Introduction

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Clinically, atypical neuroleptics are defined as drugs active in the treatment of schizophrenia but with a lesser propensity than conventional neuroleptics to induce extrapyramidal side effects. Furthermore, some neuroleptics, such as clozapine, are considered atypical because of their therapeutic efficacy in the treatment of schizophenic patients resistant to conventional neuroleptics.

Most neuroleptics display high affinity for the dopamine D₂ receptor subtype in direct relation to their therapeutic potency or plasma concentration at therapeutically active doses (Seeman, 1992). It has thus been suggested that the atypical characteristics of certain neuroleptics necessarily derive from additional pharmacological properties, such as antagonism toward 5-HT_2A or 5-HT_2C, muscarinic cholinergic or alpha-1 adrenergic receptors (Meltzer, 1991; Schmidt et al., 1995), or an interaction with  recognition sites (Ferris et al., 1991).

Molecular biological techniques have recently provided evidence that the dopamine D₁ receptor comprises a class of receptors, positively coupled to adenylate cyclase, that includes, besides the classical D₁ receptor (also termed D_1A), the D₅ or D_1B receptor (Sunahara et al., 1991) and possibly the mammalian equivalents of the D_1C (Sugamori et al., 1994) and D_1D (Demchyshyn et al., 1995) receptors. Similarly, the dopamine D₂ receptor family is now thought to include the D₂, D₃ (Sokoloff et al., 1990) and D₄ (Van Tol et al., 1991) subtypes. In particular, the D₃ and D₄ receptor subtypes have generated recent interest as potential therapeutic targets in the treatment of schizophrenia because of their preferential limbic localization (Sokoloff et al., 1990; Van Tol et al., 1991). The D₃ subtype is selectively recognized by several agonists and antagonists known for their selectivity for the dopamine autoreceptor (Sokoloff et al., 1992a, c), although it is present mainly on dopaminoceptive cells, in particular in the limbic system (Sokoloff et al., 1990, 1992c). Thus it has been suggested that stimulation of a postsynaptic dopamine D₃ receptor might be inhibitory to rat locomotor activity (Svensson et al., 1994; Waters et al., 1993b). The D₄ receptor represents the subtype of D₂ receptors for which clozapine shows the highest affinity (Van Tol et al., 1991).

Amisulpride (fig. 1) is a substituted benzamide derivative with dopamine receptor antagonist properties in vitro and in vivo (Chivers et al., 1988; Perrault et al., 1997; Scatton et al., 1994; Sokoloff et al., 1990). Although in clinical studies its efficacy in the treatment of schizophrenia has been clearly demonstrated, its most notable characteristic is its atypical profile. Thus, although amisulpride is efficacious in treating the positive symptoms of schizophrenia, it does so at doses (400-1200 mg/day) that, according to present experience, have only a low propensity to induce extrapyramidal side effects. In addition, amisulpride possesses disinhibitory effects at lower doses (50-300 mg/day) and is successfully used, at these doses, in treatment of the negative symptoms of schizophrenia and of dysthymia, a form of chronic depression (Boyer et al., 1990; Boyer et al., 1995; Delcker et al., 1990; Paillere Martinot et al., 1995; Saletu et al., 1994). These atypical therapeutic characteristics of amisulpride may be a reflection of its atypical neuropharmacological profile. Thus, amisulpride, similarly to classical neuroleptics, antagonizes the hyperactivity or stereotypies that result from the direct or indirect activation of postsynaptic dopaminergic receptors by (high doses of) dopaminomimetics such as apomorphine or amphetamine in the rat (Perrault et al., 1997). Nevertheless, amisulpride does not provoke catalepsy in the rat, which is characteristic of postsynaptic D₂ blockade, even at doses maximally effective in these experimental paradigms (Perrault et al., 1997). At lower doses, amisulpride potentiates apomorphine- and amphetamine-induced stereotyped behavior in mice, particularly favoring a transition from sniffing to gnawing behavior (Vasse et al., 1985). Furthermore, low doses of amisulpride inhibit hypokinesia induced by the administration of low doses of apomorphine, 7-OH-DPAT or quinpirole in rats (Perrault et al., 1997). Classical neuroleptics such as haloperidol are devoid of such prostereotypic effects and inhibit the diverse behavioral effects of dopamine agonists within the same dose range.

