doi: /j x

Transcription

1 Journal of Neurochemistry, 2004, 89, doi: /j x Blockade of substance P (neurokinin 1) receptors enhances extracellular serotonin when combined with a selective serotonin reuptake inhibitor: an in vivo microdialysis study in mice Bruno P. Guiard,* Cédric Przybylski,* Jean-Philippe Guilloux,* Isabelle Seif,* Nicolas Froger, Carmen De Felipe,à Stephen P. Hunt, Laurence Lanfumey and Alain M. Gardier* *Laboratoire de Neuropharmacologie EA 3544 MJENR, Faculté de Pharmacie IFR75 Institut de Signalisation et d Innovation Thérapeutique, Université Paris-Sud, Châtenay-Malabry, France INSERM U288, CHU Pitié-Salpêtrière, Paris, France àinstituto de Neurociencias, University Miguel Hernandez, Alicante, Spain Department of Anatomy and Developmental Biology, University College, London, UK Abstract Substance P antagonists of the neurokinin-1 receptor type (NK1) are gaining growing interest as new antidepressant therapies. It has been postulated that these drugs exert this putative therapeutic effect without direct interactions with serotonin (5-HT) neurones. Our recent microdialysis experiment performed in NK1 receptor knockout mice suggested evidence of changes in 5-HT neuronal function (Froger et al. 2001). The aim of the present study was to evaluate the effects of coadministration of the selective 5-HT reuptake inhibitor (SSRI) paroxetine with a NK1 receptor antagonist (GR or L733060), given either intraperitoneally (i.p.) or locally into the dorsal raphe nucleus, on extracellular levels of 5-HT ([5-HT]ext) in the frontal cortex and the dorsal raphe nucleus using in vivo microdialysis in awake, freely moving mice. The systemic or intraraphe administration of a NK1 receptor antagonist did not change basal cortical [5-HT]ext in mice. A single systemic dose of paroxetine (4 mg/kg; i.p.) resulted in a statistically significant increase in [5-HT]ext with a larger extent in the dorsal raphe nucleus (+ 138% over basal AUC values), than in the frontal cortex (+ 52% over basal AUC values). Co-administration of paroxetine (4 mg/kg; i.p.) with the NK1 receptor antagonists, GR (30 mg/kg; i.p.) or L (40 mg/kg; i.p.), potentiated the effects of paroxetine on cortical [5-HT]ext in wild-type mice, whereas GR (30 mg/kg; i.p.) had no effect on paroxetine-induced increase in cortical [5-HT]ext in NK1 receptor knock-out mice. When GR (300 lmol/l) was perfused by reverse microdialysis into the dorsal raphe nucleus, it potentiated the effects of paroxetine on cortical [5-HT]ext, and inhibited paroxetine-induced increase in [5-HT]ext in the dorsal raphe nucleus. Finally, in mice whose 5-HT transporters were first blocked by a local perfusion of 1 lmol/l of citalopram into the frontal cortex, a single dose of paroxetine (4 mg/kg i.p.) decreased cortical 5-HT release, and GR (30 mg/kg i.p.) reversed this effect. The present findings suggest that NK1 receptor antagonists, when combined with a SSRI, augment 5-HT release by modulating substance P/5 HT interactions in the dorsal raphe nucleus. Keywords: depression, frontal cortex, intracerebral microdialysis, NK1 receptor antagonist, selective serotonin reuptake inhibitor, substance P. J. Neurochem. (2004) 89, The clinical efficacy of antidepressant drugs such as selective serotonin re-uptake inhibitors (SSRIs) is based on facilitation of central serotonergic neurotransmission subsequent to the selective blockade of the serotonin (5-hydroxytryptamine, 5-HT) transporter. SSRIs are attractive drugs because they produce fewer adverse-effects than the first class of antidepressants, the tricyclics. However, SSRIs have in common with all other antidepressant drugs several drawbacks such as the length of delay to achieve clinical benefits in depressed Received April 9, 2003; revised manuscript received November 19, 2003; accepted November 24, Address correspondence and reprint requests to Alain M. Gardier, Laboratoire de Neuropharmacologie Tour D1, 2ème étage, EA 3544 MJENR, Faculté de Pharmacie, Université Paris-Sud, 5 rue J-B. Clément, Châtenay-Malabry cedex, France. alain.gardier@cep.u-psud.fr Abbreviations used: AUC, area under the curve; 5-HT, serotonin; NK, neurokinin; NA, noradrenaline; SSRI, selective serotonin reuptake inhibitor. 54

2 SSRI and NK1R antagonist effects on 5-HT release 55 patients and that 30% of patients are resistant to the treatment. These limitations led to consideration of alternative strategies to improve their therapeutic efficacy. One of the latest developments in the field of antidepressant drug therapy is an increased interest in neuropeptides such as substance P, which belongs to the family of tachykinins with two others neuropeptides, neurokinins A and B. Three types of tachykinin receptors, neurokinin-1 (NK1), NK2 and NK3 exhibiting preferences for substance P, neurokinin A and neurokinin B, respectively, have been identified (Regoli et al. 1994). The tachykinins have the peculiarity of being not highly selective for any given receptor type. Indeed, substance P binds and activates the G-protein coupled NK1 receptor, but also NK2 and NK3 receptors with a lower affinity (Maggi et al. 1993). In rodents, NK1 receptors have been identified in the hypothalamus (Liu et al. 2002), in the limbic structures (Beaujouan et al. 2000), in the locus coeruleus (Chen et al. 2000) and the dorsal raphe nucleus (Froger et al. 2001; Commons and Valentino 2002). It has been reported that the NK1 receptor antagonist MK-869 displayed antidepressant effects in a placebo-controlled trial performed in patients with a major depressive disorder (Kramer et al. 1998; Ranga and Krishnan 2002). Thus, these clinical data suggest that NK1 receptor antagonists may represent a putative new pharmacological class of antidepressant drugs. The precise central effects of NK1 receptor antagonists now need to be clarified, as it has been claimed that these latter drugs do not act directly on monoamine systems like all other major types of antidepressant treatments already available for humans (Kramer et al. 1998; Rupniak et al. 2001). This assertion seems to be surprising since central interactions between substance P and serotonergic system have already been suggested in preclinical studies (Walker et al. 1991; Compan et al. 1997). It is thus tempting to presume that NK1 receptor antagonists could interact indirectly with neuronal 5-HT and/or noradrenergic systems in the brain (Haddjeri and Blier 2000). We recently investigated this hypothesis using several approaches. For example, using in vivo microdialysis, we found evidence for alterations in 5-HT neuronal function in the frontal cortex of NK1 receptor knockout mice (Froger et al. 2001). The increase in the extracellular levels of 5-HT ([5-HT]ext) in the frontal cortex caused by a systemic administration of the SSRI, paroxetine, was four- to sixfold higher in freely moving NK1 receptor knock-out mice than in wild-types (Froger et al. 2001), suggesting the existence of critical interactions between 5-HT and substance P in the