Effects of continuous administration of paroxetine on ligand binding site and expression of serotonin transporter protein in mouse brain

Brain Research 1053 (2005) 154 – 161 www.elsevier.com/locate/brainres

Research Report

Effects of continuous administration of paroxetine on ligand binding site and expression of serotonin transporter protein in mouse brain Kazufumi Hirano a, Takahiro Seki b, Norio Sakai b, Yasuhiro Kato c, Hisakuni Hashimoto c, Shinya Uchida a, Shizuo Yamada a,* a

Department of Biopharmaceutical Sciences and Center of Excellence (COE) Program in the 21st Century, School of Pharmaceutical Sciences, University of Shizuoka, 52-1 Yada, Shizuoka 422-8526, Japan b Department of Molecular and Pharmacological Neuroscience, Graduate School of Biomedical Sciences, Hiroshima University, 1-2-3 Kasumi, Minami-ku, Hiroshima 734-8551, Japan c Department of Hospital Pharmacy, Hamamatsu University of School of Medicine, 1-20-1 Handayama, Hamamatsu 431-3192, Japan Accepted 15 June 2005 Available online 26 July 2005

Abstract Selective serotonin reuptake inhibitors (SSRIs), such as paroxetine, are utilized in the treatment of depression and anxiety disorders. Although SSRIs potently interfere with the activity of brain serotonin transporter (SERT) after acute treatment, clinical improvement of psychiatric diseases is observed only after the repeated administration for several weeks (2 – 6 weeks). The present study was undertaken to investigate the effects of continuous administration of paroxetine on specific [3H]paroxetine binding sites and expression of SERT protein in mouse brain. Mice continuously and subcutaneously received paroxetine at doses of 2.67 or 13.3 Amol/kg/day for 21 days by using osmotic minipumps, and the steady-state plasma drug levels were within the range of reported concentrations in the clinical therapy. Continuous administration of paroxetine at theses doses produced significant (25 – 46%) reduction of [3H]paroxetine binding in each brain region (cerebral cortex, striatum, hippocampus, thalamus, midbrain) of mice. In Western blot analysis, expression levels of SERT protein in the thalamus and midbrain of mice were significantly (51% and 61%, respectively) decreased on day 21 after the implantation of minipumps at the higher dose. In conclusion, this study has firstly shown that continuous administration of paroxetine induces significant reduction of not only ligand binding sites of SERT but the protein expression level in mouse brain. Such down-regulation of SERT may partly underlie the therapeutic effect of long-term treatment with SSRIs in human. D 2005 Elsevier B.V. All rights reserved. Theme: Neurotransmitters, modulators, transporters and receptors Topic: Uptake and transporters Keywords: Selective serotonin reuptake inhibitor; Serotonin transporter; Chronic treatment

1. Introduction Selective serotonin reuptake inhibitors (SSRIs), such as paroxetine, are one of the most frequently prescribed therapeutic agents in all medicines, and they show diverse therapeutic actions on various psychiatric disorders includ-

* Corresponding author. Fax: +81 54 264 5635. E-mail address: [email protected] (S. Yamada). 0006-8993/$ - see front matter D 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2005.06.038

ing depression, obsessive – compulsive disorder, panic disorder and other conditions as well [15,29]. Serotonin (5-hydroxytryptamine, 5-HT) has long been known to have a multitude of different physiological actions (e.g., mood, anxiety, sleep, temperature, appetite, sexual behavior and eating behavior) due to the wide variety of 5-HT receptors [2]; and serotonin transporter (SERT) predominantly regulates the synaptic concentration of released 5-HT [28]. It is considered that depression and other psychiatric disorders are caused by chronically low levels of seroto-

