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Antidepressant-like effects of selegiline in the forced swim test
European Neuropsychopharmacology 15 (2005) 563 – 571 www.elsevier.com/locate/euroneuro
Antidepressant-like effects of selegiline in the forced swim test Seiichiro ShimazuT,1, Akiko Minami1, Haruko Kusumoto, Fumio Yoneda Research Institute, Fujimoto Pharmaceutical Corporation, 1-3-40 Nishiotsuka, Matsubara, Osaka 580-0011, Japan Received 25 November 2004; received in revised form 10 February 2005; accepted 15 February 2005
Abstract Although selegiline, a monoamine oxidase (MAO)-B inhibitor, is reported to exert antidepressant effects in depressant patients, evidence in rodents for effects of selegiline is quite limited. The purpose of the present study was to assess effects of selegiline in the forced swim test (FST) and on locomotor activity, and to investigate whether MAO inhibition or stimulation of receptors contributes to antidepressant-like effects of selegiline. Drugs were subcutaneously administrated. The single administration of reference drug nortriptyline at 5 mg/kg reduced locomotor activity without effects in FST and brain MAO activities. But nortriptyline repeatedly given 24, 5 and 1 h before behavioral tests significantly decreased an immobility time in FST without effects in motor activities, and showed weak brain MAO-B inhibition. Single and following repeated (24, 5 and 1 h before behavioral tests) administrations of selegiline at 10 mg/kg significantly decreased the immobility time in FST, with little motor stimulant effect. In contrast, (+)-methamphetamine caused a marked decrease in immobility time and an increase in locomotor activity. Selegiline at 1 and 3 mg/kg, which failed to decrease immobility time, markedly inhibited brain total-MAO and MAO-B activities. A dopamine D1 receptor antagonist SCH 23390 completely blocked antidepressant-like effects of selegiline, but not dopamine D2, serotonergic or noradrenergic receptor antagonists. These results suggest that selegiline exerts the antidepressant-like effects by prolonging escape-directed behavior rather than by a motor stimulant effect and D1 receptor activation contributes to its effect. D 2005 Elsevier B.V. and ECNP. All rights reserved. Keywords: Selegiline; Forced swim test; Locomotor activity; Antidepressant; Monoamine oxidase
1. Introduction Selegiline, a selective and irreversible monoamine oxidase (MAO)-B inhibitor, is widely used for the therapy of Parkinson’s disease (PD) (Birkmayer et al., 1977). In addition, several studies have been shown that selegiline has antidepressant effects (Varga and Tringer, 1967; Mann and Gershon, 1980; Birkmayer et al., 1984). In a study by Birkmayer et al. (1984), about 70% of depressed patients improved following the administration of selegiline with ( )-phenylalanine, the precursor of phenylethylamine (PEA). Selegiline at high doses (40–60 mg/day) exerts marked antidepressant effect (Lees, 1991; Kuhn and Mu¨ller, 1996; Ritter and Alexander, 1997). Also, selegiline improves atypical depression, major depression and depresT Corresponding author. Tel.: +81 723 32 5151; fax: +81 723 32 8482. E-mail address: [email protected] (S. Shimazu). 1 These authors contributed equally to this work.
sion in PD (Youdim, 1980; Mendlewicz and Youdim, 1983; Quitkin et al., 1984; McGrath et al., 1989; Mayberg and Solomon, 1995; Tom and Cummings, 1998). However, there are a few reports about the antidepressant-like effects of selegiline using animal models, such as behavioral despair, learned helplessness and chronic mild stress (Porsolt and Jalfre, 1978; Willner, 1984). Several laboratories have demonstrated that selegiline decreases immobility time in the forced swim test (FST), a widely used model of depression (Porsolt et al., 1977, Zebrowska-Lupina et al., 1997, Ferigolo et al., 1998, Nowakowska et al., 2001). Furthermore, Fozard et al. (1985) suggested that antidepressant-like effect of selegiline was unrelated to MAO-B inhibition. Thus, antidepressant-like effect of selegiline and its mechanism are unclear. The aim of the present study is to evaluate antidepressant-like effects of selegiline in FST and locomotor activity, and to investigate whether antidepressant-like effects of selegiline involve its MAO inhibitory action and exerted through monoaminergic receptors.
0924-977X/$ - see front matter D 2005 Elsevier B.V. and ECNP. All rights reserved. doi:10.1016/j.euroneuro.2005.02.003
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2. Materials and methods 2.1. Animals Male ddY mice (8 weeks old, Nihon SLC, Shizuoka, Japan) were maintained in a humidity (55 F 10%)- and temperature (23 F 2 8C)-controlled facility under a 12/12-h light/dark cycle (light on at 7:00 a.m.) with free access to food (MF, Oriental Yeast, Tokyo, Japan) and water. Mice were allowed to adapt to the environments for 7 days before testing. The following animal studies were performed according to the bGuiding Principles for the Care and Use of Laboratory AnimalsQ approved by The Japanese Pharmacological Society. 2.2. Chemicals Following compounds were used: selegiline hydrochloride, ( )-methamphetamine hydrochloride (Fujimoto Pharmaceutical, Osaka, Japan), (+)-methamphetamine hydrochloride (Dainippon Pharmaceutical, Osaka, Japan), yohimbine hydrochloride (Nacalai tesque, Kyoto, Japan), S-( )-eticlopride hydrochloride (Research Biochemicals International, Natick, MA, USA), nortriptyline hydrochloride, R-(+)-7-chloro-8-hydroxy-3-methyl-1-phenyl-2,3,4,5tetrahydro-1H-3-benzazepine hydrochloride (SCH 23390), prazosin hydrochloride, propranolol hydrochloride, ketanserin tartrate, N-(2-(4-(2-methoxyphenyl)-1-piperazinyl)ethyl)-N-(2-pyridinyl) cyclohexanecarboxamide maleate
(WAY 100,635) (Sigma, St. Louis, MO, USA). The doses of ketanserin and WAY 100,635 were expressed as those of free base, and the doses of other drugs were expressed as those of hydrochloride salt. All drugs were dissolved in saline and administered subcutaneously (s.c.) in a constant volume of 10 ml/kg. Control animals were injected with saline. 2.3. Locomotor activity Locomotor activity was assessed using an infrared (Model NS-AS01)-linked activity sensor system (Model AB system-24A, Neuroscience, Tokyo, Japan). Counts of motor activity were recorded on a computer system during consecutive 5-min intervals. The motor activity during the 2-h observation period was measured starting 1 h after drug treatment. 2.4. FST FST was performed as described by Porsolt et al. (1977). Briefly, mice were placed 1 h after drug treatment in black Plexiglas cylinder (45 cm height 15 cm diameter, NSM507M, Neuroscience) filled with water at 25 8C at a depth of 35 cm. Immediately after placing the animals, immobility time (s) and active path (a distance of swimming in centimeters) were measured for 6 min by a CCD-camera linking to a computer system (Video Image Motion Analyzer System AXIS-60, Neuroscience), and were used for drug evaluation during the last 4 min.
