Abstract
S33005 displayed marked affinity for native, rat, and cloned human serotonin (5-HT) transporters (SERT) and less pronounced affinity for norepinephrine (NE) transporters (NET), while its affinity at dopamine (DA) transporters and >50 other sites was negligible. Reuptake of 5-HT and (less potently) NE into cerebral synaptosomes was inhibited by S33005, whereas DA reuptake was little affected. In vivo, S33005 prevented depletion of cerebral pools of 5-HT by parachloroamphetamine. Furthermore, it decreased electrical activity of raphe-localized serotonergic neurones, an action abolished by the 5-HT1A antagonist WAY100,635. At higher doses, S33005 blocked firing of locus ceruleus-localized adrenergic neurones, an action abolished by the α2-adrenergic antagonist idazoxan. In contrast, S33005 did not inhibit ventrotegmental dopaminergic neurones. In frontal cortex of freely moving rats, S33005 dose dependently elevated dialysate levels of 5-HT, NE,and DA. In hippocampus, levels of 5-HT and NE were similarly elevated, while in nucleus accumbens and striatum, levels of 5-HT were increased whereas DA was unaffected. Upon chronic (2 weeks) administration, basal levels of NE were elevated in frontal cortex and, therein, 5-HT2A receptor density was decreased. Comparative studies with clinically used antidepressants showed that venlafaxine possessed a profile similar to S33005 but was less potent. Clomipramine likewise interacted with SERTs and NETs but also with several other receptors types, while citalopram and reboxetine were preferential ligands of SERTs and NETs, respectively. In conclusion, S33005 interacts potently with SERTs and, less markedly, with NETs. It enhances extracellular levels of 5-HT and NE throughout corticolimbic structures and selectively elevates dialysis levels of DA in frontal cortex versus subcortical regions.
Extensive experimental and clinical evidence indicates that a perturbation of monoaminergic transmission is involved in depressive states (Maes and Meltzer, 1995;Willner, 1995; Ressler and Nemeroff, 1999; Anand and Charney, 2000). Correspondingly, currently used antidepressant agents exert their therapeutic actions via a reinforcement in monoaminergic transmission, although the relative contribution of serotonergic, adrenergic, and dopaminergic mechanisms remains to be clarified (Broekkamp et al., 1995; Burke and Preskorn, 1995; Frazer, 1997; Millan et al., 2000b). Monoamine oxidase inhibitors exert antidepressant actions by preventing the degradation of 5-HT and NE, while mianserin and mirtazapine display antagonist properties at α2-adrenoceptors (AR) and 5-HT2C receptors inhibitory to serotonergic, adrenergic, and/or dopaminergic pathways (Burke and Preskorn, 1995;Frazer, 1997; Millan et al., 2000a). Nevertheless, the majority of antidepressant agents reinforce monoaminergic transmission by blocking neuronal uptake of 5-HT and/or NE via actions at SERTs and NETs, respectively (Barker and Blakely, 1995; Frazer, 1997; Sambunaris et al., 1997; Goodnick and Goldstein, 1998; Blakely and Bauman, 2000).
Despite subtle differences among selective 5-HT reuptake inhibitors (SSRIs), such as fluoxetine and the highly selective agent citalopram (Owens et al., 1997; Sánchez and Meier, 1997; Tatsumi et al., 1997; Goodnick and Goldstein, 1998; Popik, 1999), all elevate extracellular levels of 5-HT in corticolimbic structures upon acute and chronic administration (Blier and de Montigny, 1994; Goodnick and Goldstein, 1998; Millan et al., 2000b). On the other hand, the recently introduced morpholine derivative, reboxetine, interacts preferentially with NETs versus SERTs in vitro and markedly increases extracellular levels of NE (Burrows et al., 1998; Riva et al., 1999; Sacchetti et al., 1999). In this respect, reboxetine resembles the first-generation, tricylic agent desipramine (Owens et al., 1997). However, other tricyclic antidepressants, such as clomipramine, interact with both NETs and with SERTs (Burke and Preskorn, 1995; Tatsumi et al., 1997). This dual activity is likewise displayed by the cyclohexanol derivative, venlafaxine, which lacks the undesirable histaminergic, muscarinic, and α1-AR antagonist properties of clomipramine and other tricyclic agents (Muth et al., 1991; Schweizer et al., 1997;Harvey et al., 2000). Clinical studies have demonstrated the efficacy of venlafaxine in major depressive states, including resistant and, probably, bipolar patients (Amsterdam, 1998; Poirier and Boyer, 1999). It may also possess a rapid onset of activity (Derivan et al., 1995) and be associated with a high remission rate (Ferrier, 1999), although this remains to be confirmed. In addition, venlafaxine improves generalized anxiety disorders (Rickels et al., 2000) and social phobia (Altamura et al., 1999). These observations have triggered a resurgence of interest in antidepressants uniting activity at SERTs andNETs in vivo, with the aim of optimizing clinical efficacy, achieving more rapid action, and enlarging therapeutic reach (Broekkamp et al., 1995; Frazer, 1997; Sambunaris et al., 1997).
Venlafaxine, however, displays only modest affinity for SERTs, and its affinity for NETs is weak (Muth et al., 1991; Owens et al., 1997;Tatsumi et al., 1997; Béı̈que et al., 1999, 2000). It would thus be of interest to obtain agents of greater potency. In this light, we have generated a series of benzocyclobutane derivatives possessing affinity for both SERTs and NETs. In the present and accompanying papers, the pharmacological profile of one of these, S33005 (Fig. 1), is compared with venlafaxine, citalopram, reboxetine, and clomipramine.
First, we determined their affinities at native, rat, and cloned human (h) SERTs, NETs, and DA transporters (DAT) and evaluated uptake of 5-HT, NE, and DA into cerebral rat synaptosomes. Second, we examined the ability of drugs to prevent in vivo depletion of cerebral pools of 5-HT by parachloroamphetamine (PCA), which exerts its actions following entry into serotonergic terminals via SERTs (Fuller et al., 1991). Third, serotonergic perikarya localized in the dorsal raphe nucleus (DRN) possess both SERTs and inhibitory 5-HT1Aautoreceptors. Blockade of 5-HT uptake therefore suppresses the electrical activity of serotonergic neurones via activation of 5-HT1A autoreceptors (Blier and de Montigny, 1994; Gartside et al., 1997). Thus, we examined the influence of S33005 and other antidepressants on the firing rate of serotonergic neurones. Similarly, we evaluated their actions at adrenergic neurones in the locus ceruleus (LC), which bear NETs and inhibitory α2-AR autoreceptors (Scuvee-Moreau and Dresse, 1979; Muth et al., 1991), and at dopaminergic cell bodies in the ventrotegmental area, which possess DATs and inhibitory D2/D3 autoreceptors (Millan et al., 2000b). Fourth, the influence of S33005 and the other agents upon extracellular levels of 5-HT, NE, and DA was simultaneously quantified in single dialysis samples of the frontal cortex, dorsal hippocampus, nucleus accumbens, and striatum of freely moving rats. Finally, several—although not all—classes of antidepressant diminish the density of cortical 5-HT2A and β-ARs upon long-term treatment (Okada and Tokumitsu, 1994; Newman-Tancredi et al., 1996; Yatham et al., 1999a,b). Thus, the influence of S33005 as compared with venlafaxine upon frontocortical levels of 5-HT2A and β-ARs, as well as dialysis levels of 5-HT, NE, and DA, was evaluated following their administration for 2 or 3 weeks.