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nergic neurotransmission, and SSRIs potently and selectively interfere with the activity of brain SERT under in vitro [23] and in vivo [12,13] conditions, resulting in the enhancement of serotonergic neurotransmission. Although SSRIs inhibit brain SERT activity after acute treatment, it is known that clinical improvement of psychiatric diseases is observed only after the repeated treatment for several weeks (2 – 6 weeks) [5,6]. This delayed therapeutic response is a common property of all antidepressants, suggesting that acute uptake inhibition initiates the cascade of events that eventually bring about clinical alleviation. Therefore, it is predicted that adaptive processes in brain serotonergic system would underlie the antidepressive effect of SSRIs. It is well known that serotonergic neurotransmission is regulated by not only SERT but also its autoreceptors. Namely, two types of serotonergic autoreceptors with different neuronal locations have been identified. One is somatodendritic 5-HT1A autoreceptors in the raphe nuclei, which play an important role in the regulation of serotonergic cell firing and neurotransmitter release into the synaptic clefts [11,34], and the other is 5-HT1B autoreceptors that are located on axon terminals in the serotonergic projection areas such as hippocampus, hypothalamus and frontal cortex [20,34]. To date, several studies have been undertaken to elucidate the mechanism of antidepressant actions. It has been reported that the 5-HT1A agoniststimulated [35S]GTPgS binding in the dorsal raphe was attenuated after chronic treatment of SSRIs without changes in receptor density [24,27], suggesting that somatodendritic 5-HT1A receptors are desensitized through the changes in Gprotein expression level and/or regulatory process of the receptor (e.g., phosphorylation) [11]. Similarly, the sustained administration of SSRI also induced subsensitivity of 5-HT1B autoreceptors in the serotonergic projection areas [22]. These findings were also supported by the clinical study that therapeutic efficacy of SSRIs was augmented by coadministration of 5-HT1A receptor antagonists such as pindolol [1]. Thus, the lag time between onset of medication and therapeutic efficacy is hypothesized to involve the progressive desensitization of 5-HT autoreceptors. In case of brain SERT, chronic antidepressant treatment has been reported to induce significant reduction of radioligand binding sites and SERT function in rat brain [3,4,25,32], while no change [10] and increase [14] were also reported. Thus, the results in the alteration of brain SERT are inconsistent, and it is probable that such discrepancy stems from the distinction of treatment regimen. Because the half-lives of antidepressants in human are considerably longer than those in rodents [7,13], daily oral administration of SSRIs makes it possible to produce sustained pharmacological effects throughout the day under the clinical condition but not in animals. Thus, it is important to make efforts in rodent studies to simulate serum levels after treatment with SSRIs reported in clinical studies. Previously, less well studied is the effect of long-

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term SSRI treatment on the ligand binding sites and protein expression of SERT. Therefore, we have simultaneously measured specific [3H]paroxetine binding and expression levels of SERT protein in the brain of mice maintained at the clinically relevant steady-state plasma concentration of paroxetine. In fact, such steady-state paroxetine level was achieved by subcutaneous implantation of the drugcontaining osmotic minipumps.

2. Materials and methods 2.1. Materials [3H]Paroxetine (706.7 GBq/mmol) was purchased from Dupont-NEN Co. Ltd. (Boston, MA). Paroxetine hydrochloride was kindly donated by GlaxoSmithKline Pharmaceuticals (West Sussex, UK). Rabbit polyclonal antiSERT antibody (against carboxyl-terminal region of SERT) was made as described previously [33], and all other drugs and materials were obtained from commercial sources. 2.2. Drug treatment Male ICR strain mice at 5 weeks of age (SLC, Shizuoka, Japan) were housed five per cage in the laboratory with free access to food and water and maintained on a 12-h dark/ light cycle in a room with controlled temperature (24 T 1 -C) and humidity (55 T 5%). All animal procedures were in strict accordance with the guidelines approved by the Experimental Animal Ethical Committee of University of Shizuoka. Mice were administered paroxetine (2.67 or 13.3 Amol/kg/day) for 5, 10, 15 or 21 days subcutaneously by means of osmotic minipumps (model 2004, Durect, Cupertino, CA), and control animals received vehicle (50% ethanol in distilled water). Subcutaneous implantation of osmotic minipumps was performed under aseptic condition. Briefly, a small incision was made in the skin between the scapulae under light anesthesia with diethyl ether, and a small pocket was formed by spreading the subcutaneous connective tissues apart with a hemostat. The pump was inserted into the pocket, and the skin incision was closed with sutures on day 0. After the procedure mice were housed separately. 2.3. Measurement of [3H]paroxetine binding in mouse brain Osmotic minipumps were removed from mice on day 21, and specific [3H]paroxetine binding in mouse brain was measured at 72 and 120 h (washout period) after cessation of drug treatment. Mice were exsanguinated from descending aorta under light anesthesia with diethyl ether, and brain was perfused with 0.9% NaCl from the aorta. Then, the whole brain tissue was removed and divided into five brain regions (cerebral cortex, hippocampus, striatum, thalamus

