activity with NSD1015, S18327 modestly elevated hippocampal levels of the NE precursor, l-DOPA (Fig. 7D).
Influence of S18327 on Firing Rate of VTA-Localized Dopaminergic Neurons.
The electrical activity of dopaminergic neurons in the VTA was markedly inhibited by the dopaminergic agonist, apomorphine (31 μg/kg i.v.; Fig. 8A). S18327 dose-dependently abolished this action of apomorphine with an ID50 (95% CL) value of 300 (100–900) μg/kg i.v. (Fig. 8A). Administered alone, S18327 did not alter firing rate (Fig. 8A). Furthermore, S18327 did not alter the firing pattern (regular or “bursts”; not shown).
Influence of S18327 on Cerebral DA Synthesis.
S18327, administered over a broad dose range and up to high doses, modestly enhanced ratios of the DA metabolite DOPAC to DA in the nucleus accumbens (Fig. 8B), the FCX (Fig. 8C), the olfactory tubercles (Fig.8D), and the striatum (Fig. 8E). Similarly, after inhibition of decarboxylase with NSD1015, S18327 increased levels of the DA precursorl-DOPA in the striatum (Fig. 8F).
Influence of S18327 on Firing Rate of DRN-Localized Serotonergic Neurons.
S18327 dose-dependently inhibited the firing rate of DRN-localized serotonergic neurons with an ID50(95% CL) value of 90 (60–160) μg/kg i.v. (Fig.9A). The selective 5-HT1A antagonist WAY100,635 at a dose (31 μg/kg i.v.) that abolishes the inhibitory influence of 5-HT1A agonists on DRN firing (Lejeune et al., 1997) failed to modify the inhibitory influence of S18327 (250 μg/kg i.v.; Fig. 9B). In distinction, pretreatment with the α1-AR agonist cirazoline, at a dose (5 μg/kg i.v.) that markedly reduces the inhibitory influence of prazosin and clozapine on DRN firing (Lejeune et al., 1994), significantly attenuated the action of S18327 (Fig. 9B).
Influence of S18327 on Cerebral 5-HT Synthesis and Dialysate Levels of 5-HT in Striatum, Hippocampus, and Nucleus Accumbens.
After administration of the decarboxylase inhibitor NSD1015, S18327 elicited a modest but significant decrease in levels of the 5-HT precursor 5-HTP in the striatum (innervated by the DRN) but not the dorsal hippocampus (innervated by the median raphe nucleus; Fig. 9, C and E, respectively). Similarly, in the striatum, but not the dorsal hippocampus, S18327 decreased dialysate levels of 5-HT (Fig. 9, D and F, respectively). Dialysate levels of 5-HT were not modified by S18327 (10.0 mg/kg) in nucleus accumbens (not shown).
Influence of S18327 on Dialysate Levels of DA in FCX Compared with Nucleus Accumbens and Striatum.
S18327 elicited a dose-dependent and pronounced elevation in dialysate levels of DA in the FCX of freely moving rats (Fig. 10A). In the accumbens, although S18327 elicited a significant increase in DA levels, its magnitude was significantly less marked than that in FCX (Fig. 10, A and B). Similarly, the facilitatory influence of S18327 on extracellular levels of DA in the striatum was significantly less pronounced than that in the FCX (Fig. 10, A and C).
Influence of S18327 on Levels of NE and 5-HT in FCX.
Levels of 5-HT in the FCX were unaffected by S18327 (Fig.11A), whereas S18327 dose-dependently increased levels of NE therein (Fig. 11B).
Influence of the α2-AR Agonist, S18616, Compared with the 5-HT1A Antagonist, WAY100,635, on Modulation by S18327 of FCX Levels of DA and NE.
The novel and potent α2-AR agonist, S18616 (0.00063 mg/kg s.c.;Millan, 1998), suppressed FCX levels of DA and NE and abolished their elevation by S18327 (Fig. 12, left). S18616 itself also decreased levels of 5-HT (Fig. 12, left). In contrast, the selective 5-HT1A receptor antagonist WAY100,635 (0.16 mg/kg s.c.) did not significantly modify the influence of S18327 (1.25 mg/kg s.c.) on FCX levels of DA and NE or 5-HT (Fig. 12, right). Administered alone, WAY100,635 did not itself modify FCX levels of these monoamines (Fig. 12, right).
Discussion
Antagonist Actions at Dopaminergic Receptors.