Materials and Methods
Animals.
These studies used male Wistar rats of 200 to 250 g (Iffa Credo, L'Arbresles, France) housed in sawdust-lined cages with unrestricted access to standard chow and water. There was a 12-h/12-h light/dark cycle with lights on at 7:30 AM. Laboratory temperature and humidity were 21 ± 0.5°C and 60 ± 5%, respectively. Animals were adapted to laboratory conditions for at least 1 week prior to testing. All animal use procedures conformed to international European ethical standards (86/609-EEC) and the French National Committee (décret 87/848) for the care and use of laboratory animals.
Binding at Native Rat SERTs.
Binding affinity was determined by competition with [3H]paroxetine (PerkinElmer Life Sciences, Les Ulis, France). Freshly prepared membranes of rat frontal cortex were homogenized with a Polytron and then centrifuged twice at 20,000g. The pellet was resuspended each time in incubation buffer. Membranes were incubated in triplicate with 2 nM [3H]paroxetine and competing ligand in a final volume of 0.4 ml for 2 h at 25°C. The incubation buffer contained 50 nM Tris-HCl (pH 7.4), 120 nM NaCl, and 5 mM KCl. Nonspecific binding was defined with 10 μM citalopram.
Binding at hSERTs.
Binding affinity was determined by competition with [3H]paroxetine (PerkinElmer Life Sciences). Membranes prepared from HEK293 cells stably expressing recombinant hSERTs were purchased from Receptor Biology (Beltsville, MD) and incubated in triplicate with 2 nM [3H]paroxetine and competing ligand in a final volume of 0.4 ml for 1.5 h at 25°C. The incubation buffer contained 50 mM Tris-HCl (pH 7.4), 120 mM NaCl, and 5 mM KCl. Nonspecific binding was defined with 10 μM citalopram.
Binding at Native Rat NETs.
Binding affinity was determined by competition with [3H]nisoxetine (Amersham, Les Ulis, France). Freshly prepared membranes of rat cortex were homogenized with a Polytron and then centrifuged twice at 20,000g. The pellet was resuspended each time in incubation buffer. Membranes were incubated in triplicate with 2 nM [3H]nisoxetine and competing ligand in a final volume of 0.5 ml for 4 h at 4°C. The incubation buffer contained 50 mM Tris-HCl (pH 7.4), 120 mM NaCl, and 5 mM KCl. Nonspecific binding was defined with 10 μM desipramine.
Binding at hNETs.
Binding affinity was determined by competition with [3H]nisoxetine (2.0 nM, Amersham). Membranes prepared from Madin-Darby canine kidney cells expressing hNETs were purchased from Receptor Biology and incubated in triplicate with 2 nM [3H]nisoxetine and competing ligand in a final volume of 0.5 ml for 1 h at 4°C. The incubation buffer contained 50 mM Tris-HCl (pH 7.4), 120 mM NaCl, and 5 mM KCl. Nonspecific binding was defined with 10 μM desipramine.
Binding at Native Rat DATs.
Binding affinity was determined by competition with [3H]GBR12909 (PerkinElmer Life Sciences). Freshly prepared membranes of rat striata were homogenized with a Polytron and then centrifuged twice at 20,000g. The pellet was resuspended each time in incubation buffer. Membranes were incubated in triplicate with 2 nM [3H]GBR12909 and competing ligand in a final volume of 1 ml for 1 h at 4°C. The incubation buffer contained 50 mM Tris-HCl (pH 7.4), 120 mM NaCl and 4 mM MgCl2. Nonspecific binding was defined with 10 μM GBR12909.
Binding at hDATs.
Binding affinity was determined by competition with [3H]GBR12909 (PerkinElmer Life Sciences). Membranes prepared from CHO cells stably expressing recombinant hDATs were purchased from Receptor Biology and incubated in triplicate with 2 nM [3H]GBR12909 and competing ligand in a final volume of 0.5 ml for 2 h at 4°C. The incubation buffer contained 50 mM Tris-HCl (pH 7.4) and 120 mM NaCl. Nonspecific binding was defined with 10 μM GBR12909.
Data Analysis for Binding Studies.
For all of the above protocols, at the end of the incubation period, membranes were filtered through Whatman (Packard, Meriden, CT) GF/B filters pretreated with 0.1% polyethylenimine. Radioactivity retained on the filters was determined by scintillation counting. Binding isotherms were analyzed by nonlinear regression using Prism software (GraphPad Software Inc., San Diego, CA) to determine IC50 values. These were converted to inhibition constants (Ki) by use of the Cheng-Prusoff equation: Ki = IC50 / [(L/KD) − 1], whereL is the concentration of 3H-labeled ligand and KD is its dissociation constant determined in saturation binding experiments. TheKD values were as follows: 0.13 nM for [3H]paroxetine at both native rat SERTs and hSERTs; 1.2 and 2.2 nM for [3H]nisoxetine at native rat NETs and hNETs, respectively; and 1.6 and 1.0 nM for [3H]GBR12909 at native rat DATs and hDATs, respectively.
Interaction with other Binding Sites.
The potential interaction of S33005 at diverse (>50) binding sites was evaluated by using standard procedures (see Results for several key sites) detailed elsewhere (Millan et al., 2000a). Inasmuch as S33005 showed negligible (pKi < 5.0) affinity at all sites examined, these protocols are not further described herein. Data analysis was as described above.
Influence upon [3H]Monoamine Uptake by Rat Brain Synaptosomes.
[3H]Monoamine uptake assays were carried out on synaptosomes prepared from rat cortex ([3H]5-HT), rat hypothalamus ([3H]NE), and rat striatum ([3H]DA), essentially as described previously (Janowsky et al., 1986). Synaptosomes were incubated with the radiolabeled neurotransmitter and drug for 15 min at 37°C before rapid filtration. [3H]Monoamine uptake into synaptosomes was determined by liquid scintillation counting.
Influence upon Depletion of Cerebral Pool of 5-HT by PCA.
Levels of 5-HT were determined by high-performance liquid chromatography and electrochemical detection as previously described (Millan et al., 2000a) in the frontal cortex and hippocampus of rats 60 min after s.c. administration of S33005, vehicle, or other drugs and 30 min after injection of the vehicle or PCA (5.0 mg/kg, i.p.). Data were analyzed by analysis of variance (ANOVA) followed by Dunnett's test. ID50 values plus 95% confidence limits (CL) were calculated.
Influence upon the Electrical Activity of Serotonergic, Adrenergic, and Dopaminergic Cell Bodies.