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and midbrain). Each brain region was homogenized with 19 volumes of 50 mM Tris – HCl buffer (120 mM NaCl, 5 mM KCl, pH 7.4) with a Polytron homogenizer, and the homogenate was centrifuged at 40,000  g for 15 min. The pellet was resuspended in 24 volumes of the buffer. All steps for the tissue preparation were performed at 4 -C. The binding assay for SERT in brain homogenates from mice was performed by using [3H]paroxetine, as previously described [13]. Briefly, the homogenate (400 Ag of protein) of each brain region was incubated with [3H]paroxetine (2.0 nM) for 2 h at 20 -C in the buffer. Since this radioligand concentration is about 15 times higher than its K d value (0.13 nM), specific binding in each brain region approximates B max values for [3H]paroxetine. The reaction was terminated by rapid filtration (Cell Harvester, Brandel, Gaithersburg, MD, US) through Whatman GF/B glass fiber filters, and filters were rinsed three times with 2 ml of icecold buffer. Tissue-bound radioactivity was extracted from filters overnight in scintillation fluid (2 l of toluene, 1 l of Triton X-100, 15 g of 2,5-diphenyloxazole, 0.3 g of 1,4bis[2-(5-phenyloxazolyl)]benzene) and determined in a liquid scintillation counter. Nonspecific binding of [3H]paroxetine was determined in the presence of 10 AM fluoxetine. All assays were conducted in duplicate. Every binding experiment was performed using fresh tissues. Protein concentration was measured according to the previous method [18] using bovine serum albumin as a standard. 2.4. Immunoblotting for brain SERT On day 5, 10, 15 or 21 after the implantation of osmotic minipumps, mice were exsanguinated from descending aorta without washout periods under light anesthesia with diethyl ether, and brain was perfused with 0.9% NaCl from the aorta. The plasma from mouse blood was isolated by centrifugation and stored at 80 -C until the drug concentration was determined. The brain tissue was removed, and thalamus and midbrain were used for Western blot analysis. Western blotting was performed according to the methods described in the previous report [33]. Briefly, the brain tissues (thalamus and midbrain) of mice were homogenized in 20 volumes of 20 mM Tris – HCl buffer (250 mM sucrose, 2 mM EDTA, 10 mM EGTA, 1 mM PMSF, 20 Ag/ml leupeptin, pH 7.4) with a Teflon-glass homogenizer and centrifuged at 800  g for 5 min. The supernatant was collected and centrifuged again at 18,000  g for 40 min. The pellet was resuspended in homogenizing buffer, and protein concentration was determined with Coomassie Protein Assay reagent (Pierce). The homogenates were mixed with 0.12 mM Tris –HCl buffer (6% SDS, 10% glycerol, pH 6.8) and stored at 4 -C overnight without boiling. Aliquots (20 Ag of protein) were subjected to SDS –PAGE (7.5% SDS) under nonreducing conditions, and the separated protein was transferred to polyvinylidene difluoride membrane. The membrane was

