Introduction

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A dysregulation of activity at 5-HT_1A receptors is implicated in the pathophysiology of anxiety, depression and other psychiatric disorders (see Broekkamp et al., 1995; Coplan et al., 1995; Maes and Meltzer, 1995; Roth and Meltzer, 1995). Further, chemically diverse 5-HT_1A receptor ligands exert anxiolytic and AD properties both in experimental models and in man; for example, the azaspirone, buspirone, the benzodioxane, flesinoxan, the naphthylpiperazine, S 14671 and the aminotetralin, 8-OH-DPAT (Fabre, 1990; Lucki et al., 1994; Schreiber et al., 1994; see Broekkamp et al., 1995; Coplan et al., 1995; Maes and Meltzer, 1995; Roth and Meltzer, 1995). There is also evidence that 5-HT_1A receptors are involved in the expression of the AD properties of fluoxetine and other SSRIs (Detke et al., 1995; Gambarana et al., 1995). Actions at both pre- and postsynaptic 5-HT_1A receptors appear to play a role in these therapeutic actions of 5-HT_1A receptor ligands, although their relative contribution remains to be clarified (see Coplan et al., 1995; Lucki et al., 1994; Maes and Meltzer, 1995; Roth and Meltzer, 1995; Thiébot and Martin, 1991).

In light of the above, 5-HT_1A receptor ligands offer an attractive target for the development of novel treatments for anxiety, depression and related psychiatric disorders. We have recently discovered a chemically novel benzodioxopiperazine, S 15535 (under clinical development for the treatment of anxiety), which displays exceptional selectivity for 5-HT_1A receptors in rodents (Millan et al., 1994b). This is of importance because, with the exception of the phenylpiperazine antagonist, WAY 100,635 (Pike et al., 1995), virtually all 5-HT_1A ligands (or their metabolites) exhibit marked antagonist properties at other receptor types in vivo; in particular, DA D₂ receptors (e.g., binospirone, zalospirone and buspirone), ₁-AR receptors (flesinoxan and NAN-190), ₂-AR receptors (NAN-190, buspirone and ipsapirone) and -AR receptors (()-pindolol and other alkylarylamines) (Millan et al., 1994b; Van Wijngaarden et al., 1990). Further, S 15535 behaves in vivo as an agonist at 5-HT_1A autoreceptors and as an antagonist (or weak partial agonist) at postsynaptic 5-HT_1A receptors (Newman-Tancredi et al., 1996a; Peglion et al., 1995). This pattern of activity of S 15535 may be compared with high-efficacy agonists at pre- and postsynaptic 5-HT_1A receptors, such as 8-OH-DPAT, S 14671 and flesinoxan (Millan et al., 1992); ligands acting as full/partial agonists at pre/postsynaptic 5-HT_1A receptors, such as buspirone, ipsapirone and zalospirone (Glennon and Dukat, 1995; Gobert et al., 1995a; Millan et al., 1992; Thiébot and Martin, 1991) and ligands which behave as antagonists at both pre- and postsynaptic 5-HT_1A receptors, such as WAY 100,635, ()-alprenolol and ()-tertatolol (Millan et al., 1994b; Pike et al., 1995).

The combination of marked selectivity for 5-HT_1A receptors and differential efficacy at pre (agonist)- and post (antagonist)-synaptic populations appears to impart anxiolytic properties of S 15535 in the relative absence of the disruptive motor, endocrine and amnesic actions provoked by the activation of postsynaptic sites (Millan et al., 1997, accompanying paper). The purpose of the studies reported in the present paper was to evaluate the activity of S 15535 in several models of potential AD properties. Complementary AD activity would be of particular interest for an anxiolytic agent in the light of the increasing diagnosis of co-morbid anxious and depressive states (Coplan et al., 1995).

S 15535 possesses high affinity at rat 5-HT_1A versus DA D₂ and _2D-AR receptors (Millan et al., 1994b) and, herein, we examine its interaction with recombinant h5-HT_1A, hD₂ and hD₃ and h_2A-AR (the human homolog of rat _2D) receptors. Serotonin_1A, D₂/D₃ and _2A-AR sites are present as autoreceptors on serotoninergic, dopaminergic and adrenergic pathways, respectively (Gobert et al., 1995a, b; Millan et al., 1995). In this respect, we focused on a possible modulation by S 15535 of the activity of dopaminergic and adrenergic projections to the FCX. This is of interest inasmuch as a deficit in prefrontal cortical dopaminergic transmission may be a common feature of depressive states (Carlson et al., 1993; Karoum et al., 1994; Zacharko and Anisman, 1991). Indeed, mechanistically diverse AD drugs, such as SSRIs, tricyclics and the 5-HT_1A partial agonist, buspirone, enhance dialysate levels of DA in rat FCX (Arborelius et al., 1993; Jordan et al., 1994; Tanda et al., 1994; Wong et al., 1995). Moreover, adrenergic pathways innervating the cortex play a facilitatory role in cognito-attentional processes, which may be compromised in depressive states, and an enhancement in their activity may be associated with AD effects (Aston-Jones et al., 1991b; Broekkamp et al., 1995). Thus, we examined the influence of S 15535 on dialysate levels of DA, NAD and 5-HT in the FCX of freely moving rats and compared these actions with those in two further structures in which dopaminergic pathways are implicated in the control of mood and cognition: the nucleus accumbens and the striatum (Willner, 1995). As a second and related approach, we examined the influence of long-term administration of S 15535 on the density of -AR and 5-HT_2A receptors in the cortex. There is evidence implicating 5-HT_2A receptors in the control of mood, and their activation may be related to the pathophysiology of depressive, as well as anxiolytic and psychotic, states (see Coplan et al., 1995; Maes and Meltzer, 1995; Roth and Meltzer, 1995). As a third model of potential AD activity, the LH procedure was selected inasmuch as the escape deficits evoked by inescapable shock are considered to reflect a state of decreased motivation, resignation and psychomotor retardation, key elements of depressive conditions in man (Thiébot and Martin, 1991). Further, this model is responsive to both SSRIs, such as fluoxetine, and to agonists at 5-HT_1A receptors, such as 8-OH-DPAT and buspirone (Martin et al., 1990a, b, 1991). Finally, we also examined the influence of S 15535 on the behavior of rats in a forced swim paradigm (Porsolt et al., 1979). This model displays certain similarities to the LH paradigm and is sensitive to high-efficacy 5-HT_1A agonists, although it is poorly responsive to low-efficacy 5-HT_1A agonists and SSRIs (Lucki et al., 1994; Schreiber and De Vry, 1993; Schreiber et al., 1994).

