J Pharmacol Sci 105, 000 – 000 (2007) Journal of Pharmacological Sciences ©2007 The Japanese Pharmacological Society Full Paper

Effects of on the Freezing Behavior in the Elevated Open-Platform Test in Mice

Shigeo Miyata1, Toshiko Shimoi1, Shoko Hirano1, Naoko Yamada1, Yoko Hata1, Naoki Yoshikawa1, Masahiro Ohsawa1, and Junzo Kamei1,* 1Department of Pathophysiology and Therapeutics, School of Pharmacy and Pharmaceutical Sciences, Hoshi University, Tokyo 142-8501, Japan

Received February 6, 2007; Accepted September 21, 2007

Abstract. Freezing behavior is thought to be a sign of fear in animals. We examined whether the freezing behavior during the elevated open-platform stress, which is a psychological stressor without painful stimulus, is modulated by serotonergic neurotransmission and would be a useful marker for screening and/or . Male ICR mice (6 – 8-week-old) were individually placed on an elevated open-platform and the duration of freezing behavior of mouse was measured for 10 min. and citalopram, selective (5-HT) reuptake inhibitors, markedly decreased the duration of freezing. , a 5-HT releaser, and 8- OH-DPAT, a potent 5-HT1A- , also significantly decreased the duration of freez- ing. In contrast, the 5-HT-synthesis inhibitor p-chlorophenylalanine significantly increased the duration of freezing. , a anxiolytic, had no effect on the duration of freezing at doses having no effect on locomotor activity. and , tricyclic , also did not affect the duration of freezing. Reboxetine, a selective noradrenaline reuptake inhibitor, significantly increased the duration of freezing. These results indicate that the activation of serotonergic neurotransmission attenuates the fear-related behavior in the elevated open-platform test, while the activation of noradrenergic neurotransmission increases the fear- related behavior. In addition, this test is convenient for assaying anxiolytic drugs that affect serotonergic neurotransmission.

Keywords: screening method, serotonin (5-HT), anxiolytic, selective 5-HT reuptake inhibitor (SSRI), antidepressant

Introduction used to assess the state of animals and to screen the anxiolytic effect of drugs. Although the anxiolytic- Various drugs affecting serotonin (5-HT) neurotrans- like effect of drugs that act on γ-aminobutyric acid mission, such as selective 5-HT reuptake inhibitors (GABA)/benzodiazepine neurotransmission is clearly (SSRIs) and 5-HT1A-receptor , are effective in demonstrated in the elevated plus-maze test, the drugs the treatment of anxiety disorders. In animal studies, that affect serotonergic neurotransmission vary greatly researchers have, however, reported conflicting observa- depending on the report (1 – 7). tions: that is, serotonergic agents induce anxiolytic-like The response to punishment, which is defined as the or anxiogenic-like responses. One potential interpreta- presentation of a noxious stimulus that results in the tion of these findings is that the nature of stress in suppression of behavior, is also used as an index of the different animal models differs qualitatively (1, 2). anxiety/fear state of animals (8). The four-plate test, Methods that are used to assess natural aversion in shock-probe burying test and conditioned fear test animals, such as the elevated plus-maze test, are widely measure the punished responses of animals. These tests are sensitive to the anxiolytic effects of serotonergic *Corresponding author. [email protected] agents such as SSRIs and 5-HT1A-receptor agonists (1, Published online in J-STAGE: 9 – 11). However, painful electric shocks are generally doi: 10.1254/jphs.FP0070314 required to elicit the punished responses and ‘pain’ is