immersed in phosphate-buffered saline containing 0.03% Triton-X and 5% skim milk for 1 h and then incubated with primary anti-SERT antibody (diluted 1:1000) overnight at 4 -C. After washing, the membrane was incubated with a peroxidase-labeled secondary antibody (goat antirabbit IgG) (1:10,000) for 1 h, and immunoreactive bands were visualized by the enhanced chemiluminescence system (ECL plus kit, Amersham Biosciences, UK) with a chemical imager (Fluor-Si MultiImager, BIO-RAD, Tokyo, Japan). The homogenate of human embryo kidney cells (HEK293 cells) stably expressing SERT [21] was utilized as a positive control, and SERT expression level was represented as relative intensity (SERT immunoreactivity of brain homogenates was normalized by that of positive control). 2.5. Determination of paroxetine in plasma The concentration of paroxetine in mouse plasma was determined by column-switching high-performance liquid chromatography (HPLC) with fluvoxamine as an internal standard as previously described [13]. Briefly, 50 Al of internal standard and 50 Al of 2 M NaOH were added to 450 Al of plasma. Samples were then vortex mixed and extracted into 2 ml of n-heptan/3-methyl-1-butanol (985/ 15, v/v) on a reciprocating shaker for 20 min. Then, the upper organic layer was transferred and dried under a gentle stream of nitrogen. The residue reconstituted in 110 Al of mobile phase, and it was analyzed by HPLC. The HPLC system consisted of two pumps (model LC-9A, Shimadzu, Tokyo, Japan), a six-port switching valve (model FCV-2AH, Shimadzu, Tokyo, Japan), a system controller (model SCL-6B, Shimadzu, Tokyo, Japan), an injector (SIL-6B, Shimadzu, Tokyo, Japan) and a variable wavelength UV detector (Hitachi, Tokyo, Japan). The detector was set at 295 nm. Integration of peak area was performed by a computing integrator (model Chromatopac C-R4A, Shimadzu, Tokyo, Japan). Chromatography was performed on a reversed phase. Column I was a Shim-pack SPC-RP3, 9 Am polyvinylalcohol resin, 30 mm  4 mm inside diameter, pretreatment column (Shimadzu, Tokyo, Japan). Column II was a Shim-pack CLC-CN (M), 5 Am cyanopropyl group, 250 mm  4.6 mm inside diameter, analytical column (Shimadzu, Tokyo, Japan). The columns were maintained at 50 -C. The mobile phase consisted of 0.01 M phosphate buffer (pH 6.8), acetonitrile and methanol (100:123:40). The sensitivity limit of plasma assay was 2.5 ng/ml. 2.6. Statistical analysis The results were presented as mean T standard error (SE). Statistical analysis was performed by one-way analysis of variance followed by Dunnett’s test for multiple comparisons. A value of P < 0.05 was considered significant.

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3. Results 3.1. Steady-state levels of paroxetine in mouse plasma Paroxetine concentrations in mouse plasma were measured on days 5, 10, 15 and 21 after implantation of paroxetine-containing osmotic minipumps. As shown in Fig. 1, plasma level of paroxetine became the steady-state from at least day 5 at the dose of 2.67 Amol/kg/day, and respective drug concentrations on days 5, 10, 15 and 21 were 48.7 T 24.4, 66.1 T 10.2, 49.4 T 3.0 and 56.1 T 2.6 ng/ ml, respectively. In case of 13.3 Amol/kg/day, the paroxetine concentration in mouse plasma reached a steady-state level from day 10, and respective plasma concentrations on days 10, 15 and 21 were 325 T 32, 361 T 23 and 343 T 51 ng/ml, respectively. 3.2. Effects of continuous administration of paroxetine on specific [ 3H]paroxetine binding in mouse brain Mice received paroxetine subcutaneously for 21 days, and specific [3H]paroxetine (2 nM) binding in each brain region was measured at 72 or 120 h after the cessation of drug treatment (Fig. 2). Both washout periods were enough for the mouse plasma paroxetine to be below the sensitivity limit by HPLC analysis. Continuous administration of paroxetine (2.67 Amol/kg/day) brought about significant (27.2 –36.0%) decreases in specific [3H]paroxetine binding in the mouse cerebral cortex, striatum, hippocampus, thalamus and midbrain after 72-h washout period compared with the control values. Such reduction of radioligand binding was no longer observed at 120-h washout period. In the treatment with this drug of higher dose (13.3 Amol/kg/ day) for 21 days, there were greater and longer-lasting

Fig. 1. Time courses of plasma paroxetine concentration in mice after implantation of the drug-containing osmotic minipumps. Mice received paroxetine (2.67 or 13.3 Amol/kg/day) subcutaneously on day 0, and they were exsanguinated on day 5, 10, 15 or 21. Concentrations of paroxetine were measured by column-switching HPLC. Each point represents mean T SE (n = 3 – 4).