Fig. 1. Effects of a single administration of selegiline on FST and locomotor activity in mice. Panels (A) and (B) show the immobility time and active path in the FST, respectively. The duration of immobility time and active path were recorded during the last 4 min of the 6-min test period. Panel (C) shows the cumulative locomotor activities for 2 h of action of compounds, respectively. Selegiline (1–10 mg/kg), nortriptyline (5 mg/kg) and saline were subcutaneously given 1 h before the behavioral test. Each value represents as mean F S.E.M. (n = 16, 8, 16, 16 and 16 for saline, nortriptyline, 1, 3 and 10 mg/kg selegilinetreated groups, respectively). *P b 0.05, **P b 0.01 and ***P b 0.001 vs. saline-treated control.
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potassium phosphate containing 1% EDTA (pH 7.5). The total-MAO and MAO-B activities were determined with Amplex Red Monoamine Oxidase assay Kit (Molecular Probes, Eugene, OR, USA). The protein contents were measured using Lowry’s method (Lowry et al., 1951). 2.6. Statistics
Fig. 2. Effects of a single administration of selegiline on time course changes in locomotor activity in mice. Data are presented as the averages of locomotion taken at each 5-min interval, over a period of 2 h. S.E.M. are not shown for the sake of clarity. Selegiline (1–10 mg/kg), nortriptyline (5 mg/ kg) and saline were subcutaneously given 1 h before the behavioral test (n = 16, 8, 16, 16 and 16 for saline, nortriptyline, 1, 3 and 10 mg/kg selegilinetreated groups, respectively). *P b 0.05, **P b 0.01 and ***P b 0.001 vs. saline-treated control.
Statistical analyses for FST and MAO activities were performed with the Kruskal–Wallis’ analysis of variance followed by post hoc Wilcoxon’s test (SAS preclinical package ver 5.0, SAS Institute, Cary, NC, USA). For time course data, statistical analyses were performed with twoway ANOVA followed by post hoc Dunnett’s test. The differences were considered significant at P b 0.05.
3. Results 3.1. Effect of a single injection of selegiline
2.5. Measurement of MAO activities Mice were sacrificed by cervical dislocation 1 h after FST and whole brain were isolated. Isolated whole brains were homogenized in 10 volume of ice-cold 0.2 M potassium phosphate containing 1% EDTA (pH 7.5). Homogenates were centrifuged at 900 g for 10 min at 4 8C, and thereafter the resulting supernatant was recentrifuged at 11,500 g for 20 min at 4 8C. The precipitations were resuspended in 0.2 M
A single treatment of selegiline significantly reduced the immobility time and increased the active path at the dose of 10 mg/kg, but not at 1 and 3 mg/kg (Kruskal–Wallis’ test, immobility time: df = 4, P = 0.0151; active path: df = 4, P = 0.0099) (Fig. 1A and B). However, a selective noradrenaline reuptake inhibitor nortriptyline (5 mg/kg), as a reference drug, had no effect on the immobility time and the active path in the FST.
Fig. 3. Effects of repeated administrations of selegiline on FST and locomotor activity in mice. Panels (A) and (B) show the immobility time and active path in FST, respectively. The duration of immobility time and active path were recorded during the last 4 min of the 6-min test period. Panel (C) shows the cumulative locomotor activities for 2 h of action of compounds, respectively. Selegiline (1–10 mg/kg), nortriptyline (5 mg/kg) and saline were subcutaneously given 24, 5 and 1 h before the behavioral test. Each value represents as mean F S.E.M. (n = 16 for FST and n = 8 for locomotor activities). *P b 0.05 and *P b 0.01 vs. saline-treated control.
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ities in almost measuring points of the initial phase until 40 min (Fig. 2). Also, selegiline at 1 and 10 mg/kg caused significant increases in locomotor activities at several measuring points (1 mg/kg: 110–115 min, 10 mg/kg: 60–65, 75–80, 80–85 and 100–105 min). 3.2. Repeated injections of selegiline
Fig. 4. Effects of repeated administrations of selegiline on time course changes in locomotor activity in mice. Data are presented as the averages of locomotion taken at each 5-min interval, over a period of 2 h. S.E.M. are not shown for the sake of clarity. Selegiline (1–10 mg/kg), nortriptyline (5 mg/kg) and saline were subcutaneously given 24, 5 and 1 h before the behavioral test (n = 8). *P b 0.05 and **P b 0.01 vs. saline-treated control.
Selegiline had no effect on cumulative locomotor activity for 2 h, but nortriptyline significantly decreased cumulative locomotor activity (Kruskal–Wallis’ test, df = 4, P = 0.0013) (Fig. 1C). Fig. 2 showed time course changes of locomotor activities. Two-way ANOVA analyses on the data of time course changes resulted in F = 15.56, df = 4, P b 0.0001 (Fig. 2). The animals injected with vehicle displayed locomotion related to active exploration during the initial phase that was usually followed by a decline in activity due to habituation. Nortriptyline and 10 mg/kg selegiline groups significantly decreased locomotor activ-
The three times injections (24, 5 and 1 h before behavioral test) of selegiline resulted in a significant decrease in the immobility time and an increase in the active path at the dose of 10 mg/kg, but not at 1 and 3 mg/kg (Kruskal–Wallis3 test, immobility time: df = 4, P = 0.0086, active path: df = 4, P = 0.0042) (Fig. 3A and B). Nortriptyline significantly reduced the immobility time and increased the active path. However, neither selegiline nor nortriptyline affected the cumulative locomotor activity (Kruskal–Wallis3 test, df = 4, P = 0.1052) (Fig. 3C). Two-way ANOVA analyses on the time course data resulted in F = 5.68, df = 4, P = 0.0002 (Fig. 4). Locomotor activities of 10 mg/kg selegiline group significantly decreased in the initial phase (at 5–10, and 10–15 min measuring points), and significantly increased at points of 35–40 and 50–55 min. 3.3. Effect of a single injection of methamphetamine To determine whether the antidepressant-like effects of selegiline are mediated by ( )-methamphetamine that is a major metabolite of selegiline, we investigated effects of ( )-
Fig. 5. Effects of a single administration of methamphetamine on FST in mice. Panels (A) and (C), and panels (B) and (D) show effects of ( )methamphetamine and (+)-methamphetamine, respectively. The duration of immobility time (A and B) and active path (C and D) were recorded during the last 4 min of the 6-min test period. ( )-Methamphetamine (1–10 mg/kg), (+)-methamphetamine (1–10 mg/kg) and saline were subcutaneously given 1 h before the behavioral test. Each value represents as mean F S.E.M. (n = 12 for panels (A) and (C), and n = 8 for panels (B) and (D)). **P b 0.01 and ***P b 0.001 vs. saline-treated control.