1 2 S Miyata et al one of the problems in using these tests as a screening Drugs method. For example, if the novel drug has an anti- The drugs used in this study were fluoxetine hydro- nociceptive effect, further investigations would be chloride (LKT Laboratories, Inc., St. Paul, MN, USA); required to negate the possibility that the drug-induced DL-p-chlorophenylalanine methyl ester hydrochloride anti-punishment effects were due to changes in pain (PCPA), citalopram hydrobromide, clomipramine hydro- sensitivity. Therefore, investigators need a new method chloride, (±)-8-hydroxy-2-(di-n-propylamino) tetralin that is simple and highly sensitive to the effects of hydrobromide (8-OH-DPAT), and imipramine hydro- serotonergic anxiolytics, but does not require a painful chloride (Sigma Chemical Co., St. Louis, MO, USA); stimulus. diazepam and (±)-fenfluramine hydrochloride (Wako Elevated open-platform stress is often used as a Pure Chemical Industries, Ltd., Osaka); and reboxetine psychological stressor without a painful stimulus (12, mesylate (Tocris Cookson, Ltd., Bristol, UK). Other 13). Rodents display characteristic behavioral responses chemicals were purchased from Sigma and Wako. that consist of stress-related behaviors (freezing Fluoxetine and citalopram are SSRIs. Fenfluramine is a behavior, urination, defecation) along with exploratory 5-HT releaser. 8-OH-DPAT is a potent 5-HT1A-receptor behaviors (walking, rearing, sniffing, head-dipping) on agonist. PCPA is a synthesis inhibitor of 5-HT. Diazepam the elevated open-platform. Freezing behavior is is a benzodiazepine-receptor agonist. Imipramine and characterized by a crouching posture with no movement clomipramine are tricyclic antidepressants. Reboxetine except for respiratory movement and uses it to cope is a selective noradrenaline-reuptake inhibitor. PCPA with a fearful stimulus (14). Therefore, we hypothesized was dissolved in distilled water. Diazepam was dis- that elevated open-platform stress-induced freezing solved in ethanol and then diluted in saline with one behavior might be a useful index of the anxiety/fear drop of Tween-80 (final ethanol concentration is state in animals. In addition, since conditioned fear 0.12%). The other drugs were dissolved in saline. Each stress-induced freezing behavior is suppressed by sero- drug was administered at a volume of 0.1 mL/10 g of tonergic anxiolytics (11), we speculated that freezing body weight. The dose ranges of drugs were based on behavior during elevated open-platform stress might our pilot study and the previous reports (15 – 22). All also be suppressed by serotonergic anxiolytics. drugs except for PCPA were injected 30 min before To clarify these hypotheses, we examined the effects testing. PCPA was injected i.p. twice daily for 3 days, of several anxiolytics and antidepressants on the dura- and the experiment was conducted 15 h after the last tion of freezing in the elevated open-platform test. In injection. addition, we also investigated the effect of 5-HT deple- tion on the duration of freezing. Elevated open-platform test The experiment was conducted in a room illuminated Materials and Methods by white fluorescent light (300 lux). The transparent acrylic cylinder (11.4-cm diameter, 18-cm-high) was This study was carried out in accordance with the placed upside-down and mice were placed individually Guiding Principles for the Care and Use of Laboratory on the top (open-platform) for 10 min. When the mouse Animals, Hoshi University, as adopted by the Commit- slipped off the platform, it was immediately replaced on tee on Animal Research of Hoshi University, which is the open-platform and the measurement was continued. accredited by the Ministry of Education, Culture, Sports, The behavior of mice was recorded by a video camera Science, and Technology of Japan. Every effort was and an observer blindly measured the duration of freez- made to minimize the number of animals used in the ing. Freezing behavior was defined as the absence of following experiments. movement excluding respiration. The duration of freez- ing was the total amount of time that the animal showed Animals freezing. PCPA-treated mice and control mice were Male ICR mice (Tokyo Laboratory Animals Science decapitated immediately after the behavioral experi- Co., Ltd., Tokyo), 6 – 8 weeks of age, were used. They ment, and the whole brain was removed and stored at were housed 5 per cage and had free access to food and −80°C for measurement of brain monoamine contents. water. The animal room was maintained at 24 ± 1°C and 55 ± 5% humidity with a 12-h light-dark cycle (light Locomotor activity on at 8:00, light off at 20:00). The experiments were The locomotor activity of mice was measured by a performed between 11:00 and 17:00 each day. Each digital counter with an infrared sensor (NS-AS01; animal was used only once. Neuroscience, Inc., Tokyo). The apparatus detects the movement of animals based on the release of infrared Elevated Open-Platform Test 3 rays associated with their temperature and records a digital count. A mouse was placed in a transparent plastic cage (27 × 17 × 13 cm), a transparent plastic ceiling was installed, and an infrared sensor was placed at the center of the ceiling. Mice were placed in the measurement cage and then recording was started. Total activity counts were recorded automatically for 10 min, which was the same duration as the measurement period in the elevated open-platform test.