The influence of S33005 compared with other ligands upon the firing rate of DRN-localized serotonergic perikarya, LC-localized adrenergic perikarya, and ventrotegmental area-localized dopaminergic perikarya was determined by using a procedure described in detail previously (Millan et al., 2000a). Briefly, following anesthesia with chloral hydrate (400 mg/kg, i.p.), rats were placed in a stereotaxic apparatus, and a tungsten microelectrode was lowered into the DRN, LC, or ventrotegmental area. Coordinates were as follows: DRN, AP = −7.8 from bregma, L = 0.0, and H = −5/−6.5 from dura; LC, AP = −1.2 from zero, L = 1.2, and H = −5.5/−6.5 from dura; and ventrotegmental area, AP = −5.5 from bregma, L = 0.7, and H = −7/−8.5 from dura. As detailed elsewhere (Millan et al., 2000a), serotonergic, adrenergic, and dopaminergic neurones in the DRN, LC, and ventrotegmental area, respectively, were recognized by their distinctive waveforms. Following baseline recording over 5 min, vehicle, S33005, or other agents were administered i.v. (in a volume of 0.5 ml/kg) in cumulative doses every 2 to 3 min. After vehicle or drug administration, a further injection of WAY100,635 (0.031 mg/kg, i.v.) or idazoxan (0.063 mg/kg, i.v.) was made for the DRN and LC, respectively. Drug effects were quantified over the 60-s bin corresponding to their time of peak action. Spike2 software (CED, Cambridge, UK) was used for data acquisition and analysis. Data are expressed as percentage of change from baseline firing rate (defined as 0%). Data were analyzed by ANOVA followed by Newman-Keuls test, and ID50 values (95% CL) were calculated.
Influence upon Extracellular Levels of 5-HT, NE, and DA.
Quantification of extracellular levels of 5-HT, NE, and DA in single dialysate samples of the frontal cortex, dorsal hippocampus (NE and 5-HT), nucleus accumbens (DA and 5-HT), and striatum (DA and 5-HT) was achieved by using a protocol extensively described previously (Millan et al., 2000a). The guide cannulae were implanted under pentobarbital anesthesia (40.0 mg/kg., i.p.) at the following coordinates 1 week prior to experimentation: frontal cortex, AP = +2.2 from bregma, L = ±0.6, and H = −0.2 from dura; dorsal hippocampus, AP = −3.6 from bregma, L = ±1.2, and H = −2.3 from dura; nucleus accumbens, AP = +0.8 from bregma, L = +0.6, and H = −4.5 from dura; and striatum, AP = +0.5 from bregma, L = −2.8, and H = −3.0 from dura. A cuprophane CMA/11 probe (4 mm in length for the frontal cortex and striatum, 2 mm in length for the hippocampus and nucleus accumbens, and, in each case, 0.24-mm outer diameter; Carnegie Medicine, Stockholm, Sweden) was lowered into position. Three basal samples of 20 min each were taken. Vehicle, S33005, or other drugs were administered s.c., and samples were taken for a further 3 h. NE, 5-HT, and DA levels were quantified by high-performance liquid chromatography followed by coulometric detection as previously described (Millan et al., 2000a). The assay limit of sensitivity was 0.1 to 0.2 pg/sample for 5-HT, NE, and DA in each case. Data were analyzed by ANOVA, with sampling time as the repeated within-subject factor.
Influence upon DRN Firing Rate and Dialysis Levels of 5-HT, NE, and DA in the Frontal Cortex: Chronic Administration.
Rats were treated daily with a single injection of either vehicle, S33005 (10.0 mg/kg, s.c.), or venlafaxine (10.0 mg/kg, s.c.) for 14 days. On the 15th day, rats either received an additional injection of the same drug or of vehicle. Thereafter, exactly as described above, extracellular levels of 5-HT, NE, and DA were determined in the frontal cortex of freely moving rats. In a parallel study, following a chronic 14-day treatment, the influence of S33005 or venlafaxine (day 15) upon the electrical activity of serotonergic cell bodies in the DRN was determined.
Influence upon Cortical 5-HT2A Receptors and β-ARs: Chronic Administration.
Rats were treated with S33005 (10.0 mg/kg, s.c.), venlafaxine (10.0 mg/kg, s.c.), or vehicle once daily for 2 or 3 weeks. One day following the final injection, as described previously (Newman-Tancredi et al., 1996), using [3H]CGP-12177 as a radioligand, β-ARs were examined in homogenates of cortex. Furthermore, using [3H]ketanserin as a radioligand, 5-HT2A receptors were examined in frontal cortex.Bmax andKD values were determined through conventional procedures (see Newman-Tancredi et al., 1996).
Drugs.
Actions of drugs in each of the individual studies described herein were evaluated concurrently. For binding studies, drugs were dissolved in dimethyl sulfoxide (10−2M) and dilutions made in the buffer as appropriate. For in vivo studies, drugs were dissolved in sterile water, plus a few drops of lactic acid if necessary, and pH adjusted to as close to normality (>5.0) as possible. Drug salts and sources were as follows. S33005 HCl [(−)1-(1-dimethylaminomethyl 5-methoxybenzocyclobutan-1-yl) cyclohexanol], WAY100,635 fumarate [(N-{2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl}-N-(2-pyridinyl)cyclohexanecarboxamine], citalopram HCl, idazoxan HCl, reboxetine methane sulfonate, and venlafaxine HCl were all synthesized internally (G. Lavielle and J.-L. Peglion). GBR12935 2HCl [1-[2-(diphenylmethoxy)ethyl]-4-(3-phenylpropyl)-piperazine] was purchased from Sigma/RBI (Natick, MA), and PCA HCl and clomipramine were purchased from Sigma (Saint Quentin-Fallavier, France).
Results
Characterization of S33005 as Compared with Its Isomer, S33004, and Racemic S32647.
S33005 is the optically pure (−)-isomer of racemic (±)-S32647, whereas S33004 is the optically pure (+)-isomer. S32647 potently interacted with native rat SERTs and, less potently, with native rat NETs: pKi values (Ki in nM) = 8.64 ± 0.02 (2.3) and 6.58 ± 0.09 (263), respectively. Similarly, S33005 displayed marked affinity for these sites: pKi values (Ki in nM) = 8.71 ± 0.06 (1.9) and 6.75 ± 0.10 (178), respectively. In contrast, S33004 was substantially less active at both SERTs and NETs: pKi values (Ki in nM) = 6.36 ± 0.05 (437) and 5.12 ± 0.10 (7586), respectively. These observations led us to select S33005 for further study.
Interaction of S33005 as Compared with Reference Antidepressant Agents at Cerebral Rat SERTs, NETs, and DATs.
As mentioned above, S33005 possessed pronounced affinity for cerebral rat SERTs and less marked affinity for cerebral rat NETs. A similar pattern of preferential interaction with native SERTs versus NETs was acquired with venlafaxine. However, its affinity at these sites was, respectively, 11- and 6-fold lower than those of S33005 (Fig. 2; Table 1). Clomipramine also resembled S33005 in its dual interaction at SERTs and native NETs, with a clear preference for the former. In distinction to S33005, citalopram revealed pronounced affinity for rat SERTs and negligible affinity for NETs. On the other hand, reboxetine displayed an opposite pattern of preference with superior affinity for native NETs as compared with SERTs. S33005 and all other drugs manifested low affinity for cerebral rat DATs.