View larger version (9K): [in this window] [in a new window]   Fig. 1.   Amisulpride, (±)-4-amino-N-1((1-ethyl-2-pyrrolidinyl)methyl)-5-(ethylsulphonyl)-2-methoxybenzamide.

Various hypotheses have been put forward to explain the spectrum of animal behaviors provoked by dopaminomimetics and the dichotomy in their antagonism by atypical neuroleptics. These include the presence of behaviorally suppressant dopamine receptors in the nucleus accumbens (Waters et al., 1993b) or frontal cortex (Carter and Pycock, 1980) and the presence of inhibitory dopamine autoreceptors (Di Chiara et al., 1986). Thus, in addition to postsynaptic dopamine receptors, D₂-like autoreceptors have been amply demonstrated and are thought to be involved in the regulation of neuronal firing (Lejeune and Millan, 1995), dopamine release (Suaud-Chagny et al., 1991) and tyrosine hydroxylase activation (Claustre et al., 1985; Walters and Roth, 1976). Their molecular identity, however, remains subject to debate. Alternatively, drug activity at nondopaminergic receptors, such as alpha-1 adrenoceptors (Wiszniowska-Szafraniec et al., 1983), 5-HT receptor subtypes (Carter and Pycock, 1981; Mogilnicka et al., 1977), beta adrenergic receptors (Costall et al., 1978), muscarinic cholinergic receptors (Christensen et al., 1976) and histamine H₁ receptors (Dadkar et al., 1976), may modulate the expression of dopamine receptor activation or blockade.

In view of the established atypical profile of amisulpride in the treatment of schizophrenia and dysthymia, and in view of current hypotheses with respect to dopamine receptor subtypes and the possible contribution of nondopaminergic effects to neuroleptic atypicity, we have sought to define its neurochemical characteristics and mechanism of action, largely in comparison with that of the typical neuroleptic haloperidol. To this end, we studied the interaction of amisulpride with a variety of drug receptor and recognition sites in vitro, which demonstrated that amisulpride is highly specific and selective for both the dopamine D₂ and D₃ receptor subtypes. Its antagonist character was demonstrated in vitro by its inhibition of dopamine D₃ receptor-mediated mitogenesis and by its pre- and postsynaptic effects on neurotransmitter release and was demonstrated in vivo by its effects on indices of dopamine synthesis, release and metabolism and on ACh levels. These studies show that amisulpride displays a degree of limbic selectivity and a preferential effect, at low doses, on D₂/D₃ autoreceptors as compared with the classical neuroleptic, haloperidol.

The behavioral characteristics of amisulpride are described in a companion paper (Perrault et al., 1997).

Some of the data presented here have previously been reported in abstract form (Scatton et al., 1994; Schoemaker et al., 1995).

	Materials and Methods

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Animals. Unless otherwise indicated, adult male Sprague-Dawley rats (OFA or COBS, Iffa Credo, St. Germain sur l'arbresle, France, or Charles River, St. Aubin-les-Elbeus, France), Dunkin Hartley guinea pigs (Iffa Credo) and Fauves de Bourgogne rabbits (ESD, Romans, France) were used.

Materials and drugs. Pig choroid plexi and bovine caudate nuclei were obtained from CollectOrgane (Paris, France). Cell lines expressing the human dopamine receptor subtypes or membrane preparations thereof were obtained from: D₁ and D₅ (New England Nuclear, Boston, MA), D_2S and D₃ (Dr. J.-C. Schwartz, INSERM, Paris, France), D_4.4 (Receptor Biology, Baltimore, MD).

