Center of Excellence, Division of Molecular Metabolism and Diabetes, Tohoku University Graduate School of Medicine, Japan 2

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1 International Journal of Neuropsychopharmacology (27), 1, Copyright f 26 CINP doi:1.117/s Fluvoxamine, a selective serotonin reuptake inhibitor, and 5-HT 2C receptor inactivation induce appetite-suppressing effects in mice via 5-HT 1B receptors BRIEF REPORT CINP Katsunori Nonogaki 1,2,3, Kana Nozue 1, Yukiko Takahashi 2,3, Nobuyuki Yamashita 4, Shuichi Hiraoka 4, Hiroaki Kumano 2, Tomifusa Kuboki 2 and Yohsitomo Oka 1 1 Center of Excellence, Division of Molecular Metabolism and Diabetes, Tohoku University Graduate School of Medicine, Japan 2 Department of Psychosomatic Medicine, Faculty of Medicine, University of Tokyo, Japan 3 Laboratory of Molecular Nutrition, National Institute of Health and Nutrition, Japan 4 Exploratory Pharmacology Research Laboratories, Pharmaceutical Research Department, Meiji Seika Kaisha Ltd, Japan Abstract Serotonin (5-hydroxytryptamine; 5-HT) 2C receptors and the downstream melanocortin pathway are suggested to mediate the appetite-suppressing effects of 5-HT drugs such as m-chlorophenylpiperazine (mcpp) and fenfluramine. Here, we report that fluvoxamine (3 3 mg/kg), a selective serotonin reuptake inhibitor (SSRI), in the presence of SB (1 2 mg/kg), a selective 5-HT 2C receptor antagonist, exerts appetite-suppressing effects while fluvoxamine or SB alone has no effect. The appetite-suppressing effects were attenuated in the presence of SB (5 mg/kg), a selective 5-HT 1B receptor antagonist. Moreover, CP (5 1 mg/kg), a selective 5-HT 1B receptor agonist, exerted appetite-suppressing effects and significantly increased hypothalamic pro-opiomelanocortin (POMC) and cocaine- and amphetamine-regulated transcript (CART) gene expression and decreased hypothalamic orexin gene expression. These results suggest that fluvoxamine and inactivation of 5-HT 2C receptors exert feeding suppression through activation of 5-HT 1B receptors, and that 5-HT 1B receptors up-regulate hypothalamic POMC and CART gene expression and down-regulate hypothalamic orexin gene expression in mice. Received 27 June 26; Reviewed 26 July 26; Revised 28 July 26; Accepted 1 August 26; First published online 7 September 26 Key words: Appetite, POMC, SSRI, 5-HT 2C receptor, 5-HT 1B receptor. Introduction Drugs that increase central serotonin (5-hydroxytryptamine; 5-HT) activity have been used as appetite suppressants (Cruzon et al., 1997; Halford and Blundell, 2; Simansky, 1995). Several lines of evidence indicate that 5-HT 2C receptors contribute to the anorexic actions of 5-HT agonists such as m-chlorophenylpiperazine (mcpp) and 5-HT reuptake inhibitors and 5-HT releasers such as fenfluramine and norfenfluramine (Tecott et al., 1995; Vickers et al., 1999, 21). The hypothalamic distribution of 5-HT 2C receptors on pro-opiomelanocortin (POMC) neurons Address for correspondence: Katsunori Nonogaki, M.D., Ph.D., Associate Professor, Center of Excellence, Division of Molecular Metabolism and Diabetes, Tohoku University Graduate School of Medicine, 2-1 Seiryo-machi, Aoba-ku, Sendai, , Japan. Tel.: Fax: knonogaki-tky@umin.ac.jp indicates that 5-HT 2C receptors and the melanocortin (MC) pathway might interact in the regulation of feeding (Heisler et al., 22). Fenfluramine and mcpp activate POMC neurons that express 5-HT 2C receptors in the arcuate nucleus (ARC) in vitro (Heisler et al., 22). These findings suggest that appetite-suppressing effects of 5-HT drugs such as mcpp and fenfluramine require activation of 5-HT 2C receptors and the