Interaction of S33005 as Compared with Reference Antidepressant Agents at Cloned hSERTs, hNETs, and hDATs.
In analogy to native rat SERTs, S33005 showed high affinity for heterologously expressed hSERTs (Table 1). However, its affinity for hNETs was about 10-foldlower than at native rat NETs. Venlafaxine similarly showed (∼8-fold) lower affinity for hNETs than for native NETs. In addition, its affinity for hSERTs was somewhat less pronounced than at native SERTs. In distinction, clomipramine showed more pronounced affinity for hSERTs versus SERTs and slightly inferior affinity for hNETs versus native NETs. Citalopram presented ∼3-fold lower affinity at hSERTs versus native SERTs. Its affinity was low at hNETs. Finally, the affinity of reboxetine for hSERTs was less pronounced than its affinity at hNETs. At both native DATs and cloned hDATs, the affinity of S33005 and other ligands for hDAT sites was low.
Interaction of S33005 as Compared with Reference Antidepressant Agents at Additional Sites.
S33005 displayed negligible affinity (pKi < 5.0) for multiple monoaminergic receptors, including diverse sites shown in Table 1 that, as autoreceptors and heteroceptors, modulate corticolimbic serotonergic, adrenergic, and dopaminergic transmission (see Millan et al., 2000b). It also showed negligible (pKi < 5.0) affinity for histaminergic (H)1 receptors labeled with [3H]pyralamine (1.0 nM), and for cloned, human, muscarinic h(M)1, hM2, hM3, and hM4 receptors labeled with [3H]N-methyl-scopolamine (1.0 nM) (not shown). S33005 did not bind (pKi< 5.0) to either monoamine oxidase A or monoamine oxidase B (not shown). At a broad diversity of other receptors, enzymes, and ion channels (>50 sites), S33005 also showed low affinity (pKi < 5.0). S33005 was thus highly selective for SERTs and NETs. Like S33005, venlafaxine showed low affinity (pKi < 5.0) for various monoaminergic receptors and H1 and hM1 receptors. The affinity of clomipramine at h5-HT1B, h5-HT1D, and 5-HT3 receptors was negligible, although it showed marked affinity for h5-HT2A and h5-HT2C receptors, as well as modest affinity for 5-HT3 receptors. At native α1-ARs and cloned hα1A-ARs, its affinity was marked, whereas its affinity for native rat α2-ARs and cloned hα2A-ARs was low. At hD2and hD3 receptors, the affinity of clomipramine was modest. Clomipramine also displayed high affinity for both H1 and hM1 receptors: pKi values (Ki in nM), 8.0 (10) and 7.5 (31.6), respectively. Citalopram manifested negligible affinity for monoaminergic receptors, with the exception of h5-HT2C sites for which it showed mild affinity. It showed modest affinity for H1 receptors: pKi values (Ki in nM) = 6.3 (501). Finally, reboxetine showed modest affinity for h5-HT2Csites but negligible affinity for all other sites.
Influence of S33005 as Compared with Reference Antidepressant Agents upon Uptake of 5-HT, NE, and DA into Rat Synaptosomes.
In line with its high affinity at SERTs, S33005 potently and concentration dependently inhibited the uptake of [3H]5-HT into cerebral rat synaptosomes (Table 2). It also inhibited [3H]NE uptake at higher concentrations, in line with its lower affinity for NETs. In distinction, only very high concentrations of S33005 modified [3H]DA uptake. A similar pattern of data was acquired for venlafaxine although, in line with its lower affinity than S33005 for native SERTs and NETs, venlafaxine was less potent than S33005 in inhibiting uptake of [3H]5-HT and [3H]NE. Clomipramine also preferentially inhibited [3H]5-HT versus [3H]NE uptake and weakly inhibited [3H]DA uptake. In contrast, in line with its selective interaction with native SERTs versus NETs, citalopram selectively inhibited synaptosomal uptake of [3H]5-HT versus [3H]NE (and [3H]DA). On the other hand, in analogy to its binding profile, reboxetine behaved as a preferential inhibitor of [3H]NE versus [3H]5-HT uptake. It failed to modify [3H]DA uptake.
Influence of S33005 as Compared with Reference Antidepressant Agents upon Depletion of Cerebral Pools of 5-HT by PCA.
The administration of PCA (5.0 mg/kg, i.p.) elicited a pronounced reduction of levels of 5-HT in both the frontal cortex and the hippocampus (Fig.3): frontal cortex, vehicle/vehicle (n = 12), 3.61 ± 0.24 ng/mg of protein versus vehicle/PCA (n = 10), 0.87 ± 0.09,P < 0.001; hippocampus, vehicle/vehicle (n = 12), 4.23 ± 0.22 ng/mg of protein versus vehicle/PCA (n = 10), 0.43 ± 0.08,P < 0.001. Preadministration of S33005 dose dependently, potently, and completely blocked the influence of PCA upon 5-HT levels in both frontal cortex and hippocampus. Administered alone, S33005 did not significantly modify 5-HT levels. For a dose of 10.0 mg/kg, s.c (which abolished the action of PCA): frontal cortex, vehicle/vehicle (n = 12), 3.61 ± 0.24 ng/mg of protein versus S33005/vehicle (n = 10), 3.76 ± 0.21, P > 0.05; hippocampus, vehicle/vehicle (n = 12), 4.23 ± 0.22 ng/mg of protein versus S33005/vehicle (n = 10) 4.54 ± 0.2,P > 0.05. Venlafaxine also inhibited the influence of PCA upon levels of 5-HT in frontal cortex and hippocampus, although it was less potently active than S33005. Citalopram was also highly active, whereas clomipramine less potently attenuated the action of PCA. Reboxetine (10.0 mg/kg, s.c.) did not significantly modify the action of PCA (not shown). Administered alone, venlafaxine, citalopram, clomipramine, and reboxetine did not significantly affect basal levels of 5-HT in either frontal cortex or hippocampus (not shown).
Influence of S33005 as Compared with Reference Antidepressant Agents upon the Electrical Activity of Monoaminergic Cell Bodies.
S33005 potently, dose dependently, and completely inhibited the firing rate of serotonergic perikarya localized in the DRN of anaesthetized rats (Figs. 4 and 5). This action was abolished by the selective 5-HT1Areceptor antagonist, WAY100,635. Expressed relative to baseline values (0%), S33005 (0.125 mg/kg, i.v.) + vehicle = −100.0 ± 0.0% versus S33005 + WAY100,635 (0.031 mg/kg, i.v.) = −1.1 ± 7.6%, P < 0.001. In line with previous work (Millan et al., 2000b), WAY100,635 did not modify the electrical activity of serotonergic neurones alone (not shown). Over a higher dose range, S33005 dose dependently reduced the firing rate of adrenergic neurones of the LC, an action abolished by the α2-AR antagonist idazoxan. S33005 (2.0 mg/kg, i.v.) + vehicle = −98.8 ± 0.3% versus S33005 + idazoxan (0.063 mg/kg, i.v.) = −13.3 ± 5.2%, P < 0.001. Administered alone, idazoxan elicited a significant increase in firing rate. Idazoxan (0.063) = +42.0 ± 14.0% versus vehicle = +1.3 ± 2.2%, P < 0.05. In contrast to serotonergic and adrenergic neurones, S33005 exerted little influence upon dopaminergic neurones of the ventrotegmental area. A slight tendency toward an increase in firing rate was seen at modest doses, which transformed into a tendency for inhibition at high doses, but neither effect attained statistical significance. In addition, S33005 did not modify the firing pattern (regular or burst) of dopaminergic neurones (not shown). In contrast, as a positive internal control, the selective DA reuptake inhibitor, GBR12935, dose dependently inhibited firing of ventrotegmental area dopaminergic neurones: ID50 = 0.8 (0.6–1.1) mg/kg, i.v. Its actions were blocked by the D2/D3 antagonist, haloperidol (0.016 mg/kg, i.v.). Vehicle + GBR12935 (4.0 mg/kg, i.v.) = −83.4 ± 12.5 versus haloperidol + GBR12935 = −2.2 ± 2.2%, P < 0.001.