	Methods

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Animals. Male Wistar rats weighing 160 to 220 g (Iffa-Credo, L'Arbresle, France) were used for all studies except the LH model (male Wistar rats of 250-300 g, R. Janvier, Le Genest-St-Isle, France). They were allowed at least 5 days adaptation to laboratory standard conditions before experimentation and were housed in sawdust-lined cages with free access to rat chow and water. There was a 12 h/12 h light-dark cycle (lights on from 7:30 A.M. to 7:30 P.M.). Temperature was 21 ± 1°C and humidity was 60 ± 5%.

Binding to cloned, h5-HT_1A, hD₂, hD₃ and h_2A-AR receptors. The procedures used have been described in detail previously and are summarized in table 1. Inhibitory Concentration₅₀ (IC₅₀) were calculated by nonlinear regression analysis and the K_i was computed according to K_i = IC₅₀/(1 + L/K_d), where L is the concentration of radiolabeled ligand and K_d is its apparent dissociation constant. S 15535 and fluoxetine were dissolved in incubation buffer and diluted as appropriate.

Release of 5-HT, DA and NAD in FCX, nucleus accumbens and striatum of conscious rats. Surgical procedures were performed under pentobarbital anesthesia (60 mg/kg i.p.). As described previously (Gobert et al., 1995b), rats were mounted in a David Kopf stereotaxic frame and guide cannulas (intracerebral guide, Carnegie Medicine, Stockholm, Sweden) implanted either in the striatum and contralateral nucleus accumbens or in the frontal/cingulate (frontal) cortex with coordinates, according to the atlas of Paxinos and Watson (1986), as follows: nucleus accumbens (CMA/12, AP: +1.6, L: ±1.4, DV: 5.7); striatum (CMA/12, AP: +0.5, L: ±2.8, DV: 3) and FCX (CMA/11, AP: +2.2, L: ±0.6, DV: 0.2). After surgery, rats were housed singly and adapted to handling and experimental conditions for 1 week before dialysis. On the day of dialysis, rats were kept in their own cages throughout the experiment. Polycarbonate CMA/12 probes (striatum: 3 mm length, 0.5 mm O.D.; nucleus accumbens: 2 mm length, 0.5 mm O.D.) or a cuprophan CMA/11 probe (FCX, 4 mm length, 0.24 mm O.D.) were slowly lowered into position and secured to the guides. Probes were perfused at 1 µl/min with a phosphate-buffered solution of 147.2 mM NaCl, 4 mM KCl, 2.3 mM CaCl₂ (pH 7.3). Two hours after probe insertion, 20-min dialysate samples were collected on ice for 4 h. Three basal samples were collected before drug administration. Twenty-microliter dialysate samples were diluted with 20 µl of mobile phase (75 mM NaH₂PO₄, 20 µM EDTA, 1 mM sodium decanesulfonate, 17.5% methanol, 0.01% triethylamine, pH 5.70). Thirty-three-microliter samples were analyzed immediately by high-performance liquid chromatography with a reverse-phase column (hypersil C18, 150 × 4.6 mm, 5 µm, Thermo Separation Products, Les Ulis, France) maintained at 45°C for separation and a coulometric detector (ESA 5014, Coulochem II, Bedford, MA) used for quantification. The first electrode of the detector was set at 90 mV (reduction) and the second at +280 mV (oxidation). The mobile phase was delivered at a flow rate of 2 ml/min. The sensitivity of the assay for DA, NAD and 5-HT was 0.55 fmol/sample. At the end of the experiment, rats were decapitated and the brains removed and frozen in cold isopentane. Serial frozen sections were cut at 100-µm intervals and stained with cresyl violet, and the dialysis probe placements verified. For evaluation of drug effects, fluoxetine (10.0 mg/kg s.c.), S 15535 (0.08, 0.63 and 5.0 mg/kg s.c.) or corresponding pH-matched vehicles were injected and dialysates collected every 20 min. For antagonist studies, WAY 100,635 (0.16 mg/kg s.c.) or corresponding pH-matched vehicle were injected before S 15535 (0.63 mg/kg s.c.) or corresponding pH-matched vehicle and their effects on the action of S 15535 were analyzed 60 to 120 min later (during which time levels were stable). Their own, intrinsic effects were evaluated 20 min after injection, before the second administration. DA, 5-HT and NAD levels were expressed as a function of mean basal preinjection values (= 100%). ANOVA with sampling time as the repeated within-subject factor was performed. In an additional experiment on separate rats, to further verify the selective influence of S 15535 on serotoninergic as compared with dopaminergic pathways (see "Results"), we examined its influence on extracellular dialysate levels of their respective metabolites, 5-HIAA and DOPAC, in the nucleus accumbens and striatum of freely moving rats. The techniques used and the expression of 5-HIAA and DOPAC levels were as discussed above, except that the high-performance liquid chromatography elution conditions were as follows: mobile phase (100 mM KH₂PO₄, 20 µM EDTA, 0.5 mM sodium octyldodecyl sulfate, 5% methanol, pH 3.42) delivered at a low rate of 1 ml/min onto the reverse phase column (as above) maintained at 30°C. ANOVA (with the factor time as a repeated measure) was performed.

Modulation of -AR and 5-HT_2A receptor density in the rat. The density of -AR receptors and 5-HT_2A receptors in cerebral cortex was determined by saturation binding with the selective radioligands, [³H]CGP-12177 and [³H]ketanserin. Desipramine (15.0 mg/kg s.c.), fluoxetine (10.0 mg/kg s.c.) or S 15535 methanesulfonate salt (10.0 mg/kg s.c.) were administered to the animals, once a day, for 14 days. Twenty-four hours after the last injection, the animals were sacrificed by decapitation, the brains rapidly removed and the cerebral cortex dissected and stored at 80°C. Each rat was analyzed individually. Homogenates of whole cortex for -AR receptors and of FCX for 5-HT_2A receptors were prepared with a Polytron (Kinematica, Basle, CH) as described previously (Newman-Tancredi et al., 1996b). They were incubated with eight different concentrations of [³H]CGP-12177 or [³H]ketanserin. Nonspecific binding was determined in the presence of 10 µM alprenolol or 10 µM spiperone for -AR and 5-HT_2A sites, respectively. Incubation conditions and buffers are shown in table 1. At the end of the incubation periods, all samples were filtered rapidly through Whatman GF/B filter paper with a Brandel cell harvester. The filters were washed twice with 5 ml of cold buffer, and the radioactivity retained on the filters was determined by liquid scintillation counting on a Packard Tri-Carb 1500 counter. Saturation binding isotherms were analyzed by Scatchard analysis for the determination of maximum radioligand binding (B_max) and dissociation constant (K_d) with the program LUNDON1 (Lundon Software Inc. Cleveland, OH). For all treatments, the saturation isotherms fitted best to a model which assumed the presence of a single class of binding sites (runs test value, P > .05; decreases in residual variance when the data were modeled to two sites were not significant in the F-test). Data were analyzed by ANOVA followed by Dunnett's test.