downstream MC pathway. On the other hand, pharmacological and/or genetic inactivation of 5-HT 2C receptors enhances the efficacy of SSRIs in rats and mice (Cremers et al., 24). SB 24284, a selective 5-HT 2C receptor antagonist, enhances the increase in extracellular 5-HT levels induced by the SSRI citapran in the hippocampus in rats while SB alone has no significant effects (Cremers et al., 24). In addition, increases in extracellular 5-HT levels induced by the SSRI fluoxetine in the prefrontal cortex are augmented in 5-HT 2C Downloaded from on 5 February 218

2 676 K. Nonogaki et al. receptor-null mutant mice (Cremers et al., 24). Moreover, inactivation of 5-HT 2C receptors enhances the antidepressive effects of SSRIs (Cremers et al., 24). Fluvoxamine, a selective serotonin reuptake inhibitor (SSRI), is prescribed to treat depression (Zanardi et al., 23). Fluvoxamine is also used to treat bingeeating disorder and bulimia nervosa (Ayuso-Gutierrez et al., 1994; Fichter et al., 1996; Hudson et al., 1988), and it has none of the cardiotoxic effects associated with fenfluramine. In the present study, to determine the effects of fluvoxamine on appetite in relation to 5-HT 2C receptors, we examined: (1) the effects of fluvoxamine on appetite following pretreatment with or without SB 24284, a selective 5-HT 2C receptor antagonist; (2) the effects of SB , a selective 5-HT 1B receptor antagonist, on appetite-suppressing effects induced by fluvoxamine in the presence of SB 24284; and (3) the effects of CP 94253, a selective 5-HT 1B receptor agonist, on food intake and the expression of hypothalamic genes involved in the regulation of feeding behavior in mice. Materials and methods Animals and drug treatment Four-week-old male C57BL6J mice (body weight g) were purchased from Japan CLEA (Tokyo, Japan). Mice were housed in individual cages with free access to water and chow pellets in a 12 h light- (lights on: 8: hours, lights off: 2: hours) and temperature-controlled environment (2 22 xc). Animals were acclimatized for 1 wk before the experiment. Drugs were administered between 1: and 12: hours. Fluvoxamine, SB dihydrochloride (a selective blood brain barrier-penetrating 5-HT 2C receptor antagonist), SB hydrochloride (a selective blood brain barrier penetrating 5-HT 1B receptor antagonist), and CP hydrochloride (a selective 5-HT 1B receptor agonist), were purchased from Sigma Chemical Co., Japan. Fluvoxamine and CP hydrochloride were dissolved in saline; SB was dissolved in distilled water and suspended in saline and SB was dissolved in DMSO and suspended in saline. The dose of SB (1 mg/kg) eliminates 5-HT 2C receptor functions in vivo as described previously (Kennet et al., 1997; Yamauchi et al., 24). The dose of SB dihydrochloride (5 mg/kg) eliminates 5-HT 1B receptor functions in vivo (Lee et al., 24a,b). The doses of fluvoxamine (3 3 mg/kg) were chosen from the range at which the drug exerts biological effects without inducing toxicity in rodents (Fletcher et al., 22; Kamei et al., 23; Van Dijken et al., 1992; Yamauchi et al., 24). The 3 mg/kg dose of fluvoxamine elicits antidepressantlike behavioral effects in rodents (Kamei et al., 23; Yamauchi et al., 24). At first, animals were deprived of food for 23 h and were injected intraperitoneally (i.p.) with saline or SB dihydrochloride (1 mg/kg). SB dihydrochloride has anxiolytic-like activity, but does not have hyperphagic properties like 5-HT 2C receptor mutant mice (Kennet et al., 1997). Thirty minutes later, animals were injected with saline or fluvoxamine (3 3 mg/kg) i.p., and after a further 3 min they were given chow pellets; intake of pellets was measured