Monoamine contents The contents of 5-HT and its metabolite 5-hydroxy- indoleacetic acid (5-HIAA), noradrenaline and its metabolite 3-methoxy-4-hydroxyphenylethyleneglycol (MHPG), and and its metabolites 3,4- dihydroxyphenylacetic acid (DOPAC) and 3-methoxy- 4-hydroxyphenylacetic acid (HVA) were determined by HPLC-ECD equipped with an SC-5ODS column (Eicom, Co., Kyoto). Whole brains were homogenized in 2.5 ml of solution containing 0.2 M perchloric acid with 100 µM EDTA (2Na) and 1 mg/l isoproterenol as an internal standard. The homogenates were centrifuged at 20,000 × g for 15 min at 4°C, and the supernatants were obtained and maintained at pH 3.0 using 1 M sodium acetate. The samples of 40 µl were analyzed. The mobile phase consisted of sodium acetate (0.1 M)/citric acid (0.1 M) buffer, pH 3.5, containing 15% (v/v) methanol, sodium 1-octanesulfonate, and Fig. 1. Effects of fluoxetine, citalopram, fenfluramine, and 8-OH- EDTA (2Na). The flow rate was set to 0.5 ml/min with DPAT on the duration of freezing in the elevated open-platform test a column temperature of 25°C. (A) and locomotor activity (B). A: Each column represents the mean ± S.E.M. of 9 – 20 mice. *P<0.05, **P<0.01, ***P<0.001 vs saline-treated group (Dunnett test). B: Each column represents the Statistics mean ± S.E.M. of 4 – 7 mice. The data are expressed as means ± S.E.M. Significant differences were determined by one-way analysis of variance (ANOVA) for factorial comparisons and the PCPA (250 mg/kg, i.p. twice daily for 3 days) Dunnett test for multiple comparisons. Aspin-Welch’s significantly increased the duration of freezing in mice t-test was used to evaluate the significance of differences (Fig. 2A). PCPA (250 mg/kg, i.p. twice daily for between groups. Probability values of less than 0.05 3 days), however, did not affect locomotor activity in (P<0.05) were considered statistically significant. mice (Fig. 2B). The 5-HT and 5-HIAA contents were significantly decreased in PCPA-treated mice without Results changing the contents of noradrenaline, dopamine, or their respective metabolites (Table 1). Fluoxetine (0.1 – 10 mg/kg, i.p.) and citalopram Diazepam (0.03 – 1 mg/kg, i.p.) had no significant (0.1 – 10 mg/kg, i.p.) dose-dependently and significantly effect on the duration of freezing in the elevated open- decreased the duration of freezing in mice (Fig. 1A). platform test (Fig. 3A) or locomotor activity in mice Fenfluramine (0.1 – 10 mg/kg, i.p.) also dose-depen- (Fig. 3B). However, i.p. treatment with diazepam at dently and significantly decreased the duration of freez- 3mg/kg significantly increased the duration of freezing ing (Fig. 1A). Furthermore, 8-OH-DPAT (3 – 30 µg in the elevated open-platform test (Fig. 3A). Diazepam /kg, i.p.) dose-dependently and significantly decreased at 3 mg/kg significantly decreased locomotor activity in the duration of freezing (Fig. 1A). Fluoxetine, citalo- mice (Fig. 3B). pram, fenfluramine, or 8-OH-DPAT had no significant Imipramine (0.1 – 10 mg/kg, i.p.) and clomipramine effect on locomotor activity in mice at the doses (0.1–10mg/kg, i.p.) did not affect the duration of examined (Fig. 1B). freezing in the elevated open-platform test (Fig. 4A). 4 S Miyata et al

Fig. 2. Effects of PCPA on the duration of freezing in the elevated open-platform test (A) and locomotor activity (B). A: Each column represents the mean ± S.E.M. of 10 mice. *P<0.01 vs saline-treated group (Aspin-Welch’s t-test). B: Each column represents the mean ± S.E.M. of 5 mice. Fig. 3. Effects of diazepam on the duration of freezing in the elevated open-platform test (A) and locomotor activity (B). A: Each column represents the mean ± S.E.M. of 10 – 20 mice. B: Each column represents the mean ± S.E.M. of 5 – 10 mice. ***P<0.001 vs vehicle-treated group (Dunnett test). Reboxetine (0.1 – 1 mg/kg, i.p.) dose-dependently and significantly increased the duration of freezing (Fig. 4A). Discussion Imipramine (0.1 – 10 mg/kg, i.p.), clomipramine (0.1 – 10 mg/kg, i.p.), or reboxetine (0.1 – 1 mg/kg, i.p.) had Fluoxetine, citalopram, fenfluramine, and 8-OH- no effect on locomotor activity (Fig. 4B). DPAT markedly decreased the duration of freezing in