Radioligand binding studies in vitro. Studies of radioligand binding to various receptor and/or drug recognition sites were performed essentially as described by the authors indicated: the human dopamine D₂ receptor expressed in CHO cells (1.0 nM [¹²⁵I]iodosulpride; Sokoloff et al., 1992a), the human dopamine D₃ receptor expressed in CHO cells (0.2 nM [¹²⁵I]iodosulpride; Sokoloff et al., 1992a), the human dopamine D_4.4 receptor expressed in CHO cells (0.5 nM [³H]spiperone; Van Tol et al., 1991), the human dopamine D₁ receptor in Sf9 cells (1.6 nM [³H]SCH23390; Sunahara et al., 1991), the human dopamine D₅ receptor expressed in Sf9 cells (1.8 nM [³H]SCH23390; Sunahara et al., 1991), the dopamine D₁ receptor in rat striatum (0.3 nM [³H]SCH23390; Billard et al., 1984), the dopamine D₂ receptor in rat striatum (0.3 nM [³H]spiperone; Briley and Langer, 1978), the dopamine D₃ receptor in bovine caudate nucleus (0.8 nM [³H]7-OH-DPAT in the presence of 0.2 µM eliprodil; Schoemaker, 1993), the plasma membrane dopamine transporter from the rat striatum (1 nM [³H]GBR12935; Janowsky et al., 1986), the alpha-1A adrenoceptor in the rat salivary gland (0.2 nM [³H]prazosin; Faure et al., 1994), the alpha-1B adrenoceptor in the rat liver (0.1 nM [³H]prazosin; Faure et al., 1994), the alpha-2 adrenoceptor in the rat cerebral cortex (5 nM [³H]clonidine; Pimoule and Langer, 1982), the beta adrenoceptor in the rat cerebral cortex (2 nM [³H]dihydroalprenolol; Mogilnicka et al., 1980), the plasma membrane noradrenaline transporter from the rat vas deferens (2 nM [³H]desipramine; Raisman et al., 1982), the 5-HT_1A receptor in rat hippocampus (1 nM [³H]8-OH-DPAT in the presence of 3 µM paroxetine; Sanger and Schoemaker, 1992; Schoemaker and Langer, 1986), the 5-HT_1B receptor in rat striatum (5 nM [³H]5-HT in the presence of 100 nM 8-OH-DPAT and 100 nM mesulergine; Herrick-Davis et al., 1988; Heuring and Peroutka, 1987; Peroutka, 1986), the 5-HT_1D receptor in bovine caudate nucleus (2 nM [³H]5-HT in the presence of 100 nM 8-OH-DPAT and 100 nM mesulergine; Herrick-Davis et al., 1988; Heuring and Peroutka, 1987), the 5-HT_2A receptor in rat cerebral cortex (0.4 nM [³H]spiperone; Hicks et al., 1984), the 5-HT_2C receptor in the pig choroid plexus (1 nM [³H]mesulergine; Pazos et al., 1984; Yagaloff and Hartig, 1986), the 5-HT₃ receptor in the rat cerebral cortex (0.8-0.9 nM [³H]quipazine in the presence of 10 nM ketanserin and 100 nM paroxetine; Angel et al., 1993), the 5-HT₄ receptor in the guinea pig striatum (0.1 nM [³H]GR 113808; Grossman et al., 1993), the muscarinic cholinergic receptor in the rat cortex (0.3 nM [³H]quinuclidinyl benzylate; Yamamura and Snyder, 1974), the histamine H₁ receptor in the guinea pig cerebellum (1 nM [³H]pyrilamine; Tran et al., 1978), the GABA_A receptor in