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Differences between mice strains in response to paroxetine in the forced swimming test: Involvement of serotonergic or noradrenergic systems
European Journal of Pharmacology 672 (2011) 121–125
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European Journal of Pharmacology journal homepage: www.elsevier.com/locate/ejphar
Behavioural Pharmacology
Differences between mice strains in response to paroxetine in the forced swimming test: Involvement of serotonergic or noradrenergic systems Yumi Sugimoto a,⁎, Masami Yamamoto b, Noriko Tagawa c, Yoshiharu Kobayashi c, Kumiko Mitsui-Saitoh d, Yoshihiro Hotta d, Jun Yamada a a
Laboratory of Pharmacology, Department of Clinical Pharmacy, Yokohama College of Pharmacy, 601 Matano-cho, Totsuka-ku, Yokohama 245-0066, Japan Department of Pharmacology, Kobe Pharmaceutical University, Motoyamakita-machi, Higashianda-ku, Kobe 658-8558, Japan Department of Medical Biochemistry, Kobe Pharmaceutical University, Motoyamakita-machi, Higashianda-ku, Kobe 658-8558, Japan d College of Pharmacy, Kinjo Gakuin University, Omori, Moriyama-ku, Nagoya, 463-8521, Japan b c
a r t i c l e
i n f o
Article history: Received 31 March 2011 Received in revised form 23 September 2011 Accepted 2 October 2011 Available online 10 October 2011 Keywords: Paroxetine Forced swimming test Strain difference 5-HT1A receptor α1-Adrenoceptor Fluvoxamine
a b s t r a c t We studied the effect of the selective serotonin reuptake inhibitor (SSRI) paroxetine on the immobility time in the forced swimming test using different strains of mice (ICR, ddY, C57BL/6, BALB/c and DBA/2). There was a difference between strains in the response to paroxetine (although it induced anti-immobility effects in all strains of mice used). The mouse strain most sensitive to paroxetine was DBA/2; the ICR strain showed the lowest sensitivity. We previously demonstrated variations in the responses to another SSRI, fluvoxamine, in different strains of mice, which was in agreement with the present findings. In DBA/2 and ICR mice, the anti-immobility effects of paroxetine were significantly antagonized by the selective 5-HT1A receptor antagonist N-[2-[4-(2methoxyphenyl)-1-piperazinyl]ethyl]-N-(2-pyridinyl)cyclohexanecarboxamide (WAY 100635). The noradrenergic α1-adrenoceptor antagonist prazosin significantly reduced the anti-immobility effects elicited by a high dose (5 mg/kg) of paroxetine in DBA/2 and ICR mice. However, prazosin did not affect the anti-immobility effects of a lower dose of paroxetine (1 mg/kg) in DBA/2 mice. This suggests that the anti-immobility effects of a higher dose of paroxetine in mice are associated with serotonergic and noradrenergic neurons. Prazosin did not the affect anti-immobility effects of fluvoxamine. These results suggest that there are differences between mice strains in the antidepressant-like effects of paroxetine (which are similar to those elicited by fluvoxamine). Moreover, involvement of the noradrenergic system was partly related to the anti-immobility effects of paroxetine (which are different to those elicited by fluvoxamine). © 2011 Elsevier B.V. All rights reserved.
1. Introduction The forced swimming test established by Porsolt et al. (1977) is widely used for evaluation of the efficacy of antidepressants in mice or rats. The forced swimming test in mice is an easy and reliable method because many antidepressants can shorten the duration of immobility (termed “behavioral despair”) (Porsolt et al., 1977, 1979). For example, tricyclic antidepressants such as imipramine and desipramine or atypical antidepressants such as mianserin reduce the immobility time in this test (Porsolt et al., 1977; Sugimoto et al., 2002; Yamada and Sugimoto, 2001). We reported that the baseline immobility time is different in various strains of mice (ICR, ddY, C57BL, DBA/2 and BALB/c) (Sugimoto et al., 2008). This difference between mice strains with respect to immobility time was reported in another evaluation method for antidepressants: the tail suspension test (Crowley et al., 2005; Ripoll et al., 2003). We found that differences between strains in baseline immobility ⁎ Corresponding author. Tel.: + 81 45 859 1300; fax: + 81 45 859 1301. E-mail address: [email protected] (Y. Sugimoto). 0014-2999/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.ejphar.2011.10.002
in the forced swimming test were associated with levels of serotonin (5-hydroxytryptamine (5-HT)) transporter binding, and not with those of noradrenaline transporter binding (Sugimoto et al., 2008). Moreover, we demonstrated that there was a variation in responses to the selective serotonin reuptake inhibitor (SSRI) fluvoxamine in different strains of mice, but not to the noradrenaline reuptake inhibitor desipramine (Sugimoto et al., 2008). The differences between strains with respect to responses to fluvoxamine are also related to 5-HT transporter binding (Sugimoto et al., 2008). The SSRI paroxetine is widely used in the treatment of depression, panic disorder or obsessive compulsive disorder (Wagstaff et al., 2002). Paroxetine has greater affinity with the 5-HT transporter than fluvoxamine (Sanchez and Hyttel, 1999). We previously demonstrated that there were distinctly different responses to fluvoxamine in five strains of mice (Sugimoto et al., 2008). However, it is not clear if the responses to paroxetine are different in these strains of mice. Our previous report indicated that the anti-immobility effects of fluvoxamine are mediated by 5-HT1A receptors (Sugimoto et al., 2010a). Therefore, we studied the effects of paroxetine in five strains of mice and examined the involvement of 5-HT receptor subtypes.
Y. Sugimoto et al. / European Journal of Pharmacology 672 (2011) 121–125
It has been reported that paroxetine has moderate affinity for noradrenaline transporters (Kelly and Leonnard, 1995; Owens et al., 2001). Some reports show that paroxetine can increase extracellular noradrenaline levels in the cortex of the brains of mice and rats (Beyer et al., 2002; David et al., 2003). The antidepressant effects of paroxetine may be related to noradrenergic systems. Thus, we examined the involvement of noradrenaline in the anti-immobility effects of paroxetine in mice. 2. Materials and methods Experiments were carried out in accordance with the Guiding Principles for Care and Use of Laboratory Animals set by the Japanese Pharmacological Society. The study protocol was approved by the Ethics Committee of the Yokohama College of Pharmacy (Yokohama, Japan). 2.1. Animals Male ICR, ddY, C57BL/6Cr, DBA/2Cr and BALB/cCr mice (age, 5–7 weeks) were purchased from SLC Japan Incorporated (Shizuoka, Japan). Mice were housed in groups of five per cage under a controlled 12-h/12-h light–dark cycle (light from 7 am to 7 pm), at a room temperature of 23± 1 °C and humidity of 55 ± 5%. Mice had free access to food and water. Each mouse was used only once for the experiments. 2.2. Forced swimming test The forced swimming test was undertaken according to the methods described by Porsolt et al. (1977) and our reports (Sugimoto et al., 2008; Yamada and Sugimoto, 2002; Yamada et al., 2004). Each mouse was placed in a 25-cm glass cylinder (diameter, 10 cm) containing 10 cm of water at 23± 1 °C. Immobility was recorded during a 6-min swimming test. Mice were judged to be immobile if they floated, their hindlimbs were immobile, and if only small movements of the forepaws were made to keep the head above the water level. 2.3. Drugs and treatment Paroxetine maleate, N-[2-[4-(2-methoxyphenyl)-1-piperazinyl] ethyl]-N-(2-pyridinyl)cyclohexanecarboxamide maleate (WAY 100635), 6-methyl-1-(methylethyl)-ergoline-8beta-carboxylic acid 2-hydroxy-1methylpropyl ester maleate (LY 53857), ondansetron HCl and 4-amino5-chloro-2-methoxy-benzoic acid 2-(diethylamino)ethyl ester HCl (SDZ 205,557) were obtained from Sigma-Aldrich (St Louis, MO, USA). 3-[3(Dimethylamino)propyl]-4-hy-droxy-N-[4-(4-pyridinyl)phenyl]benzamide dihydrochloride (GR55562) was purchased from Tocris Bioscience (Bristol, UK). All drugs were dissolved in saline. Paroxetine (0.5–5 mg/kg) was injected i.p. Mice in the control group received saline. 5-HT receptor antagonists were administered i.p. 30 min before paroxetine was given. Thirty minutes after treatment with paroxetine, the forced swimming test was carried out. 2.4. Measurement of locomotor activity The locomotor activity of mice was measured using a digital counter with an infrared sensor (NS-AS01, Neuroscience Incorporated, Tokyo, Japan) following a previously described method (Yamada et al., 2004). An infrared sensor was set over an open-top, clear polycarbonate cage (22.5 × 33.8 × 14.0 cm) into which each mouse was placed. Locomotor activity was determined over 10 min. The apparatus was used to detect beam breaks and record a digital count of the horizontal movements of animals.