brain. In an attempt to find the brain region in which these interactions may occur, double immunocytochemical labelling experiments were performed by Moratalla s group (Madrid, Spain) and others. No colocalization of 5-HTand NK1 receptors in the dorsal raphe nucleus cell bodies of wild-type mice was reported (Froger et al. 2001; Santarelli et al. 2001). Our knowledge of the central effects of NK1 receptor antagonists is further limited by some negative results obtained following administration of a single dose of NK1 receptor antagonists to rodents. Low doses of these compounds did not modify by themselves the firing activity of 5-HT neurones located in the dorsal raphe nucleus in rats (Haddjeri and Blier 2000), although acute higher doses or sustained pharmacological blockade of NK1 receptors increased spontaneous firing activity of the dorsal raphe nucleus 5-HT neurones (Haddjeri and Blier 2001; Conley et al. 2002). These latter results disagree with an intracerebral in vivo microdialysis study suggesting that NK1 receptor antagonists did not modify basal [5-HT]ext in the rat prefrontal cortex (Millan et al. 2001). In this study, we show that the substance P system play a key role in the modulation of the brain serotonergic neurotransmission, since a single dose of a NK1 receptor antagonist [given either intraperitoneally (i.p.) or perfused locally into the dorsal raphe nucleus] potentiated the paroxetine response on dialysate 5-HT levels in the frontal cortex of mice. Materials and methods Animals Male C57BL/6 wild-type and substance P (neurokinin-1) receptor knockout mice, 5 6 weeks old, weighing g, were used in this study. Mutant mice derived from heterozygote crossings NK1 +/ couples raised on 129/Sv C57BL/6 genetic background were made at the animal facility of University College, London (De Felipe et al. 1998). The founders were shipped to France and their offspring were bred and reared on a C57BL/6 genetic background as heterozygote colonies and genotyped by PCR as already described (Froger et al. 2001). All animals were housed in animal care facility in groups of 3 6 and kept under standard conditions (room temperature of C, 12 h : 12 h light dark cycle, food and water ad libitum). Procedures involving animals and their care were conducted in conformity with the institutional guidelines that are in compliance with national and international laws and policies (council directive , 19 October 1987, Ministère de l Agriculture et de la Forêt, Service Vétérinaire de la Santé et de la Protection Animale, permissions # to A.M. Gardier). Drugs and treatment GR was a generous gift from GlaxoSmithKline laboratory (Marly le-roi, France). L and L were purchased from Sigma-Aldrich (Saint Quentin Fallavier, France). The selective 5-HT reuptake inhibitors, paroxetine hydrochloride was a generous gift from GlaxoSmithKline (Harlow, UK) and citalopram from Lundbeck laboratory (Copenhagen, Denmark). All chemical compounds such as GR205171, L733060, L733061, paroxetine and citalopram were dissolved in distilled water. Paroxetine was administered at the dose of 4 mg/kg and the NK1 receptor antagonists at the dose of 10, 30 or 40 mg/kg by the i.p. route. Control animals were injected using the appropriate vehicle and the

3 56 B. P. Guiard et al. same administration route. For their local administration into the dorsal raphe nucleus or the frontal cortex, GR (30 or 300 lmol/l) and citalopram (1 lmol/l) were dissolved in artificial cerebrospinal fluid (CSF) and perfused at a flow rate of 1.5 ll/min and 0.5 ll/min, respectively. Microdialysis procedure Concentric dialysis probes were made of cuprophan fibres and set up as described previously (Malagie et al. 1996). All probes presented an active length of 2 and 1 mm for the frontal cortex and the dorsal raphe nucleus, respectively ( 0.30 mm outer diameter). Animals were anaesthetized with chloral hydrate (400 mg/kg, i.p.). They were placed in a stereotaxic frame and were implanted with a probe according to the Mouse brains atlas (Hof et al. 2000): coordinates from Bregma (in mm), the frontal cortex: anterior ¼ 1.6, lateral ¼ 1.3, ventral ¼ ) 1.6; the dorsal raphe nucleus: anterior ¼ 4.5, lateral ¼ 0, ventral ¼ ) 3. Animals were allowed to recover from surgery overnight. The next day, 20 h after the surgery, the probe was continuously perfused with artificial CSF (composition in mmol/l: NaCl 147, KCl 3.5, CaCl , MgCl 2 1.2, NaH 2 PO 4 1.0, ph 7.4 ± 0.2) at a flow rate of 1.5 ll/min in the frontal cortex and 0.5 ll/min in the dorsal raphe nucleus using CMA/100 pump (Carnegie Medicin, Stockholm, Sweden). Dialysates were collected every 15 min for the frontal cortex and every 30 min for the dorsal raphe nucleus in a small Eppendorf tube for the measurements of their 5-HT contents using a high performance liquid chromatography (HPLC) system (XL-ODS, mm, particle size 3 lm; Beckman) coupled to an amperometric detector (1049 A, Hewlett-Packard, Les Ulis, France). Four fractions were collected to measure basal values (means ± SEM) before systemic administration of the drugs. The limit of sensitivity for [5-HT]ext was 0.5 fmol per sample (signal-to-noise ratio ¼ 2). At the end of the experiments, the placement of microdialysis probes was verified histologically. Histological verification of microdialysis probes implantation After the microdialysis study, mice were killed by cervical dislocation, brains were removed and stored at ) 40 C. To determine the exact probe implantation in the frontal cortex or dorsal raphe nucleus, brains were placed in a Kryomat apparatus and kept at ) 25 C. Brain regions were identified according to Hof et al. (2000) Mouse brains atlas and coronal frozen section of brain sliced serially at 40-lm intervals. Slices were realized from AP 1 to 2 mm and AP ) 4 to ) 5 mm for the frontal cortex and the dorsal raphe nucleus, respectively. Each slice was photographed using a digital camera (PowerShot G1, Canon) and the implantation of the probe estimated in comparison to the corresponding slices obtained by the Mouse brains atlas software (Hof et al. 2000). Only mice with probes confined to either the frontal cortex or the dorsal raphe nucleus were used for subsequent data analysis (examples of a probe implantation in the frontal cortex and the dorsal raphe nucleus are given in Fig. 6a,b, respectively). Experimental protocols In this study, all experiments were performed to determine the effects of substance P (neurokinin-1) receptor antagonists on extracellular 5-HT levels ([5-HT]ext) in the frontal cortex and the dorsal raphe nucleus of mice. Experiment 1. Effect of NK1 receptor antagonists administered i.p. on cortical [5-HT]ext of wild-type mice. Following collection of four baseline dialysate samples, freely moving wild-type mice were administered with either the vehicle or various NK1 receptor antagonists, GR (30 mg/kg; i.p.) or L (40 mg/kg; i.p.). Dialysate samples were collected for a min posttreatment period. Experiment 2. Effect of NK1 receptor antagonists