rat brain (4 nM [³H]GABA; Langer et al., 1985), the ₁/benzodiazepine receptor in the rat cerebellum (1 nM [³H]flumazenil; Arbilla et al., 1985), the ₂/benzodiazepine receptor in the rat spinal cord (1 nM [³H]flumazenil; Arbilla et al., 1985), the ₅/benzodiazepine receptor in the rat hippocampus (1 nM [³H]flumazenil in the presence of 5 µM zolpidem; Tan and Schoemaker, 1993), the picrotoxin site of the GABA_A receptor channel in the rat cerebral cortex (2 nM [³H]TBOB; Van Rijn et al., 1990), the GABA_B receptor in rat brain (10 nM [³H]GABA; Hill and Bowery, 1981), the presynaptic GABA transporter in the rat cerebral cortex (5 µM [³H]nipecotic acid; Vargas et al., 1993), the strychnine-sensitive glycine receptor in the rat spinal cord (2 nM [³H]strychnine; Young and Snyder, 1974; Young and Snyder, 1973), the glutamate recognition site of the NMDA receptor in whole rat brain (1.5 nM [³H]CGP 39653; Sills et al., 1991), the strychnine-insensitive glycine recognition site of the NMDA receptor in the whole rat brain (14-17 nM [³H]glycine; Kishimoto et al., 1981; Zukin et al., 1974), the polyamine-sensitive modulatory site of the NMDA receptor complex in the rat cerebral cortex (1 nM [³H]ifenprodil in the presence of 3 µM GBR 12909; Schoemaker et al., 1991), the NMDA receptor-ion channel site in well-washed membranes from the whole rat brain (2 nM [³H]MK801; Reynolds et al., 1987; Wong et al., 1988), the quisqualate/AMPA subtype of glutamate receptors in the rat brain (4 nM [³H]AMPA; Honoré et al., 1982; Honoré and Drejer, 1988), the kainate subtype of glutamate receptors in the rat brain (2 nM [³H]kainate; Simon et al., 1976), the adenosine A₁ receptor in rat hippocampal membranes (3 nM ()N⁶-R[G-³H]-phenylisopropyladenosine; Schwabe and Trost, 1980), the adenosine A₂ receptor in rat striatal membranes (4 nM 5-N-ethylcarboxamido[8(n)-³H]-adenosine in the presence of 50 nM N⁶-cyclopentyladenosine; Bruns et al., 1986), the angiotensin II AT₁ receptor in rabbit adrenocortical membranes (2 nM [³H]angiotensin II; Glossmann et al., 1974), the angiotensin II AT₂ receptor in membranes from the rat adrenal medulla (0.1 nM [¹²⁵I]angiotensin II; Glossmann et al., 1974), the L-type Ca⁺⁺ channel in rat cerebral cortex (0.1 nM [5-methyl-³H]-nitrendipine; Schoemaker and Langer, 1989), the Na⁺ channel in rat cerebral cortex (2 nM [benzoyl-2,5-³H]-batrachotoxinin A 20--benzoate in the presence of 1 µM tetrodotoxin and scorpion toxin; Pauwels et al., 1986),  recognition sites in rat cortex (0.5 nM [³H]ifenprodil; Schoemaker et al., 1991), the p-site (peripheral benzodiazepine receptor) in the rat kidney (0.5 nM [³H]Ro 5-4864; Schoemaker et al., 1983), the imidazoline I₂ recognition site in the whole rat brain (1 nM [³H]idazoxan in the presence of 10 µM ()adrenaline; Le Rouzic et al., 1995).