Venlafaxine likewise inhibited both serotonergic and, less potently, adrenergic cell bodies without affecting dopaminergic neurones. However, its potency was less pronounced than that of S33005. Its influence upon serotonergic and adrenergic neurones was reversed by WAY100,635 and idazoxan, respectively. For DRN, venlafaxine (1.0 mg/kg, i.v.) + vehicle = −100.0 ± 0.0% versus venlafaxine + WAY100,635 (0.031 mg/kg, i.v.) = −14.3 ± 12.8%,P < 0.01. For LC, venlafaxine (4.0 mg/kg, i.v.) + vehicle = −93.1 ± 4.7% versus venlafaxine + idazoxan (0.063 mg/kg, i.v.) = −18.7 ± 8.5%, P < 0.001. Clomipramine inhibited serotonergic neurones less potently than S33005, an action blocked by WAY100,635. It only decreased the firing rate of adrenergic neurones at a high dose, an effect antagonized by idazoxan. For DRN, clomipramine (0.5 mg/kg, i.v.) + vehicle = −90.7 ± 6.5% versus clomipramine + WAY100,635 (0.031 mg/kg, i.v.) = −1.5 ± 11.2%, P < 0.001. For LC, clomipramine (8.0 mg/kg, i.v.) + vehicle = −63.3 ± 15.5% versus clomipramine + idazoxan (0.063 mg/kg, i.v.) = +35.1 ± 10.1, P < 0.01. In contrast, citalopram selectively and WAY100,635-reversibly suppressed the activity of serotonergic versus adrenergic neurones. For DRN, citalopram (0.5 mg/kg, i.v.) + vehicle = −100.0 ± 0.0% versus citalopram + WAY100,635 (0.031 mg/kg, i.v.) = +58.6 ± 32.5%, P < 0.001. It slightly enhanced the activity of LC adrenergic cell bodies and marginally excited ventrotegmental area dopaminergic neurones at high doses. On the other hand, reboxetine selectively and idazoxan-reversibly inhibited adrenergic neurones. For LC, reboxetine (0.5 mg/kg, i.v.) + vehicle = −100.0 ± 0.0% versus reboxetine + idazoxan (0.063 mg/kg, i.v.) = −5.8 ± 8.6%,P < 0.001. Reboxetine significantlyincreased the activity of serotonergic neurones, whereas dopaminergic perikarya were unaffected.
Influence of S33005 as Compared with Reference Antidepressant Agents upon Extracellular Levels of 5-HT, NE, and DA in Dialysates of the Frontal Cortex of Freely Moving Rats.
S33005 elicited a rapid, sustained, and dose-dependent elevation in extracellular levels of 5-HT in frontal cortex of freely moving rats (Figs.6through 8; Table 3). In the same dialysis samples, levels of NE and DA were likewise augmented. Area under the curve (AUC) analysis revealed that the most pronounced influence of S33005 was, in fact, upon extracellular levels of NE, with those of 5-HT and DA displaying a less marked increase. A similar pattern of data was acquired with both venlafaxine and clomipramine, although they were less potent than S33005. In distinction, citalopram preferentially elevated levels of 5-HT versus NE and DA and only evoked a slight rise in levels of NE and DA even at the highest dose tested. On the contrary, reboxetine potently increased levels of NE and, less markedly, DA without significantly modifying levels of 5-HT. Surprisingly, and for reasons remaining to be clarified, the induction of NE levels by reboxetine was less pronounced at the highest dose evaluated (40.0 mg/kg, s.c.). Upon i.p. and p.o. administration, S33005 also dose dependently (0.63–40.0 mg/kg, in each case), significantly (P < 0.01, in each case), and markedly elevated dialysate levels of 5-HT, NE, and DA in frontal cortex (not shown).
Influence of S33005 as Compared with Reference Antidepressant Agents upon Extracellular Levels of 5-HT, NE, and DA in Dialysates of the Dorsal Hippocampus, Nucleus Accumbens, and Striatum of Freely Moving Rats.
S33005 elicited a pronounced and sustained elevation in levels of 5-HT and NE in the dorsal hippocampus (Fig. 9). For 5-HT AUC analysis, vehicle = 99.2 ± 2.5% versus S33005 = 207.8 ± 6.1%. For NE AUC analysis, vehicle = 97.8 ± 2.5% versus S33005 = 231.7 ± 8.1%. Venlafaxine similarly increased extracellular levels of 5-HT and NE in this structure. For 5-HT AUC analysis, vehicle = 99.2 ± 2.5% versus venlafaxine = 205.2 ± 10.5%. For NE AUC analysis, vehicle = 97.8 ± 2.5% versus venlafaxine = 204.1 ± 7.0%. Clomipramine also enhanced levels of both 5-HT and NE (not shown). For 5-HT,F(1,16) = 30.8, P < 0.01; and AUC analysis, vehicle = 99.2 ± 2.5% versus clomipramine = 165.3 ± 5.9%. For NE, F(1,16) = 34.4,P < 0.01; and AUC analysis, vehicle = 97.8 ± 2.5% versus clomipramine = 189.2 ± 7.4%. In contrast, citalopram selectively elevated levels of 5-HT as compared with NE (not shown). For 5-HT, F(1,15) = 87.0, P < 0.01; and AUC analysis, vehicle = 99.2 ± 2.5% versus citalopram = 193.0 ± 9.0%. For NE, F(1,15) = 0.7, P > 0.05; and AUC analysis, vehicle = 97.8 ± 2.5% versus citalopram = 102.1 ± 3.5%. On the other hand, reboxetine selectively increased levels of NE as compared with 5-HT. For 5-HT, F(1,16) = 0.2, P> 0.05; and AUC analysis, vehicle = 99.2 ± 2.5% versus reboxetine = 101.5 ± 3.0%. For NE, F(1,16) = 32.6, P < 0.01; and AUC analysis, vehicle = 97.8 ± 2.5% versus reboxetine = 215.0 ± 8.3%.