2.5. Statistical analyses Results are means ± S.E.M. of 5–8 mice in the behavioral studies. The effects of paroxetine on immobility time in each mouse strain were analyzed by one-way ANOVA followed by Dunnet's multiple comparison post-hoc test. The effects of paroxetine on immobility time were also determined by two-way ANOVA with paroxetine treatment and mice strains. The effects of receptor antagonists on the antiimmobility effects of paroxetine were analyzed by two-way ANOVA, with pretreatment and paroxetine treatment showing anti-immobility effects as the main factors. Pairwise follow-up comparisons of individual treatment groups were analyzed by Tukey's multiple comparison post-hoc test. 3. Results 3.1. Effects of paroxetine on immobility time and locomotor activity in five strains of mice Fig. 1 shows the immobility times in five strains of mice after treatment with paroxetine. Paroxetine induced significant anti-immobility effects in the five strains of mice (ICR: F(3, 25) =40.16, P≤0.0001. ddY: F (3, 25)= 19.73, P≤ 0.0001. C57BL/6: F(3, 26)= 12.59, P≤ 0.0001. DBA/2: F(3, 26) =125.86, P ≤0.0001. BALB/c: F(3, 26)= 21.11, P ≤0.0001. Twoway ANOVA: Treatment: F(3, 129)= 122.72, P≤0.0001. Strain: F(4, 129) = 360.83, P ≤ 0.0001. Treatment × strain interaction: F(12, 129) = 8.35, P ≤ 0.0001). However, the anti-immobility effects of paroxetine were elicited in DBA/2 and BALB/c mice at lower doses than in C57BL, ddY and ICR strains of mice. ICR mice showed the lowest sensitivity to paroxetine among the five strains of mice evaluated. Fig. 2 shows the locomotor activity in five strains of mice after treatment with paroxetine. Paroxetine did not alter locomotor activity in any mouse strain (ICR: F(3, 21) = 0.18, P ≥ 0.05. ddY: F(3, 21) = 0.18, P ≥ 0.05. C57BL/6: F(3, 21) = 1.64, P ≥ 0.05. DBA/2: F(3, 20) = 3.34, P ≥ 0.05. BALB/c: F(3, 29) = 1.25, P ≥ 0.05. Two-way ANOVA: Treatment: F(3, 112)= 0.4152, P ≥ 0.05. Strain: F(4, 129)= 133.81, P ≤ 0.0001. Treatment× strain interaction: F(12, 112) = 1.86, P ≤ 0.05). 3.2. Effects of 5-HT receptor antagonists and the α1-adrenoceptor antagonist prazosin on the anti-immobility effects of paroxetine at 5 mg/kg The effects of 5-HT receptor antagonists and the α1-adrenoceptor antagonist prazosin on the anti-immobility effects of paroxetine at 5 mg/kg in DBA/2 and ICR mice are shown in Figs. 3 and 4, respectively. The 5-HT1A receptor antagonist WAY 100635 significantly attenuated
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Fig. 1. Effects of paroxetine on immobility in the forced swimming test in five mice strains. Results are shown as mean ± S.E.M. (N = 6–8). Paroxetine was given i.p. 30 min before the test. *P b 0.05, ** P b 0.01.
Y. Sugimoto et al. / European Journal of Pharmacology 672 (2011) 121–125
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Fig. 4. Effects of several 5-HT receptor antagonists and prazosin on paroxetine at 5 mg/ kg-induced anti-immobility in ICR mice. Results are shown as mean ± S.E.M. (N = 5–8). Paroxetine at 5 mg/kg was given i.p. 5-HT receptor antagonists and prazosin were injected (i.p.) 30 min before paroxetine. ***P b 0.001 vs. saline of the respective group. ### P b 0.001 vs. saline + paroxetine-treated group.
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the anti-immobility effects of paroxetine in DBA/2 and ICR mice. However, the 5-HT1B receptor antagonist GR55562, the 5-HT2 receptor antagonist LY 53857, the 5-HT3 receptor antagonist ondansetron, and the 5-HT4 receptor antagonist SDZ205,557 did not influence paroxetineinduced anti-immobility effects. Two way ANOVA analysis on results in DBA/2 mice (WAY100635: Pretreatment: F(2, 30)=25.17, P≤0.0001.
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Fig. 2. Effects of paroxetine on locomotor activity in five strains of mice. Results are shown as mean ± S.E.M. (N = 5–7). The axis represents the total digital counts based on beam breaks.
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Fig. 3. Effects of several 5-HT receptor antagonists and prazosin on paroxetine-induced anti-immobility in DBA/2 mice. Results are shown as mean ± S.E.M. (N = 6–7). Paroxetine at 5 mg/kg was given i.p. 5-HT receptor antagonists (the 5-HT1A receptor antagonist WAY 100635, the 5-HT1B receptor antagonist GR 55562, the 5-HT2 receptor antagonist LY53857, the 5-HT3 receptor antagonist ondansetron and the 5-HT4 receptor antagonist SDZ205,557) and the α1-adrenoceptor antagonist prazosin were injected i.p. 30 min before paroxetine. ***P b 0.001 vs. saline of respective group. ###P b 0.001 vs. saline + paroxetine-treated group.