for the next hour as described previously (Tecott et al., 1995). In the second experiment, animals were deprived of food for 23 h and were injected i.p. with saline or SB dihydrochloride (.5 2. mg/kg). Thirty minutes later, animals were injected i.p. with saline or fluvoxamine (1 mg/kg), and after a further 3 min they were given chow pellets; intake of pellets was measured for the next hour. In the third experiment, animals were deprived of food for 23 h and were injected i.p. with saline or SB dihydrochloride (1 mg/kg). Thirty minutes later, animals were injected i.p. with saline or fluvoxamine (3 mg/kg), and after a further 3 min they were given chow pellets; intake of pellets was measured for the next 6 h. In the fourth experiment, animals were deprived of food for 23 h and were injected i.p. with saline or SB dihydrochloride (2 mg/kg) plus SB hydrochloride (5 mg/kg). Thirty minutes later, animals were injected i.p. with saline or fluvoxamine (1 mg/kg), and after a further 3 min they were given chow pellets; intake of pellets was measured for the next 2 h. In the fifth experiment, animals were deprived of food for 23 h and were injected i.p. with saline or CP (2.5, 5, 1 mg/kg). Thirty minutes later, they were given chow pellets and intake of pellets was measured for next 2 h. Finally, 5-wk-old male C57BL6J mice were injected i.p. with saline or CP (1 mg/kg). Sixty minutes later, the animals were decapitated; animals were not fed. The hypothalamus was removed for RNA extraction. Real-time quantitative reverse transcription polymerase chain reaction (RT PCR) Total RNA was isolated from mouse hypothalamic tissue using the RNeasy Midi kit (Qiagen, Hilden, Germany) according to the manufacturer s Downloaded from on 5 February 218

3 Fluvoxamine, 5-HT receptors and appetite suppression in mice 677 instructions, and cdna synthesis was performed using a SuperScript III First-Strand Synthesis System for RT-PCR kit (Invitrogen, Rockville, MD, USA) using 1 mg total RNA. cdna synthesized from total RNA was evaluated in a real-time PCR quantitative system (Light Cycler Quick System 35S; Roche Diagnostics, Mannheim, Germany). The primers used were as follows. For mouse POMC, sense: 5k-ATA GAT GTG TGG AGC TGG TG-3k, antisense: 5k-GGC TGT TCA TCT CCG TTG-3k; for mouse cocaine- and amphetamine-regulated transcript (CART), sense: 5k-CTG GAC ATC TAC TCT GCC GTG G-3k, antisense: 5k-GTT CCT CGG GGA CAG TCA CAC AGC-3k; for mouse neuropeptide Y (NPY), sense: 5k-GCT TGA AGA CCC TTC CAT TGG TG-3k, antisense: 5k-GGC GGA GTC CAG CCT AGT GG-3k; for mouse agouti-related protein (AGRP), sense: 5k-CAG ACC GAG CAG AAG AAG-3k, antisense: 5k-GAC TCG TGC AGC CTT ACA-3k; for mouse corticotropin-releasing hormone (CRH), sense: 5k-CCG GGC AGA GCA GTT AGC-3k, antisense: 5k-CAA CAT TTC ATT TCC CGA TAA TCT C-3k; for mouse orexin, sense: 5k-CTC CTT CAG GCC AAC GGT A-3k, antisense: 5k-GTG GTA GTT ACG GTC GGA CA-3; for mouse 5-HT 1B receptor, sense: 5k-TGC CTG CTG GTT TCA CAT-3k,5k-ATA GAT GTG TGG AGC TGG TG-3k, antisense: 5k-GCG CAC TTA AAG CGT ATC A-3k; for mouse b-actin, sense: 5k-TTG TAA CCA ACT GGG ACG ATA TGG-3k, antisense: 5k-GAT CTT GAT CTT CAT GGT GCT AGG-3k. The relative amount of mrna was calculated using b-actin mrna as the invariant control. The data are shown as the fold change of the mean value of the control group, which received saline. Statistical methods Data are presented as the mean value S.E.M. Statistical significance of differences between two groups was determined using Student s t test. Comparisons among more than two groups were done by ANOVA using Bonferroni s test. p<.5 was considered statistically significant. The