Dopamine D₃ receptor-stimulated mitogenic activity in vitro. The functional effects of amisulpride at the dopamine D₃ receptor subtype were assessed as described by Pilon et al. (1994) and Sautel et al. (1995). Briefly, the mitogenic response elicited in NG108-15 neuroblastoma-glioma cells stably transfected with human dopamine D₃ receptor cDNA by the addition of 10 nM quinpirole in the presence of 1 µM forskolin was quantified by the incorporation of [³H]thymidine. Antagonism of quinpirole-induced mitogenesis was measured in the presence of increasing (0.1-100 nM) concentrations of amisulpride.

Radioligand binding studies in vivo. In vivo [³H]raclopride binding to rat brain structures was measured according to a previously described procedure (Köhler et al., 1985). The radioligand (9 µCi/200 µl) was injected into the tail vein of male Sprague-Dawley rats 45 min before sacrifice. Test drug or vehicle was administered in a final volume of 1 ml 75 min before [³H]raclopride. Brain structures (striatum, limbic system and cerebellum) were dissected by hand, and the incorporated radioactivity was measured after overnight digestion in 0.5 ml of Soluene. The radioactivity incorporated into the cerebellum was taken as nonspecific binding.

Neurotransmitter release in vitro. The modulation of electrically evoked [³H]dopamine and [¹⁴C]ACh release from slices (1 × 1 × 0.3 mm) of the rat striatum and nucleus accumbens was studied essentially as described by Arbilla and Langer (1984). Briefly, slices were incubated with 0.1 µM [³H]dopamine and 19 µM [¹⁴C]choline for 30 min at 37°C in Krebs buffer (mM: NaCl, 118; KCl, 4.7; CaCl₂, 1.3; MgCl₂, 1.2; NaH₂PO₄, 1.0; NaHCO₃, 25.0; glucose, 11.0; EDTA, 0.04, equilibrated with 5% CO₂/95% O₂). Slices were then washed, transferred to superfusion chambers (1 slice/chamber) and superfused with Krebs buffer supplemented with 10 µM hemicholinium-3 for 60 min at a flow rate of 0.7 or 1 ml/min. After this period, fractions (7 and 6 min, respectively) were collected until the end of the experiment. Slices were initially stimulated electrically (2 min, 3 Hz, 2 ms, 16 mA) in the absence of amisulpride or 7-OH-DPAT, 74 min after the beginning of the superfusion, and were stimulated 40 min thereafter in their presence. Amisulpride or 7-OH-DPAT was added to the superfusion buffer 20 min before the second stimulation period. When the interaction between amisulpride and 7-OH-DPAT was studied, amisulpride was present in the superfusion buffer as of 20 min before the first stimulation period. In each case, the stimulation-evoked [³H] and [¹⁴C] overflow (S₁ and S₂, respectively) was calculated with respect to the spontaneous outflow (sp₁ and sp₂, respectively) in the fractions immediately before stimulation. Although both the neurotransmitter and derived metabolites probably contribute to stimulation-evoked [³H] and [¹⁴C] overflow (see, for instance, Parker and Cubeddu, 1985), the terms [³H]dopamine and [¹⁴C]ACh are used for convenience.

Microdialysis studies. Adult male Sprague-Dawley rats were anesthetized with chloral hydrate (400 mg/kg), and guide cannulae were stereotaxically implanted onto the dura mater above the striatum and the nucleus accumbens (+0.7 mm anterior, +3 mm lateral and +1.7 mm anterior, +1.0 mm lateral from bregma, respectively) (Paxinos and Watson, 1982). At least 5 days after surgery, microdialysis probes (Carnegy Medicine) 250 µm in diameter with an exposed membrane length of 4 (striatum) and 2 (nucleus accumbens) mm were positioned within the guide cannulae (vertical coordinates: 7 mm and 8 mm, respectively, from the dura surface) and perfused with artificial CSF (mM: NaCl, 147; KCl, 4; CaCl₂, 1.2; MgCl₂, 1.0) using a CMA/100 pump (BAS) at a flow rate of 2 µl/min. Twenty-minute dialysate fractions were collected in a valve loop and analyzed online using HPLC with electrochemical detection. The average concentration of five stable fractions immediately preceding drug administration was defined as the 100% control value.

Electrically evoked dopamine release in vivo. Presynaptic autoregulation of electrically evoked dopamine release in the rat olfactory tubercle was studied in vivo as described by Suaud-Chagny et al. (1991). Briefly, dopamine release was monitored every 1 s with electrochemically treated 12-µm-diameter carbon-fiber electrodes (1.6 mm lateral, 1.7 mm anterior to bregma and 9.0 mm below the cortical surface) combined with differential pulse amperometry with the final potential adjusted to +85 mV. Bipolar stimulating electrodes were implanted in the ascending dopaminergic pathway at 1.2 mm lateral and 4.0 mm posterior to bregma, at a depth adjusted for each experiment so that the stimulation-evoked dopamine response was maximal. Electrical stimulation was by twenty 1-s trains of six square wave form current pulses (pulse interval 70 ms, 250 µA, 0.5 ms) and was repeated every 10 min.

Monoamines and metabolites in vivo. Two hours after drug administration, animals were sacrificed by decapitation, and the brain structures (frontal cortex, striatum and limbic system) were dissected on ice. Samples were then weighed, homogenized in 20 volumes of 0.1 M HClO₄ and centrifuged at 10,000 × g for 10 min. Dopamine and DOPAC levels were measured in the supernatant by HPLC with electrochemical detection as described previously (Sermerdjian-Rouquier et al., 1981). ACh and choline levels were measured in aliquots of a buffered supernatant (0.5 M Tris-citrate, pH 4) by HPLC with electrochemical detection using platinum electrodes (Asano et al., 1986).