Treatment: F(1, 30) = 33.13, P ≤ 0.0001. Pretreatment× treatment interaction: F(2, 30) = 23.42, P ≤ 0.0001. GR 55562: Pretreatment: F(2, 36) = 0.77, P ≥ 0.05. Treatment: F(1, 36) = 321.18, P ≤ 0.0001. Pretreatment× treatment interaction: F(2, 36) = 0.10, P ≥ 0.05. LY53857: Pretreatment: F(2, 32) = 1.74, P ≥ 0.05. Treatment: F(1, 32) = 288.44, P ≤ 0.0001. Pretreatment× treatment interaction: F(2, 32) = 0.51, P ≥ 0.05. Ondansetron: Pretreatment: F(2, 31) = 0.36, P ≥ 0.05. Treatment: F(1, 31) = 144.7, P ≤ 0.0001. Pretreatment× treatment interaction: F(2, 31) =0.31, P≥0.05. SDZ205,557: Pretreatment: F(2, 32) = 0.02, P≥0.05. Treatment: F(1, 32) =190.94, P≤0.0001. Pretreatment × treatment interaction: F(2, 32) =0.96, P ≥0.05). Two way ANOVA analysis on results in ICR mice (WAY100635: Pretreatment: F(2, 36) =6.01, P≤0.01. Treatment: F(1, 36)= 66.52, P≤0.0001. Pretreatment×treatment interaction: F(2, 36)= 8.63, P ≤0.001. GR 55562: Pretreatment: F(2, 29) =0.69, P≥0.05. Treatment: F(1, 29) =216.81, P≤0.0001. Pretreatment× treatment interaction: F(2, 29) =0.59, P≥0.05. LY53857: Pretreatment: F(2, 36) =0.10, P≥0.05. Treatment: F(1, 36) =159.42, P≤0.0001. Pretreatment× treatment interaction: F(2, 36) =0.17, P≥0.05. Ondansetron: Pretreatment: F(2, 29) =0.07, P≥0.05. Treatment: F(1, 29) =226.68, P≤0.0001. Pretreatment ×treatment interaction: F(2, 29) =0.22, P≥0.05. SDZ205,557: Pretreatment: F(2, 28) = 0.31, P≥0.05. Treatment: F(1, 28) =81.39, P≤0.0001. Pretreatment× treatment interaction: F(2, 28) =0.12, P ≥0.05). The α1-adrenoceptor antagonist prazosin inhibited the anti-immobility effects elicited by paroxetine in DBA/2 and ICR mice (DBA/2 mice: Pretreatment: F(2, 31) = 10.74, P ≤ 0.0001. Treatment: F(1, 31) = 232.21, P ≤ 0.0001. Pretreatment×treatment interaction: F(2, 31)=9.41, P≤0.0001. ICR mice: Pretreatment: F(2, 28)=3.39, P ≤0.05. Treatment: F(1, 28)=80.28,
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P≤0.0001. Pretreatment×treatment interaction: F(2, 28)=10.82, P≤0.001). 3.3. Effects of prazosin on the anti-immobility effects of paroxetine at 1 mg/kg in DBA/2 mice The effects of prazosin on the anti-immobility effects of paroxetine at 1 mg/kg in DBA/2 mice are shown in Fig. 5A. The anti-immobility effects of paroxetine at 1 mg/kg in DBA/2 mice were not antagonized by prazosin (Pretreatment: F(2, 34) = 0.9735, P ≥ 0.05. Treatment: F(1, 34) = 397.81, P ≤ 0.0001. Pretreatment × treatment interaction: F(2, 34) = 0.68, P ≥ 0.05.) 3.4. Effects of prazosin on the anti-immobility effects of fluvoxamine in DBA/2 mice Prazosin did not affect the fluvoxamine-induced anti-immobility effects in DBA/2 mice (Fig. 5B). (Pretreatment: F(2, 34) = 0.9735, P ≥ 0.05. Treatment: F(1, 34) = 397.81, P ≤ 0.0001. Pretreatment × treatment interaction: F(2, 34) = 0.68, P ≥ 0.05.) 4. Discussion The forced swimming test is an evaluation method for the efficacy of antidepressants. However, a lower sensitivity to SSRIs in the forced swimming test has been suggested (Sanchez and Meier, 1997). We previously demonstrated that there is an apparent difference between five strains of mice with respect to responses to fluvoxamine (Sugimoto et al., 2008). In DBA/2 and BALB/c mice, fluvoxamine induced clear antiimmobility effects. However, fluvoxamine had no effects in ICR, ddY and C57BL/6 mice. These differences between strains derive from variations of 5-HT transporter binding in the brain (Sugimoto et al., 2008). Paroxetine is a well-known SSRI used widely for the treatment of depression (Wagstaff et al., 2002). Paroxetine has a higher affinity for the serotonin transporter than fluvoxamine, which leads to its potent antidepressant effects (Sanchez and Hyttel, 1999; Wagstaff et al., 2002). However, it is not clear if there is a difference between mice strains with respect to the anti-immobility effects of paroxetine. We therefore examined the effect of paroxetine on immobility time in five strains of mice. Paroxetine induced significant anti-immobility effects in all five strains of mice. However, there was a difference in the sensitivity to paroxetine between mice strains. Paroxetine even at a low dose (0.5 mg/kg) elicited apparent anti-immobility effects in DBA/2 and BALB/c mice. In ddY and C57BL mice, paroxetine induced reduced immobility above the dose of 1 mg/kg. In ICR mice, only a high dose of 5 mg/kg caused significant anti-immobility effects. In our previous
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Fig. 5. Effects of prazosin on paroxetine (1 mg/kg)- and fluvoxamine-induced antiimmobility in DBA/2 mice. (A) Effects of prazosin on paroxetine (1 mg/kg)-induced anti-immobility effects. Results are shown as mean ± S.E.M. (N = 5–6). Paroxetine (1 mg/kg) was given i.p. Prazosin were injected i.p. 30 min before paroxetine. ***P b 0.001 vs. saline of the respective group. (B) Effects of prazosin on fluvoxamineinduced anti-immobility in DBA/2 mice. Results are mean ± S.E.M. (N = 5–6). Fluvoxamine (10 mg/kg) was given (i.p.). Prazosin was injected (i.p.) 30 min before fluvoxamine. ***P b 0.001 vs. saline of the respective group.
report, fluvoxamine caused strong anti-immobility effects in DBA/2 and BALB/c mice, whereas fluvoxamine did not alter the immobility time in C57BL/6, ddY and ICR mice (Sugimoto et al., 2008). Therefore, differences between strains with respect to responses to paroxetine were similar to those seen with fluvoxamine. Paroxetine did not affect locomotor activity in the five strains of mice, but it elicited antiimmobility effects. This indicated that locomotor activity is not related to anti-immobility effects. It has been reported that the 5-HT1A receptor is involved in antidepressant-like effects in the forced swimming test (ChojnackaWojcik et al., 1991). We also observed that the anti-immobility effects of fluvoxamine in DBA/2 mice are mediated by the 5-HT1A receptor because the selective 5-HT1A receptor antagonist WAY 100635 abolished them (Sugimoto et al., 2010a). Therefore, we studied the effects of 5-HT receptor antagonists (including WAY 100635) on the antiimmobility effects of paroxetine. We examined the effects of 5-HT receptor antagonists in DBA/2 and ICR mice; these strains showed the highest and lowest sensitivity to paroxetine, respectively. Pretreatment with WAY 100635 antagonized the paroxetine-induced antiimmobility effects in DBA/2 and ICR mice. It has been suggested that other 5-HT receptor subtypes (e.g., 5-HT1B, 5-HT2, 5-HT3, and 5-HT4) may also be related to the anti-immobility effects in rats and mice (Lucas et al., 2007; Redrobe and Bourin, 1997; Redrobe et al., 1996). However, the 5-HT1B receptor antagonist GR55562 and the nonselective 5-HT2 receptor antagonist LY 53857 did not modify the antiimmobility effects of paroxetine in DBA/2 and ICR mice. The 5-HT3 and 5-HT4 receptor antagonists ondansetron and SDZ205,557, respectively, were also ineffective in both mice strains. These results suggest that the anti-immobility effects of paroxetine are mediated by the 5HT1A receptor, which is in accordance with our previous results using fluvoxamine. Thus, the inhibitory effects of paroxetine on 5-HT reuptake elicit elevation of 5-HT concentrations in the synaptic cleft. It is likely that elevated levels of 5-HT by paroxetine activate the 5-HT1A receptor, leading to anti-immobility effects. We previously reported that the selective 5-HT1A receptor agonist 8-OH-DPAT elicits anti-immobility effects, and that its effects are antagonized by WAY 100635 (Sugimoto et al., 2010b). Chojnacka-Wojcik