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methamphetamine on the FST and on the locomotor activity. ( )-Methamphetamine (1–10 mg/kg) decreased the immobility time and increased the active path in a dose-dependent manner (Kruskal–Wallis’ test, immobility time: df = 3, P = 0.0045; active path: df = 3, P = 0.0005) (Fig. 5A and C). However, ( )-methamphetamine did not affect the locomotor activity at any doses (two-way ANOVA: F = 1.44, df = 3, P = 0.2294) (Fig. 6A). In addition, we evaluated effects of (+)methamphetamine, which is an active enantiomer and psychostimulant. (+)-Methamphetamine (1–10 mg/kg) reduced the immobility time and increased the active path (Kruskal–Wallis’ test, immobility time: df = 3, P b 0.0001; active path: df = 3, P b 0.0001) (Fig. 5B and D). (+)Methamphetamine at 1 and 3 mg/kg markedly increased the locomotor activity, and showed long-lasting enhancement of locomotor activity until 2 h (two-way ANOVA, F = 171.06,
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df = 3, P b 0.0001) (Fig. 6B). In contrast, 10 mg/kg (+)methamphetamine significantly decreased locomotor activities in the initial phase until 25 min, and significantly increased at several measuring points after 55 min. Two-way ANOVA at saline vs. 10 mg/kg (+)-methamphetamine did not show a significance ( F = 0.17, df = 1, P = 0.6782). Lower doses (0.03–0.3 mg/kg) of (+)-methamphetamine did not affect the immobility time, the active path and the locomotor activity (data not shown). 3.4. Effects of several receptor antagonists against selegiline-induced antidepressant-like effects Next, we examined the effects of selective receptor antagonists on the selegiline-induced behavioral effects. The treatment of SCH 23390 completely inhibited selegiline-
Fig. 6. Effects of a single administration of methamphetamine on time course changes in locomotor activity in mice. Panels (A) and (B) show effects of ( )methamphetamine and (+)-methamphetamine, respectively. ( )-Methamphetamine (1–10 mg/kg), (+)-methamphetamine (1-10 mg/kg) and saline were subcutaneously given 1 h before the behavioral test. Data are presented as the averages of locomotion taken at each 5–min interval, over a period of 2 h. S.E.M. are not shown for the sake of clarity (n = 8). *P b 0.05, **P b 0.01 and ***P b 0.001 vs. saline-treated control.
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induced decrease in immobility time and increase in active path (Table 1). In contrast, a dopamine D2 receptor antagonist eticlopride, a 5-HT1A receptor antagonist WAY 100,635, a 5-HT2A receptor antagonist ketanserin, an a1 adrenergic receptor antagonist prazosin, an a2 adrenergic receptor antagonist yohimbine and a h receptor antagonist propranolol did not influence on antidepressant-like effects of selegiline. 3.5. Effects of selegiline on MAO activity To determine if MAO inhibition contributes to antidepressant-like effects of selegiline, we measured the totalMAO and MAO-B activities in whole brain 1 h after behavioral tests. Selegiline at all doses had same inhibitory effects on MAO-B activity in single (Kruskal–Wallis’ test, total-MAO: df = 4, P b 0.0001; MAO-B: df = 4, P = 0.0001) and repeated injections (Kruskal–Wallis’ test, total-MAO: df = 4, P = 0.0002; MAO-B: df = 4, P = 0.0008) (Fig. 7). The repeated injections of nortriptyline significantly decreased MAO-B activity by 18%, but the single injection had no inhibitory effects.
4. Discussion The present study demonstrated that selegiline reduced immobility time and increased active path in single or following repeated injections. The effects of a single dose of selegiline confirm the finding of the previous study in rats reported by Zebrowska-Lupina et al. (1997). In the present study, 10 mg/kg selegiline caused decreases in motor activities in the initial phase of active exploration, and weak increases in activities in the phase after initial exploration periods, but did not affect cumulative motor activities. Thus, the analyses at detail revealed that selegiline hardly stimulated motor activities, comparing with a psychostimulant like (+)-methamphetamine (Figs. 2, 4, and 6). It is noteworthy that selegiline, at a single dose which lacked stimulant effects, had an antidepressant effect, indicating that its effect is not merely due to a stimulation of motor activity. On the other hand, repeated administration of a selective noradrenaline reuptake inhibitor nortriptyline at 5 mg/kg caused a reduction in immobility time and an increase in active path, but the single administration was ineffective.