Table 1. Effects of PCPA on the contents of 5-HT, noradrenaline (NA), dopamine (DA), and their major metabo- lites in the whole brain Saline PCPA Contents (ng/mg tissue) 5-HT 0.2806 ± 0.0064 0.0803 ± 0.0056*** 5-HIAA 0.1395 ± 0.0030 0.0372 ± 0.0015*** NA 0.1193 ± 0.0066 0.1160 ± 0.0032 MHPG 0.2812 ± 0.0103 0.2747 ± 0.0107 DA 0.6811 ± 0.0131 0.6827 ± 0.0209 DOPAC 0.0559 ± 0.0014 0.0541 ± 0.0025 HVA 0.0797 ± 0.0013 0.0760 ± 0.0033

Data represent the mean with S.E.M. of 10 mice. ***P<0.001 vs saline-treated mice. Elevated Open-Platform Test 5

cant prolongation of duration of freezing in diazepam (3 mg/kg, i.p.)-treated mice is likely to be reflected in the hypolocomotor activity. We previously reported that diazepam at 0.3 mg/kg exerted the significant anxiolytic-like effect in the mouse hole-board test (20). Therefore, the freezing behavior in the elevated open- platform test may not be affected by diazepam at the dose having no effect on locomotor activity. The elevated plus-maze test is a popular method for screen- ing anxiolytics and examining their mechanisms of action. This test is reported to be suitable for detecting anxiolytics acting on GABAergic systems but not sero- tonergic ones (1, 2). On the other hand, the elevated open-platform test detects the anxiolytics that affects serotonergic neurotransmission. Therefore, the elevated open-platform test is more sensitive than the elevated plus-maze test to screen serotonergic anxiolytics and to study the serotonergic function in the brain, although it needs to be accompanied by the measurement of loco- motor activity as an additional behavior test. In the elevated plus-maze test, increase of 5-HT levels in the hippocampus induces the anxiety-like behavior in rats (24) possibly mediated by the activation of 5-HT2C receptors (25). In contrast, the increase of 5-HT levels by SSRI exerts the anti-freezing effect in the elevated open-platform test. Since the activation of 5-HT1A receptors decreased the duration of freezing, the anti- Fig. 4. Effects of imipramine, clomipramine, and reboxetine on freezing effect of SSRIs would be mediated by the the duration of freezing in the elevated open-platform test (A) and activation of 5-HT1A receptors. Therefore, it is possible ± locomotor activity (B). A: Each column represents the mean S.E.M. that 5-HT receptor subtypes associating with the regula- of 9 – 10 mice. ***P<0.001 vs saline-treated group (Dunnett test). B: Each column represents the mean ± S.E.M. of 5 – 8 mice. tion of mouse behavior are different between the elevated plus-maze test and the elevated open-platform test. the elevated open-platform test in mice without affecting In the present study, reboxetine significantly in- locomotor activity. In addition, PCPA-induced depletion creased the duration of freezing without affecting of 5-HT significantly increased the duration of freezing. locomotor activity. Reboxetine selectively inhibits the These results indicate that the freezing behavior in the reuptake of synaptic noradrenaline without any marked elevated open-platform test is modulated by the influence on other neurotransmissions (19, 26). Several serotonergic activity in the brain. In addition, the acti- lines of evidence indicate that noradrenergic and sero- vation of 5-HT1A receptors produces the anti-freezing tonergic neurotransmissions may play opposite roles in effect in the elevated open-platform test. Recently, we the mechanisms of fear learning and fear expression. reported that streptozotocin-induced diabetic mice Noradrenergic neurotransmission elicits the fear-related exhibit a prolonged duration of freezing in the elevated behavior in animals while serotonergic neurotransmis- open-platform test (23). In the microdialysis study, the sion suppresses the fear-related behavior (11, 27). elevated open-platform stress-elicited 5-HT release was Therefore, the reboxetine-induced activation of nor- markedly decreased in the prefrontal cortex, but not adrenergic neurotransmission may cause the prolonged amygdala, of diabetic mice (23). Therefore, the duration duration of freezing in the elevated open-platform test. of freezing in the elevated open-platform test could be Imipramine and clomipramine did not affect the duration affected by the serotonergic neurotransmission in the of freezing in the elevated open-platform test. In prefrontal cortex. microdialysis studies, imipramine and clomipramine Diazepam failed to affect the duration of freezing in increased equally the extracellular levels of both 5-HT the elevated open-platform test at the doses that did not and noradrenaline (28, 29). Therefore, the absence of significantly affect locomotor activity in mice. Signifi- anti-freezing effects for tricyclic antidepressants may be 6 S Miyata et al due to their dual effects on the serotonergic and nor- 10 Lopez-Rubalcava C. Pre- or postsynaptic activity of 5-HT1A adrenergic neurotransmissions. compounds in mice depends on the anxiety paradigm. Pharmacol The cliff-avoidance reaction on the elevated open- Biochem Behav. 1996;54:677–686. 11 Hashimoto S, Inoue T, Koyama T. Serotonin reuptake inhibitors platform is known to be an index of behavioral terato- reduce conditioned fear stress-induced freezing behavior in rats. logy in rodents, which can be impaired by motor, Psychopharmacology. 