et al. (1991) also reported that the 5-HT1A receptor agonist gepirone induced anti-immobility effects in rats. Taken together, our results with paroxetine in the present study further support the role of the 5-HT1A receptor in antidepressant-like effects in mice. Noradrenaline, as well as 5-HT, has an important role in depression. Paroxetine has a moderate affinity for the noradrenaline transporter in addition to its affinity for the 5-HT transporter (Owens et al., 2001; Sanchez and Hyttel, 1999). In contrast, fluvoxamine has no affinity for the noradrenaline transporter (Owens et al., 2001; Sanchez and Hyttel, 1999). Owens et al. (2001) reported that the Ki values of paroxetine and fluvoxamine for the inhibition of noradrenaline uptake are 156 nM and 1119 nM, respectively, in HEK-293 cells derived from humans. Furthermore, in the microdialysis technique, paroxetine elicited noradrenaline release in the cortex in Swiss mice at doses of 4 mg/kg and 8 mg/kg and Sprague–Dawley rats at 30 mg/kg (Beyer et al., 2002; David et al., 2003). It is assumed that noradrenaline may be related to the pharmacological effects of paroxetine. It has been reported that the antidepressant-like effects in rodents induced by several drugs are mediated by the α1-adrenoceptor (Danysz et al., 1986; Kaster et al., 2007; Takeda et al., 2003); these data are based on antagonism by α1-adrenoceptor antagonists. We therefore studied the effects of the α1-adrenoceptor antagonist prazosin on the anti-immobility effects of paroxetine in DBA/2 and ICR mice. Prazosin significantly antagonized the antidepressant-like effects elicited by a high dose (5 mg/kg) of paroxetine in DBA/2 and ICR mice. This indicated that the α1-adrenoceptor may be related to the anti-immobility effects induced by 5 mg/kg paroxetine. However, the anti-immobility effects of 1 mg/kg paroxetine in DBA/2 mice were not antagonized by prazosin. This result suggests that the antidepressant-
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like effects of a high dose of paroxetine are involved in the α1-adrenoceptor, whereas the effects of a lower dose of paroxetine are not. David et al. (2003) reported that, in Swiss mice using microdialysis, paroxetine (1–8 mg/kg) elicited 5-HT release in the cortex. They also demonstrated that paroxetine increased noradrenaline outflow in the cortex at 4 mg/kg and 8 mg/kg, but significant increases were not observed at 1 mg/kg (David et al., 2003). These observations of dose-dependent elevation of noradrenaline by microdialysis are closely related to our results, showing that noradrenaline is associated with the antiimmobility effects of 5 mg/kg paroxetine but not to those of 1 mg/kg paroxetine. Therefore, it is likely that, at higher doses, the inhibitory effects of the noradrenaline transporter of paroxetine may contribute to the anti-immobility effects in the forced swimming test. In contrast, prazosin did not affect fluvoxamine-induced anti-immobility effects in DBA/2 mice. This indicates that the α1-adrenoceptor is not associated with the antidepressant-like effects of fluvoxamine (which is different from the effects seen with paroxetine). It was suggested that the affinity of fluvoxamine for the noradrenaline transporter is low compared with that for paroxetine (Kelly and Leonnard, 1995; Owens et al., 2001; Sanchez and Hyttel, 1999). Therefore, involvement of noradrenergic neurons in the anti-immobility effects of fluvoxamine may be excluded. Stone and Quartemain (1999) reported that the α1-adrenoceptor is involved in the anti-immobility effects of noradrenaline. Doze et al. (2009) reported that prazosin reversed the anti-immobility effects in mice expressing constitutively active mutant α1-adrenoceptors. These findings support those in the present study showing the involvement of α1-adrenoceptors in the antidepressant-like effects of paroxetine. Prazosin has an affinity for α1A and α1B receptors. It is not clear which receptor subtype is involved, and further studies are required. Guzzetti et al. (2008) reported that paroxetine elicited antiimmobility effects in C57BL mice but not in DBA/2 and BALB/c mice, but this finding is not in accordance with our results. They suggested that these strain differences are related to serotonergic transmission because co-administration with paroxetine and the 5-HT precursor tryptophan elicited anti-immobility effects in DBA/2 and BALB/c mice. In the forced swimming test, the experimental condition may affect the immobility time or the anti-immobility effects of drugs. Guzzetti et al. (2008) used a deeper water depth in the glass cylinder. Detke and Lucki (1996) reported that the depth of water decreases the basal immobility time in rats. Therefore, water depth may cause a different sensitivity to paroxetine, and we are now investigating this theme. In summary, we demonstrated that there are differences in the sensitivity to paroxetine in five strains of mice, and that these differences are similar to those seen with fluvoxamine. The anti-immobility effects of paroxetine are mediated by the 5-HT1A receptor. In addition, the α1-adrenoceptor is associated with the effects of paroxetine at a high dose, whereas the α1-adrenoceptor is not associated with the antidepressant-like effects of fluvoxamine. These results suggest that the inhibitory effects of the reuptake of noradrenaline are related (at least in part) to the antidepressant-like effects of paroxetine. Redrobe et al. (1998) reported that the anti-immobility effects of a high dose of paroxetine are resistant to the depletion of 5-HT induced by p-chlorophenylalanine, and they speculated on the involvement of the noradrenergic system in explaining the effects of paroxetine. Therefore, our results indicate that the strong antidepressant-like effects of paroxetine may be caused by its effects on serotonergic and noradrenergic neurons. References Beyer, C.E., Boikess, S., Luo, B., Dawson, L.A., 2002. Comparison of the effects of antidepressants on norepinephrine and serotonin concentrations in the rat frontal cortex: an in-vivo microdialysis study. J. Psychopharmacol. 16, 297–304. Chojnacka-Wojcik, E., Tatarczynska, E., Golembiowska, K., Przegalinski, E., 1991. Involvement of 5-HT1A receptors in the antidepressant-like activity of gepirone in the forced swimming test in rats. Neuropharmacology 30, 711–717.
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Crowley, J., Blendy, J.A., Lucki, I., 2005. Strain-dependent antidepressant-like effects of citalopram in the mouse tail suspension test. Psychopharmacology 183, 257–264. Danysz, W., Kostowski, W., Kozak, W., Hauptmann, M., 1986. On the role of noradrenergic neurotransmission in the action of desipramine and amitriptyline in animal models of depression. Pol. J. Pharmacol. Pharm. 38, 285–298. David, D.J.P., Bourin, M., Jego, G., Przybylski, C., Jolliet, P., Gardier, A.M., 2003. Effects of acute treatment with paroxetine, citalopram and venlafaxine in vivo on noradrenaline and serotonin outflow: a microdialysis study in Swiss mice. Br. J. Pharmacol. 140, 1128–1136. Detke, M.J., Lucki, I., 1996. Detection of serotonergic and noradrenergic antidepressants in the rat forced swimming test: the effects of water depth. Behav. Brain Res. 73, 43–46. Doze, V.A., Handel, E.M., Jensen, K.A., Darsie, B., Luger, E.J., Haselton, J.R., Talbot, J.N., Rorabaugh, B.R., 2009. α1Α- and α1B-adrenergic receptors differentially modulate antidepressant-like behavior in the mouse. Brain Res. 1285, 148–157. Guzzetti, S., Calcagno, E., Canetta, A., Sacchetti, G., Fracasso, C., Cassia, S., Cervo, L., Invernizzi, R.W., 2008. Strain differences in paroxetine-induced reduction of immobility time in the forced swimming test in mice: role of serotonin. Eur. J. Pharmacol. 