administered i.p. on cortical [5-HT]ext of paroxetine-treated wild-type mice. Following collection of four baseline dialysate fractions, freely moving wild-type mice were administered first with paroxetine (4 mg/kg; i.p.), then 1 h later, with either vehicle, GR (10 and 30 mg/kg; i.p.), L (active enantiomer) or L (low-affinity enantiomer) (40 mg/kg; i.p.) and dialysates were collected for an additional 1 h. Experiment 3. Effect of NK1 receptor antagonist administered i.p. on cortical [5-HT]ext of paroxetine-treated NK1 receptor knockout mice. Following collection of four baseline dialysate fractions, freely moving NK1 receptor knock-out mice received first paroxetine (4 mg/kg; i.p.), then 1 h later, either vehicle or GR (30 mg/kg; i.p.) and dialysate samples were collected for an additional 1 h. Experiment 4. Effect of intraraphe perfusion of NK1 receptor antagonist on [5-HT]ext levels in the dorsal raphe nucleus of paroxetine-treated wild-type mice. Following collection of four baseline dialysate fractions, freely moving wild-type mice received first vehicle or paroxetine (4 mg/kg; i.p.), then 1 h later, a continuous perfusion of vehicle or GR (30 or 300 lmol/l) locally into the dorsal raphe nucleus for 1 h, and dialysate samples were collected for this additional 1 h. Experiment 5. Effect of intraraphe perfusion of selective NK1 receptor antagonist on cortical [5-HT]ext of paroxetine-treated wildtype mice. Following collection of four baseline dialysate fractions, freely moving wild-type mice received first vehicle or paroxetine (4 mg/kg; i.p.), then 1 h later, a continuous perfusion of vehicle or GR (30 or 300 lmol/l) locally into the dorsal raphe nucleus for 1 h, and dialysate samples were collected for this additional 1 h. Experiment 6. Effect of a NK1 receptor antagonist administered i.p. on cortical 5-HT release of wild-type mice. The 5-HT transporter was blocked in the frontal cortex by a local continuous perfusion of citalopram (1 lmol/l). Following collection of four baseline dialysate fractions, freely moving wild-type mice received first vehicle or paroxetine (4 mg/kg; i.p.), then 1 h later, either vehicle or GR (30 mg/kg; i.p.), and dialysate samples were collected for an additional 1 h. Data analysis and statistics The basal value of [5-HT]ext was calculated from the mean of the four first samples collected. All subsequent sample values calculated as the amount of 5-HT outflow collected during the min period from the frontal cortex are expressed as a percentage of basal values. Statistical analyses, using the computer software StatView

4 SSRI and NK1R antagonist effects on 5-HT release for IBM compatible (Abacus Concepts, Inc, Berkley, CA, USA), were performed on the area under the curve (AUC; mean ± SEM) values for the amount of 5-HT outflow collected during the min period after vehicle or substance P (neurokinin-1) receptor antagonist administration (experiment 1 only), and during the 0 60 and min period post paroxetine or substance P (neurokinin-1) receptor antagonist administration, respectively (experiment 2 5). To compare different AUC values in each group of treated animals, statistical analysis was performed using a one-way ANOVA with drugs treatment as the main factor, followed by Fisher Protected Least Significance Difference post hoc test. Results Effects of systemic injection of the NK1 receptor antagonists GR or L on extracellular 5-HT levels in the frontal cortex of wild-type mice Basal cortical extracellular levels of 5-HT in groups of mice treated with either vehicle, GR (30 mg/kg; i.p.) or L (40 mg/kg; i.p.) were (in fmol/20 ll) 3.38 ± 0.48 (n ¼ 5); 3.50 ± 0.92 (n ¼ 5) and 4.26 ± 0.61 (n ¼ 5), respectively, and did not significantly differ between the three groups of mice [F 2,12 ¼ 1.84, p ¼ 0.19]. Neither the vehicle nor NK1 receptor antagonists such as GR (30 mg/kg) or L (40 mg/kg) administered intraperitoneally modified the basal cortical [5-HT]ext during the min post-treatment period [F 2,12 ¼ 0.37, p ¼ 0.69] (Fig. 1a,b). Effects of systemic injection of the NK1 receptor antagonists GR205171, L or L on extracellular 5-HT levels in the frontal cortex of paroxetine-treated wild-type mice Basal cortical extracellular levels of 5-HT in mice treated with either paroxetine/vehicle, paroxetine/gr (10 and 30 mg/kg; i.p.), paroxetine/l (40 mg/kg; i.p.) or paroxetine/l (40 mg/kg; i.p.) were (in fmol/20 ll) 2.85 ± 0.35 (n ¼ 9); 4.66 ± 0.34 (n ¼ 5); 3.18 ± 0.25 (n ¼ 8); 3.01 ± 0.17 (n ¼ 7) and 3.63 ± 0.39 (n ¼ 6), respectively. These values did not significantly differ between these various paroxetine-treated groups [F 4,30 ¼ 1.24, p ¼ 0.31]. In the frontal cortex, the paroxetine/vehicle treatment, as compared with a vehicle/vehicle treatment, increased significantly the [5-HT]ext during the t 0 t 60 period (AUC values were: 159 ± 20% versus 90 ± 8%, respectively) [F 1,12 ¼ 7.18, p ¼ 0.02], but only marginally during the t 60 t 120 period (AUC values were: 135 ± 16% versus 99 ± 10%, respectively) [F 1,12 ¼ 2.08, p ¼ 0.17: not statistically significant]. Before the intraperitoneal administration of NK1 receptor antagonists (from t 0 to t 60 ), increases in cortical [5-HT]ext were not significantly different in the various paroxetine-treated groups [F 4,31 ¼ 1.53, p ¼ 0.22]. Fig. 1 Effects of intraperitoneal administration of the selective NK1 receptor antagonists GR or L alone on extracellular 5-HT levels in the frontal cortex of freely moving wild-type mice. (a) Data are means ± SEM of [5-HT]ext expressed as percentages of basal values. Mice received (arrow) either vehicle (e) or GR (30 mg/kg; i.p.) (n) or L (40 mg/kg; i.p.) (m). The dotted line ( ) correspond to the 100% value. (b) Data are area under the curve (AUC; mean ± SEM) values calculated for the amount of 5-HT outflow measured during the min post-treatment with NK1 receptor antagonists and expressed as percentages of baseline (n ¼ 5 mice per group). i.p., intraperitoneal. No statistical differences in cortical [5-HT]ext were observed between these groups of mice [one-way ANOVA followed by a Fischer protected least significant difference (PLSD) post hoc test]. Then, Analysis of AUC values, calculated for the amount of 5-HT collected in the frontal cortex during the min post-nk1 receptor antagonists treatment, were significantly higher in groups which received a single dose of paroxetine (4 mg/kg; i.p.) and GR (30 mg/kg; i.p.; p < 0.05) or L (40 mg/kg; i.p.; p < 0.05) than the AUC value of the paroxetine (4 mg/kg; i.p.)/vehicle-treated group [F 5,35 ¼ 5.41, p ¼ 0.001] (Fig. 2b,d, respectively). The AUC value in the group of mice receiving paroxetine and the low-affinity enantiomer L was not statistically different as compared with the paroxetine/vehicle-treated group (p ¼ 0.49). Thus, the NK1 receptor antagonists GR and L enhanced cortical extracellular 5-HT levels after paroxetine injection in wild-type mice. This effect might be the consequence of either a reduced reuptake or an increased release of 5-HT in the frontal cortex.