1996;123:182–186. arousal, or cognitive dysfunction (30, 31). Impairment of 12 Balfour DJ, Reid A. Effects of betamethasone on the stimulation the cliff-avoidance reaction is defined as falling (or of corticosterone secretion in rats. Arch Int Pharmacodyn Ther. jumping) from the edge of the open-platform. In the 1979;237:67–74. present study, we did not observe disturbance of either 13 Degroot A, Wade M, Salhoff C, Davis RJ, Tzavara ET, Nomikos the cliff-avoidance reaction (data not shown) or loco- GG. Exposure to an elevated platform increases plasma motor activity in mice. Therefore, the anti-freezing corticosterone and hippocampal acetylcholine in the rat: reversal by . Eur J Pharmacol. 2004;493:103–109. effects of serotonergic agents are not related to motor or 14 Koolhaas JM, Korte SM, De Boer SF, Van Der Vegt BJ, Van cognitive impairment. Reenen CG, Hopster H, et al. Coping styles in animals: current In conclusion, the present findings indicate that the status in behavior and stress-physiology. Neurosci Biobehav freezing behavior in the elevated open-platform test is Rev. 1999;23:925–935. modulated oppositely by serotonergic and noradrenergic 15 Sarkissian CF, Wurtman RJ, Morse AN, Gleason R. Effects of neurotransmissions. In addition, anxiolytics that affect fluoxetine or D-fenfluramine on serotonin release from, and serotonergic neurotransmission can be assayed by the levels in, rat frontal cortex. Brain Res. 1990;529:294–301. 16 Tanda G, Carboni E, Frau R, Di Chiara G. Increase of extra- elevated open-platform test. cellular dopamine in the prefrontal cortex: a trait of drugs with antidepressant potential? Psychopharmacology. 1994;115:285– Acknowledgements 288. 17 Invernizzi R, Velasco C, Bramante M, Longo A, Samanin R. We thank Mr. N. Kaneda and Ms. A. Kurihara for Effect of 5-HT1A receptor antagonists on citalopram-induced their excellent technical assistances. increase in extracellular serotonin in the frontal cortex, striatum and dorsal hippocampus. Neuropharmacology. 1997;36:467– 473. References 18 Hatanaka K, Yatsugi S, Yamaguchi T. Effect of acute treatment with YM992 on extracellular serotonin levels in the rat frontal 1 Hascoet M, Bourin M, Nic Dhonnchadha BA. The influence of cortex. Eur J Pharmacol. 2000;395:23–29. , and its metabolite 1-PP, on the activity of paroxetine 19 Page ME, Lucki I. Effects of acute and chronic reboxetine in the mouse light/dark paradigm and four plates test. Pharmacol treatment on stress-induced monoamine efflux in the rat frontal Biochem Behav. 2000;67:45–53. cortex. Neuropsychopharmacology. 2002;27:237–247. 2 Pinheiro SH, Zangrossi-Jr H, Del-Ben CM, Graeff FG. Elevated 20 Kamei J, Ohsawa M, Tsuji M, Takeda H, Matsumiya T. mazes as animal models of anxiety: effects of serotonergic Modification of the effects of on the explor- agents. An Acad Bras Cienc. 2007;79:71–85. atory behaviors of mice on a hole-board by diabetes. Jpn J 3 Dunn RW, Corbett R, Fielding S. Effects of 5-HT1A receptor Pharmacol. 2001;86:47–54. agonists and NMDA receptor antagonists in the social inter- 21 Miyata S, Hirano S, Kamei J. Diabetes attenuates the anti- action test and the elevated plus maze. Eur J Pharmacol. depressant-like effect mediated by the activation of 5-HT1A 1989;169:1–10. receptor in the mouse tail suspension test. Neuropsycho- 4 Moser PC. An evaluation of the elevated plus-maze test using pharmacology. 2004;29:461–469. the novel anxiolytic buspirone. Psychopharmacology. 1989;99: 22 Mayorga AJ, Dalvi A, Page ME, Zimov-Levinson S, Hen R, 48–53. Lucki I. Antidepressant-like behavioral effects in 5-hydroxy- 5 Kostowski W, Dyr W, Krzascik P, Jarbe T, Archer T. 5- tryptamine1A and 5-hydroxytryptamine1B receptor mutant mice. Hydroxytryptamine1A receptor agonists in animal models of J Pharmacol Exp Ther. 2001;298:1101–1107. depression and anxiety. Pharmacol Toxicol. 1992;71:24–30. 23 Miyata S, Yamada N, Hirano S, Tanaka S, Kamei J. Diabetes 6 Treit D, Robinson A, Rotzinger S, Pesold C. Anxiolytic effects attenuates psychological stress-elicited 5-HT secretion in the of serotonergic interventions in the shock-probe burying test and prefrontal cortex but not in the amygdala of mice. Brain Res. the elevated plus-maze test. Behav Brain Res. 1993;54:23–34. 2007;1147:233–239. 7 Koks S, Beljajev S, Koovit I, Abramov U, Bourin M, Vasar E. 8- 24 Rex A, Voigt JP, Fink H. Anxiety but not arousal increases 5- OH-DPAT, but not deramciclane, antagonizes the anxiogenic- hydroxytryptamine release in the rat ventral hippocampus in like action of paroxetine in an elevated plus-maze. Psycho- vivo. Eur J Neurosci. 2005;22:1185–1189. pharmacology. 2001;153:365–372. 25 Alves SH, Pinheiro G, Motta V, Landeira-Fernandez J, Cruz AP. 8 Tye NC, Everitt BJ, Iversen SD. 5-Hydroxytryptamine and Anxiogenic effects in the rat elevated plus-maze of 5-HT2C punishment. Nature. 1977;268:741–743. agonists into ventral but not dorsal hippocampus. Behav 9 Conti LH, Maciver CR, Ferkany JW, Abreu ME. Footshock- Pharmacol. 2004;15:37–43. induced freezing behavior in rats as a model for assessing 26 Wong EH, Sonders MS, Amara SG, Tinholt PM, Piercey MF, anxiolytics. Psychopharmacology. 1990;102:492–497. Hoffmann WP, et al. Reboxetine: a pharmacologically potent, Elevated Open-Platform Test 7