In the nucleus accumbens and striatum, S33005 and venlafaxine likewise provoked a pronounced increase in dialysis levels of 5-HT, whereas levels of DA were only marginally affected. Clomipramine and citalopram likewise significantly elevated levels of 5-HT (not shown). Accumbens, citalopram, F(1,11) = 22.3, P < 0.01; and AUC analysis, vehicle = 97.7 ± 2.0% versus citalopram = 178.0 ± 7.3%. Clomipramine,F(1,10) = 89.9, P < 0.01; and AUC analysis, vehicle = 99.2 ± 2.5% versus clomipramine = 196.5 ± 7.4%. Striatum, citalopram, F(1,11) = 19.9, P < 0.01; and AUC analysis, vehicle = 102.6 ± 2.9% versus citalopram = 185.9 ± 7.9%. Clomipramine, F(1,11) = 105.3, P < 0.01; and AUC analysis, vehicle = 102.6 ± 2.9% versus clomipramine = 191.2 ± 6.0%. However, they did not significantly affect levels of DA (not shown). Accumbens, citalopram,F(1,11) = 0.4, P > 0.05; and AUC analysis, vehicle = 99.0 ± 1.6% versus citalopram = 101.2 ± 1.3%. Clomipramine, F(1,10) = 1.6,P > 0.05; and AUC analysis, vehicle = 99.0 ± 1.6% versus clomipramine = 108.4 ± 2.9%. Striatum, citalopram, F(1,12) = 0.1, P > 0.05; and AUC analysis, vehicle = 97.4 ± 1.2% versus citalopram = 96.3 ± 2.2%. Clomipramine,F(1,12) = 1.4, P > 0.05; and AUC analysis, vehicle = 97.4 ± 1.2% versus clomipramine = 106.0 ± 2.4%. Reboxetine did not affect levels of 5-HT and DA in either the accumbens or the striatum (data not shown).
Used as a positive, internal control, the selective DA uptake inhibitor, GBR12935, selectively elevated DA as compared with 5-HT levels in both nucleus accumbens and striatum (not shown). Nucleus accumbens: for 5-HT, F(1,10) = 1.4, P> 0.05; and AUC analysis, vehicle = 97.7 ± 2.0% versus GBR12935 = 104.6 ± 2.9%. For DA, F(1,10) = 18.8, P < 0.01; and AUC analysis, vehicle = 99.0 ± 1.6% versus GBR12935 = 326.4 ± 18.6%. Striatum: for 5-HT, F(1,9) = 0.3, P > 0.05; and AUC analysis, vehicle = 102.6 ± 2.9% versus GBR12935 = 105.4 ± 2.8%. For DA, F(1,11) = 44.5, P < 0.01; and AUC analysis, vehicle = 97.4 ± 1.2% versus GBR12935 = 254.6 ± 10.5%.
Influence of Chronic Administration of S33005 as Compared with Venlafaxine upon Extracellular Levels of 5-HT, NE, and DA in Frontal Cortex and the Electrical Activity of DRN Serotonergic Neurones.
Following administration of S33005 for 2 weeks, there was no significant modification in basal dialysate levels of DA in frontal cortex (Fig. 10). Levels of 5-HT showed a (nonsignificant) tendency for an increase. Furthermore, there was a significant elevation in basal levels of NE. Expressed relative to basal levels, an additional injection of S33005 at the dose used for chronic treatment (10.0 mg/kg, s.c.) evoked an elevation in frontocortical dialysate levels of 5-HT, NE, and DA similar to that seen in rats treated chronically with vehicle (Fig. 11). A comparable pattern of data was obtained for venlafaxine inasmuch as the influence of its acute administration upon 5-HT, NE, and DA levels in frontal cortex was not significantly modified by chronic administration (not shown). Nevertheless, in distinction to S33005, venlafaxine did not show an increase in basal levels of NE following chronic administration (Fig.10).
The potency of S33005 for inhibition of the firing rate of DRN serotonergic neurones was not modified following its administration for 2 weeks: ID50 (mg/kg, i.v.; 95% CL) for chronic vehicle = 0.18 (0.14–0.21) versus chronic S33005 = 0.18 (0.14–0.20). Similar findings were acquired for venlafaxine: chronic vehicle = 0.09 (0.6–0.14) versus chronic venlafaxine = 0.07 (0.05–0.10).
Influence Upon Cortical Levels of 5-HT2A Receptors and β-ARs.
Following chronic (2 weeks) administration of S33005, relative to vehicle treatment (Bmax, 22.9 ± 1.3 fmol/mg of tissue = 100.0 ± 5.5%), there was a significant (P < 0.05) reduction in the density (Bmax) of 5-HT2Areceptors in frontal cortex (85.0 ± 2.5%) in the absence of an alteration of affinity (vehicle, KD = 1.64 ± 0.12 nM versus S33005, KD= 1.39 ± 0.08 nM) (Fig. 12). Venlafaxine tended to decrease the density of 5-HT2A receptors, although this effect did not attain statistical significance (92.2 ± 3.7%, P> 0.05); the KD was also not affected (1.64 ± 0.14 nM). At 3 weeks, relative to vehicle (Bmax, 28.0 ± 1.4 fmol/mg of tissue = 100.0 ± 5.0%), both S33005 and venlafaxine yielded a significant decrease in Bmax: 79.6 ± 2.8 and 77.8 ± 2.8%, respectively,P < 0.01 in each case.KD values were unaffected: vehicle = 2.27 ± 0.11 nM, S33005 = 2.03 ± 0.19 nM, and venlafaxine = 2.18 ± 0.21 nM. Compared with vehicle-treated controls (Bmax, 5.58 ± 0.27 fmol/mg of tissue = 100.0 ± 4.6%), following 2 weeks of treatment, S33005 tended to decrease levels of β-ARs in cortex (86.6 ± 4.2%), but this effect just failed to reach statistical significance (P = 0.056). Venlafaxine, in distinction, exerted little influence upon β-ARs (97.7 ± 5.3%). Following 3 weeks of administration, S33005 and venlafaxine did not significantly modify β-AR density or affinity (not shown).
Discussion
Receptor Binding Profile.
S33005 possessed pronounced affinity for native rat and cloned human SERTs, yielding pKi values similar to citalopram and clomipramine and superior to venlafaxine and reboxetine (Muth et al., 1991; Owens et al., 1997; Tatsumi et al., 1997; Béı̈que et al., 1999; Riva et al., 1999). Citalopram was ∼3-foldless potent at human versus rat SERTs. This observation, obtained with [3H]paroxetine, may be compared with ratios of 2- and 10-fold for binding studies performed with [3H]citalopram at rat SERTs as compared with [3H]citalopram and [3H]5-HT, respectively, at hSERTs (Owens et al., 1997). On the contrary, clomipramine shows ∼10-foldhigher affinity at human versus rat SERTs (Table 1; Owens et al., 1997). Such contrasting affinities (Barker and Blakely, 1995; Sur et al., 1998) emphasize the importance of determining drug affinities in both species.