5 58 B. P. Guiard et al. Fig. 2 Effects of intraperitoneal administration of the selective NK1 receptor antagonists GR (a), L (active enantiomer), L (low affinity enantiomer) (c) on paroxetine-induced changes in extracellular 5-HT levels ([5-HT]ext) in the frontal cortex of freely moving wild-type mice. (a) and (c) Data are means ± SEM of [5-HT]ext expressed as percentages of the respective basal values. Mice received (arrows) either vehicle/vehicle (e) or paroxetine (4 mg/kg; i.p.)/vehicle (n) or paroxetine (4 mg/kg; i.p.)/gr (10 mg/kg; i.p.) (d) or (30 mg/kg; i.p.) (m) or paroxetine (4 mg/kg; i.p.)/l (40 mg/kg; i.p.) (m) or L (m). (b) and (d) Data are area under the curve (AUC; mean ± SEM) values calculated for the amount of 5-HT outflow measured during the min post-treatment period and expressed as percentages of baseline (n ¼ 9 12 mice per group). *p < 0.05; **p < significantly different from vehicle/vehicle treated group; p < 0.05; significantly different from paroxetine/vehicle treated group (one-way ANOVA followed by a PLSD post hoc test). Prx, paroxetine; i.p., intraperitoneal; ns, not statistically significant. Effect of systemic injection of the NK1 receptor antagonist GR on extracellular 5-HT levels in the frontal cortex of paroxetine-treated NK1 receptor knockout mice Basal cortical extracellular levels of 5-HT in knockout mice treated with either paroxetine/vehicle or paroxetine/ GR (30 mg/kg; i.p.) were (in fmol/20 ll) 3.57 ± 0.40 (n ¼ 7) and 3.28 ± 0.13 (n ¼ 8), respectively. These values did not significantly differ between these two paroxetine-treated groups [F 1,13 ¼ 0.52, p ¼ 0.48]. Before the intraperitoneal administration of either vehicle or GR (30 mg/kg) (from t 0 to t 60 ), increases in [5-HT]ext were not significantly different in the various paroxetine-treated groups of mutant mice [F 1,13 ¼ 0.46, p ¼ 0.50)]. Then, analysis of AUC values calculated for the amount of 5-HT collected in the frontal cortex during the min post-vehicle or GR (30 mg/kg; i.p.) were not significantly different in these two groups of mutant mice receiving the paroxetine/vehicle and paroxetine/gr (30 mg/kg; i.p.) treatments [F 1,13 ¼ 0.055, p ¼ 0.81] (Fig. 3a time course study and Fig. 3b on AUC values). Thus, the enhancing effect of GR on cortical extracellular 5-HT levels after paroxetine injection in wildtype mice appears to be NK1 receptor specific. Effects of intraraphe perfusion of the NK1 receptor antagonist GR on extracellular 5-HT levels in the dorsal raphe nucleus of paroxetine-treated wild-type mice Basal extracellular levels of 5-HT in the dorsal raphe nucleus of mice treated with either vehicle/vehicle, vehicle/gr (300 lmol/l), paroxetine/vehicle or paroxetine/gr (30 or 300 lmol/l) were (in fmol/10 ll) 9.97 ± 2.20 (n ¼ 5); 5.84 ± 0.37 (n ¼ 5); 7.90 ± 1.09 (n ¼ 9); 6.13 ± 0.71 (n ¼ 8) and 7.64 ± 0.80 (n ¼ 10), respectively. These values did not significantly differ between these groups of mice [F 4,32 ¼ 1.32, p ¼ 0.28].