594, 117–124. Kaster, M.P., Raupp, I., Binfaré, R.W., Andreatini, R., Rodrigues, A.L., 2007. Antidepressantlike effect of lamotrigine in the mouse forced swimming test: evidence for the involvement of the noradrenergic system. Eur. J. Pharmacol. 565, 119–124. Kelly, J.P., Leonnard, B.E., 1995. The contribution of pre-clinical drug evaluation in predicting clinical profile of the selective serotonin reuptake inhibitor paroxetine. J. Ser. Res. 1, 27–46. Lucas, G., Rymar, V., Mnie-Filali, O., Bosgaard, C., Manta, S., Lambas-Senas, L., Wilborg, O., Haddjeri, N., Pineyro, G., Sadikoto, A.F., Debonnel, G., 2007. Serotonin(4)(5-HT4) receptor agonists are putative antidepressnts with a rapid onset of action. Neuron 55, 712–725. Owens, M.J., Knight, D.L., Nemeroff, C.B., 2001. Second-generation SSRIs: human monoamine transporter binding profile of escitalopram and R-fluoxetine. Biol. Psychiatry 50, 345–350. Porsolt, R.D., Bertin, A., Jalfre, M., 1977. Behavioural despair in mice:a primary screening test for antidepressants. Arch. Int. Pharmacodyn. 229, 327–336. Porsolt, R.D., Bertin, A., Blavert, N., Denie, M., Jalfre, M., 1979. Immobility induced by forced swimming in rats:Effects of agents which modify central catecholamine and serotonin activity. Eur. J. Pharmacol. 57, 201–210. Redrobe, J.P., Bourin, M., 1997. Partial role of 5-HT2 and 5-HT3 receptors in the activity of antidepressants in the mouse forced swimming test. Eur. J. Pharmacol. 325, 129–135. Redrobe, J.P., MacSweeney, C.P., Bourin, M., 1996. The role of 5-HT1A and 5-HT1B receptors in antidepressant drug actions in the mouse forced swimming test. Eur. J. Pharmacol. 318, 213–220. Redrobe, J.P., Bourin, M., Colombel, M.C., Baker, G.B., 1998. Psychopharmacological profile of the selective serotonin reuptake inhibitor, paroxetine: implication of noradrenergic and serotonergic mechanisms. J. Psychopharmacol. 12, 348–355. Ripoll, N., David, D.J.P., Dailly, E., Hascoet, M., Bourin, M., 2003. Antidepressant-like effects in various mice strains in the tail suspension test. Behav. Brain Res. 143, 193–200. Sanchez, C., Hyttel, J., 1999. Comparison of the effects of antidepressants and their metabolites on reuptake of biogenic amines and on receptor binding. Cell. Mol. Neurobiol. 19, 467–489. Sanchez, C., Meier, E., 1997. Behavioral profiles of SSRIs in animal models of depression, anxiety and aggression. Are they all alike? Psychopharmacology 129, 197–205. Stone, E.A., Quartemain, D., 1999. Alfa-1-noradrenergic neurotransmission, corticosterone, and behavioral depression. Biol. Psychiatry 46, 1287–1300. Sugimoto, Y., Yamada, S., Yamada, J., 2002. The 5-HT2 receptor antagonist reduces immobility of mice treated with the atypical antidepressant mianserin in the forced swimming test. Biol. Pharm. Bull. 25, 1479–1481. Sugimoto, Y., Kajiwara, Y., Hirano, K., Yamada, S., Tagawa, N., Kobayashi, Y., Hotta, Y., Yamada, J., 2008. Mouse strain differences in immobility and sensitivity to fluvoxamine and desipramine in the forced swimming test:Analysis of serotonin and noradrenaline transporter binding. Eur. J. Pharmacol. 592, 116–122. Sugimoto, Y., Furutani, S., Kajiwara, Y., Hirano, K., Yamada, S., Tagawa, N., Kobayashi, Y., Hotta, Y., Yamada, J., 2010a. Involvement of the 5-HT1A receptor in the antiimmobility effects of fluvoxamine in the forced swimming test and mouse strain differences in 5-HT1A receptor binding. Eur. J. Pharmacol. 629, 53–57. Sugimoto, Y., Furutani, S., Nishimura, K., Itoh, A., Tanahashi, T., Oshiro, H., Sun, S., Yamada, J., 2010b. Antidepressant-like effects of neferine in the forced swimming test involve the serotonin1A (5-HT1A) receptor in mice. Eur. J. Pharmacol. 634, 62–67. Takeda, H., Tsuji, M., Miyamoto, J., Masuya, J., Iimori, M., Matsumiya, T., 2003. Caffeic acid produces antidepressive- and/or anxiolytic-like effects through indirect modulation of the α1A-adrenoceptor system in mice. Neuroreport 14, 1047–1070. Wagstaff, A., Cheer, S., Matheson, A., Ormrod, D., Goa, K., 2002. Paroxetine: an update of its use in psychiatric disorders in adults. Drugs 62, 655–703. Yamada, J., Sugimoto, Y., 2001. Effects of 5-HT2 receptor antagonists on the antiimmobility effects of imipramine in the forced swimming test with mice. Eur. J. Pharmacol. 427, 221–225. Yamada, J., Sugimoto, Y., 2002. 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European Journal of Pharmacology journal homepage: www.elsevier.com/locate/ejphar
Behavioural Pharmacology
Differences between mice strains in response to paroxetine in the forced swimming test: Involvement of serotonergic or noradrenergic systems Yumi Sugimoto a,⁎, Masami Yamamoto b, Noriko Tagawa c, Yoshiharu Kobayashi c, Kumiko Mitsui-Saitoh d, Yoshihiro Hotta d, Jun Yamada a a
Laboratory of Pharmacology, Department of Clinical Pharmacy, Yokohama College of Pharmacy, 601 Matano-cho, Totsuka-ku, Yokohama 245-0066, Japan Department of Pharmacology, Kobe Pharmaceutical University, Motoyamakita-machi, Higashianda-ku, Kobe 658-8558, Japan Department of Medical Biochemistry, Kobe Pharmaceutical University, Motoyamakita-machi, Higashianda-ku, Kobe 658-8558, Japan d College of Pharmacy, Kinjo Gakuin University, Omori, Moriyama-ku, Nagoya, 463-8521, Japan b c
a r t i c l e
i n f o
Article history: Received 31 March 2011 Received in revised form 23 September 2011 Accepted 2 October 2011 Available online 10 October 2011 Keywords: Paroxetine Forced swimming test Strain difference 5-HT1A receptor α1-Adrenoceptor Fluvoxamine
a b s t r a c t We studied the effect of the selective serotonin reuptake inhibitor (SSRI) paroxetine on the immobility time in the forced swimming test using different strains of mice (ICR, ddY, C57BL/6, BALB/c and DBA/2). There was a difference between strains in the response to paroxetine (although it induced anti-immobility effects in all strains of mice used). The mouse strain most sensitive to paroxetine was DBA/2; the ICR strain showed the lowest sensitivity. We previously demonstrated variations in the responses to another SSRI, fluvoxamine, in different strains of mice, which was in agreement with the present findings. In DBA/2 and ICR mice, the anti-immobility effects of paroxetine were significantly antagonized by the selective 5-HT1A receptor antagonist N-[2-[4-(2methoxyphenyl)-1-piperazinyl]ethyl]-N-(2-pyridinyl)cyclohexanecarboxamide (WAY 100635). The noradrenergic α1-adrenoceptor antagonist prazosin significantly reduced the anti-immobility effects elicited by a high dose (5 mg/kg) of paroxetine in DBA/2 and ICR mice. However, prazosin did not affect the anti-immobility effects of a lower dose of paroxetine (1 mg/kg) in DBA/2 mice. This suggests that the anti-immobility effects of a higher dose of paroxetine in mice are associated with serotonergic and noradrenergic neurons. Prazosin did not the affect anti-immobility effects of fluvoxamine. These results suggest that there are differences between mice strains in the antidepressant-like effects of paroxetine (which are similar to those elicited by fluvoxamine). Moreover, involvement of the noradrenergic system was partly related to the anti-immobility effects of paroxetine (which are different to those elicited by fluvoxamine). © 2011 Elsevier B.V. All rights reserved.