Although S33005 showed relatively modest affinity at rat NETs, its actions at these sites are functionally important (see below). Indeed, this “dual” profile may be distinguished from citalopram (SERT-selective) and reboxetine (NET-preferential) and resembles clomipramine and venlafaxine, although the latter was a substantially weaker ligand. In fact, the affinity of venlafaxine (22 nM) at rat SERTs corresponds well to the studies of Owens et al. (1997),Béı̈que et al. (1999), and Muth et al. (1986). Although its affinity at rat NETs was only 998 nM, this value is close to those of 1260 and 1067 reported by Béı̈que et al. (1999) andOwens et al. (1997), respectively. Similarly, the affinity of venlafaxine for hSERTs (76 nM) coincides with previous values (Owens et al., 1997; Tatsumi et al., 1997), while its lower affinity for hNETs (6310 nM) bears comparison to values of 2269 and 1644 obtained with [3H]nisoxetine and [3H]norepinephrine, respectively, by Owens et al. (1997), and of 1060 with [3H]nisoxetine byTatsumi et al. (1997). Indeed the 83-fold preference of venlafaxine for hSERTs versus hNETs seen herein corresponds well to values of 283 and 118 for Owens et al. (1997) and Tatsumi et al. (1997), respectively.
While Owens et al. (1997) found 2-fold lower affinity of venlafaxine at human versus rat NETs, the ratio was 6-fold herein and 10-fold for S33005. Venlafaxine and S33005 are chemically related, and this human/rat difference is a common feature of such derivatives (A. Newman-Tancredi and M. J. Millan, unpublished observations). Apart from methodological aspects (tissue versus cloned sites), species differences or contrasting interactions withmultiple binding sites on NETs may be involved (Barker and Blakely, 1995; Sur et al., 1998). In addition, several pharmacologically distinct isoforms of NETs are differentially localized in the central nervous system (Hughes and Stanford, 1998;Kitayama et al., 1999). Their existence may also be pertinent to the disparity between the weak activity of venlafaxine at rat NETs as compared with its potent influence upon extracellular levels of NE in vivo (Bolden-Watson and Richelson, 1993; Hughes and Stanford, 1998;Béı̈que et al., 1999, 2000) (see below).
The affinity of S33005 for >50 receptors, enzymes, and ion channels was negligible, supporting attribution of its functional actions to SERTs and NETs. In corroboration of previous studies, citalopram and clomipramine displayed modest affinity for h5-HT2C sites, at which they displayantagonist properties (Pälvimäki et al., 1996;Millan et al., 2000a), and reboxetine also revealed mild affinity for h5-HT2C receptors. While blockade (or down-regulation) of 5-HT2C sites favorably influences mood, it may also elicit hyperphagia (see Millan et al., 2000b). Moreover, antagonism by clomipramine of H1 receptors contributes to weight gain, while its cardiovascular autonomic and sedative side effects reflect actions at α1-ARs, H1, and M1 receptors (Burke and Preskorn, 1995; Owens et al., 1997). The absence of affinity of S33005 for these sites is, thus, important.
Prevention of PCA-Induced 5-HT Depletion.
Following access to serotonergic terminals via SERTs, PCA triggers 5-HT release (Fuller et al., 1991). Correspondingly, S33005, venlafaxine, citalopram, and clomipramine suppressed depletion of cerebral pools of 5-HT by PCA, in analogy to their attenuation of its behavioral actions (accompanying paper), whereas reboxetine was inactive. The high potency of S33005 versus venlafaxine in this model underpins in vitro observations discussed above.
Electrical Activity of Monoaminergic Neurones.
Reflecting its marked activity at SERTs, S33005 potently and WAY100,635-reversibly inhibited the firing rate of serotonergic perikarya, and, in line with its lower activity at NETs, higher doses of S33005 idazoxan-reversibly inhibited the electrical activity of adrenergic cell bodies. In correspondence with binding studies discussed above, such actions were expressed less potently by venlafaxine (Muth et al., 1991;Gartside et al., 1997; Béı̈que et al., 1999). Clomipramine likewise inhibited electrical activity of the DRN but only weakly affected the LC, probably since its actions at sites other than NETs influence the activity of adrenergic cell bodies (Scuvee-Moreau and Dresse, 1979; Gartside et al., 1997). This combined inhibition of serotonergic and adrenergic neurones by S33005 contrasted with their selective inhibition by citalopram and reboxetine, respectively (Popik, 1999; Riva et al., 1999). Interestingly, the potency of S33005 as compared with citalopram and reboxetine for inhibition of serotonergic and adrenergic neurones, respectively, was pronounced when compared with their relative affinities for SERTs (S33005 versus citalopram) and NETs (S33005 versus reboxetine). This particularly high activity of S33005 in vivo, underpinned by dialysis studies considered below, may reflect pharmacokinetic factors or, possibly, the existence of specific cerebral isoforms of SERTs and NETs for which S33005 has higher affinity than revealed by binding studies (see below). Notably, ventrotegmental area-localized dopaminergic cell bodies were not inhibited by S33005, consistent with its low activity at DATs. Indeed, although a modest inhibitory influence of fluoxetine upon dopaminergic neurones was suggested to underlie (rare) cases of akathisia (Prisco and Esposito, 1995), we have not found fluoxetine or citalopram to inhibit dopaminergic neurones in our studies (Fig. 5; Millan et al., 2000b). Rather, the latter slightly excited dopaminergic perikarya at high doses, an observation also acquired with clomipramine and reboxetine and justifying further characterization.
In a previous study, Wong et al. (2000) showed that high doses of reboxetine (2.0–10.0 mg/kg, i.v.) inhibit the electrical activity of serotonergic neurones, presumably due to a loss of selectivity versus SERTs. They did not, however, report on the effects of lower, NET-selective doses of reboxetine corresponding to those inhibiting adrenergic neurones. Indeed, an intriguing observation of the present study was that modest doses of reboxetine excited serotonergic perikarya. This finding probably reflects actions of reboxetine at NETs localized on adrenergic terminals innervating the DRN, inasmuch as serotonergic dendrites therein bear excitatory α1-ARs (Day et al., 1997; Haddjeri et al., 1995; Millan et al., 2000b). We are currently further examining this issue using other NARIs and selective α1-AR antagonists.
Influence upon Extracellular Levels of Monoamines.
In support of its potent interaction at SERTs, S33005 markedly and dose dependently elevated dialysate levels of 5-HT in frontal cortex, hippocampus, and nucleus accumbens, structures implicated in the etiology and treatment of depressive states. Comparable, dose-dependent actions of venlafaxine and citalopram were observed, extending previous studies (Béı̈que et al., 1999, 2000; Popik, 1999;Millan et al., 2000b). Clomipramine acted similarly, whereas reboxetine (a weak ligand of SERTs) was ineffective and, in amplification of a single-dose study by Sacchetti et al. (1999), reboxetine dose dependently elevated frontocortical levels of NE. Most striking, however, was the pronounced influence of S33005, venlafaxine, and clomipramine upon dialysate levels of NE despite their preferential interaction with SERTs versus NETs in vitro. The reasons underlying this marked influence upon adrenergic transmission in vivo (Béı̈que et al., 1999, 2000; Dawson et al., 1999) require clarification. However, as alluded to above, S33005, venlafaxine, and clomipramine may potently interact with a distinctive population of NETs controlling NE levels in corticolimbic structures (Hughes and Stanford, 1998; Béı̈que et al., 1999, 2000; Kitayama et al., 1999). This may also be pertinent to the mild influence of high doses of citalopram (and other SSRIs) upon NE levels, an action mediated independently of serotonergic mechanisms (Millan et al., 2000b).