6 SSRI and NK1R antagonist effects on 5-HT release 59 cell bodies located in the dorsal raphe nucleus following paroxetine injection. Fig. 3 Effects of intraperitoneal administration of the selective NK1 receptor antagonist GR on paroxetine-induced changes in extracellular 5-HT levels [5-HT]ext in the frontal cortex of freely moving NK1 receptor knock-out mice (NK1-R KO). (a) Data are means ± SEM of [5-HT]ext expressed as percentages of the respective basal values. Mice received (arrows) either paroxetine (4 mg/kg; i.p.)/vehicle (e) or paroxetine (4 mg/kg; i.p.)/gr (30 mg/kg; i.p.) (n). (b) Data are area under the curve (AUC; mean ± SEM) values calculated for the amount of 5-HT outflow measured during the min post-treatment period and expressed as percentages of baseline (n ¼ 7 8 mice per group). No statistical differences in cortical [5-HT]ext were observed between these two groups of KO NK1-R of mice (one-way ANOVA followed by a PLSD post hoc test). KO NK1-R, NK1 receptor knockout mice, Prx, paroxetine, i.p., intraperitoneal. In the dorsal raphe nucleus, the paroxetine/vehicle, as compared with a vehicle/vehicle treatment, increased significantly the [5-HT]ext during the t 0 t 60 period (AUC values were: 185 ± 15% versus 119 ± 3%, respectively) [F 1,12 ¼ 9.75, p ¼ 0.008] and during the t 60 to t 120 period (307 ± 29% versus 107 ± 6%) [F 1.12 ¼ 24.25, p ¼ 0.001]. Before the intraraphe perfusion of GR (30 or 300 lmol/l) (from t 0 to t 60 ), increases in extracellular 5-HT levels in the dorsal raphe nucleus were not significantly different in the various paroxetine-treated groups [F 2,24 ¼ 0.015, p ¼ 0.98]. Then, analysis of AUC values, calculated for the amount of 5-HT collected in the dorsal raphe nucleus during the min period of intraraphe GR perfusion, revealed that the administration of a single dose of paroxetine (4 mg/kg; i.p.) and GR lmol/l had no effect on extracelluar 5-HT levels as compared with the paroxetine/vehicle-treated group. However, when administered with GR at a 10-fold higher concentration (300 lmol/l), paroxetineinduced increase in [5-HT]ext was inhibited as compared with the paroxetine/vehicle-treated group [F 4,32 ¼ 10.70, p < ]. We verified that, when given alone, GR (300 lmol/l) did not modify basal extracellular 5-HT levels in the dorsal raphe nucleus as compared with the AUC value of vehicle/vehicle-treated mice (p ¼ 0.97) (Fig. 4a,b). Thus, the enhancing effect of GR on cortical extracellular 5-HT levels could originate in a lower [5-HT]ext increase occurring in the vicinity of serotonergic Effects of intraraphe perfusion of the NK1 receptor antagonist GR on extracellular 5-HT levels in the frontal cortex of paroxetine-treated wild-type mice Basal cortical extracellular levels of 5-HT in mice treated with either vehicle/vehicle, vehicle/gr (300 lmol/l), paroxetine/vehicle or paroxetine/gr (30 or 300 lmol/l) were (in fmol/20 ll) 3.50 ± 0.61 (n ¼ 5); 3.18 ± 0.34 (n ¼ 8); 3.29 ± 0.57 (n ¼ 6); 4.87 ± 0.52 (n ¼ 6) and 5.49 ± 0.73 (n ¼ 8), respectively. These values did not significantly differ between these groups of mice [F 4,28 ¼ 1.46, p ¼ 0.075]. Before the intraraphe perfusion of GR (30 and 300 lmol/l) (from t 0 to t 60 ), changes in [5-HT]ext in the frontal cortex were not significantly different in the various paroxetine-treated groups [F 2,17 ¼ 0.59, p ¼ 0.94]. Then, the analysis of AUC values, calculated for the amount of 5-HT collected in the frontal cortex during the min period of intraraphe GR perfusion, revealed that the administration of a single dose of paroxetine (4 mg/kg; i.p.) and GR lmol/l (but not 30 lmol/l), significantly potentiated the effect of paroxetine on cortical [5-HT]ext as compared with the AUC value of paroxetine/vehicle-treated group [F 4,28 ¼ 3.16, p ¼ 0.03] (Fig. 4c,d). When given alone, intraraphe perfusion of GR (300 lmol/l) did not modify basal cortical [5-HT]ext in mice. Thus, the enhancing effect of GR on cortical extracellular 5-HT levels after paroxetine injection appears to be mediated by brainstem cells. Effects of systemic injection of the NK1 receptor antagonist GR on 5-HT release in the frontal cortex of paroxetine-treated wild-type mice In the presence of a local perfusion of citalopram (1 lmol/l) by reverse microdialysis into the frontal cortex, basal cortical extracellular level of 5-HT in mice treated with either vehicle/vehicle, vehicle/gr (30 mg/kg; i.p.), paroxetine/vehicle or paroxetine/gr (30 mg/kg; i.p.) were (in fmol/20 ll) ± 1.51 (n ¼ 13); ± 2.16 (n ¼ 12); ± 1.2 (n ¼ 11) and 9.45 ± 0.57 (n ¼ 11), respectively. These values did not significantly differ between these groups of mice [F 3,42 ¼ 0.92, p ¼ 0.43]. In the presence of 1 lmol/l of citalopram, acute administration of paroxetine (4 mg/kg; i.p.)/vehicle decreased dialysate 5-HT in the frontal cortex over the min of dialysate collection (Fig. 5). Before administration of either the vehicle or GR (from t 0 to t 60 ), decreases in cortical [5-HT]ext were not significantly different in both paroxetine-treated groups [F 1,20 ¼ 0.60, p ¼ 0.44)]. Administration of a single dose of GR (30 mg/kg; i.p.) 1 h after paroxetine, reduced paroxetine-induced decrease in cortical 5-HT levels (from t 75 to t 120 ) [F 7,145 ¼ 7.83, p < 0.001]. Differences

7 60 B. P. Guiard et al. Fig. 4 Effects of the selective NK1 receptor antagonist GR205171, perfused locally via a microdialysis probe implanted into the dorsal raphe nucleus (DRN), on the effects of paroxetine on extracellular 5-HT levels in the frontal cortex and dorsal raphe nucleus of freely moving wild-type mice. (a) and (c) Data are means ± SEM of [5-HT]ext levels in the dorsal raphe nucleus (a) and frontal cortex (c), respectively, expressed as percentages of the respective basal values following exposure to vehicle/vehicle (e) or vehicle/gr (300 lmol/l) ( ) or paroxetine (4 mg/kg; i.p.)/vehicle (n) or paroxetine (4 mg/kg; i.p.)/ GR (30 lmol/l) (d) or paroxetine (4 mg/kg; i.p.)