Measurement of dopamine and 5-HT synthesis rates. The rate of dopamine and 5-HT synthesis was estimated by measuring the accumulation of dopa and 5-HTP, respectively, 30 min after the administration of NSD-1015 (100 mg/kg) (Claustre et al., 1985). The presynaptic modulation of dopamine synthesis (Walters and Roth, 1976) was studied by co-administration of -hydroxy-butyric acid (750 mg/kg) 45 min before sacrifice. Amisulpride and 7-OH-DPAT were given 75 and 15 min, respectively, before -hydroxy-butyric acid. Dopa and 5-HTP levels were measured by HPLC with electrochemical detection according to Sermerdjian-Rouquier et al. (1981).

Statistical analyses. Statistical differences between groups were assessed using the ANOVA test, followed by Dunnett's or Duncan's test where appropriate. Simple statistical comparisons were done using a two-tailed Student's t test. ED₅₀ values for drug inhibition of in vivo radioligand binding in the striatum and limbic system were tested for statistical identity using the partial F test.

	Results

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In vitro radioligand binding studies. Amisulpride shows high affinity toward the cloned and stably transfected human dopamine D₂ (K_i = 2.8 ± 0.4 nM; n = 7) and D₃ (K_i = 3.2 ± 0.3 nM; n = 7) receptor subtypes labeled with [¹²⁵I]iodosulpiride and fails to recognize the D₁, D₄ and D₅ receptor subtypes. Both haloperidol and clozapine recognize all human dopamine receptor subtypes (table 1).

In vivo radioligand binding studies. [³H]Raclopride selectively recognizes D₂ and D₃ receptors in vitro and may be used as a label for D₂-like receptors in vivo (Köhler et al., 1985). Amisulpride displaces [³H]raclopride binding in vivo (fig. 2) with an ED₅₀ value of 17.3 ± 1.86 mg/kg in the rat limbic system but is significantly (P < .05) less active in displacing binding in the striatum (ED₅₀ = 43.6 ± 6.2 mg/kg; table 3). Like amisulpride, sulpiride is more potent in displacing [³H]raclopride binding in the limbic system, whereas remoxipride and haloperidol show similar activity in both brain regions.

View larger version (17K): [in this window] [in a new window]   Fig. 2.   Displacement by amisulpride of [3H]raclopride binding to the rat striatum and limbic system in vivo. Data represent specific [3H]raclopride binding, the cerebellum being used as a measure of nonspecific binding, and are the mean with S.E.M. of results obtained from six rats per group. Test drugs were administered 75 min before the radioligand.

Dopamine D₃ receptor-mediated mitogenesis in vitro. Quinpirole (10 nM) stimulated [³H]thymidine incorporation, a measure of mitogenesis, in NG108-15 cells stably transfected with the human D₃ dopamine receptor, to 171.9 ± 5.0% of controls (100.0 ± 4.6%; n = 3; P < .01). Although amisulpride (100 nM) failed to stimulate [³H]thymidine incorporation (95.8 ± 7.1%; n = 3; P > .05 vs. control), it inhibited quinpirole-elicited [³H]thymidine incorporation with an IC₅₀ value of 22 ± 3 nM (n = 3).

Neurotransmitter release studies in vitro. Dopamine D₂/D₃ receptor antagonism can be demonstrated in vitro by studying the modulation of neurotransmitter release. Thus the electrically stimulated [³H]dopamine release from slices of the striatum or the nucleus accumbens is subject to inhibitory modulation through a D₂-like (D₂/D₃) terminal autoreceptor and is inhibited by the D₂/D₃ agonist 7-OH-DPAT with EC₅₀ values of 13.9 ± 0.7 and 4.7 ± 0.6 nM, respectively. At maximally effective concentrations, 7-OH-DPAT reduces the evoked release to 3.7% and 13.8% of controls in the striatum and nucleus accumbens, respectively.