1. Introduction The forced swimming test established by Porsolt et al. (1977) is widely used for evaluation of the efficacy of antidepressants in mice or rats. The forced swimming test in mice is an easy and reliable method because many antidepressants can shorten the duration of immobility (termed “behavioral despair”) (Porsolt et al., 1977, 1979). For example, tricyclic antidepressants such as imipramine and desipramine or atypical antidepressants such as mianserin reduce the immobility time in this test (Porsolt et al., 1977; Sugimoto et al., 2002; Yamada and Sugimoto, 2001). We reported that the baseline immobility time is different in various strains of mice (ICR, ddY, C57BL, DBA/2 and BALB/c) (Sugimoto et al., 2008). This difference between mice strains with respect to immobility time was reported in another evaluation method for antidepressants: the tail suspension test (Crowley et al., 2005; Ripoll et al., 2003). We found that differences between strains in baseline immobility ⁎ Corresponding author. Tel.: + 81 45 859 1300; fax: + 81 45 859 1301. E-mail address: [email protected] (Y. Sugimoto). 0014-2999/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.ejphar.2011.10.002
in the forced swimming test were associated with levels of serotonin (5-hydroxytryptamine (5-HT)) transporter binding, and not with those of noradrenaline transporter binding (Sugimoto et al., 2008). Moreover, we demonstrated that there was a variation in responses to the selective serotonin reuptake inhibitor (SSRI) fluvoxamine in different strains of mice, but not to the noradrenaline reuptake inhibitor desipramine (Sugimoto et al., 2008). The differences between strains with respect to responses to fluvoxamine are also related to 5-HT transporter binding (Sugimoto et al., 2008). The SSRI paroxetine is widely used in the treatment of depression, panic disorder or obsessive compulsive disorder (Wagstaff et al., 2002). Paroxetine has greater affinity with the 5-HT transporter than fluvoxamine (Sanchez and Hyttel, 1999). We previously demonstrated that there were distinctly different responses to fluvoxamine in five strains of mice (Sugimoto et al., 2008). However, it is not clear if the responses to paroxetine are different in these strains of mice. Our previous report indicated that the anti-immobility effects of fluvoxamine are mediated by 5-HT1A receptors (Sugimoto et al., 2010a). Therefore, we studied the effects of paroxetine in five strains of mice and examined the involvement of 5-HT receptor subtypes.
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It has been reported that paroxetine has moderate affinity for noradrenaline transporters (Kelly and Leonnard, 1995; Owens et al., 2001). Some reports show that paroxetine can increase extracellular noradrenaline levels in the cortex of the brains of mice and rats (Beyer et al., 2002; David et al., 2003). The antidepressant effects of paroxetine may be related to noradrenergic systems. Thus, we examined the involvement of noradrenaline in the anti-immobility effects of paroxetine in mice. 2. Materials and methods Experiments were carried out in accordance with the Guiding Principles for Care and Use of Laboratory Animals set by the Japanese Pharmacological Society. The study protocol was approved by the Ethics Committee of the Yokohama College of Pharmacy (Yokohama, Japan). 2.1. Animals Male ICR, ddY, C57BL/6Cr, DBA/2Cr and BALB/cCr mice (age, 5–7 weeks) were purchased from SLC Japan Incorporated (Shizuoka, Japan). Mice were housed in groups of five per cage under a controlled 12-h/12-h light–dark cycle (light from 7 am to 7 pm), at a room temperature of 23± 1 °C and humidity of 55 ± 5%. Mice had free access to food and water. Each mouse was used only once for the experiments. 2.2. Forced swimming test The forced swimming test was undertaken according to the methods described by Porsolt et al. (1977) and our reports (Sugimoto et al., 2008; Yamada and Sugimoto, 2002; Yamada et al., 2004). Each mouse was placed in a 25-cm glass cylinder (diameter, 10 cm) containing 10 cm of water at 23± 1 °C. Immobility was recorded during a 6-min swimming test. Mice were judged to be immobile if they floated, their hindlimbs were immobile, and if only small movements of the forepaws were made to keep the head above the water level. 2.3. Drugs and treatment Paroxetine maleate, N-[2-[4-(2-methoxyphenyl)-1-piperazinyl] ethyl]-N-(2-pyridinyl)cyclohexanecarboxamide maleate (WAY 100635), 6-methyl-1-(methylethyl)-ergoline-8beta-carboxylic acid 2-hydroxy-1methylpropyl ester maleate (LY 53857), ondansetron HCl and 4-amino5-chloro-2-methoxy-benzoic acid 2-(diethylamino)ethyl ester HCl (SDZ 205,557) were obtained from Sigma-Aldrich (St Louis, MO, USA). 3-[3(Dimethylamino)propyl]-4-hy-droxy-N-[4-(4-pyridinyl)phenyl]benzamide dihydrochloride (GR55562) was purchased from Tocris Bioscience (Bristol, UK). All drugs were dissolved in saline. Paroxetine (0.5–5 mg/kg) was injected i.p. Mice in the control group received saline. 5-HT receptor antagonists were administered i.p. 30 min before paroxetine was given. Thirty minutes after treatment with paroxetine, the forced swimming test was carried out. 2.4. Measurement of locomotor activity The locomotor activity of mice was measured using a digital counter with an infrared sensor (NS-AS01, Neuroscience Incorporated, Tokyo, Japan) following a previously described method (Yamada et al., 2004). An infrared sensor was set over an open-top, clear polycarbonate cage (22.5 × 33.8 × 14.0 cm) into which each mouse was placed. Locomotor activity was determined over 10 min. The apparatus was used to detect beam breaks and record a digital count of the horizontal movements of animals.
2.5. Statistical analyses Results are means ± S.E.M. of 5–8 mice in the behavioral studies. The effects of paroxetine on immobility time in each mouse strain were analyzed by one-way ANOVA followed by Dunnet's multiple comparison post-hoc test. The effects of paroxetine on immobility time were also determined by two-way ANOVA with paroxetine treatment and mice strains. The effects of receptor antagonists on the antiimmobility effects of paroxetine were analyzed by two-way ANOVA, with pretreatment and paroxetine treatment showing anti-immobility effects as the main factors. Pairwise follow-up comparisons of individual treatment groups were analyzed by Tukey's multiple comparison post-hoc test. 3. Results 3.1. Effects of paroxetine on immobility time and locomotor activity in five strains of mice Fig. 1 shows the immobility times in five strains of mice after treatment with paroxetine. Paroxetine induced significant anti-immobility effects in the five strains of mice (ICR: F(3, 25) =40.16, P≤0.0001. ddY: F (3, 25)= 19.73, P≤ 0.0001. C57BL/6: F(3, 26)= 12.59, P≤ 0.0001. DBA/2: F(3, 26) =125.86, P ≤0.0001. BALB/c: F(3, 26)= 21.11, P ≤0.0001. Twoway ANOVA: Treatment: F(3, 129)= 122.72, P≤0.0001. Strain: F(4, 129) = 360.83, P ≤ 0.0001. Treatment × strain interaction: F(12, 129) = 8.35, P ≤ 0.0001). However, the anti-immobility effects of paroxetine were elicited in DBA/2 and BALB/c mice at lower doses than in C57BL, ddY and ICR strains of mice. ICR mice showed the lowest sensitivity to paroxetine among the five strains of mice evaluated. Fig. 2 shows the locomotor activity in five strains of mice after treatment with paroxetine. Paroxetine did not alter locomotor activity in any mouse strain (ICR: F(3, 21) = 0.18, P ≥ 0.05. ddY: F(3, 21) = 0.18, P ≥ 0.05. C57BL/6: F(3, 21) = 1.64, P ≥ 0.05. DBA/2: F(3, 20) = 3.34, P ≥ 0.05. BALB/c: F(3, 29) = 1.25, P ≥ 0.05. Two-way ANOVA: Treatment: F(3, 112)= 0.4152, P ≥ 0.05. Strain: F(4, 129)= 133.81, P ≤ 0.0001. Treatment× strain interaction: F(12, 112) = 1.86, P ≤ 0.05). 3.2. Effects of 5-HT receptor antagonists and the α1-adrenoceptor antagonist prazosin on the anti-immobility effects of paroxetine at 5 mg/kg The effects of 5-HT receptor antagonists and the α1-adrenoceptor antagonist prazosin on the anti-immobility effects of paroxetine at 5 mg/kg in DBA/2 and ICR mice are shown in Figs. 3 and 4, respectively. The 5-HT1A receptor antagonist WAY 100635 significantly attenuated
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Fig. 2. Effects of paroxetine on locomotor activity in five strains of mice. Results are shown as mean ± S.E.M. (N = 5–7). The axis represents the total digital counts based on beam breaks.