selective, and specific norepinephrine reuptake inhibitor. Biol Threlkeld PG, Nelson DL, et al. Comparison of effects of dual Psychiatry. 2000;47:818–829. transporter inhibitors on monoamine transporters and extra- 27 Inoue T, Nakagawa S, Izumi T, Kitaichi Y, Koyama T. Effect of cellular levels in rats. Neuropharmacology. 2003;45:935–944. combined treatment with noradrenaline and serotonin reuptake 30 Yoshida S, Numachi Y, Matsuoka H, Sato M. The absence of inhibitors on conditioned freezing. Eur J Pharmacol. 2006; impairment of cliff avoidance reaction induced by subchronic 540:91–95. methamphetamine treatment in inbred strains of mice. Tohoku J 28 Mochizuki D, Tsujita R, Yamada S, Kawasaki K, Otsuka Y, Exp Med. 2000;190:205–212. Hashimoto S, et al. Neurochemical and behavioural character- 31 Matsuoka Y, Furuyashiki T, Yamada K, Nagai T, Bito H, ization of milnacipran, a serotonin and noradrenaline reuptake Tanaka Y, et al. Prostaglandin E receptor EP1 controls impulsive inhibitor in rats. Psychopharmacology. 2002;162:323–332. behavior under stress. Proc Natl Acad Sci U S A. 2005;102: 29 Koch S, Hemrick-Luecke SK, Thompson LK, Evans DC, 16066–16071.