[35S]GTPγS binding, which provides a measure of G protein coupling (Missale et al., 1998; Newman-Tancredi et al., 1999), revealed the antagonist activity of S18327 at hD2 receptors. In vivo, antagonist properties of S18327 at postsynaptic D2 receptors were reflected by blockade of rotation elicited by quinpirole in unilateral substantia nigra-lesioned rats (see Results), whereas antagonist actions at presynaptic D2 sites were indicated by reversal of the inhibitory influence of apomorphine on VTA-localized dopaminergic neurons (Fig. 8; Lejeune et al., 1997;Gobert et al., 1998; Koeltzow et al., 1998). The latter action may also involve a (minor) population of colocalized D3autoreceptors (Gobert et al., 1995; Tepper et al., 1997). Furthermore, the attenuation by S18327 of hypothermia elicited by the preferential D3 agonist PD128,907 (see Results) may involve antagonism of (postsynaptic) D2 and D3 receptors (Millan et al., 1995; Xu et al., 1999). Indeed, in analogy to hD2 receptors, S18327 antagonized DA-stimulated [35S]GTPγS binding at hD3 receptors (Fig. 2; Newman-Tancredi et al., 1999). Moreover, S18327 also abolished DA activation of MAP kinase (Fig. 2), a downstream component of the transduction cascade initiated by hD3 receptor activation (Cussac et al., 1998). Although the importance of D2receptor blockade for antipsychotic agents is well established, the significance of D3 receptor antagonism remains unclear (Gurevich et al., 1997; Levant, 1997). Similarly, the potential role of D4 receptor blockade awaits clinical confirmation, and a clinical study with the selective D4 antagonist L745,870 found that it was ineffective in acutely ill, neuroleptic-responsive inpatients (Kramer et al., 1997). However, Gazi et al. (1998) recently questioned the antagonist properties of this ligand at human hD4receptors. Furthermore, on the basis of alterations in D4 receptor density in schizophrenic patients, it has been argued that preferential D4 versus D2 receptor blockade may afford an improved antipsychotic profile (Roth et al., 1995; Lahti et al., 1998; Millan et al., 1998c; Wilson et al., 1998). In fact, the “atypical” antipsychotic, clozapine, possesses higher affinity for hD4 versus hD2 sites (Millan et al., 1998c; Wilson et al., 1998), and this preference was more pronounced for S18327, which likewise behaved as a D4 antagonist (Fig. 2), as shown by [35S]GTPγS binding (Newman-Tancredi et al., 1997).
The comparable (modest) affinity of S18327 for hD1 and hD5 sites (Table 1) is consistent with their similar recognition profiles (Missale et al., 1998). Although the role of D5 receptors remains unclear, blockade by S18327 of SKF38393-induced rotation in substantia nigra-lesioned rats presumably reflects antagonist properties at D1 receptors. A dysequilibrium in D1 versus D2 receptor blockade may exacerbate extrapyramidal symptoms (Gerlach and Hansen, 1992). Notably, then, S18327 shows similar affinity at hD1 and hD2 sites and blocks SKF38393- and quinpirole-induced rotation at similar doses. Antagonist actions at limbic and/or cortical populations of D1 receptor may, furthermore, participate in antipsychotic actions (Martin et al., 1994; Josselin et al., 1997;Darracq et al., 1998), although selective D1receptor blockade may not itself be effective in acutely ill psychotic patients (De Beaurepaire et al., 1995; Karlsson et al., 1995).
Antagonist Action at α1- and α2-ARs.
S18327 displayed high affinity for various α1-AR subtypes and potently antagonized the NE-induced increase in [Ca2+]i levels at hα1A-ARs. Pronounced, central α1-AR antagonist actions may contribute to (improved) antipsychotic properties (see the introduction). However, peripheral α1-AR antagonism is associated with orthostatic hypotension. Indeed, in analogy to the α1-AR antagonist prazosin and to clozapine and other antipsychotics possessing pronounced α1-AR antagonist properties (see accompanying paper; Cunningham-Owens, 1996), S18327 reduces arterial blood pressure in rats (M.J.M. and M.B., unpublished observations). Such cardiovascular actions in humans can complicate drug use, but they generally are moderated by drug titration and subside on repetitive administration (Cunningham-Owens, 1996). We have systematically examined the actions of prazosin in the functional models described in the present and accompanying paper (M.J.M. and M.B., unpublished observations). With the exception of certain, specific responses known to involve central α1-AR blockade (discussed herein and in the accompanying paper), there is no evidence that a hypotension due to peripheral α1-AR receptor blockade can account for the influence of S18327 on dopaminergic and adrenergic transmission—not for its antipsychotic and other functional properties in vivo. The affinity of S18327 for rat/hα2A-ARs was comparable with that at rat/hD2 sites. A [35S]GTPγS protocol revealed antagonist properties of S18327 at hα2A-ARs (Fig. 4), and S18327 similarly blocked α2-AR-mediated [35S]GTPγS binding in rat amygdala, as visualized with the use of quantitative autoradiography (Fig. 5). Although it is currently unclear whether this (amygdala) population of α2-ARs is presynaptic or postsynaptic to adrenergic neurons, the antagonist actions of S18327 at α2A-autoreceptors (Fig. 7; Gobert et al., 1998) were expressed in its reinforcement of corticolimbic adrenergic transmission (see later).