View larger version (19K): [in this window] [in a new window]   Fig. 3.   Effect of amisulpride on electrically evoked [3H]dopamine release from the rat striatum and nucleus accumbens in vitro. The effect of amisulpride on electrically evoked [3H]dopamine release was studied using slices prepared from the rat striatum and nucleus accumbens. Slices were initially stimulated electrically (2 min, 3 Hz, 16 mA) in the absence of amisulpride or 7-OH-DPAT and, 40 min thereafter, in their presence. Amisulpride or 7-OH-DPAT (30 nM for the striatum, 10 nM for the nucleus accumbens) was added to the superfusion buffer 20 min before the second stimulation period. When the interaction between amisulpride and 7-OH-DPAT was studied, amisulpride was present in the superfusion buffer as of 20 min before the first stimulation period. In each case, the stimulation-evoked [3H]-overflow (S1 and S2, respectively) was calculated with respect to the spontaneous outflow in the fractions immediately before stimulation (sp1 and sp2, respectively). Data are shown as the mean and S.E.M. of 3 to 11 observations. * and : P < .05 compared with control and 7-OH-DPAT, respectively.

View larger version (24K): [in this window] [in a new window]   Fig. 4.   Effect of amisulpride on electrically evoked [3H]dopamine and [14C]ACh release from the rat striatum in vitro. The effects of amisulpride () on the 7-OH-DPAT (30 nM)-induced () inhibition of electrically evoked [3H]dopamine and [14C]ACh release were studied using slices prepared from the rat striatum. Data are shown as a percentage of the corresponding S2/S1 control () ratio ([3H]dopamine: 0.92 ± 0.02, n = 32; [14C]ACh: 0.90 ± 0.01, n = 37).

Effects on dopaminergic neurotransmission in vivo. Tissue dopamine and DOPAC levels. The effects of amisulpride, haloperidol, sulpiride and clozapine on regional dopamine and DOPAC tissue levels are shown in table 4. The doses of the latter three reference compounds were chosen as those previously shown to have maximal effects on dopamine turnover in the striatum (Scatton et al., 1977).

View this table: [in this window] [in a new window]   TABLE 6 Effects of amisulpride on dopamine synthesis in the rat brain after blockade of impulse flow Rats received NSD-1015 (100 mg/kg) 90 min after the administration of amisulpride or its vehicle control and were sacrificed 30 min thereafter. -Hydroxy-butyric acid was administered 45 min before sacrifice. Because the data for amisulpride derive from multiple experiments, they are expressed as a percentage of the control value for each experiment. Mean control dopa levels varied from 1038 ± 80 to 1451 ± 86 and from 592 ± 19 to 710 ± 33 ng/g wet tissue weight for the striatum and limbic system, respectively, in the case of amisulpride. In the case of haloperidol, control dopa levels were 1038 ± 80 and 619 ± 30 ng/g wet tissue weight for the striatum and limbic system, respectively. Shown are the mean and S.E.M. (n = 3-7/group).

View this table: [in this window] [in a new window]   TABLE 7 Effects of 7-OH-DPAT and amisulpride on dopamine synthesis in the rat brain after blockade of impulse flow Rats received NSD-1015 (100 mg/kg) 90 min after the administration of amisulpride or its vehicle control and were sacrificed 30 min thereafter. -Hydroxy-butyric acid was administered 45 min before sacrifice. Data are expressed as the mean and S.E.M. (n = 3-7/group). Dopa levels calculated as a percentage of the corresponding control groups are shown in parentheses.

View larger version (18K): [in this window] [in a new window]   Fig. 5.   Effects of amisulpride on extracellular dopamine and DOPAC levels, as measured by microdialysis, in the rat striatum and nucleus accumbens. Extracellular dopamine and DOPAC levels in the rat striatum (n = 4) and nucleus accumbens (n = 6) were measured after the administration of amisulpride (10 mg/kg i.p.), using the microdialysis technique. Dialysates were collected as 20-min fractions and analyzed by online HPLC coupled to electrochemical detection. Data are shown as a percentage of basal outflow, taken over five stable fractions immediately before drug administration, and represent the mean and S.E.M.

View larger version (18K): [in this window] [in a new window]   Fig. 6.   Effects of haloperidol on extracellular dopamine and DOPAC levels, as measured by microdialysis, in the rat striatum and nucleus accumbens. Extracellular dopamine and DOPAC levels in the rat striatum (n = 6) and nucleus accumbens (n = 6) were measured after the administration of haloperidol (0.03 mg/kg i.p.), using the microdialysis technique. Dialysates were collected as 20-min fractions and analyzed by on-line HPLC coupled to electrochemical detection. Data are shown as a percentage of basal outflow, taken over five stable fractions immediately before drug administration, and represent the mean and S.E.M.