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Fig. 3. Effects of several 5-HT receptor antagonists and prazosin on paroxetine-induced anti-immobility in DBA/2 mice. Results are shown as mean ± S.E.M. (N = 6–7). Paroxetine at 5 mg/kg was given i.p. 5-HT receptor antagonists (the 5-HT1A receptor antagonist WAY 100635, the 5-HT1B receptor antagonist GR 55562, the 5-HT2 receptor antagonist LY53857, the 5-HT3 receptor antagonist ondansetron and the 5-HT4 receptor antagonist SDZ205,557) and the α1-adrenoceptor antagonist prazosin were injected i.p. 30 min before paroxetine. ***P b 0.001 vs. saline of respective group. ###P b 0.001 vs. saline + paroxetine-treated group.
Treatment: F(1, 30) = 33.13, P ≤ 0.0001. Pretreatment× treatment interaction: F(2, 30) = 23.42, P ≤ 0.0001. GR 55562: Pretreatment: F(2, 36) = 0.77, P ≥ 0.05. Treatment: F(1, 36) = 321.18, P ≤ 0.0001. Pretreatment× treatment interaction: F(2, 36) = 0.10, P ≥ 0.05. LY53857: Pretreatment: F(2, 32) = 1.74, P ≥ 0.05. Treatment: F(1, 32) = 288.44, P ≤ 0.0001. Pretreatment× treatment interaction: F(2, 32) = 0.51, P ≥ 0.05. Ondansetron: Pretreatment: F(2, 31) = 0.36, P ≥ 0.05. Treatment: F(1, 31) = 144.7, P ≤ 0.0001. Pretreatment× treatment interaction: F(2, 31) =0.31, P≥0.05. SDZ205,557: Pretreatment: F(2, 32) = 0.02, P≥0.05. Treatment: F(1, 32) =190.94, P≤0.0001. Pretreatment × treatment interaction: F(2, 32) =0.96, P ≥0.05). Two way ANOVA analysis on results in ICR mice (WAY100635: Pretreatment: F(2, 36) =6.01, P≤0.01. Treatment: F(1, 36)= 66.52, P≤0.0001. Pretreatment×treatment interaction: F(2, 36)= 8.63, P ≤0.001. GR 55562: Pretreatment: F(2, 29) =0.69, P≥0.05. Treatment: F(1, 29) =216.81, P≤0.0001. Pretreatment× treatment interaction: F(2, 29) =0.59, P≥0.05. LY53857: Pretreatment: F(2, 36) =0.10, P≥0.05. Treatment: F(1, 36) =159.42, P≤0.0001. Pretreatment× treatment interaction: F(2, 36) =0.17, P≥0.05. Ondansetron: Pretreatment: F(2, 29) =0.07, P≥0.05. Treatment: F(1, 29) =226.68, P≤0.0001. Pretreatment ×treatment interaction: F(2, 29) =0.22, P≥0.05. SDZ205,557: Pretreatment: F(2, 28) = 0.31, P≥0.05. Treatment: F(1, 28) =81.39, P≤0.0001. Pretreatment× treatment interaction: F(2, 28) =0.12, P ≥0.05). The α1-adrenoceptor antagonist prazosin inhibited the anti-immobility effects elicited by paroxetine in DBA/2 and ICR mice (DBA/2 mice: Pretreatment: F(2, 31) = 10.74, P ≤ 0.0001. Treatment: F(1, 31) = 232.21, P ≤ 0.0001. Pretreatment×treatment interaction: F(2, 31)=9.41, P≤0.0001. ICR mice: Pretreatment: F(2, 28)=3.39, P ≤0.05. Treatment: F(1, 28)=80.28,
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P≤0.0001. Pretreatment×treatment interaction: F(2, 28)=10.82, P≤0.001). 3.3. Effects of prazosin on the anti-immobility effects of paroxetine at 1 mg/kg in DBA/2 mice The effects of prazosin on the anti-immobility effects of paroxetine at 1 mg/kg in DBA/2 mice are shown in Fig. 5A. The anti-immobility effects of paroxetine at 1 mg/kg in DBA/2 mice were not antagonized by prazosin (Pretreatment: F(2, 34) = 0.9735, P ≥ 0.05. Treatment: F(1, 34) = 397.81, P ≤ 0.0001. Pretreatment × treatment interaction: F(2, 34) = 0.68, P ≥ 0.05.) 3.4. Effects of prazosin on the anti-immobility effects of fluvoxamine in DBA/2 mice Prazosin did not affect the fluvoxamine-induced anti-immobility effects in DBA/2 mice (Fig. 5B). (Pretreatment: F(2, 34) = 0.9735, P ≥ 0.05. Treatment: F(1, 34) = 397.81, P ≤ 0.0001. Pretreatment × treatment interaction: F(2, 34) = 0.68, P ≥ 0.05.) 4. Discussion The forced swimming test is an evaluation method for the efficacy of antidepressants. However, a lower sensitivity to SSRIs in the forced swimming test has been suggested (Sanchez and Meier, 1997). We previously demonstrated that there is an apparent difference between five strains of mice with respect to responses to fluvoxamine (Sugimoto et al., 2008). In DBA/2 and BALB/c mice, fluvoxamine induced clear antiimmobility effects. However, fluvoxamine had no effects in ICR, ddY and C57BL/6 mice. These differences between strains derive from variations of 5-HT transporter binding in the brain (Sugimoto et al., 2008). Paroxetine is a well-known SSRI used widely for the treatment of depression (Wagstaff et al., 2002). Paroxetine has a higher affinity for the serotonin transporter than fluvoxamine, which leads to its potent antidepressant effects (Sanchez and Hyttel, 1999; Wagstaff et al., 2002). However, it is not clear if there is a difference between mice strains with respect to the anti-immobility effects of paroxetine. We therefore examined the effect of paroxetine on immobility time in five strains of mice. Paroxetine induced significant anti-immobility effects in all five strains of mice. However, there was a difference in the sensitivity to paroxetine between mice strains. Paroxetine even at a low dose (0.5 mg/kg) elicited apparent anti-immobility effects in DBA/2 and BALB/c mice. In ddY and C57BL mice, paroxetine induced reduced immobility above the dose of 1 mg/kg. In ICR mice, only a high dose of 5 mg/kg caused significant anti-immobility effects. In our previous
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Fig. 5. Effects of prazosin on paroxetine (1 mg/kg)- and fluvoxamine-induced antiimmobility in DBA/2 mice. (A) Effects of prazosin on paroxetine (1 mg/kg)-induced anti-immobility effects. Results are shown as mean ± S.E.M. (N = 5–6). Paroxetine (1 mg/kg) was given i.p. Prazosin were injected i.p. 30 min before paroxetine. ***P b 0.001 vs. saline of the respective group. (B) Effects of prazosin on fluvoxamineinduced anti-immobility in DBA/2 mice. Results are mean ± S.E.M. (N = 5–6). Fluvoxamine (10 mg/kg) was given (i.p.). Prazosin was injected (i.p.) 30 min before fluvoxamine. ***P b 0.001 vs. saline of the respective group.