Learned helplessness test in the rat. The procedure was as described previously (Martin et al., 1990a, b). On the afternoon of day 1 of testing, rats were removed from their home cages and placed in a small (20 × 10 × 10 cm) box equipped with a stainless steel grid floor (LH group). They were exposed to a 15-s inescapable scrambled shock (0.8 mA) every minute for 1 h, delivered by a constant current shocker (Letica LE 10026, Barcelona, Spain). An additional control group of rats was similarly placed in the boxes, but did not receive shocks. Two days later, both groups of rats were submitted to an avoidance task (session 1) with use of a shuttle-box (Letica LE 916, Barcelona, Spain). This box (50 cm × 23 cm × 25 cm) comprised two equally sized compartments divided by a stainless steel partition equipped with a gate (7 × 7 cm). A photoelectric system permitted passage of shock through the stainless steel grid floor to be terminated as soon as the animal went through the gate into the other compartment. Each animal was placed singly into the shuttle-box. After a 5-min adaptation period, 30 stimulus-shock trials were presented over 15 min (2/min). During the first 3 s of each trial, a light signal appeared, which was followed by a shock (0.8 mA) of 3-s maximal duration and then by a 24-s resting period. For each trial, the rat could terminate the shock by escaping into the other compartment. This procedure was repeated on days 4 (session 2) and 5 (session 3). In each of these three sessions, the numbers of escape failures and of intertrial crossings were recorded. An escape failure was counted for every trial on which the rat received the shock by not crossing into the other compartment. Intertrial crossings were the number of transfers of the rat through the gate from one compartment to the other during each 24-s resting period of the session. Fluoxetine (i.p.), S 15535 (p.o.) or vehicle (i.p. or p.o.) were administered to the stressed LH animals throughout the 5-day period. On day 1, the drug was given as a single bolus 6 h after exposure to the inescapable shocks. On days 2 to 5, S 15535 was administered b.i.d., with half the dose 45 min before the avoidance control session (12:00 P.M.) and the other half at 6:00 P.M. Fluoxetine was given once daily (after the avoidance session). Vehicle was administered to nonstressed animals. Data are expressed as escape failures and intertrial crossings recorded during avoidance sessions 1, 2 and 3. On each of the 3 days of avoidance testing, differences between nonstressed (control) and stressed (LH) rats were evaluated by Student's t test (P  .05). Data obtained with drugs in LH rats were analyzed by ANOVA followed by Dunnett's test (P  .05).

Forced swim test in the rat. As described previously (Schreiber et al., 1994), on the first day of the experiment, rats were immersed for 15 min in glass cylinders (20 cm diameter × 30 cm height) filled to a depth of 15 cm with water maintained at 25°C. The following day, rats were again placed in the water and the duration of immobility was recorded over 5 min. Immobility was defined as the rat remaining almost motionless floating in the water in an upright position while making only small movements to keep its head above the surface. Fluoxetine (s.c.), S 15535 (s.c.), desipramine (i.p.) or vehicle (i.p, s.c.) were administered 30 min before the test on day 2. The duration of immobility (seconds) in drug-treated animals was compared with that of vehicle-treated animals with ANOVA followed by Dunnett's test (P  .05).

Drugs. All drug doses are in terms of the base. Drugs were dissolved in sterile water, plus a few drops of lactic acid if necessary, and pH adjusted to as close to neutrality (75.0) as possible. Drugs were injected s.c. and i.p. as a solution (injection volume = 1 ml/kg, unless specified) or administered orally by gavage as a suspension in water with a few drops of Tween 80 (injection volume, 10 ml/kg). S 15535 base was used except in the chronic studies of -AR and 5-HT_2A receptor modulation where the methanesulfonate salt of S 15535 was preferred because of its greater solubility. Other drug structures, salts and sources were as follows: desipramine HCl, ()-alprenolol tartrate, haloperidol and yohimbine HCl (Sigma, Chesnes, France); 8-OH-DPAT HBr, spiperone HCl, ()-propanolol HCl and WB 4101 HCl (Research Biochemicals, Natick, MA); (+)-7-OH-DPAT HCl (J. Besselièvre, INSERM, Paris, France); raclopride tartrate (Astra Lab., Sodertälje, Sweden); fluoxetine HCl, S 15535, WAY 100,635 3HCl and MDL 100,907 (C.C. Malen, J.-L. Peglion and G. Lavielle, Servier, Paris, France), [³H]ketanserin (62 Ci/mmol, NEN-Dupont S.A., Les Ulis, France); [¹²⁵I] iodosulpride (2,000 Ci/mmol, Amersham S.A., Les Ulis, France); [³H]8-OH-DPAT (228 Ci/mmol, Amersham S.A., Les Ulis, France); [³H]MK 912 (76.5 Ci/mmol, NEN-Dupont S.A., Les Ulis, France); and [³H]CGP 12177 (53 Ci/mmol, Amersham S.A., Les Ulis, France). Drug structures are as follows: 8-OH-DPAT (8-hydroxy-2-(di-n-propylamino)-tetralin); 7-OH-DPAT (7-hydroxy-2-(di-n-propylamino)-tetralin), WB 4101 {2-(2,6-dimethoxyphenoxyethyl)-amino-methyl-1,4-benzodioxane}, WAY 100,635 (N-{2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl}-N(2-pyridinyl) cyclohexanecarboxamide; MDL 100,907, [R(+)--(2,3-dimethoxyphenyl)-1-[2-(4-fluorophenylethyl)]-4-piperidine-methanol]; [³H]CGP-12177 ()-4-(3-t-butylamino-2-hydroxypropoxy)-5,7-[³H]benzimidazol-2-one); S 15535, 4-(benzodioxan-5-yl)1-(indan-2-yl)piperazine.