View larger version (24K): [in this window] [in a new window]   Fig. 7.   Effects of amisulpride on the amplitude of evoked dopamine release in the rat olfactory tubercle in vivo. The effect of amisulpride was studied on dopamine release evoked by electrical stimulations of the ascending dopaminergic pathway, which were repeated every 10 min. Dopamine release was measured using an electrochemically treated carbon-fiber electrode implanted in the olfactory tubercle and was recorded every 1 s. Data are expressed as a percentage of the mean evoked dopamine release during the three stimulations immediately before s.c. drug administration, and represent the mean and S.E.M. of four (amisulpride) or six (vehicle) rats per group.

View larger version (12K): [in this window] [in a new window]   Fig. 8.   Dose-response curve of the effect of amisulpride on stimulation-evoked dopamine release in the rat olfactory tubercle in vivo. The effect of amisulpride, administered at the dose of 0.5, 1, 3, 5, 10 and 15 mg/kg s.c., on dopamine release was studied as described in fig. 7. Shown is the effect, expressed as a percentage of the mean evoked dopamine release in vehicle-treated controls, of amisulpride 90 min after drug treatment (mean ± S.E.M.; n = 4/group).

Effects on striatal choline and ACh levels. Amisulpride, sulpiride, haloperidol and clozapine did not affect striatal choline levels at the doses tested. Amisulpride decreased striatal ACh levels significantly at 30 and 100 mg/kg (87.5% and 56.3% of control levels, respectively). Haloperidol (2 mg/kg) and sulpiride (100 mg/kg) decreased striatal ACh levels to a similar extent (68% and 62% of controls, respectively), whereas clozapine (10 mg/kg) was without significant effect (table 8).

	Discussion

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The present studies indicate that amisulpride, in comparison with other neuroleptics both typical and atypical, possesses complex neurochemical characteristics and can largely be defined as a specific dopamine receptor antagonist with a high degree of selectivity for the D₂ and D₃ receptor subtypes that it recognizes in vitro with similar affinity. In vivo, these properties translate into a degree of selectivity for the presynaptic dopamine autoreceptors that control the dopaminergic system and for its limbic projections.

Selectivity for the dopaminergic system. The present data confirm and extend previous reports (Chivers et al., 1988) in showing that amisulpride is highly selective for the D₂ receptor family. Within this class of receptors, it recognizes the cloned human and native D₂ (rat) and D₃ (bovine) subtypes with similar and low-nanomolar (K_i = 3 nM) affinity in vitro. In this respect, amisulpride is similar to drugs such as sulpiride, AJ76 and UH232 that show a D₂/D₃ affinity ratio close to unity and is different from the classical neuroleptics, such as haloperidol, which generally show higher affinity for the D₂ than for the D₃ receptor in vitro (Sokoloff et al., 1990, 1992a). Previous studies have shown that amisulpride recognizes the two isoforms of the human D₂ receptor (D_2S and D_2L) with equal affinity (Malmberg et al., 1993).

Presynaptic dopamine autoreceptor selectivity. As stated in the Introduction, amisulpride, like most neuroleptics, antagonizes the hyperactivity and stereotypies that result from the activation of postsynaptic dopaminergic receptors by (high doses of) direct- or indirect-acting dopaminomimetics such as apomorphine or amphetamine (Perrault et al., 1997). However, a characteristic feature of amisulpride is that at lower doses, it potentiates apomorphine- and amphetamine-induced stereotyped behavior (Vasse et al., 1985) and inhibits hypokinesia induced by the administration of low (presynaptic) doses of apomorphine, 7-OH-DPAT or quinpirole (Perrault et al., 1997). The aim of the present study was to examine whether the effects of low and high doses of amisulpride could be neurochemically discriminated at the level of dopaminergic neurotransmission.

Selectivity for limbic D₂/D₃ receptors. It has been suggested that the atypical character of certain neuroleptics arises from a greater effect on the limbic system, which is thought to be involved in emotional and cognitive processes, than on the extrapyramidal system, which is intimately related to the control of motor behavior (Bischoff, 1992; Meltzer, 1993