animal studies were conducted under protocols in accordance with the institutional guidelines for animal experiments at Tohoku University, National Institute of Health and Nutrition, and Meiji Seika Kaisha. Results Dose response effects of fluvoxamine on food intake after pretreatment with or without SB Fluvoxamine (3 3 mg/kg) without SB had no effect on food intake during the first hour of the feeding period after a 23-h fast in C57BL6J mice. All non-zero doses tested had the same response (Figure 1a). In contrast, fluvoxamine (3 3 mg/kg) following pretreatment with SB (1 mg/kg) significantly reduced food intake compared with saline-treated controls during the same period [to y74% (3 mg/kg), 7% (1 mg/kg), 7% (3 mg/kg) that of controls; p<.5] (Figure 1a). In addition, the group treated with fluvoxamine (3 3 mg/kg) following pretreatment with SB (1 mg/kg) significantly reduced food intake compared with the group treated with fluvoxamine without SB at each dose (Figure 1a). There were no differences in food intake between groups treated with saline and SB without fluvoxamine. These findings demonstrate that regardless of dose, fluvoxamine following treatment with SB (1 mg/kg) suppresses food intake during the first hour of the feeding period after a 23-h fast, while fluvoxamine alone has no effect. To confirm the results, we examined the effect of various doses of SB (.5 2. mg/kg) on fluvoxamine (1 mg/kg)-induced feeding suppression. The group treated with fluvoxamine (1 mg/kg) following pretreatment with SB ( and 2. mg/kg) had significantly reduced food intake compared with the group treated with saline or fluvoxamine without SB (Figure 1b). Time-course of the effects of fluvoxamine on food intake following pretreatment with or without SB To determine the time-course of the anorexic effects of fluvoxamine following treatment with SB 24284, we examined the effect of fluvoxamine (3 mg/kg) following treatment with SB (1 mg/kg) on food intake during a 4-h feeding period after a 23-h fast. The anorexic effects of fluvoxamine following pretreatment with SB were sustained for 2 h (to y7% that of controls), but no significant anorexic effects were observed after 3 h (Figure 1c). These findings indicate that fluvoxamine suppresses food intake when 5-HT 2C receptor signalling is blocked in mice. Effects of fluvoxamine on food intake following treatment with SB plus SB To determine the role of 5-HT 1B receptors in the appetite-suppressing effects induced by fluvoxamine following pretreatment with SB 24284, we examined the effects of SB (5 mg/kg), a selective 5-HT 1B receptor antagonist, on feeding suppression induced by fluvoxamine (3 mg/kg) in the presence of SB Downloaded from on 5 February 218

4 678 K. Nonogaki et al. (a) 1.5 (b) Fluvoxamine (mg/kg) SB (mg/kg) (c) Saline SB Fluvo SB+Fluvo Time (h) 3 4 Figure 1. (a) Dose response effects of fluvoxamine (3 3 mg/kg) after pretreatment with or without SB (1 mg/kg) on food intake in mice. Open circles: saline with or without fluvoxamine; filled circles: SB with or without fluvoxamine. p<.5 vs. SB (1 mg/kg) without fluvoxamine. (b) Dose response effects of SB (.5 2 mg/kg) before administration of fluvoxamine (1 mg/kg) on food intake in mice. (c) Time-course of the effects of fluvoxamine (3 mg/kg) following pretreatment with or without SB (1 mg/kg) on food intake in mice. Drugs were administered as described in the Materials and Methods section. Values and each column and bar represents the mean value S.E.M. of 7 8 mice. SB, SB 24284; Fluvo, Fluvoxamine ( p<.5) (2 mg/kg) in mice. Fluvoxamine following pretreatment with SB plus SB reversed the feeding suppression