Antagonist Actions at Serotonergic Receptors.
S18327 displayed modest affinity for 5-HT1A sites, at which it blocked 5-HT-induced [35S]GTPγS binding indicating, in contrast to clozapine (Millan et al., 1998b), predominantly antagonist properties. In contrast to 5-HT1A sites, S18327 itself suppressed basal (“constitutive”) activity at h5-HT1B and h5-HT1D receptors, suggesting inverse agonist properties. Although this effect is shared with clozapine (A.N.-T. and D.C., unpublished observations), its significance remains unclear. Indeed, like 5-HT1A autoreceptors, 5-HT1B and 5-HT1Dautoreceptors on serotonergic neurons are tonically silent, and neither antagonists nor inverse agonists at these sites markedly modify serotonergic transmission (Gobert et al., 1998). Similarly, although 5-HT1A and/or 5-HT1Breceptor agonists modulate frontocortical and/or mesolimbic dopaminergic transmission, antagonists (inverse agonists) at these receptors do not affect corticolimbic DA release in vivo (Boulenguez et al., 1998; Gobert et al., 1998).
S18327 was a potent antagonist of 5-HT2Areceptors as revealed by 1) blockade of h5-HT2Areceptor-mediated increases in [Ca2+]i levels and 2) inhibition of DOI-induced CS secretion (Fig. 6; Boess and Martin, 1994;Rivet et al., 1997, 1999). In analogy to clozapine (Millan et al. 1998b), these 5-HT2A receptor antagonist actions of S18327 underlie its activity in several models of potential antipsychotic activity (accompanying paper), and a pronounced ratio of 5-HT2A versus D2 antagonist properties may afford a favorable window of antipsychotic versus extrapyramidal properties (Maurel-Remy et al., 1995; Roth and Meltzer, 1995; Schmidt et al., 1995). The marked affinity of S18327 for 5-HT7 (Table 3) sites is notable inasmuch as clozapine is also a potent ligand at these sites, which are enriched in limbic tissues and implicated in the control of mood (Sleight et al., 1997). However, it has been questioned whether a preference for 5-HT7 versus D2 receptors is associated with an improved antipsychotic profile (Roth and Meltzer, 1995).
Modulation of Adrenergic Transmission.
Tonically active α2A-AR autoreceptors inhibit adrenergic transmission (Gobert et al., 1998), and antagonist properties of S18327 at α2A-autoreceptors were expressed in an excitation of adrenergic cell bodies in the LC, an acceleration of hippocampal NE turnover, and an elevation in dialysate levels of NE in hippocampus and nucleus accumbens (Fig. 7). Similar actions have been documented for selective α2-AR antagonists, in analogy to which (Gobert et al., 1998), S18327 markedly augmented extracellular levels of NE in FCX (Fig. 7). This observation is of note inasmuch as frontocortical adrenergic mechanisms control cognitive-attentional processes, which are perturbed in psychotic patients (Arnsten, 1997; Coull et al., 1997). Indeed, S18327 improves cognitive-attentional function in rats (accompanying article), although a potential implication of adrenergic mechanisms in these actions requires evaluation.
Modulation of Dopaminergic Transmission.
Notwithstanding its (modest) antagonist properties at D2/D3 autoreceptors, S18327 did not itself modify the firing rate of VTA-localized dopaminergic neurons. This lack of excitatory influence may reflect its marked α1-AR antagonist properties that stabilize VTA firing and suppress the excitatory influence of D2/D3 antagonists (Lane et al., 1990; Svensson et al., 1995). Although clozapine shares the α1-AR antagonist properties of S18327, yet slightly enhances VTA firing, this difference from S18327 may reflect its additional antagonist properties at 5-HT2Creceptors inhibitory to frontocortical and mesolimbic dopaminergic pathways (Di Matteo et al., 1998; Millan et al., 1998a; Gobert et al., in press). This lack of influence of S18327 on VTA-localized dopaminergic neurons was paralleled by its relatively mild influence on cerebral DA synthesis and release in the nucleus accumbens (and olfactory tubercles; Fig. 8), actions presumably reflecting antagonism of terminal-localized D2(D3) autoreceptors (Gobert et al., 1998; Koeltzow et al., 1998). The mild increase in extracellular levels of DA elicited by S18327 in the striatum (Fig. 8), reported in certain studies with clozapine (Gray and Connick, 1998), might offset the functional consequences of postsynaptic striatal D2 receptor blockade and thereby palliate extrapyramidal symptoms. In any case, this mild induction of striatal DA synthesis underpins the argument (accompanying article) that, in analogy to clozapine (Millan et al., 1998b), S18327 possesses a benign extrapyramidal profile.