While S33005 dose dependently elevated frontocortical levels of DA, an action mimicked by venlafaxine, clomipramine, and reboxetine, citalopram—in line with a local injection study (Pozzi et al., 1999)—was little effective. This increase in frontocortical levels of DA by S33005 is important since a deficiency in frontocortical (and subcortical) dopaminergic transmission contributes to depressive states (Zacharko and Anisman, 1991; Willner, 1995; Di Chiara and Tanda, 1997;Merriam et al., 1999), while a common property of antidepressants is an elevation in extracellular levels of DA in frontal cortex (see Millan et al., 2000b). S33005 and the other antidepressants evaluated herein show weak affinity for DATs and little modified subcortical levels of DA. An indirect influence upon DA release via serotonergic mechanisms is also unlikely to underlie elevations in extracellular levels of DA in frontal cortex since (low doses of) citalopram selectively elevate 5-HT levels without affecting DA. Furthermore, lesions of serotonergic neurones and 5-HT antagonists fail to block the influence of SSRIs upon DA levels in frontal cortex (Pozzi et al., 1999; Millan et al., 2000b). It is probable, therefore, that increases in frontocortical levels of DA elicited by S33005 follow those of NE due to the key role of NETs in clearing extracellular DA in this region (Yamamoto and Novotney, 1998; Millan et al., 2000b).
Chronic Administration.
Chronic administration of tricyclics reduces cortical levels of β-ARs, but the significance of this change to the evolution of antidepressant efficacy has been challenged since it occurs rapidly (1–3 days) (Okada and Tokumitsu, 1994;Newman-Tancredi et al., 1996; Anand and Charney, 2000). Moreover, several classes of antidepressant, including SSRIs, do not reliably down-regulate β-ARs (Okada and Tokumitsu, 1994; Millan et al., 1997). Although venlafaxine desensitized pineal β-ARs, it failed to reduce central β-ARs (Muth et al., 1986; Schweizer et al., 1997; Nalepa et al., 1998), an observation confirmed herein, and extended to S33005. In contrast to β-ARs, down-regulation of cortical 5-HT2A receptors gradually increases over several (2–3) weeks of administration, paralleling clinical progression of therapeutic activity (Maes and Meltzer, 1995; Newman-Tancredi et al., 1996). Furthermore, antidepressant treatment of depressed patients likewise decreases levels of 5-HT2A receptors (Yatham et al., 1999a,b). Thus, it is important that S33005 and venlafaxine reduced the density of 5-HT2Areceptors in frontal cortex. Interestingly, S33005, but not venlafaxine, significantly diminished 5-HT2Areceptor density at 2 weeks. Adrenergic mechanisms have been implicated in the influence of antidepressants upon 5-HT2Asites (Gravel and de Montigny, 1987; Yatham et al., 1999a,b), so this difference may reflect the elevation in extracellular NE levels observed with S33005 but not venlafaxine at this time. Although this rapid reduction in 5-HT2A receptor density with S33005 provides an interesting distinction to venlafaxine, and hints at a more rapid onset of action, it should be regarded as a “marker” rather than as a mechanism of antidepressant action. Indeed, the relationship between activity at 5-HT2A receptors and depressive states is still under experimental and clinical exploration, and decreased transmission at 5-HT2A sites is unlikely to be sufficient for expression of antidepressant properties (Yatham et al., 1999a,b;Meyer et al., 2001).
This increase in frontocortical NE levels with S33005 may reflect (partial) desensitization of α2-AR autoreceptors (Mongeau et al., 1994; Invernizzi et al., 2001), although the identical response to additional administration of S33005 after 2 weeks of pretreatment provides no further evidence for this possibility. Moreover, notwithstanding the potential importance of 5-HT1A autoreceptor desensitization (Blier and de Montigny, 1994), 2 weeks of administration of S33005 or venlafaxine was insufficient to provoke changes inasmuch as basal levels of 5-HT and NE were not significantly modified (Béı̈que et al., 2000). The (acute) influence of S33005 and venlafaxine upon frontocortical levels of 5-HT and DRN firing rate was also not attenuated. Similarly, chronic (3 weeks) administration of venlafaxine did not modify basal levels (or in vitro release) of 5-HT and NE in a previous study of the hippocampus (Béı̈que et al., 2000). Notably, the latter observations were obtained by using administration of venlafaxine by osmotic minipumps, which allows for a sustained, high, steady-state level of drug. Herein, although the duration of action of S33005 and venlafaxine in the rat is modest (4–8 h), we preferred the approach of repeated injections since this most clearly resembles the clinical condition. Additional studies with S33005 using both injection and minipump treatment studies, longer treatment times, autoreceptor agonists, and behavioral parameters will be necessary to further characterize potential adaptive changes in monoaminergic transmission following its long-term administration (Béı̈que et al., 2000; Kalsner, 2000).
Summary and Conclusions.
In conclusion, the novel cyclobutane derivative, S33005, potently interacts with SERTs and, less markedly, with NETs. In freely moving rats, S33005 markedly elevates extracellular levels of 5-HT and NE throughout corticolimbic structures, as well as DA levels in frontal cortex. S33005 may, thus, be differentiated from the SSRI, citalopram, and from the NE reuptake inhibitor, reboxetine. Although S33005 resembles venlafaxine, it displays higher potency. This preferential SERT > NET profile also bears comparison with clomipramine, but S33005 is devoid of the latter's affinity for adrenergic, histaminergic, and muscarinic receptors. In line with these observations, S33005 displays robust activity in behavioral paradigms suggestive of antidepressant activity (accompanying paper).
Acknowledgments
We thank Laurence Verrièle, Christine Chaput, Manuelle Touzard, Valérie Pasteau, Laetitia Cistarelli, Christophe Melon, Loretta Iob, and Jimmy Mullot for technical assistance.
Footnotes
- Abbreviations:
- 5-HT
- serotonin
- AUC
- area under the curve
- AR
- adrenoceptor
- DA
- dopamine
- DAT
- dopamine transporter
- DRN
- dorsal raphe nucleus
- GBR12935
- 1-[2-(diphenylmethoxy)ethyl]-4-(3-phenylpropyl)-piperazine
- ANOVA
- analysis of variance
- CL
- confidence limit
- AP
- lateral
- L
- anteroposterior
- H
- height
- LC
- locus ceruleus
- NE
- norepinephrine
- NET
- norepinephrine transporter
- PCA
- parachloroamphetamine
- S33005
- (−)1-(1-dimethylaminomethyl) 5-methoxybenzocyclobutan-1-yl) cyclohexanol
- SERT
- serotonin transporter
- SSRI
- selective serotonin reuptake inhibitor
- WAY100,635
- N-{2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl}-N-(2-pyridinyl) cyclo hexanecarboxamide
- VTA
- ventrotegmental area
- h
- human
- CHO
- Chinese hamster ovary
- M
- muscarinic
- Received December 26, 2000.
- Accepted March 29, 2001.
- The American Society for Pharmacology and Experimental Therapeutics