induced by fluvoxamine following SB for a 2-h feeding period after a 23-h fast (Figure 2a). These results indicate that 5-HT 1B receptors contribute to appetite suppression induced by fluvoxamine in the presence of SB SB plus SB had no effect on food intake compared with saline controls for a 2-h feeding period after a 23-h fast (Figure 2b). Effects of SB on hypothalamic 5-HT 1B receptor mrna levels To determine whether inactivation of 5-HT 2C receptors can alter 5-HT 1B receptor gene expression, we examined the effects of SB on hypothalamic 5-HT 1B receptor mrna levels in mice. SB (1 mg/kg) significantly increased hypothalamic 5-HT 1B receptor mrna levels compared with saline controls (1.7-fold, Figure 2b). These results suggest Downloaded from on 5 February 218

5 Fluvoxamine, 5-HT receptors and appetite suppression in mice 679 (a) (b) h Saline SB1+SB2 1 h Saline SB1+SB2 2 h (c) 5-HT 1B receptor mrna 2 h Saline Fluvo SB1+Fluvo SB1+SB2+Fluvo SB Figure 2. (a) Effects of fluvoxamine (3 mg/kg) following treatment with SB (2 mg/kg) plus SB (5 mg/kg) on food intake, (b) effects of SB (2 mg/kg) plus SB (5 mg/kg) on food intake, and (c) effects of SB (1 mg/kg) on hypothalamic 5-HT 1B receptor mrna levels in mice. Drugs were administered as described in the the Materials and Methods section. Each column and bar represents the mean value S.E.M. of several mice: (a) n=13 for each group, (b) n=6 for each group, (c) n=5 for each group. Fluvo, Fluvoxamine; SB1, SB 24284; SB2, SB ( p<.5). that inactivation of 5-HT 2C receptors increases 5-HT 1B receptor gene expression in the hypothalamus of mice. Effects of CP on food intake and hypothalamic POMC, CART, CRH, NPY, AGRP and orexin mrna levels To determine the role of 5-HT 1B receptors in food intake and the expression of hypothalamic genes involved in the regulation of feeding and energy homeostasis, we examined the effects of CP 94253, a selective 5-HT 1B receptor agonist, on food intake and mrna levels of the hypothalamic peptides POMC, CART, CRH (anorexigenic peptides) and NPY, AGRP, orexin (orexigenic peptides) (Friedman, 24). CP (5 1 mg/kg) significantly suppressed food intake for 2 h (Figure 3a). CP (1 mg/kg) significantly increased hypothalamic POMC (2.7-fold) and CART (2-fold) mrna levels compared with saline controls and decreased hypothalamic orexin mrna levels (.54-fold), while having no significant effects on hypothalamic CRH, NPY and AGRP mrna levels (Figure 3b). These results suggest that activation of 5-HT 1B receptors increases hypothalamic POMC and CART gene expression and decreases hypothalamic orexin gene expression, leading to hypophagic effects in mice. Discussion Fluvoxamine and fenfluramine have different actions on 5-HT systems; fenfluramine inhibits 5-HT reuptake and stimulates 5-HT release from nerve terminals whereas fluvoxamine selectively inhibits 5-HT reuptake but does not directly stimulate 5-HT receptors or 5-HT release. Pharmacological and/or genetic inactivation of 5-HT 2C receptors attenuates the anorexic effects of fenfluramine in rats and mice (Tecott et al., 1995; Vickers et al., 1999, 21). On the other hand, the present results demonstrate that pharmacological inactivation of 5-HT 2C receptors elicits anorexic effects of fluvoxamine in mice. Thus, inactivation of 5-HT 2C receptors exerts opposite effects on the actions of fenfluramine and fluvoxamine. Pharmacological and/or genetic inactivation of 5-HT 2C receptors, however, enhances the increases in extracellular 5-HT levels induced by the SSRIs citapran and fluoxetine in the hippocampus and the prefrontal cortex in rats and Downloaded from on 5 February 218