	Results

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Interaction of S 15535 with cloned, h5-HT_1A, hD₂/hD₃ receptors and h_2A-AR receptors. Like the prototypical 5-HT_1A receptor agonist, 8-OH-DPAT, S 15535 displayed high affinity for cloned, human 5-HT_1A receptors stably transfected into CHO cells (fig. 1, table 2). It monophasically inhibited the binding of [³H]8-OH-DPAT, consistent with an interaction at a single population of 5-HT_1A receptors. In distinction to 5-HT_1A receptors, S 15535 possessed only low affinity for cloned, DA hD₂ and hD₃ receptors, as compared with the prototypical antagonist, haloperidol (fig. 1, table 1). Likewise, the affinity of S 15535 at cloned, h_2A-AR receptors was low as compared with the prototypical antagonist, yohimbine (fig. 1, table 2). Fluoxetine possessed low affinity (K_i > 1,000 nM) at all sites examined (not shown).

View larger version (K): [in this window] [in a new window]   Fig. 1.   Binding profile of S 15535 at cloned, h5-HT1A as compared with dopamine hD2, dopamine hD3 and h2A-adrenergic receptors. Data are representative of at least three experiments per receptor, each of which was performed in triplicate. See table 2 for further analysis.

View this table: [in this window] [in a new window]   TABLE 2 Interaction of S 15535 at cloned, human h5-HT1A as compared with DA hD2, DA hD3 and 2A-AR receptors

Modulation of dialysate levels of 5-HT, DA and NAD in FCX by S 15335 as compared with fluoxetine. As shown in figures 2 and 3, treatment with vehicle elicited a mild, nonsignifcant and variable elevation in dialysis levels of 5-HT and DA in dialysates of the FCX of freely moving rats. In contrast, those of NAD were consistently and significantly elevated upon vehicle injection (figs. 2 and 3). This modest influence of vehicle on NAD, which was significant only in the sample immediately after injection, presumably reflects the "arousal" of manipulation (see "Discussion"). In contrast to vehicle, S 15535 elicited a marked, dose-dependent and sustained diminution in levels of 5-HT which remained stable throughout the duration (3 h) of sampling. Similarly, S 15535 provoked a marked, dose-dependent and rapid increase in dialysate levels of both DA and NAD; these increases remained significant throughout the sampling, although the magnitude of the increase tended to decline during the final hour of measurement. The influence of fluoxetine on 5-HT levels was opposite to that of S 15535 in that it elicited a rapid increase which was most marked within the first hour of sampling (fig. 4). However, fluoxetine mimicked the action of S 15535 on DA and NAD levels inasmuch as their levels rapidly increased after its injection. Nevertheless, in contrast to S 15535, the fluoxetine-evoked increase in DA levels was no longer significant, with the exception of a solitary time point, by the second hour of sampling.

View larger version (K): [in this window] [in a new window]   Fig. 2.   Influence of s.c. injection of vehicle as compared with S 15535 upon dialysate levels of serotonin, dopamine and noradrenaline in the frontal cortex of freely moving rats. Data are means ± S.E.M.; n = 5-11 per value. They are expressed as a percentage of basal, preinjection values which were defined as 100%. These were 0.80 ± 0.07, 1.48 ± 0.13 and 1.46 ± 0.21 pg/20 min dialysate for 5-HT, DA and NAD, respectively, for vehicle-treated animals (n = 13). For comparison of individual values with the vehicle-treated group, ANOVA with drug as between factor and time as within factor, was performed. 5-HT: influence of dose, F(3,22) = 12.5, P < .001; influence of time, F(8,240) = 5.1, P < .001 and interaction, F(24,176) = 1.1, P > .05. DA: influence of dose, F(3,31) = 23.3, P < .001; influence of time, F(8,240) = 19.7, P < .001 and interaction, F(24,248) = 2.2, P < .01. NAD: Influence of dose, F(3,30) = 6.9, P < .01; influence of time, F(8,240) = 32.2, P < .001 and interaction, F(24,240) = 2.8, P < .001. For vehicle-treated rats, comparison with basal preinjection values: 5-HT: F(9,45) = 2.0, P > .05. DA: F(9,90) = 2.0, P > .05. NAD: F(9,72) = 3.3, P < .01.

View larger version (K): [in this window] [in a new window]   Fig. 3.   Influence of s.c. injection of fluoxetine on dialysate levels of serotonin, dopamine and noradrenaline in the frontal cortex of freely moving rats. Data are means ± S.E.M.; n = 7 per value. They are expressed as a percentage of basal, preinjection values, which were defined as 100%. These are as given in figure 2 in which the corresponding values for vehicle-treated animals are presented. For comparison of individual values with vehicle-treated group, ANOVA with drug as between factor and time as within factor, was performed. 5-HT: influence of fluoxetine, F(1,12) = 15.6, P < .01; influence of time, F(8,96) = 1.3, P > .05 and interaction, F(8,96) = 1.6, P > .05. DA: influence of fluoxetine, F(1,19) = 28.7, P < .001; influence of time, F(8,152) = 7.3, P < .001 and interaction, F(8,152) = 2.6, P < .05. NAD: influence of fluoxetine, F(1,16) = 17.9, P < .001; influence of time, F(8,128) = 1.5, P > .05 and interaction, F(8,128) = 1.6, P > .05.

View larger version (K): [in this window] [in a new window]   Fig. 4.   Influence of WAY 100,635 (0.16 mg/kg s.c.) on the modulation of cortical dialysate levels of serotonin (left panels), dopamine (middle panels) and noradrenaline (right panels) elicited by S 15535 (0.63 mg/kg s.c.) in the frontal cortex of freely moving rats. Data are means ± S.E.M.; n = 8-9 per value. They are expressed as a percentage of basal, preinjection values which were defined as 100%. These were 0.58 ± 0.06, 1.26 ± 0.08 and 0.87 ± 0.09 pg/20 min dialysate for 5-HT, DA and NAD, respectively, for vehicle-treated animals. ANOVA with drug as between factor and time as within factor, was performed over 60 to 120 min. (upper panel) 5-HT: influence of WAY 100,635, F(1,16) = 30.7, P < .001, influence of time; F(3,48) = 1.0, P > .05 and interaction, F(3,48) = 0.9, P > .05. DA: influence of WAY 100,635, F(1,15) = 28.4, P < .001, influence of time; F(3,45) = 4.4, P < .01 and interaction, F(3,45) = 2.2, P > .05. NAD: Influence of WAY 100,635, F(1,16) = 19.3, P < .001, influence of time; F(3,48) = 8.2, P < .001 and interaction, F(3,48) = 0.9, P > .05. (lower panel) 5-HT: influence of WAY 100,635, F(1,16) = 0.3, P > .05, influence of time; F(3,48) = 1.4, P > .05 and interaction, F(3,48) = 1.1, P > .05. DA: influence of WAY 100,635, F(1,15) = 1.3, P > .05, influence of time; F(3,45) = 2.7, P > .05 and interaction, F(3,45) = 1.7, P > .05. NAD: influence of WAY 100,635, F(1,15) = 0.1, P > .05, influence of time; F(3,45) = 1.3, P > .05 and interaction, F(3,45) = 0.6, P > .05. The intrinsic effect of WAY 100,635 alone was evaluated 20 min after injection by use of the Students' two tailed-t test (* P  .05).