report, fluvoxamine caused strong anti-immobility effects in DBA/2 and BALB/c mice, whereas fluvoxamine did not alter the immobility time in C57BL/6, ddY and ICR mice (Sugimoto et al., 2008). Therefore, differences between strains with respect to responses to paroxetine were similar to those seen with fluvoxamine. Paroxetine did not affect locomotor activity in the five strains of mice, but it elicited antiimmobility effects. This indicated that locomotor activity is not related to anti-immobility effects. It has been reported that the 5-HT1A receptor is involved in antidepressant-like effects in the forced swimming test (ChojnackaWojcik et al., 1991). We also observed that the anti-immobility effects of fluvoxamine in DBA/2 mice are mediated by the 5-HT1A receptor because the selective 5-HT1A receptor antagonist WAY 100635 abolished them (Sugimoto et al., 2010a). Therefore, we studied the effects of 5-HT receptor antagonists (including WAY 100635) on the antiimmobility effects of paroxetine. We examined the effects of 5-HT receptor antagonists in DBA/2 and ICR mice; these strains showed the highest and lowest sensitivity to paroxetine, respectively. Pretreatment with WAY 100635 antagonized the paroxetine-induced antiimmobility effects in DBA/2 and ICR mice. It has been suggested that other 5-HT receptor subtypes (e.g., 5-HT1B, 5-HT2, 5-HT3, and 5-HT4) may also be related to the anti-immobility effects in rats and mice (Lucas et al., 2007; Redrobe and Bourin, 1997; Redrobe et al., 1996). However, the 5-HT1B receptor antagonist GR55562 and the nonselective 5-HT2 receptor antagonist LY 53857 did not modify the antiimmobility effects of paroxetine in DBA/2 and ICR mice. The 5-HT3 and 5-HT4 receptor antagonists ondansetron and SDZ205,557, respectively, were also ineffective in both mice strains. These results suggest that the anti-immobility effects of paroxetine are mediated by the 5HT1A receptor, which is in accordance with our previous results using fluvoxamine. Thus, the inhibitory effects of paroxetine on 5-HT reuptake elicit elevation of 5-HT concentrations in the synaptic cleft. It is likely that elevated levels of 5-HT by paroxetine activate the 5-HT1A receptor, leading to anti-immobility effects. We previously reported that the selective 5-HT1A receptor agonist 8-OH-DPAT elicits anti-immobility effects, and that its effects are antagonized by WAY 100635 (Sugimoto et al., 2010b). Chojnacka-Wojcik et al. (1991) also reported that the 5-HT1A receptor agonist gepirone induced anti-immobility effects in rats. Taken together, our results with paroxetine in the present study further support the role of the 5-HT1A receptor in antidepressant-like effects in mice. Noradrenaline, as well as 5-HT, has an important role in depression. Paroxetine has a moderate affinity for the noradrenaline transporter in addition to its affinity for the 5-HT transporter (Owens et al., 2001; Sanchez and Hyttel, 1999). In contrast, fluvoxamine has no affinity for the noradrenaline transporter (Owens et al., 2001; Sanchez and Hyttel, 1999). Owens et al. (2001) reported that the Ki values of paroxetine and fluvoxamine for the inhibition of noradrenaline uptake are 156 nM and 1119 nM, respectively, in HEK-293 cells derived from humans. Furthermore, in the microdialysis technique, paroxetine elicited noradrenaline release in the cortex in Swiss mice at doses of 4 mg/kg and 8 mg/kg and Sprague–Dawley rats at 30 mg/kg (Beyer et al., 2002; David et al., 2003). It is assumed that noradrenaline may be related to the pharmacological effects of paroxetine. It has been reported that the antidepressant-like effects in rodents induced by several drugs are mediated by the α1-adrenoceptor (Danysz et al., 1986; Kaster et al., 2007; Takeda et al., 2003); these data are based on antagonism by α1-adrenoceptor antagonists. We therefore studied the effects of the α1-adrenoceptor antagonist prazosin on the anti-immobility effects of paroxetine in DBA/2 and ICR mice. Prazosin significantly antagonized the antidepressant-like effects elicited by a high dose (5 mg/kg) of paroxetine in DBA/2 and ICR mice. This indicated that the α1-adrenoceptor may be related to the anti-immobility effects induced by 5 mg/kg paroxetine. However, the anti-immobility effects of 1 mg/kg paroxetine in DBA/2 mice were not antagonized by prazosin. This result suggests that the antidepressant-
Y. Sugimoto et al. / European Journal of Pharmacology 672 (2011) 121–125
like effects of a high dose of paroxetine are involved in the α1-adrenoceptor, whereas the effects of a lower dose of paroxetine are not. David et al. (2003) reported that, in Swiss mice using microdialysis, paroxetine (1–8 mg/kg) elicited 5-HT release in the cortex. They also demonstrated that paroxetine increased noradrenaline outflow in the cortex at 4 mg/kg and 8 mg/kg, but significant increases were not observed at 1 mg/kg (David et al., 2003). These observations of dose-dependent elevation of noradrenaline by microdialysis are closely related to our results, showing that noradrenaline is associated with the antiimmobility effects of 5 mg/kg paroxetine but not to those of 1 mg/kg paroxetine. Therefore, it is likely that, at higher doses, the inhibitory effects of the noradrenaline transporter of paroxetine may contribute to the anti-immobility effects in the forced swimming test. In contrast, prazosin did not affect fluvoxamine-induced anti-immobility effects in DBA/2 mice. This indicates that the α1-adrenoceptor is not associated with the antidepressant-like effects of fluvoxamine (which is different from the effects seen with paroxetine). It was suggested that the affinity of fluvoxamine for the noradrenaline transporter is low compared with that for paroxetine (Kelly and Leonnard, 1995; Owens et al., 2001; Sanchez and Hyttel, 1999). Therefore, involvement of noradrenergic neurons in the anti-immobility effects of fluvoxamine may be excluded. Stone and Quartemain (1999) reported that the α1-adrenoceptor is involved in the anti-immobility effects of noradrenaline. Doze et al. (2009) reported that prazosin reversed the anti-immobility effects in mice expressing constitutively active mutant α1-adrenoceptors. These findings support those in the present study showing the involvement of α1-adrenoceptors in the antidepressant-like effects of paroxetine. Prazosin has an affinity for α1A and α1B receptors. It is not clear which receptor subtype is involved, and further studies are required. Guzzetti et al. (2008) reported that paroxetine elicited antiimmobility effects in C57BL mice but not in DBA/2 and BALB/c mice, but this finding is not in accordance with our results. They suggested that these strain differences are related to serotonergic transmission because co-administration with paroxetine and the 5-HT precursor tryptophan elicited anti-immobility effects in DBA/2 and BALB/c mice. In the forced swimming test, the experimental condition may affect the immobility time or the anti-immobility effects of drugs. Guzzetti et al. (2008) used a deeper water depth in the glass cylinder. Detke and Lucki (1996) reported that the depth of water decreases the basal immobility time in rats. Therefore, water depth may cause a different sensitivity to paroxetine, and we are now investigating this theme. In summary, we demonstrated that there are differences in the sensitivity to paroxetine in five strains of mice, and that these differences are similar to those seen with fluvoxamine. The anti-immobility effects of paroxetine are mediated by the 5-HT1A receptor. In addition, the α1-adrenoceptor is associated with the effects of paroxetine at a high dose, whereas the α1-adrenoceptor is not associated with the antidepressant-like effects of fluvoxamine. These results suggest that the inhibitory effects of the reuptake of noradrenaline are related (at least in part) to the antidepressant-like effects of paroxetine. Redrobe et al. (1998) reported that the anti-immobility effects of a high dose of paroxetine are resistant to the depletion of 5-HT induced by p-chlorophenylalanine, and they speculated on the involvement of the noradrenergic system in explaining the effects of paroxetine. Therefore, our results indicate that the strong antidepressant-like effects of paroxetine may be caused by its effects on serotonergic and noradrenergic neurons. References Beyer, C.E., Boikess, S., Luo, B., Dawson, L.A., 2002. Comparison of the effects of antidepressants on norepinephrine and serotonin concentrations in the rat frontal cortex: an in-vivo microdialysis study. J. Psychopharmacol. 16, 297–304. Chojnacka-Wojcik, E., Tatarczynska, E., Golembiowska, K., Przegalinski, E., 1991. Involvement of 5-HT1A receptors in the antidepressant-like activity of gepirone in the forced swimming test in rats. Neuropharmacology 30, 711–717.
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