Table 1 The effects of receptor antagonists on selegiline-induced behavioral effects in FST in mice Drug
FST Immobility (s)
Sal Sel Sal Sel Sal Sel Sal Sel Sal Sel Sal Sel Sal Sel Sal Sel Sal Sel Sal Sel Sal Sel Sal Sel Sal Sel Sal Sel
Sal Sal SCH SCH Sal Sal Eti Eti Sal Sal WAY WAY Sal Sal Ket Ket Sal Sal Pra Pra Sal Sal Yoh Yoh Sal Sal Pro Pro
167.2 F 9.0 115.0 F 10.5*** 179.6 F 9.8 160.3 F 7.7## 181.9 F 10.7 105.9 F 26.9* 134.1 F 22.7 86.1 F 22.7** 162.9 F 11.7 80.8 F 15.1*** 148.2 F 17.0 97.1 F13.0** 119.1 F16.4 54.9 F 15.5* 135.9 F 11.9 58.4 F 17.2* 118.5 F 13.6 64.9 F 13.6** 144.2 F 12.6 76.0 F 14.2* 137.8 F 15.7 80.3 F 12.9* 113.8 F 10.6 76.8 F 11.1** 154.1 F18.8 76.2 F 21.1* 127.2 F 26.4 84.7 F 12.5*
Active path (cm) a
df = 3, P b 0.0001
df = 3, P = 0.0207
df = 3, P = 0.0017
df = 3, P = 0.0019
df = 3, P = 0.0005
df = 3, P = 0.0082
df = 3, P = 0.0363
543.0 F 70.6 1032.6 F 106.6*** 476.2 F 87.7 685.0 F 67.8# 272.6 F 49.5 740.6 F 176.2* 468.3 F 129.7 964.7 F 197.1** 341.4 F 52.7 925.3 F 109.9*** 384.1 F 71.7 795.7 F 105.2*** 646.2 F 124.2 1147.9 F 127.2* 597.7 F 77.5 1256.8 F 186.0* 564.4 F 73.4 994.2 F 102.4** 589.0 F 97.8 1178.9 F 159.6** 497.2 F 83.7 934.8 F 99.9** 533.2 F 45.3 726.8 F 51.7* 398.0 F 89.4 977.9 F 196.4* 562.8 F 166.8 825.7 F 83.1*
df = 3, P b 0.0001
df = 3, P = 0.0062
df = 3, P b 0.0001
df = 3, P = 0.0011
df = 3, P = 0.0005
df = 3, P = 0.0017
df = 3, P = 0.0349
Selegiline (Sel; 10 mg/kg) or saline (Sal) were given 1 h before the test. SCH 23390 (SCH; 0.03 mg/kg), eticlopride (Eti; 0.01 mg/kg), WAY 100,635 (WAY; 0.1 mg/kg), ketanserin (Ket; 5 mg/kg), prazosin (Pra; 2 mg/kg), yohimbine (Yoh; 5 mg/kg) and propranolol (Pro; 5 mg/kg) were administered simultaneously with selegiline or saline. Each value represents as mean F S.E.M. (n = 7–28). Post hoc Wilcoxon’s test: *P b 0.05, **P b 0.01 and ***P b 0.001 vs. saline-treated group. #P b 0.05 and ##P b 0.01 vs. selegiline-treated group. a Kruskal–Wallis’ test.
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Fig. 7. Effects of selegiline on MAO activities in whole brains. Panels (A) and (B) show total-MAO and MAO-B inhibition 2 h after a single injection of selegiline (1–10 mg/kg), nortriptyline (5 mg/kg) or saline, respectively. Panels (C) and (D) show total-MAO and MAO-B inhibition after repeated injections (3 times) of drugs, respectively. Selegiline, nortriptyline or saline was given 25, 6, and 2 h before the decapitation. MAO activities were measured using fluorometric assay. Each value represents as mean F S.E.M. (n = 10 for single saline injection group and n = 5 for other groups). **P b 0.01 and ***P b 0.001 vs. saline-treated control.
Onodera et al. (2000) demonstrated that another selective noradrenaline reuptake inhibitor desipramine had no effect in the FST in a single injection, but decreased immobility time in repeated administration. These phenomena suggest that the antidepressant effects cannot be explained solely by the change in monoaminergic tone (Nestler et al., 2002) and may involve adaptive changes in intracellular signal transduction and synaptic connectivity (Manji et al., 2001; Nestler et al., 2002). Porsolt et al. (1978) reported that antidepressants reduce open field activities. In the present study, a single treatment of nortriptyline showed a significant decrease in locomotor activities in initial phase of active exploration, though repeated administrations of nortriptyline showed no effect. Whereas, single and repeated treatments of 10 mg/kg selegiline significantly reduced motor activities in initial phase of active exploration, which may suggests its antianxious effect to unfamiliar environment. Selegiline orally treated is mainly metabolized by the liver cytochrome P-450 system and the metabolites identified are desmethylselegiline, ( )-methamphetamine and ( )-amphetamine in rats (Heinonen et al., 1994). The ratio of l-( )-methamphetamine, which is metabolized from selegiline in in vitro liver microsomal system of mice, are similar to that of rats (Levai et al., 2004). In the present study, 10 mg/kg s.c. ( )-methamphetamine, but not 1 and 3 mg/kg s.c., resulted in a significant decrease in immobility time and an increase in active path, without affecting locomotor activity. Using rats, Melega et al. (1999) have shown that the area under the curve of metabolically
generated ( )-methamphetamine after administration of 10 mg/kg s.c. selegiline was approximately 15% of that observed after administration of 2.5 mg/kg s.c. ( )methamphetamine, in rats. Taken together, ( )-methamphetamine generated after the administration of 10 mg/kg s.c. selegiline might not play an important role in the antidepressant-like effects of selegiline. Furthermore, a psychostimulant (+)-methamphetamine resulted in a marked decrease in immobility time and an increase in locomotor activity. The reduction of immobility induced by (+)methamphetamine seems to be caused by increase in general motor activity (Kitada et al., 1981). Thus, effects of selegiline and its metabolite ( )-methamphetamine on the immobility time could be clearly distinguished from those of psychostimulants such as (+)-methamphetamine. In the present study, selegiline inhibited total-MAO and MAO-B activities. Fozard et al. (1985) showed that a MAOB inhibitor [(E)-2-(3,4-dimethoxyphenyl)-3-fluoroallylamine hydrochloride, MDL 72145] failed to decrease immobility time, although its inhibitory effect of MAO-B is equivalent to that of selegiline. Gordon et al. (1999) suggested that antidepressant-like effects of selegiline were mediated by MAO-A inhibition rather than MAO-B inhibition. It has been reported that selegiline at 8 mg/kg i.p. and 10 mg/kg s.c. inhibited 20% MAO-A activities in rat striatum, and about a half of MAO-A activities in rat brains, respectively (Paterson et al., 1991, Tipton et al., 1976). The inhibition in MAO-A activities by the administration of 10 mg/kg s.c. selegiline may be contribute to antidepressant-like effects of selegiline. Repeated treatment