6 68 K. Nonogaki et al. (a) Saline CP 2.5 mg/kg CP 5 mg/kg CP 1 mg/kg 1 h 2 h 3 h (b) Fold change by RT PCR POMC CART NPY Saline CP1 mg/kg AGRP CRH Orexin Figure 3. Effects of CP (2.5 1 mg/kg) on (a) food intake and (b) hypothalamic gene expression in mice. Drugs were administered as described in the Materials and Methods section. Each column and bar represents the mean value S.E.M. of several mice. (a) n=12 for saline controls and n=6 for each group treated with CP 94253, (b) n=6 for each group ( p<.5). mice while SB alone has no significant effect (Cremers et al., 24). Accordingly, it is likely that the antagonism of 5-HT 2C receptors acts synergistically with fluvoxamine to increase extracellular 5-HT concentrations even further, leading to a 5-HT-dependent suppression of food intake. 5-HT 1B receptors, which are located on presynaptic neurons and stimulate 5-HT release, and 5-HT 2C receptors, which are located on post-synaptic neurons (Barnes and Sharp, 1999), are suggested to mediate 5-HT-induced feeding suppression (Tecott et al., 1995; Vickers et al., 1999, 21; Lee et al., 24a,b). Because a selective 5-HT 1B receptor agonist-induced hypophagia is not attenuated by pharmacological and/or genetic inactivation of 5-HT 2C receptors (Dalton et al., 26), it is unlikely that 5-HT 2C receptors mediate hypophagia induced by activation of 5-HT 1B receptors. Rather, 5-HT 2C receptor mutant mice are more sensitive to CP induced hypophagia than wild-type mice (Dalton et al., 26). The present study demonstrates that pharmacological inactivation of 5-HT 2C receptors rapidly increases hypothalamic 5-HT 1B receptor gene expression, suggesting that 5-HT 2C receptors downregulate 5-HT 1B receptor gene expression. It is likely that activated 5-HT 1B receptors act synergistically with fluvoxamine to induce appetite suppression. It is probable that 5-HT and hypothalamic neuropeptide neurons interact in the regulation of feeding. In addition to the central MC pathway (Heisler et al., 22), our previous and present studies raise the possibility that CART contributes to 5-HT-mediated signals for satiety (Nonogaki et al., 26) because CART neurons have a downstream pathway distinct from the MC pathway (Broberger, 1999; Elias et al., 21). In addition, our present results indicate that activation of 5-HT 1B receptors induces an increase in hypothalamic POMC and CART gene expression and a decrease in hypothalamic orexin gene expression, leading to hypophagic effects. The hypophagic effects induced by activation of 5-HT 1B receptors support previous results by others (Dalton et al., 26; Lee et al., 24a). In summary, the present findings suggest that fluvoxamine and 5-HT 2C receptor inactivation exerts appetite-suppressing effects via 5-HT 1B receptors. In addition, activation of 5-HT 1B receptors increases hypothalamic POMC and CART gene expression and decreases hypothalamic orexin gene expression. Thus, we demonstrate a novel interaction between 5-HT 2C and 5-HT 1B receptors and also between 5-HT 1B receptors and neuropeptides in the regulation of feeding behavior. Acknowledgements We thank Ms. Youko Hasegawa for experimental assistance, Ms. Mie Clarke for secretarial assistance, and Dr Bruce Chasten for critical reading of the manuscript. This work was supported by a Grantin-Aid for Scientific Research (C2) and Human Science Research (KH2116). Statement of Interest None. References Ayuso-Gutierrez JL, Palazon M, Ayuso-Mateos JL (1994). Open trial of fluvoxamine in the treatment of bulimia nervosa. International Journal of Eating Disorders 15, Downloaded from on 5 February 218

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