Inhibition of the actions of S 15535 in FCX by the 5-HT_1A antagonist, WAY 100,635. Figure 3 shows that, as compared with vehicle, the 5-HT_1A receptor antagonist, WAY 100,635, elicited a slight but significant increase in 5-HT levels in dialysates of FCX. This effect, opposite to that of S 15535, was transient in that it was seen only in the sample immediately after injection. In the presence of WAY 100,135, the inhibitory influence of S 15535 on 5-HT levels was blocked (fig. 3). As concerns DA levels, the mild increase seen upon injection was slightly, but not significantly, lower with WAY 100,635 than with vehicle (fig. 3). Further, in the presence of WAY 100,635, the influence of S 15535 on DA levels was significantly attenuated. For NAD, the increase in its levels provoked by injection did not significantly differ between WAY 100,635 and vehicle (fig. 3). However, the increase in NAD levels provoked by S 15535 was significantly attenuated in the presence of WAY 100,635.

Influence of S 15535 compared with fluoxetine on dialysate levels of 5-HT and DA in nucleus accumbens and striatum. The injection of vehicle little modified dialysate levels of 5-HT and DA in either nucleus accumbens or striatum, although a slight tendency for a decrease in DA release was seen in the striatum over the last three samples (140-180 min) (fig. 5). S 15535 elicited a pronounced and rapid decrease in dialysate levels of 5-HT in both accumbens and striatum at a dose of 5.0 mg/kg. In contrast, S 15535 did not significantly modify levels of DA in either structure. In contrast to S 15535, fluoxetine (10.0 mg/kg) significantly increased dialysate levels of 5-HT in both the nucleus accumbens and striatum. This action was selective inasmuch as levels of DA were not modified. Further, in confirmation of the selective influence of S 15535 on serotoninergic as compared with dopaminergic pathways, S 15535 (5.0) elicited a marked and sustained reduction in levels of the 5-HT metabolite, 5-HIAA, in the nucleus accumbens and striatum, whereas those of the DA metabolite, DOPAC, were not significantly modified in either structure (fig. 6).

View larger version (K): [in this window] [in a new window]   Fig. 5.   Influence of s.c. injection of vehicle as compared with S 15535 and fluoxetine on dialysate levels of serotonin and dopamine in the nucleus accumbens (left panels) and striatum (right panels) of freely moving rats. Data are means ± S.E.M.; n = 8-10 per value. They are expressed as a percentage of basal, preinjection values which were defined as 100%. These were 0.87 ± 0.11 and 6.56 ± 0.62 pg/20 min dialysate for 5-HT and DA, respectively, in the nucleus accumbens for vehicle-treated animals (n = 7) and 0.88 ± 0.43 and 11.18 ± 0.97 pg/20 min dialysate for 5-HT and DA, respectively, for vehicle-treated animals (n = 9) in the striatum. ANOVA with drug as between factor and time as within factor was performed. Nucleus accumbens (upper panel): influence of drugs, F(2,19) = 29.8, P < .01, influence of time; F(11,209) = 0.6, P > .05 and interaction, F(22,209) = 7.3, P < .01 and (lower panel): influence of drugs, F(2,31) = 0.6, P > .05, influence of time; F(11,341) = 2.2, P < .05 and interaction, F(22,341) = 0.7, P > .05. Striatum (upper panel): Influence of drugs, F(2,27) = 28.5, P < .01, influence of time; F(11,297) = 1.0, P > .05 and interaction, F(22,297) = 6.2, P < .01 and (lower panel): influence of drugs, F(2,33) = 4.0, P < .05, influence of time; F(11,363) = 7.4, P < .01 and interaction, F(22,363) = 1.9, P < .01.

View larger version (K): [in this window] [in a new window]   Fig. 6.   Influence of S 15535 (5.0 mg/kg s.c.) on dialysate levels of DOPAC and 5-HIAA in the nucleus accumbens (lower panel) and striatum (upper panel). Data are means ± S.E.M.; n = 5 per value. They are expressed as a percentage of basal, preinjection values which were defined as 100%. Basal levels of DOPAC were 7.8 ±1.1 and 11.0 ± 2.0 ng/20 min dialysate, and basal levels of 5-HIAA were 3.2 ± 0.4 and 3.0 ± 0.7 ng/20 min dialysate in the nucleus accumbens and striatum, respectively. DOPAC, nucleus accumbens, F(11,33) = 0.8, P > .05, DOPAC, striatum, F(11,33) = 0.5, P > .05; 5-HIAA, nucleus accumbens, F(11,33) = 41.3, P < .001 and 5-H1AA, striatum, F(11,33) = 13.6, P < .001.

Influence of chronic treatment with S 15535 on the density of 5-HT_2A compared with -AR receptors in rat cortex (fig. 7, table 3). Chronic treatment with the prototypical tricyclic AD, desipramine, for 14 days resulted in a marked and significant reduction in the density (B_max) of 5-HT_2A and -AR receptors in the cortex of rats, in the absence of a significant alteration in the apparent dissociation constant (K_d) of the respective radioligands, [³H]CGP 12177 and [³H]ketanserin. Similarly, long-term administration of S 15535 over 14 days was associated with a significant decrease in the density of 5-HT_2A receptors in FCX, in the absence of an alteration in their apparent affinity. In contrast, the density of cortical -AR receptors was not modified by S 15535. Chronic fluoxetine treatment modified levels of neither 5-HT_2A nor -AR receptors.