Enhancement of Frontocortical Dopaminergic Transmission.
Likewise in analogy to clozapine (Millan et al., 1998b; Westerink et al., 1998; Kuroki et al., 1999), S18327 markedly increased extracellular levels of DA in FCX (Fig. 10). Several mechanisms other than D2/D3 autoreceptor blockade are potentially involved in this preferential enhancement of frontocortical dopaminergic transmission of DA. First, 5-HT1A agonists disinhibit frontocortical dopaminergic pathways (Lejeune et al., 1997; Gobert et al., 1998;Millan et al., 1998b). However, the influence of S18327 on FCX levels of DA was insensitive to WAY100,635 (Fig. 12). Furthermore, S18327 is an antagonist at h5-HT1A receptors. Second, 5-HT2C receptors are inhibitory to mesocortical dopaminergic pathways (Millan et al., 1998a), but S18327 has low affinity for these sites. Third, and most likely, S18327 may antagonize inhibitory, tonically active α2-AR heteroceptors on the terminals of frontocortical dopaminergic terminals (Gobert et al., 1998). Indeed, the selective α2-AR agonist S18616 (Millan, 1998) abolished the facilitatory influence of S18327 on FCX level of DA (Fig. 12). Fourth, S18327 elevates dialysate levels of NE via blockade of α2A-AR autoreceptors. Inasmuch as extracellular DA in the FCX is synaptically cleared by NE as well as DA uptake sites (Gresch et al., 1995), competition with NE at the former may indirectly contribute to increases in levels of DA. Regardless of the underlying mechanisms, this enhancement of FCX levels of DA by S18327 is of importance because a deficiency in mesocortical dopaminergic transmission is implicated in the “hypofrontality” that underlies the negative and cognitive features of schizophrenia (Knable and Weinberger, 1997; Parellada et al., 1998).
Modulation of Serotonergic Transmission.
The inhibition by S18327 of the electrical activity of DRN-localized serotonergic neurons was associated with a reduction in the release and turnover of 5-HT in striatum (Fig. 9), a structure innervated by the DRN (Rouquier et al., 1994). Activation of 5-HT1A autoreceptors could, in theory, be implicated, but suppression of DRN firing by S18327 was unaffected by WAY100,635. In contrast, it was attenuated by the α1-AR agonist cirazoline (Fig. 9), which similarly reduces the inhibitory actions of clozapine and prazosin on DRN firing (Lejeune et al., 1994). The functional significance of this α1-AR-mediated suppression of DRN-derived serotonergic transmission by S18327 is unclear, but it may be correlated with anxiolytic action and a low propensity to elicit extrapyramidal side effects (accompanying article).
Conclusions.
S18327 possesses a broad pattern of interactions at multiple monoaminergic receptors, with prominent antagonist properties at α1-ARs and 5-HT2A and D4 receptors and less pronounced, balanced antagonist activity at α2-ARs and D2 and D1 receptors. This profile may be distinguished from haloperidol yet closely resembles that of clozapine. Such comparisons are underpinned by functional paradigms discussed in the accompanying paper. Although the antagonist properties of S18327 at α2-ARs underlie its enhancement of cerebral adrenergic transmission and its facilitation of frontocortical dopaminergic transmission, blockade of α1-ARs contributes to its inhibition of DRN-localized serotonergic pathways.
Acknowledgments
We thank C. Langaney and K. Dutartre for secretarial assistance and C. Chaput, L. Cistarelli, V. Pasteau, and M. Touzard for technical assistance.
Footnotes
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Send reprint requests to: Dr. Mark J. Millan, Institut de Recherches Servier, Centre de Recherches de Croissy, Psychopharmacology Department, 125 Chemin de Ronde, 78290-Croissy-sur-Seine, France.
- Abbreviations:
- FCX
- frontal cortex
- 5-HT
- 5-hydroxytryptamine (serotonin)
- 5-HTP
- 5-hydroxytryptophan
- AR
- adrenergic receptor
- [Ca2+]i
- intracellular Ca2+concentration
- DOI
- (±)-1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane
- CL
- confidence limits
- CS
- corticosterone
- DA
- dopamine
- DOPAC
- dihydroxyphenylalaninecarboxylic acid
- DRN
- dorsal raphe nucleus
- h
- human
- LC
- locus ceruleus
- CHO
- Chinese hamster ovary
- l-DOPA
- l-dihydroxyphenylalanine
- MAP
- mitogen-activated protein
- NE
- norepinephrine
- [35S]GTPγS
- guanosine-5′-O-(3-thio)triphosphate
- VTA
- ventrotegmental area
- Received June 7, 1999.
- Accepted September 22, 1999.
- The American Society for Pharmacology and Experimental Therapeutics