/gr (300 lmol/l) (m). (b) and (d) Area under the curve (AUC; mean ± SEM) values calculated for the amount of 5-HT in the dorsal raphe nucleus (b) and frontal cortex (d) outflow measured during the min post-treatment period and expressed as percentages of baseline (n ¼ 5 10 mice per group). **p < and ***p < significantly different from vehicle/vehicle treated group; p < 0.05 and p < 0.001; significantly different from paroxetine/vehicle treated group (one-way ANOVA followed by a PLSD post hoc test). FCX, frontal cortex; DRN, Dorsal raphe nucleus; Prx, paroxetine; i.p., intraperitoneal. between the two groups were statistically significant at t 90 (p < 0.01), t 105 (p < 0.001) and t 120 (p < 0.001) (Fig. 5). In the presence of a local perfusion of citalopram in the frontal cortex, GR alone (30 mg/kg; i.p.) did not modify cortical [5-HT]ext as compared with the group of wild-type mice treated with vehicle only (p ¼ 0.79) (Fig. 5). Thus, the enhancing effect of GR on cortical extracellular 5-HT levels after paroxetine injection could be attributable to an increase in 5-HT release, at least partly. Discussion In the present study, using in vivo microdialysis in awake, freely moving mice, we found that the effects of the SSRI, paroxetine, on cortical [5-HT]ext, were potentiated by a systemic administration of the NK1 receptor antagonists, GR and L These effects were not observed in NK1-R KO mice. In addition, the local perfusion of GR through the dialysis probe implanted into the dorsal raphe nucleus in wild-type mice reproduced the effects of its systemic administration at serotonergic nerve terminals, while opposite changes were found in the dorsal raphe nucleus with a decrease in extracellular 5-HT levels. In a first experiment, we evaluated the effects of the NK1 receptor antagonists GR and L given alone on cortical [5-HT]ext in C57BL/6 wild-type mice. Consistent with results already obtained using in vivo microdialysis in the frontal cortex of rats (Millan et al. 2001) or CD-1 mice

8 SSRI and NK1R antagonist effects on 5-HT release 61 (a) FCX FCX DRN Fig. 5 Time course of the effect of intraperitoneal administration of the selective NK1 receptor antagonist GR on paroxetine-induced 5-HT release in the frontal cortex as assessed in the presence of locally applied citalopram in the perfusion fluid, in freely moving wildtype mice. Mice under reverse microdialysis application of citalopram (1 lmol/l) into the frontal cortex received either vehicle/vehicle (e) or vehicle/gr (30 mg/kg; i.p.) ( ) or paroxetine (4 mg/kg; i.p.)/ vehicle (n) or paroxetine (4 mg/kg, i.p.)/gr (30 mk/kg; i.p.) (m). Data are means ± SEM of [5-HT]ext expressed as percentages of basal values (n ¼ 11 mice per group). **p < 0.001, ***p < significantly different from the paroxetine/vehicle-treated group at the corresponding time (one-way ANOVA followed by a PLSD post hoc test). Prx, paroxetine; i.p., intraperitoneal. (Zocchi et al. 2003), we found that the blockade of NK1 receptors did not affect basal cortical [5-HT]ext. These microdialysis data agree with the absence of modifications of the spontaneous firing activity of 5-HT neurones located in the dorsal raphe nucleus following a single, systemic administration of NK1 receptor antagonists to rats (WIN51708, CP96345: Haddjeri and Blier 2000; GR205171: Lejeune et al. 2002). Then, we tested for the effects of a coadministration of paroxetine with either an active NK1 receptor antagonist (GR205171, L733060) or a low-affinity enantiomer (L733061) on cortical [5-HT]ext in mice. A systemic administration of a single dose of GR (30 mg/kg; i.p.) significantly potentiated the effects of paroxetine on cortical [5-HT]ext. These results obtained in mice concur with studies reporting that GR is a brain penetrant compound, exhibits a high affinity for the NK1 receptors in rats (Gardner et al. 1996; Rupniak et al. 2003) and displaces the binding of [ 3 H]-SP with a similar efficacy in mice compared to rats (Zocchi et al. 2003). Based on the high affinity of L for NK1 receptors and its good brain penetration in human and the guinea-pig (Kramer et al. 1998), we also evaluated its effects on paroxetine-induced changes in cortical [5-HT]ext. L (40 mg/kg; i.p.) significantly potentiated the effects of paroxetine on cortical [5-HT]ext whereas its low-affinity enantiomer, L (40 mg/kg; i.p.) had no significant effect. However, statistical analysis failed to achieve a clear separation between L and L presumably due to the elevated dose and/or the difference of efficacy of NK1 receptor antagonists (b) DRN Fig. 6 Coronal sections of a wild-type mouse brain showing the location of the concentric microdialysis probes according to Hof et al. (2000). The arrows indicate the tip of the membrane. The Probes were implanted with the following stereotaxic coordinates in mm, in (a) the right frontal cortex (FCX); (b) the dorsal raphe nucleus (DRN): AP 1.6, L 1.3, D )1.6 and AP )4.5, L 0, D)3, respectively. between some species because L was shown to produce enantioselective central effects in gerbils or guinea pigs at the dose of 10 mg/kg after its systemic administration (Rupniak et al. 1996; Rupniak et al. 2000). Because these compounds have a lower affinity for the mouse NK1 receptors compared to human or guinea pig, we had to administer relatively high doses of NK1 receptor antagonists (30 or 40 mg/kg). Thus, unspecified effects could have occurred through the blockade of either NK2/NK3 receptors or calcium channels after their systemic administration (Rupniak et al. 1993, 1994). Here, a control study using NK1 receptor knockout mice, recently generated by De Felipe et al. (1998), assessed the selectivity of the cortical serotonergic effects of the systemic coadministration of paroxetine and GR GR administered i.p. did not alter the effects of paroxetine on cortical [5-HT]ext in these mutants. These data confirm that, in wild-type mice, the selective blockade of brain NK1 receptors, but not that of NK2 or NK3 receptors, by GR and L733060, mediated the potentiation of the effects of paroxetine on cortical [5-HT]ext. It is of interest to consider these results in light of recent studies showing that GR (30 mg/kg) displayed antidepressant-like activity (Zocchi et al. 2003) and inhibited neonatal vocalizations in mice with a marginal enantioselectivity (Rupniak et al. 2000; Rupniak et al. 2003). Taken together, our result suggest that activation of NK1 receptors by endogenous substance P in the dorsal raphe nucleus may limit the effects of a single dose of SSRI