View larger version (K): [in this window] [in a new window]   Fig. 7.   Saturation binding of -AR and 5-HT2A receptors in the cortex of rats treated for 14 days with vehicle, desipramine or S 15535. Rats were injected s.c. once daily with vehicle, desipramine (15.0 mg/kg), fluoxetine (10.0 mg/kg) or S 15535 (10.0 mg/kg). Saturation binding analysis was carried out on the cortices of individual rats with the selective antagonist [3H]CGP 12177 (-AR) or [3H]ketanserin (5-HT2A). (panel A) representative -AR saturation isotherms; (panel B) Scatchard plot of points from panel A; (panel C) representative 5-HT2A saturation isotherms; (panel D) Scatchard plot of points from panel B.

View this table: [in this window] [in a new window]   TABLE 3 Effect of chronic (14-day) treatment with desipramine, fluoxetine or S 15535 on cortical populations of -AR and 5-HT2A receptorsa

Action of S 15535 compared with fluoxetine in the LH test. As compared with control animals which had not been previously exposed to inescapable shock, vehicle-treated, preshocked ("helpless") rats performed poorly in an avoidance paradigm, displaying a significantly higher number of escape failures (fig. 8, table 4). These helpless rats also manifested a pronounced reduction in intertrial ambulatory activity as compared with nonstressed rats. On each of the three sessions (over 3 days) of testing, S 15535 elicited a marked reduction in escape deficits, which was significant over a broad dose range of 0.63 to 40.0 mg/kg p.o., although only for a comparison between the lowest and highest doses tested was a dose dependence seen. At the highest dose, the number of escape failures emitted was no higher than that seen in control nonshocked animals. In distinction to S 15535, fluoxetine manifested a markedly biphasic curve of activity on each of the 3 days of testing, with the action of the intermediate dose of 4.0 being greater than that of the doses of 2.0 and 8.0. At the dose of 4.0, escape failures were almost completely eliminated. For both S 15535 and fluoxetine, there was no indication of either a delay in onset of activity or of a reduction across the three sessions, with their activity being expressed similarly across each of the three sessions. Both S 15535 and fluoxetine significantly increased intertrial responses, although in terms of minimal effective dose and maximal percentage effect, this action could be dissociated from their influence on escape failures.

View larger version (K): [in this window] [in a new window]   Fig. 8.   Influence of S 15535 as compared with fluoxetine on escape failures in an avoidance paradigm (shuttle box) across three consecutive days of testing and after inescapable shock. Data are means ± S.E.M.; n  8 per value. NLH indicates (vehicle-treated) control animals which were not exposed to inescapable shock. All other groups were exposed and were treated with vehicle or various doses of S 15535 or fluoxetine. For the vehicle-treated shocked (LH) animals, for each panel, the difference to control values was significant (P  .05) in Students two-tailed t test. Asterisks indicate significance of drug differences to corresponding vehicle values in Dunnett's following ANOVA. * P < .05. ANOVA as follows. Fluoxetine, session 1: F(3,40) = 7.5, P < .01; session 2, F(3,40) = 7.7, P < .01; session 3, F(3,40) = 9.4, P < .001. S 15535, session 1, F(5,82) = 8.2, P < .001; session 2, F(5,82) = 7.7, P < .001; session 3, F(5,82) = 7.5, P < .001.

Influence of S 15535 on the duration of immobility in the forced swim test. As indicated in table 5, in contrast to desipramine, S 15535 did not significantly modify the duration of immobility in rats forced to swim in a water-filled cylinder. Fluoxetine also did not reduce immobility time; in fact, this significantly increased at the highest dose examined.

	Discussion

Top Abstract Introduction Methods Results Discussion References

Interaction at cloned, h5-HT_1A versus hD₂/hD₃ and h_2A-AR receptors. S 15535 displayed high affinity for cloned h5-HT_1A receptors (Newman-Tancredi et al., 1992). Consistent with this observation, radiolabeled [³H]S 15535 specifically occupies a homogeneous population of 5-HT_1A receptors both in CHO cells and in rat hippocampus (Peglion et al., 1995). The binding of [³H]S 15535 is little influenced (16% reduction) by GppNHp (10 µM) at both human and rat postsynaptic 5-HT_1A receptors, and in [³⁵S]GTPS binding studies, S 15535 behaves as a weak partial agonist with an intrinsic activity of about 25 to 30% relative to 5-HT (100%) and buspirone (65%) (Newman-Tancredi et al., 1996a; Peglion et al., 1995). This level of efficacy corresponds to its antagonist/weak partial agonist actions at postsynaptic 5-HT_1A receptors in vivo (Millan et al., 1994b). In contrast, reflecting their greater receptor reserve, S 15535 behaves as an agonist at 5-HT_1A autoreceptors in vivo in reducing both 5-HT synthesis and the firing rate of DRN-localized serotoninergic neurons (Gobert et al., 1995a; Millan et al., 1994b). As such, S 15535 may be distinguished from WAY 100,635, which acts as an antagonist at both pre- and postsynaptic populations of 5-HT_1A receptors (Hjorth et al., 1995; Pike et al., 1995). The low affinity of S 15535 for native, rodent DA D₂ and D₁ receptors (Millan et al., 1994b) is extended herein to cloned DA hD₂ and hD₃ (fig. 1) as well as to hD₄ and hD₅ (K_i values > 1000 nM, Newman-Tancredi, A., unpublished observations) receptors. Likewise in line with native, rat _2D-AR receptors (Millan et al., 1994b), the affinity of S 15535 was low at cloned, h_2A-AR receptors, their human homolog. The low efficacy of S 15535 at postsynaptic 5-HT_1A receptors, together with its comparative lack of activity at DA D₂/D₃, ₂-AR and other (Millan et al., 1994b) receptors, suggests that S 15535 should be relatively devoid of undesirable motor, endocrine and physiological side effects (Millan et al., 1997, accompanying paper).