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decreases (cerebral cortex: 45.8% and 37.1%; striatum: 40.0% and 30.5%; hippocampus: 46.2% and 24.7%; thalamus: 41.7% and 39.0%; and midbrain: 40.3% and 34.3%, respectively) in brain [3H]paroxetine binding after 72- and 120-h washout period. 3.3. Expression levels of brain SERT protein In Western blot analysis, the antibody against carboxylterminal region of SERT protein recognized the SERT with the molecular size of approximately 70 kDa in mouse thalamus and midbrain, which was distinct from that (¨80 kDa) in HEK293 cells (Figs. 3 and 4, lower panels). Both immunoreactive bands were reduced to the same molecular size by peptide N-glycosidase F (PNGase F, Biolabs) treatment, and the antibodies for SERT failed to stain any bands in the cerebellum (very low density of SERT) of mice (data not shown), suggesting that the difference of apparent molecular size of SERT between brain tissue and HEK293 cell is derived from glycosylation level as described in the previous study [33], and that the observed 70-kDa band corresponds to brain SERT of mice. As shown in Fig. 3, continuous administration of paroxetine at the dose of 2.67 Amol/kg/day for 5 –21 days had little effect on SERT protein levels in the mouse thalamus. However, there were tendency of decrease (36.9%, day 15) and significant decrease (50.8%, day 21) in SERT protein level at the higher dose, compared with the vehicle-treated group. Similarly, in the midbrain, higher dose of paroxetine produced significant (60.6%) reduction of SERT protein level on day 21 (Fig. 4).

4. Discussion Previous studies have shown that intermittent (once or twice a day) treatment with citalopram and fluoxetine had no change or increase in rat brain SERT [10,14]. More recently, quantitative autoradiographical studies have shown remarkable reduction of SERT sites in rat brain after continuous administration of paroxetine (13.3 and 26.7 Amol/kg/day for 21 days) or sertraline (21.9 Amol/kg/day for 21 days) [3], accompanied by marked reduction of 5-HT clearance from the extracellular space of brain using in vivo electrochemistry [4]. Such variability in results may be attributable in part to the difference in type of SSRIs used or dose as well as the route and frequency of drug administration. In fact, Benmansour et al. [3,4] used osmotic minipumps to maintain steady-state serum concentrations of SSRIs in rats. It has been reported that the half-life of paroxetine in mouse plasma (3.4 h) after oral administration [13] is much shorter than that in human (21 – 28 h) [5,6] and that a wide range (10 – 600 ng/ml) of steady-state plasma concentration of paroxetine in depressive patients [7] may be due to the large interindividual variability of pharmacokinetics [6]. Considering these previous observations, the present study

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Fig. 2. Effects of chronic treatment with paroxetine on specific [3H]paroxetine binding in mouse brain regions. Mice received paroxetine (2.67 or 13.3 Amol/kg/day) for 21 days subcutaneously by the drug-containing osmotic minipumps, and specific [3H]paroxetine (2 nM) binding in the cerebral cortex, striatum, hippocampus, thalamus and midbrain was measured after 72- or 120-h washout period. Each column represents mean T SE (n = 5). Asterisks show a significant difference from the control values, *P < 0.05, **P < 0.01, ***P < 0.001.

was undertaken to examine the effects of continuous application of paroxetine at steady-state concentration on brain SERT in mice. In fact, the subcutaneous implantation

of the drug-containing osmotic minipump enabled the continuous administration of paroxetine at doses of 2.67 and 13.3 Amol/kg/day, and succeeded in maintaining the

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Fig. 3. Immunoblot analysis for SERT protein in mouse thalamus. Mice received paroxetine (2.67 or 13.3 Amol/kg/day) subcutaneously by the drug-containing osmotic minipumps for 5, 10, 15 or 21 days, and the expression level of brain SERT protein was measured. Samples prepared from HEK293 cell stably expressing SERT was utilized as a positive control, and the SERT expression level was represented as relative intensity. The lower panel shows representative blots of each group as described above. Each column represents mean T SE (n = 3 – 4). Asterisk shows a significant difference from the control values, *P < 0.05.

Fig. 4. Immunoblot analysis for SERT protein in the mouse midbrain. Mice received paroxetine (2.67 or 13.3 Amol/kg/day) subcutaneously by the drugcontaining osmotic minipumps for 5, 10, 15 or 21 days, and the expression level of brain SERT protein was measured. Samples prepared from HEK293 cell stably expressing SERT was utilized as a positive control, and SERT expression level was represented as relative intensity. The lower panel shows representative blots of each group as described above. Each column represents mean T SE (n = 3 – 4). Asterisks show a significant difference from the control values, ***P < 0.001.