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of nortriptyline caused a significant inhibition of 18% in MAO-B activities, which may not contribute to its antidepressant effect. Moreover, the antidepressant-like effects of selegiline were completely inhibited by a dopamine D1 receptor antagonist SCH 23390. Nikulina et al. (1991) demonstrated a dopamine D1 receptor agonist SKF 38393 increased swimming activity and antagonist SCH 23390 reduced. Selegiline, as a bcatecholaminergic activity enhancer,Q increased the release of dopamine by 30 to 90% in striatum, substantia nigra, and tuberculum olfactorium (Knoll et al., 1996). Anti-immobility effects of selegiline may be through dopamine D1 receptor activation due to increases in dopamine level by bcatecholaminergic activity enhancerQ effects and MAO-B inhibitory effect. Electroconvulsive therapy, MAO inhibitors or an atypical depressant bupropion were applied for refractory depression that was resistance to selective serotonin reuptake inhibitor or serotonin–noradrenaline reuptake inhibitor (Stern et al., 1983; Sackeim et al., 1990; Thase et al., 1995). Elevation of extracellular dopamine level in frontal cortex, striatum and nucleus accumbens is considered as a possible mechanism for the effect of electroconvulsive therapy, MAO inhibitors and bupropion (Nomikos et al., 1989, 1991a,b; Segal et al., 1992; Yoshida et al., 1997, 1998; Curet et al., 1998; Li et al., 2002). As selegiline facilitates the activities of nigrostriatal and mesolimbic dopaminergic system (Knoll, 1987, 1996), it may be useful in the treatment of refractory depression. In conclusion, selegiline exerts the antidepressant-like effects in the FST by prolonging escape-directed behavior, rather than by a generalized motor stimulant effect observed in mice treated with (+)-methamphetamine. The antidepressant-like effects of selegiline are related to the dopamine D1 receptor activation subsequent to elevation of dopamine level. In addition, the effects of selegiline could not be explained solely by a metabolite of selegiline ( )-methamphetamine, or its inhibitory effects of MAO-B. Selegiline could be clinically useful for depression. References Birkmayer, W., Riederer, P., Ambrozi, L., Youdim, M.B.H., 1977. Implications of combined treatment with dMadoparT and l-deprenil in Parkinson’s disease. A long-term study. Lancet 26, 439 – 443. Birkmayer, W., Riederer, P., Linauer, P., Knoll, J., 1984. l-Deprenyl plus l-phenylalanine in the treatment of depression. J. Neural Transm. 59, 81 – 87. Curet, O., Damoiseau-Ovens, G., Sauvage, C., Sontag, N., Avenet, P., Depoortere, H., Caille, D., Bergis, O., Scatton, B., 1998. Preclinical profile of befloxatone, a new reversible MAO-A inhibitor. J. Affect. Disord. 51, 287 – 303. Ferigolo, M., Barros, H.M.T., Marquardt, A.R., Tannhauser, M., 1998. Comparison of behavioral effects of moclobemide and deprenyl during forced swimming. Pharmacol. Biochem. Behav. 60, 431 – 437. Fozard, J.R., Zreika, M., Robin, M., Palfreyman, M.G., 1985. The functional consequences of inhibition of monoamine oxidase type B: comparison of the pharmacological properties of l-deprenyl and MDL 72145, Naunyn. Schmiedebergs. Arch. Pharmacol. 331, 186 – 193.
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S. Shimazu et al. / European Neuropsychopharmacology 15 (2005) 563–571 Onodera, T., Watanabe, R., Tha, K.K., Hayashi, Y., Murayama, T., Okuma, Y., Ono, C., Oketani, Y., Hosokawa, M., Nomura, Y., 2000. Depressive behavior and alterations in receptors for dopamine and 5-hydroxytryptamine in the brain of the senescence accelerated mouse (SAM)-P10. Jpn. J. Pharmacol. 83, 312 – 318. Paterson, I.A., Juorio, A.V., Berry, M.D., Zhu, M.Y., 1991. Inhibition of monoamine oxidase-B by ( )-deprenyl potentiates neuronal responses to dopamine agonists but does not inhibit dopamine catabolism in the rat striatum. J. Pharmacol. Exp. Ther. 258, 1019 – 1026. Porsolt, R.D., Jalfre, M., 1978. Swimming rats and human depression. Nature 274, 512 – 513. Porsolt, R.D., Pichon, M.L., Jalfre, M., 1977. Depression: a new animal model sensitive to antidepressant treatments. Nature 266, 730 – 732. Porsolt, R.D., Anton, G., Blavet, N., Jalfre, M., 1978. Behavioural despair in rats: a new model sensitive to antidepressant treatments. Eur. J. Pharmacol. 47, 379 – 391. Quitkin, F.M., Liebowitz, M.R., Stewart, J.W., McGrath, P.J., Harrison, W., Rabkin, J.G., Markowitz, J., Davies, S.O., 1984. l-Deprenyl in atypical depressives. Arch. Gen. Psychiatry 41, 777 – 781. Ritter, J.L., Alexander, B., 1997. Retrospective study of selegilineantidepressant drug interactions and a review of the literature. Ann. Clin. Psychiatry 9, 7 – 13. Sackeim, H.A., Prudic, J., Devanand, D.P., Decina, P., Kerr, B., Malitz, S., 1990. The impact of medication resistance and continuation pharmacotherapy on relapse following response to electroconvulsive therapy in major depression. J. Clin. Psychopharmacol. 10, 96 – 104. Segal, D.S., Kuczenski, R., Okuda, C., 1992. Clorgyline-induced increases in presynaptic DA: changes in the behavioral and neurochemical effects of amphetamine using in vivo microdialysis. Pharmacol. Biochem. Behav. 42, 421 – 429. Stern, W.C., Harto-Truax, N., Bauer, N., 1983. Efficacy of bupropion in tricyclic-resistant or intolerant patients. J. Clin. Psychiatry 44, 148 – 152.