Modulation of cerebral release of 5-HT by S 15535. S 15535 decreased dialysate concentrations of 5-HT in the FCX, nucleus accumbens and striatum, an action which appears to be mediated by 5-HT_1A autoreceptors. First, S 15535 possesses high affinity at 5-HT_1A receptors and reduces the electrical and synthetic activity of central serotoninergic pathways (Gobert et al., 1995a; Millan et al., 1994b). Second, although serotoninergic neurons bear inhibitory 5-HT_1B autoreceptors (Briley and Moret, 1993), the affinity of S 15535 for these is low (>1000 nM) (Millan et al., 1994b). Further, whereas _2A-AR receptors also inhibit the release of 5-HT in the cortex (Feuerstein et al., 1993; Trendelenburg et al., 1994), S 15535 does not show significant affinity at these sites (see above). Moreover, whereas 5-HT₆ and 5-HT₇ receptors are found in the DRN (Zo et al., 1995), the affinity of S 15535 for these is low (>1000 nM, Newman-Tancredi,A., unpublished observations). Third, in the presence of WAY 100,635, S 15535 did not reduce dialysate levels of 5-HT levels in the FCX (fig. 3). Further, WAY 100,635 also abolished the S 15535-elicited reduction of 5-HT release in the hippocampus (Millan et al., 1997, accompanying paper) and blocked the inhibition of raphe firing by S 15535 (Lejeune et al., 1996). WAY 100,635 itself transiently increased dialysate levels of 5-HT in FCX, likely reflecting the interruption of a tonic autoinhibitory tone on 5-HT release. Indeed, Gurling et al. (1994) showed that WAY 100,635 increases hippocampal 5-HT release in conscious rats during the dark phase of the cycle, whereas Fornal et al. (1995) reported that WAY 100,635 increases DRN firing in conscious, but not anesthetized, cats. In addition, Mundey et al. (1996) showed that WAY 100,635 increases DRN firing rates in the guinea pig. Thus, the lack of influence of WAY 100,635 on the release of 5-HT in certain other studies (Gartside et al., 1995; Hjorth et al., 1995) may reflect the use of anesthetized rats. Indeed, two additional 5-HT_1A receptor antagonists, ()-tertatolol and spiperone, enhance the firing rate of serotoninergic neurons in the DRN (Gobert et al., 1995a; Lejeune et al., 1993). Psychological stress increases 5-HT release in FCX (Kawahara et al., 1993), and the ability of S 15535 to reduce 5 HT levels via an action at 5-HT_1A autoreceptors is consistent with its anxiolytic properties (Millan et al., 1997, accompanying paper). In line with its ability to inhibit 5-HT uptake (Wong et al., 1995), fluoxetine increased dialysate concentrations of 5-HT. However, fluoxetine also inhibits 5-HT uptake at the dendritic levels in the raphe. This will increase release of 5-HT onto inhibitory 5-HT_1A autoreceptors (Invernizzi et al., 1992), thereby attenuating its facilitatory actions on 5-HT levels in the cortex and elsewhere. Indeed, in contrast to S 15535, WAY 100,635 selectively potentiates the increase in FCX levels of 5-HT provoked by both fluoxetine and two further SSRIs, paroxetine and duloxetine (Gartside et al., 1995; Gobert et al., 1997). Correspondingly, 5-HT_1A antagonists may accelerate the onset of clinical efficacy of SSRIs by mimicking the process of gradual desensitization of 5-HT_1A autoreceptors (Fanelli and McMonagle-Strucko, 1992; Gobbi et al., 1991) which occurs on chronic SSRI (or 5-HT_1A autoreceptor agonist) treatment (Artigas et al., 1994; Blier and De Montigny, 1994; Hjorth and Auerbach, 1994; Invernizzi et al., 1994).

Modulation of FCX levels of DA by S 15535. Although DA D₂ and D₃ autoreceptors inhibit the activity of dopaminergic pathways (Gobert et al., 1995b, 1996; Kreiss et al., 1995; Lejeune and Millan, 1995; Tang et al., 1994), S 15535 possesses only low affinity at these sites (see above). Further, if a direct action of S 15535 at D₂/D₃ sites were of importance in increasing FCX dialysate levels of DA, these would likely have also augmented by S 15535 in parallel in the accumbens and striatum, but this was not the case. This distinction provides an insight into the mechanism underlying the action of S 15535 inasmuch as the subpopulation of dopaminergic neurons in the ventrotegmental area which projects to the FCX is subject to a more pronounced serotoninergic modulation than its counterpart which projects to the accumbens (Svensson et al., 1995). Thus, it is likely that the increase in cortical DA release provoked by S 15535 reflects the involvement of 5-HT_1A autoreceptors, which results in an indirect reduction in the inhibitory serotoninergic tone on mesocortical dopaminergic neurons (Arborelius et al., 1993; Chen and Reith, 1995; Lejeune et al., in press; Pessia et al., 1994). Support for this argument is provided by the present observations that: 1) the same dose range of S 15535 increased and decreased cortical levels of DA and 5-HT, respectively; 2) WAY 100,635 blocked the increase in FCX levels of DA provoked by S 15535; 3) other 5-HT_1A autoreceptor agonists, such as 8-OH-DPAT, also increase cortical dialysate levels of DA, actions blocked by WAY 100,635 (Arborelius et al., 1993; Tanda et al., 1994; Gobert, A., unpublished observations); 4) because WAY 100,635 shares the antagonist actions of S 15535 at postsynaptic sites, yet antagonizes its agonist action at 5-HT_1A autoreceptors, activation of 5-HT_1A autoreceptors by S 15535 likely underlies the increase in DA levels in FCX; 5) S 15535 increases the firing rate of dopaminergic neurons in the ventrotegmental area, an action blocked by WAY 100,635 (Lejeune et al., 1996). This observation of parallel changes in the electrical activity of dopaminergic neurons and of extracellular DA levels in dialysates of the FCX is similar to the parallel decrease in DRN firing rate and cortical levels of 5-HT discussed above. Collectively, the findings strongly suggest that an action of S 15535 at 5-HT_1A receptors underlies the increase in FCX dialysate levels of DA. Although the data are consistent with a role of 5-HT_1A autoreceptors, additional mechanistic studies would be needed to confirm this suggestion.

Significance of increased activity of DA in FCX. The influence of S 15535 on cortical levels of DA is of interest in several respects. First, a selective increase in levels of DA in the FCX may be a common property of mechanistically diverse AD drugs, including 5-HT_1A receptor ligands, tricyclics and SSRIs (Arborelius et al., 1993; Jordan et al., 1994; Tanda et al., 1994; but see Ichikawa and Meltzer, 1995). Indeed, fluoxetine also elevated dialysate levels of DA in the FCX, an action resistant to WAY 100,635 and possibly involving postsynaptic, excitatory 5-HT₃ receptors localized on dopaminergic terminals (Jordan et al., 1994; Prisco and Esposito, 1995; Tanda et al., 1995). Second, a reduction in the activity of mesocortical dopaminergic systems may contribute to the emotional and cognitive "hypofrontality" which is a unifying feature of many types of depressive conditions (Baxter et al., 1989; Carlsson et al., 1993; Drevets et al., 1992; Karoum et al., 1994; Willner, 1991, 1995; Zacharko and Anisman, 1991). Third, hypofrontality and a deficient mesocortical dopaminergic transmission are also implicated in the negative symptomatology of schizophrenia, which includes depressive-like symptoms such as blunted effect, social withdrawal and apathy (Weinberger, 1987). Further, unlike conventional neuroleptics, the atypical antipsychotic, clozapine, selectively increases the activity of mesocortical dopaminergic neurons and effectively treats these symptoms (Hand et al., 1987; Moghaddam and Bunney, 1990). Fourth, a reinforcement of dopaminergic transmission in the FCX may be associated with a restoration of a deficient cognitive performance (Sawaguchi et al., 1990), and S 15535 possesses promnesic properties (unpublished observations).