9 62 B. P. Guiard et al. on extracellular 5-HT levels at serotonergic nerve terminals in mice. However, in this inhibitory effect on neuronal 5-HT activity through the activation of NK1 receptors, we cannot exclude the involvement of the endogenous neurokinin A whose mrna was also identified in the dorsal raphe nucleus (Harlan et al. 1989). Then, to specify one possible central site of action of the NK1 receptor antagonist, we perfused GR locally into the dorsal raphe nucleus, the main source of brain serotonergic neurones. First, we found that a single dose of paroxetineinduced a higher increase in [5-HT]ext in the dorsal raphe nucleus (AUC values ¼ + 138% above baseline) than in the frontal cortex (AUC values ¼ + 52%). These results obtained in mice agree with those already described in rats following SSRI administration (Bel and Artigas 1992). When paroxetine was coadministered with GR perfused into the dorsal raphe nucleus, the lower concentration used (30 lmol/l) did not change dialysate 5-HT in both the dorsal raphe nucleus and the frontal cortex. However, intraraphe perfusion of GR at a 10 fold higher concentration (300 lmol/l), reduced the increase in raphe 5-HT induced by paroxetine and simultaneously potentiated the effect of paroxetine in the frontal cortex presumably through a less feedback inhibition of the serotonergic neurones (Artigas 1993). Finally, several hypotheses can be considered regarding the mechanism by which NK1 receptor antagonists induced changes in cortical extracellular levels of 5-HT when combined with a SSRI. In vivo intracerebral microdialysis technique is recognized as a presynaptic test measuring a balance between release and reuptake of neurotransmitters such as 5-HT (Ungerstedt 1984). Thus, to discriminate between these two processes, 5-HT transporters in wild-type mice were first blocked by a continuous reverse-dialysis perfusion of a 5-HT reuptake inhibitor (citalopram 1 lmol/l) into the 5-HT nerve terminal area, the frontal cortex (Hjorth and Auerbach 1994). Mice then received a systemic administration of paroxetine, which decreased cortical [5-HT]ext, because it blocked 5-HT reuptake in the dorsal raphe nucleus and, in turn, activated somatodendritic 5-HT 1A autoreceptors by endogenous 5-HT. The magnitude of this decrease was comparable to that previously described with the SSRI fluoxetine in rats (Rutter and Auerbach. 1993; Hervas et al. 2001). When a systemic administration of GR was given 1 h after paroxetine, the NK1 receptor antagonist likely interferes with neuronal 5-HT neurotransmission by reversing paroxetine-induced decrease in 5-HT release at brain serotonergic nerve terminals. The dorsal raphe nucleus/frontal cortex serotonergic pathway seems to mediate, at least in part, the central effects of NK1 receptor antagonists in mice. It is noteworthy that the changes in cortical [5-HT]ext observed here in mice treated with paroxetine and a NK1 receptor antagonist are similar to those reported in awake rats receiving a coadministration of SSRI and a single low dose of 5-HT 1A receptor antagonist (Artigas et al. 1996; Malagie et al. 1996). It was shown that GR exhibits micromolar affinity for the rat 5-HT 1A receptor (Gardner et al. 1996) so we cannot exclude unspecific, direct effects of GR through the blockade of the 5-HT 1A autoreceptors thus explaining partly our results. However, Lejeune et al. (2003) recently demonstrated that a pretreatment with GR did not modify the firing inhibition of 5-HT neurones in the dorsal raphe nucleus as well as the decrease in cortical 5-HT levels induced by the 5-HT 1A receptor agonist, 8-OH-DPAT. Because we administered NK1 receptor antagonists systematically, we also have to take into consideration the possible role of the brain noradrenergic system in modulating cortical 5-HT levels consecutively to the selective blockade of NK1 receptors. It has been shown that GR alone ( mg/kg, i.p.), dose-dependently elevated dialysate levels of noradrenaline (NA), in the frontal cortex of freely moving rats (Millan et al. 2001). Indeed, substance P and NK1 receptors are highly expressed in the locus coeruleus (Chen et al. 2000) and some studies suggest that the selective blockade of NK1 receptors may facilitate the activity of adrenergic pathways in the locus coeruleus (Haddjeri and Blier 2000; Maubach et al. 2002) and noradrenaline release at noradrenergic nerve terminals located in the frontal cortex and dorsal hippocampus in rats (Millan et al. 2001). Taken together, our results provided evidence that, when coadministered with a SSRI, the selective blockade of NK1 receptors by GR is associated with an enhanced 5-HT levels at serotonergic nerve terminals. These data suggest that this facilitation of 5-HT neurotransmission by NK1 receptor antagonists may be linked to their property to limit the increase in endogenous 5-HT levels in the dorsal raphe nucleus. The present preclinical approach suggests that strong interactions between the brain 5-HT transporter and NK1 receptors exist in the dorsal raphe nucleus/frontal cortex serotonergic pathway, however, whether somatodendritic 5-HT 1A autoreceptors play a key role in these interactions needs to be clarified. References Artigas F. (1993) 5-HT and antidepressants: new views from microdialysis studies. Trends Pharmacol. Sci. 14, 262. Artigas F., Romero L., de Montigny C. and Blier P. (1996) Acceleration of the effect of selected antidepressant drugs in major depression by 5-HT1A antagonists. Trends Neurosci. 19, Beaujouan J. C., Saffroy M., Torrens Y. and Glowinski J. (2000) Different subtypes of tachykinin NK(1) receptor binding sites are present in the rat brain. J. Neurochem. 75, Bel N. and Artigas F. (1992) Fluvoxamine preferentially increases extracellular 5-hydroxytryptamine in the raphe nuclei: an in vivo microdialysis study. Eur. J. Pharmacol. 229, Chen L. W., Wei L. C., Liu H. L. and Rao Z. R. (2000) Noradrenergic neurons expressing substance P receptor (NK1) in the locus coeruleus complex: a double immunofluorescence study in the rat. Brain Res. 873,