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Thase, M.E., Trivedi, M.H., Rush, A.J., 1995. MAOIs in the contemporary treatment of depression. Neuropsychopharmacology 12, 185 – 219. Tipton, K.F., Houslay, M.D., Mantle, T.J., 1976. The nature and locations of the multiple forms of monoamine oxidase. In: Bernheim, M.L.C. (Ed.), Monoamine Oxidase and its Inhibition. Elsevier of Amsterdam, NorthHolland, The Netherlands, pp. 5 – 31. Tom, T., Cummings, J.L., 1998. Depression in Parkinson’s disease. Pharmacological characteristics and treatment. Drugs Aging 12, 55 – 74. Varga, E., Tringer, L., 1967. Clinical trial of new type promptly acting psychoenergestic agent (phenyl-isopropyl-methylpropinyl-HCl, bE250Q). Acta Med. Acad. Sci. Hung. 23, 289 – 295. Willner, P., 1984. The validity of animal models of depression. Psychopharmacology 83, 1 – 16. Yoshida, K., Higuchi, H., Kamata, M., Yoshimoto, M., Shimizu, T., Hishikawa, Y., 1997. Dopamine releasing response in rat striatum to single and repeated electroconvulsive shock treatment. Prog. Neuropsychopharmacol. Biol. Psychiatry 21, 707 – 715. Yoshida, K., Higuchi, H., Kamata, M., Yoshimoto, M., Shimizu, T., Hishikawa, Y., 1998. Single and repeated electroconvulsive shocks active dopaminergic and 5-hydroxytryptaminergic neurotransmission in the frontal cortex of rats. Prog. Neuro-psychopharmacol. Biol. Psychiatry 22, 435 – 444. Youdim, M.B., 1980. Monoamine oxidase inhibitors as anti-depressant drugs and as adjunct to l-dopa therapy of Parkinson’s disease. J. Neural Transm., Suppl. 16, 157 – 161. Zebrowska-Lupina, I., Pietrasiewicz, T., Ossowska, G., Lupina, T., KlenkMajewska, B., 1997. ACTH 4-9 analogue facilitates the antiimmobility effect of antidepressants and dopamine agonists in swimming rats. J. Physiol. Pharmacol. 48, 263 – 275.
Antidepressant-like effects of selegiline in the forced swim test Seiichiro ShimazuT,1, Akiko Minami1, Haruko Kusumoto, Fumio Yoneda Research Institute, Fujimoto Pharmaceutical Corporation, 1-3-40 Nishiotsuka, Matsubara, Osaka 580-0011, Japan Received 25 November 2004; received in revised form 10 February 2005; accepted 15 February 2005
Abstract Although selegiline, a monoamine oxidase (MAO)-B inhibitor, is reported to exert antidepressant effects in depressant patients, evidence in rodents for effects of selegiline is quite limited. The purpose of the present study was to assess effects of selegiline in the forced swim test (FST) and on locomotor activity, and to investigate whether MAO inhibition or stimulation of receptors contributes to antidepressant-like effects of selegiline. Drugs were subcutaneously administrated. The single administration of reference drug nortriptyline at 5 mg/kg reduced locomotor activity without effects in FST and brain MAO activities. But nortriptyline repeatedly given 24, 5 and 1 h before behavioral tests significantly decreased an immobility time in FST without effects in motor activities, and showed weak brain MAO-B inhibition. Single and following repeated (24, 5 and 1 h before behavioral tests) administrations of selegiline at 10 mg/kg significantly decreased the immobility time in FST, with little motor stimulant effect. In contrast, (+)-methamphetamine caused a marked decrease in immobility time and an increase in locomotor activity. Selegiline at 1 and 3 mg/kg, which failed to decrease immobility time, markedly inhibited brain total-MAO and MAO-B activities. A dopamine D1 receptor antagonist SCH 23390 completely blocked antidepressant-like effects of selegiline, but not dopamine D2, serotonergic or noradrenergic receptor antagonists. These results suggest that selegiline exerts the antidepressant-like effects by prolonging escape-directed behavior rather than by a motor stimulant effect and D1 receptor activation contributes to its effect. D 2005 Elsevier B.V. and ECNP. All rights reserved. Keywords: Selegiline; Forced swim test; Locomotor activity; Antidepressant; Monoamine oxidase
1. Introduction Selegiline, a selective and irreversible monoamine oxidase (MAO)-B inhibitor, is widely used for the therapy of Parkinson’s disease (PD) (Birkmayer et al., 1977). In addition, several studies have been shown that selegiline has antidepressant effects (Varga and Tringer, 1967; Mann and Gershon, 1980; Birkmayer et al., 1984). In a study by Birkmayer et al. (1984), about 70% of depressed patients improved following the administration of selegiline with ( )-phenylalanine, the precursor of phenylethylamine (PEA). Selegiline at high doses (40–60 mg/day) exerts marked antidepressant effect (Lees, 1991; Kuhn and Mu¨ller, 1996; Ritter and Alexander, 1997). Also, selegiline improves atypical depression, major depression and depresT Corresponding author. Tel.: +81 723 32 5151; fax: +81 723 32 8482. E-mail address: [email protected] (S. Shimazu). 1 These authors contributed equally to this work.
sion in PD (Youdim, 1980; Mendlewicz and Youdim, 1983; Quitkin et al., 1984; McGrath et al., 1989; Mayberg and Solomon, 1995; Tom and Cummings, 1998). However, there are a few reports about the antidepressant-like effects of selegiline using animal models, such as behavioral despair, learned helplessness and chronic mild stress (Porsolt and Jalfre, 1978; Willner, 1984). Several laboratories have demonstrated that selegiline decreases immobility time in the forced swim test (FST), a widely used model of depression (Porsolt et al., 1977, Zebrowska-Lupina et al., 1997, Ferigolo et al., 1998, Nowakowska et al., 2001). Furthermore, Fozard et al. (1985) suggested that antidepressant-like effect of selegiline was unrelated to MAO-B inhibition. Thus, antidepressant-like effect of selegiline and its mechanism are unclear. The aim of the present study is to evaluate antidepressant-like effects of selegiline in FST and locomotor activity, and to investigate whether antidepressant-like effects of selegiline involve its MAO inhibitory action and exerted through monoaminergic receptors.