Increase in NAD release in the cortex by S 15535. Although the activity of central adrenergic networks is controlled by inhibitory _2A-AR autoreceptors (Millan et al., 1994a; Trendelenburg et al., 1993), S 15535 displays only low activity for these (table 2 and Millan et al., 1994b). Thus, they are unlikely to be involved in the increase in FCX levels of NAD elicited by S 15535. One component of this response to S 15535 may be attributed to an activation of 5-HT_1A receptors inasmuch as WAY 100,635 attenuated the increase in NAD levels. Indeed, there exists an extensive serotoninergic innervation of the locus ceruleus, and although the modulatory influence of 5-HT is complex, there is an overall inhibitory serotoninergic tone (Aston-Jones et al., 1991a; Bobker and Williams, 1989; Ferron, 1988; Gorea and Adrien, 1988). This inhibitory tone may be relieved by activation of 5-HT_1A autoreceptors. Nevertheless, the location (pre- or postsynaptic) of the population of 5-HT_1A receptors involved in the modulation of FCX NAD levels by S 15535 will require further evaluation. Further, WAY 100,635 did not abolish the influence of S 15535 on FCX levels of NAD, which suggests that another mechanism must exist via which S 15535 modulates cortical adrenergic transmission. In analogy, WAY 100,635 only partially inhibits the increase in cortical NAD levels elicited by 8-OH-DPAT (Gobert, A., unpublished observations). Possibly, the increase in extracellular DA concentrations may saturate DA reuptake mechanisms resulting in its uptake by noradrenergic neurones, thereby displacing NAD (Carboni et al., 1990). On the other hand, cortical NAD release reflects levels of arousal and this likely explains the mild, but significant and reproducible, increase in FCX dialysate levels of NAD elicited by vehicle injection in the present and previous studies (Aston-Jones et al., 1991b; Cenci et al., 1992). Indeed, inasmuch as S 15535 exerts a disinhibitory influence upon behavior (this article and Millan et al., 1997, accompanying paper), this may be associated with an enhanced activity of cortical adrenergic pathways. A "stress"-like action of S 15535 itself seems unlikely in view of its anxiolytic properties (Milan et al., 1997, accompanying paper). As concerns fluoxetine, its modest NAD-uptake inhibiting actions may contribute to the increase in cortical NAD levels (Bolden-Watson and Richelson, 1993; Wong et al., 1995). The ability of S 15535 to increase cortical release of NAD is of interest in several respects. First, levels of NAD and _2A-AR receptors are altered in the FCX and locus ceruleus of (some) depressed patients (Arango et al., 1993; González et al., 1994; Ordway et al., 1994). Second, ₂-AR receptor antagonists, which reinforce cortical adrenergic transmission by blockade of inhibitory _2A-AR autoreceptors (Millan et al., 1994a), display (mild) AD properties in animal and man (Broekkamp et al., 1995; Pollack and Hammerness, 1993; Redfern et al., 1993; Sanger, 1988). Third, animals presenting LH deficits show deficient adrenergic transmission in the cortex, changes which may be corrected by AD treatment (Kitayama et al., 1994; Nakamura, 1990). Fourth, adrenergic transmission in the cortex is implicated in processes underlying attention and memory formation (Aston-Jones et al., 1991b; Coull, 1994; Foote and Aston-Jones, 1995; Robbins and Everitt, 1995), and a reinforcement of adrenergic transmission in the FCX (by S 15535) may be associated with its potential promnesic properties (Millan et al., 1997, accompanying paper).

Modulation of cortical -AR receptors. Although many ADs down-regulate cortical -AR receptors (Broekkamp et al., 1995; Okada and Tokumitsu, 1994), there are several false negatives, notably SSRIs (Goodnough and Baker, 1994; Nelson et al., 1990; but see Byerley et al., 1988). In addition, by use of more specific ligands (such as [³H]CGP 12177) than those used in older studies ([³H]dihydroalprenolol), the down-regulation of -AR receptors by tricyclic ADs occurs rapidly (within 24 h) challenging the assumption of a parallelism to the delayed onset of AD efficacy (Riva and Creese, 1989; Newman-Tancredi et al., 1996b). Further, at least for certain tricyclics, their actions may not reflect a down-regulation of -AR receptors per se, but rather a direct interaction at the coupled G-protein (Ozawa et al., 1994). In fact, buspirone and other 5-HT_1A agonists do not consistently modify -AR density (Thiébot and Martin, 1991) such that the lack of effect of S 15535 on cortical -AR receptors is not surprising. Nevertheless, a possible influence of S 15535 on -AR receptors may justify further evaluation inasmuch as -AR receptors may be modulated only in cortical substructures (specific laminae) (Byerley et al., 1988; Okada and Tokumitsu, 1994). Further, a limited change in receptor number may be amplified at the second messenger level.

Down-regulation of 5-HT_2A receptors. Several 5-HT₂ receptor antagonists express AD properties (Broekkamp et al., 1995; Granier et al., 1985; Marek et al., 1989) and LH-"depressed" rats display an increase in the cortical density of 5-HT_2A receptors (Barone et al., 1990; Nankai et al., 1995). Further, chronic stress elevates levels of cortical 5-HT_2A but not -AR receptors in rats (Takao et al., 1995). In addition, in depressed human subjects, there is evidence for an increase in the functional activity of platelet 5-HT_2A receptors (Mikuni et al., 1991) and an increase in the density of 5-HT_2A receptors in the FCX (Arango et al., 1990; Arranz et al., 1994). For these reasons, the ability of chronic tricyclic and other ADs to down-regulate cortical 5-HT_2A receptors has attracted much interest (Burnet et al., 1994; Roth and Ciranello, 1991). Although fluoxetine and other SSRIs have not been observed to reliably down-regulate 5-HT_2A receptors (Goodnough and Baker, 1994; Hrdina, 1993