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mouse plasma paroxetine concentrations within therapeutic levels (Fig. 1). Such continuous administration of paroxetine (2.67 and 13.3 Amol/kg/day) for 21 days brought about significant dose-related decreases in specific [3H]paroxetine binding to mouse brain regions (cerebral cortex, striatum, hippocampus, thalamus and midbrain) excised after 72- and/or 120-h washout periods of this drug. Such reduction lasted for longer period when the higher dose of paroxetine was applied. This result is essentially consistent with the quantitative autoradiographical study that has shown marked reduction of SERT sites in the rat brain after continuous administration of paroxetine [3]. Inasmuch as extracellular concentrations of brain 5-HT were significantly increased by chronic treatment of SSRIs [16,19], it is considered that significant reduction of brain SERT binding sites after continuous administration of paroxetine may reflect adaptive change of SERT due to excessive exposure to 5-HT in the synaptic cleft. It is well known that SERT is synthesized in both midbrain and brainstem nuclei (e.g., dorsal raphe nucleus) where serotonergic cell bodies are predominantly located and transferred to the projection areas by axonal transport [28,33]. The levels of SERT protein in each brain region are controlled by the balance of rates for production and degradation [31]. The production rate involves in net effects of various intracellular processes such as transcription, translation, posttranslational modification, axonal transport and insertion into plasma membrane, and likewise the degradation rate includes phosphorylation, sequestration, internalization and proteolysis. Notably, the expression level of SERT protein in the mouse thalamus and midbrain was significantly decreased by the continuous administration of paroxetine at the dose of 13.3 Amol/kg/day for 21 days (Figs. 3 and 4). This means that down-regulation of brain SERT by long-term exposure to paroxetine may be accompanied by decreases not only in the ligand binding sites but also in the protein expression. Such compensatory regulation may be associated with the sequestration and internalization of SERT and/or reduction of transporter protein [26,30]. It is plausible that continuous exposure to relatively high concentration of paroxetine in the mouse brain is essential for the reduction of SERT protein expression occurring possibly as a consequence of change in production, degradation or both processes. The treatment with paroxetine at low dose (2.67 Amol/kg/day) decreased significantly specific [3H]paroxetine binding in the thalamus and midbrain but it had little effect on the SERT protein expression. It seems likely that the apparent discrepancy between ligand binding sites and protein expression of SERT is attributable partly to differences in the extent and duration of brain SERT occupancy due to paroxetine. In fact, the duration of decrease of [3H]paroxetine binding in mouse brain appeared to be significantly shorter with paroxetine at the dose of 2.67 Amol/kg/day than 13.3 Amol/ kg/day (Fig. 2). In other words, long-lasting occupancy by

high concentrations of paroxetine of brain SERT may be critical for the occurrence of down-regulation of SERT protein level following the compensatory alteration in ligand binding sites. Previous studies have shown that chronic treatment with fluoxetine in mice causes the desensitization of presynaptic 5-HT1A receptor, which occurs at the level of receptor-G protein interaction on dorsal raphe neurons [24,27]. Chronic down-expression of SERT by injection of antisense oligonucleotides of SERT into rat dorsal raphe brought about significant decrease in 5-HT1A-mediated [35S]GTPgS binding without change in receptor protein level in the same brain region [9]. Similarly, both 5-HT1A and 5-HT1Bmediated [35S]GTPgS binding were reduced due to the decrease in receptor density in SERT knockout mice compared with wild type [8,17]. Thus, it is suggested that down-expression or absence of brain SERT is closely associated with the altered function of 5-HT autoreceptors, as observed after chronic SERT blockade by SSRIs [11]. Taken together, synergistic or additive enhancement of serotonergic neurotransmission due to down-regulation of SERT sites as well as the desensitization of presynaptic 5HT autoreceptors may underlie the therapeutic efficacy of long-term treatment with SSRIs. In conclusion, the present study has firstly shown that continuous administration of paroxetine by osmotic minipumps induces significant reduction of not only ligand binding sites of mouse brain SERT but also the protein expression level. Such down-regulation of SERT may be applicable to the clinical situation, because the steady-state plasma concentration of this drug was maintained within or near the range recommended clinically.

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