0924-977X/$ - see front matter D 2005 Elsevier B.V. and ECNP. All rights reserved. doi:10.1016/j.euroneuro.2005.02.003
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2. Materials and methods 2.1. Animals Male ddY mice (8 weeks old, Nihon SLC, Shizuoka, Japan) were maintained in a humidity (55 F 10%)- and temperature (23 F 2 8C)-controlled facility under a 12/12-h light/dark cycle (light on at 7:00 a.m.) with free access to food (MF, Oriental Yeast, Tokyo, Japan) and water. Mice were allowed to adapt to the environments for 7 days before testing. The following animal studies were performed according to the bGuiding Principles for the Care and Use of Laboratory AnimalsQ approved by The Japanese Pharmacological Society. 2.2. Chemicals Following compounds were used: selegiline hydrochloride, ( )-methamphetamine hydrochloride (Fujimoto Pharmaceutical, Osaka, Japan), (+)-methamphetamine hydrochloride (Dainippon Pharmaceutical, Osaka, Japan), yohimbine hydrochloride (Nacalai tesque, Kyoto, Japan), S-( )-eticlopride hydrochloride (Research Biochemicals International, Natick, MA, USA), nortriptyline hydrochloride, R-(+)-7-chloro-8-hydroxy-3-methyl-1-phenyl-2,3,4,5tetrahydro-1H-3-benzazepine hydrochloride (SCH 23390), prazosin hydrochloride, propranolol hydrochloride, ketanserin tartrate, N-(2-(4-(2-methoxyphenyl)-1-piperazinyl)ethyl)-N-(2-pyridinyl) cyclohexanecarboxamide maleate
(WAY 100,635) (Sigma, St. Louis, MO, USA). The doses of ketanserin and WAY 100,635 were expressed as those of free base, and the doses of other drugs were expressed as those of hydrochloride salt. All drugs were dissolved in saline and administered subcutaneously (s.c.) in a constant volume of 10 ml/kg. Control animals were injected with saline. 2.3. Locomotor activity Locomotor activity was assessed using an infrared (Model NS-AS01)-linked activity sensor system (Model AB system-24A, Neuroscience, Tokyo, Japan). Counts of motor activity were recorded on a computer system during consecutive 5-min intervals. The motor activity during the 2-h observation period was measured starting 1 h after drug treatment. 2.4. FST FST was performed as described by Porsolt et al. (1977). Briefly, mice were placed 1 h after drug treatment in black Plexiglas cylinder (45 cm height 15 cm diameter, NSM507M, Neuroscience) filled with water at 25 8C at a depth of 35 cm. Immediately after placing the animals, immobility time (s) and active path (a distance of swimming in centimeters) were measured for 6 min by a CCD-camera linking to a computer system (Video Image Motion Analyzer System AXIS-60, Neuroscience), and were used for drug evaluation during the last 4 min.
Fig. 1. Effects of a single administration of selegiline on FST and locomotor activity in mice. Panels (A) and (B) show the immobility time and active path in the FST, respectively. The duration of immobility time and active path were recorded during the last 4 min of the 6-min test period. Panel (C) shows the cumulative locomotor activities for 2 h of action of compounds, respectively. Selegiline (1–10 mg/kg), nortriptyline (5 mg/kg) and saline were subcutaneously given 1 h before the behavioral test. Each value represents as mean F S.E.M. (n = 16, 8, 16, 16 and 16 for saline, nortriptyline, 1, 3 and 10 mg/kg selegilinetreated groups, respectively). *P b 0.05, **P b 0.01 and ***P b 0.001 vs. saline-treated control.
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potassium phosphate containing 1% EDTA (pH 7.5). The total-MAO and MAO-B activities were determined with Amplex Red Monoamine Oxidase assay Kit (Molecular Probes, Eugene, OR, USA). The protein contents were measured using Lowry’s method (Lowry et al., 1951). 2.6. Statistics
Fig. 2. Effects of a single administration of selegiline on time course changes in locomotor activity in mice. Data are presented as the averages of locomotion taken at each 5-min interval, over a period of 2 h. S.E.M. are not shown for the sake of clarity. Selegiline (1–10 mg/kg), nortriptyline (5 mg/ kg) and saline were subcutaneously given 1 h before the behavioral test (n = 16, 8, 16, 16 and 16 for saline, nortriptyline, 1, 3 and 10 mg/kg selegilinetreated groups, respectively). *P b 0.05, **P b 0.01 and ***P b 0.001 vs. saline-treated control.
Statistical analyses for FST and MAO activities were performed with the Kruskal–Wallis’ analysis of variance followed by post hoc Wilcoxon’s test (SAS preclinical package ver 5.0, SAS Institute, Cary, NC, USA). For time course data, statistical analyses were performed with twoway ANOVA followed by post hoc Dunnett’s test. The differences were considered significant at P b 0.05.
3. Results 3.1. Effect of a single injection of selegiline
2.5. Measurement of MAO activities Mice were sacrificed by cervical dislocation 1 h after FST and whole brain were isolated. Isolated whole brains were homogenized in 10 volume of ice-cold 0.2 M potassium phosphate containing 1% EDTA (pH 7.5). Homogenates were centrifuged at 900 g for 10 min at 4 8C, and thereafter the resulting supernatant was recentrifuged at 11,500 g for 20 min at 4 8C. The precipitations were resuspended in 0.2 M
A single treatment of selegiline significantly reduced the immobility time and increased the active path at the dose of 10 mg/kg, but not at 1 and 3 mg/kg (Kruskal–Wallis’ test, immobility time: df = 4, P = 0.0151; active path: df = 4, P = 0.0099) (Fig. 1A and B). However, a selective noradrenaline reuptake inhibitor nortriptyline (5 mg/kg), as a reference drug, had no effect on the immobility time and the active path in the FST.
Fig. 3. Effects of repeated administrations of selegiline on FST and locomotor activity in mice. Panels (A) and (B) show the immobility time and active path in FST, respectively. The duration of immobility time and active path were recorded during the last 4 min of the 6-min test period. Panel (C) shows the cumulative locomotor activities for 2 h of action of compounds, respectively. Selegiline (1–10 mg/kg), nortriptyline (5 mg/kg) and saline were subcutaneously given 24, 5 and 1 h before the behavioral test. Each value represents as mean F S.E.M. (n = 16 for FST and n = 8 for locomotor activities). *P b 0.05 and *P b 0.01 vs. saline-treated control.
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ities in almost measuring points of the initial phase until 40 min (Fig. 2). Also, selegiline at 1 and 10 mg/kg caused significant increases in locomotor activities at several measuring points (1 mg/kg: 110–115 min, 10 mg/kg: 60–65, 75–80, 80–85 and 100–105 min). 3.2. Repeated injections of selegiline
Fig. 4. Effects of repeated administrations of selegiline on time course changes in locomotor activity in mice. Data are presented as the averages of locomotion taken at each 5-min interval, over a period of 2 h. S.E.M. are not shown for the sake of clarity. Selegiline (1–10 mg/kg), nortriptyline (5 mg/kg) and saline were subcutaneously given 24, 5 and 1 h before the behavioral test (n = 8). *P b 0.05 and **P b 0.01 vs. saline-treated control.