10 SSRI and NK1R antagonist effects on 5-HT release 63 Commons K. G. and Valentino R. J. (2002) Cellular basis for the effects of substance P in the periaqueductal gray and dorsal raphe nucleus. J. Comp. Neurol. 447, Compan V., Salin P. and Daszuta A. (1997) Selective effects of partial and severe lesions of the serotonergic systems on Met-enkephalin and substance P neurons in rat basal ganglia. Brain Res. Mol. Brain Res. 50, Conley R. K., Cumberbatch M. J. et al. (2002) Substance P (neurokinin 1) receptor antagonists enhance dorsal raphe neuronal activity. J. Neurosci. 22, De Felipe C., Herrero J. F., O Brien J. A., Palmer J. A., Doyle C. A., Smith A. J., Laird J. M., Belmonte C., Cervero F. and Hunt S. P. (1998) Altered nociception, analgesia and aggression in mice lacking the receptor for substance P. Nature 392, Froger N., Gardier A. M., Moratalla R., Alberti I., Lena I., Boni C., De Felipe C., Rupniak N. M., Hunt S. P., Jacquot C. et al. (2001) 5-Hydroxytryptamine (5-HT) 1A autoreceptor adaptive changes in substance P (neurokinin 1) receptor knock-out mice mimic antidepressant-induced desensitization. J. Neurosci. 21, Gardner C. J., Armour D. R., Beattie D. T., Gale J. D., Hawcock A. B., Kilpatrick G. J., Twissell D. J. and Ward P. (1996) GR205171: a novel antagonist with high affinity for the tachykinin NK1 receptor, and potent broad-spectrum anti-emetic activity. Regul. Pept. 65, Haddjeri N. and Blier P. (2000) Effect of neurokinin-i receptor antagonists on the function of 5-HT and noradrenaline neurons. Neuroreport 11, Haddjeri N. and Blier P. (2001) Sustained blockade of neurokinin-1 receptors enhances serotonin neurotransmission. Biol. Psychiatry 50, Harlan R. E., Garcia M. M. and Krause J. E. (1989) Cellular localization of substance P- and neurokinin A-encoding preprotachykinin mrna in the female rat brain. J. Comp. Neurol. 287, Hervas I., Vilaro M. T., Romero L., Scorza M. C., Mengod G. and Artigas F. (2001) Desensitization of 5-HT(1A) autoreceptors by a low chronic fluoxetine dose effect of the concurrent administration of WAY Neuropsychopharmacology 24, Hjorth S. and Auerbach S. B. (1994) Further evidence for the importance of 5-HT1A autoreceptors in the action of selective serotonin reuptake inhibitors. Eur. J. Pharmacol. 260, Hof P. R., Young W. G., Bloom F. E., Belichenko P. V. and Celio M. R. (2000) Mouse Brains. Comparative Cytoarchitectonic Atlas of the C57BL/6 and 129/SV, CD-Rom. Elsevier Science, Amsterdam. Kramer M. S., Cutler N., Feighner J. et al. (1998) Distinct mechanism for antidepressant activity by blockade of central substance P receptors. Science 281, Lejeune F., Gobert A. and Millan M. J. (2002) The selective neurokinin (NK)(1) antagonist, GR205,171, stereospecifically enhances mesocortical dopaminergic transmission in the rat: a combined dialysis and electrophysiological study. Brain Res. 935, Lejune F., Gobert A. and Millan M. J. (2003) The neurokinin (NK) 1 agonist GR205, 171, alters the influence of serotonin (5-HT) reuptake inhibitors (SSRIs) on serotonergic neurones: a dual electrophysiological and microdialysis analysis. Neurosci. 29, Abstract Liu H. L., Cao R., Jin L. and Chen L. W. (2002) Immunocytochemical localization of substance P receptor in hypothalamic oxytocincontaining neurons of C57 mice. Brain Res. 948, Maggi C. A., Patacchini R., Rovero P. and Giachetti A. (1993) Tachykinin receptors and tachykinin receptor antagonists. J. Auton. Pharmacol. 13, Malagie I., Trillat A. C., Douvier E., Anmella M. C., Dessalles M. C., Jacquot C. and Gardier A. M. (1996) Regional differences in the effect of the combined treatment of WAY and fluoxetine: an in vivo microdialysis study. Naunyn Schmiedebergs Arch. Pharmacol. 354, Maubach K. A., Martin K., Chicchi G., Harrison T., Wheeldon A., Swain C. J., Cumberbatch M. J., Rupniak N. M. and Seabrook G. R. (2002) Chronic substance P (NK1) receptor antagonist and conventional antidepressant treatment increases burst firing of monoamine neurones in the locus coeruleus. Neuroscience 109, Millan M. J., Lejeune F., De Nanteuil G. and Gobert A. (2001) Selective blockade of neurokinin (NK) (1) receptors facilitates the activity of adrenergic pathways projecting to frontal cortex and dorsal hippocampus in rats. J. Neurochem. 76, Ranga K. and Krishnan R. (2002) Clinical experience with substance P receptor (NK1) antagonists in depression. J. Clin. Psychiatry 63, Regoli D., Boudon A. and Fauchere J. L. (1994) Receptors and antagonists for substance P and related peptides. Pharmacol. Rev. 46, Rupniak N. M., Boyce S., Williams A. R., Cook G., Longmore J., Seabrook G. R., Caeser M., Iversen S. D. and Hill R. G. (1993) Antinociceptive activity of NK1 receptor antagonists: non-specific effects of racemic RP Br. J. Pharmacol. 110, Rutter J. J. and Auerbach S. B. (1993) Acute uptake inhibition increases extracellular serotonin in the rat forebrain. J. Pharmacol. Exp. Ther. 265, Rupniak N. M. and Jackson A. (1994) Non-specific inhibition of dopamine receptor agonist-induced behaviour by the tachykinin NK1 receptor antagonist CP-99,994 in guinea-pigs. Eur. J. Pharmacol. 262, Rupniak N. M., Carlson E., Boyce S., Webb J. K. and Hill R. G. (1996) Enantioselective inhibition of the formalin paw late phase by the NK1 receptor antagonist L-733,060 in gerbils. Pain 67, Rupniak N. M., Carlson E. C., Harrison T., Oates B., Seward E., Owen S., de Felipe C., Hunt S. and Wheeldon A. (2000) Pharmacological blockade or genetic deletion of substance P(NK(1)) receptors attenuates neonatal vocalisation in guinea-pigs and mice. Neuropharmacology 39, Rupniak N. M., Carlson E. J., Webb J. K., Harrison T., Porsolt R. D., Roux S., de Felipe C., Hunt S. P., Oates B. and Wheeldon A. (2001) Comparison of the phenotype of NK1R / mice with pharmacological blockade of the substance P (NK1) receptor in assays for antidepressant and anxiolytic drugs. Behav. Pharmacol. 12, Rupniak N. M., Carlson E. J., Shepheard S. et al. (2003) Comparison of the functional blockade of rat substance P (NK1) receptors by GR205171, RP67580, SR and NKP-608. Neuropharmacology 45, Santarelli L., Gobbi G., Debs P. C., Sibille E. T., Blier P., Hen R. and Heath M. J. (2001) Genetic and pharmacological disruption of neurokinin 1 receptor function decreases anxiety-related behaviors and increases serotonergic function. Proc. Natl Acad. Sci. USA 98, Ungerstedt U. (1984) Measurement of neurotransmitter release in vivo, in: Methods In Neuroscience, Vol. 6 (Marsden, C. A., ed.), pp Wiley-Interscience Publication, New York. Walker P. D., Riley L. A., Hart R. P. and Jonakait G. M. (1991) Serotonin regulation of tachykinin biosynthesis in the rat neostriatum. Brain Res. 546, Zocchi A., Varnier G., Arban R., Griffante C., Zanetti L., Bettelini L., Marchi M., Gerrard P. A. and Corsi M. (2003) Effects of antidepressant drugs and GR , an neurokinin-1 (NK1) receptor antagonist, on the response in the forced swim test and on monoamine extracellular levels in the frontal cortex of the mouse. Neurosci. Lett. 345,