Selegiline had no effect on cumulative locomotor activity for 2 h, but nortriptyline significantly decreased cumulative locomotor activity (Kruskal–Wallis’ test, df = 4, P = 0.0013) (Fig. 1C). Fig. 2 showed time course changes of locomotor activities. Two-way ANOVA analyses on the data of time course changes resulted in F = 15.56, df = 4, P b 0.0001 (Fig. 2). The animals injected with vehicle displayed locomotion related to active exploration during the initial phase that was usually followed by a decline in activity due to habituation. Nortriptyline and 10 mg/kg selegiline groups significantly decreased locomotor activ-
The three times injections (24, 5 and 1 h before behavioral test) of selegiline resulted in a significant decrease in the immobility time and an increase in the active path at the dose of 10 mg/kg, but not at 1 and 3 mg/kg (Kruskal–Wallis3 test, immobility time: df = 4, P = 0.0086, active path: df = 4, P = 0.0042) (Fig. 3A and B). Nortriptyline significantly reduced the immobility time and increased the active path. However, neither selegiline nor nortriptyline affected the cumulative locomotor activity (Kruskal–Wallis3 test, df = 4, P = 0.1052) (Fig. 3C). Two-way ANOVA analyses on the time course data resulted in F = 5.68, df = 4, P = 0.0002 (Fig. 4). Locomotor activities of 10 mg/kg selegiline group significantly decreased in the initial phase (at 5–10, and 10–15 min measuring points), and significantly increased at points of 35–40 and 50–55 min. 3.3. Effect of a single injection of methamphetamine To determine whether the antidepressant-like effects of selegiline are mediated by ( )-methamphetamine that is a major metabolite of selegiline, we investigated effects of ( )-
Fig. 5. Effects of a single administration of methamphetamine on FST in mice. Panels (A) and (C), and panels (B) and (D) show effects of ( )methamphetamine and (+)-methamphetamine, respectively. The duration of immobility time (A and B) and active path (C and D) were recorded during the last 4 min of the 6-min test period. ( )-Methamphetamine (1–10 mg/kg), (+)-methamphetamine (1–10 mg/kg) and saline were subcutaneously given 1 h before the behavioral test. Each value represents as mean F S.E.M. (n = 12 for panels (A) and (C), and n = 8 for panels (B) and (D)). **P b 0.01 and ***P b 0.001 vs. saline-treated control.
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methamphetamine on the FST and on the locomotor activity. ( )-Methamphetamine (1–10 mg/kg) decreased the immobility time and increased the active path in a dose-dependent manner (Kruskal–Wallis’ test, immobility time: df = 3, P = 0.0045; active path: df = 3, P = 0.0005) (Fig. 5A and C). However, ( )-methamphetamine did not affect the locomotor activity at any doses (two-way ANOVA: F = 1.44, df = 3, P = 0.2294) (Fig. 6A). In addition, we evaluated effects of (+)methamphetamine, which is an active enantiomer and psychostimulant. (+)-Methamphetamine (1–10 mg/kg) reduced the immobility time and increased the active path (Kruskal–Wallis’ test, immobility time: df = 3, P b 0.0001; active path: df = 3, P b 0.0001) (Fig. 5B and D). (+)Methamphetamine at 1 and 3 mg/kg markedly increased the locomotor activity, and showed long-lasting enhancement of locomotor activity until 2 h (two-way ANOVA, F = 171.06,
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df = 3, P b 0.0001) (Fig. 6B). In contrast, 10 mg/kg (+)methamphetamine significantly decreased locomotor activities in the initial phase until 25 min, and significantly increased at several measuring points after 55 min. Two-way ANOVA at saline vs. 10 mg/kg (+)-methamphetamine did not show a significance ( F = 0.17, df = 1, P = 0.6782). Lower doses (0.03–0.3 mg/kg) of (+)-methamphetamine did not affect the immobility time, the active path and the locomotor activity (data not shown). 3.4. Effects of several receptor antagonists against selegiline-induced antidepressant-like effects Next, we examined the effects of selective receptor antagonists on the selegiline-induced behavioral effects. The treatment of SCH 23390 completely inhibited selegiline-
Fig. 6. Effects of a single administration of methamphetamine on time course changes in locomotor activity in mice. Panels (A) and (B) show effects of ( )methamphetamine and (+)-methamphetamine, respectively. ( )-Methamphetamine (1–10 mg/kg), (+)-methamphetamine (1-10 mg/kg) and saline were subcutaneously given 1 h before the behavioral test. Data are presented as the averages of locomotion taken at each 5–min interval, over a period of 2 h. S.E.M. are not shown for the sake of clarity (n = 8). *P b 0.05, **P b 0.01 and ***P b 0.001 vs. saline-treated control.
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induced decrease in immobility time and increase in active path (Table 1). In contrast, a dopamine D2 receptor antagonist eticlopride, a 5-HT1A receptor antagonist WAY 100,635, a 5-HT2A receptor antagonist ketanserin, an a1 adrenergic receptor antagonist prazosin, an a2 adrenergic receptor antagonist yohimbine and a h receptor antagonist propranolol did not influence on antidepressant-like effects of selegiline. 3.5. Effects of selegiline on MAO activity To determine if MAO inhibition contributes to antidepressant-like effects of selegiline, we measured the totalMAO and MAO-B activities in whole brain 1 h after behavioral tests. Selegiline at all doses had same inhibitory effects on MAO-B activity in single (Kruskal–Wallis’ test, total-MAO: df = 4, P b 0.0001; MAO-B: df = 4, P = 0.0001) and repeated injections (Kruskal–Wallis’ test, total-MAO: df = 4, P = 0.0002; MAO-B: df = 4, P = 0.0008) (Fig. 7). The repeated injections of nortriptyline significantly decreased MAO-B activity by 18%, but the single injection had no inhibitory effects.
4. Discussion The present study demonstrated that selegiline reduced immobility time and increased active path in single or following repeated injections. The effects of a single dose of selegiline confirm the finding of the previous study in rats reported by Zebrowska-Lupina et al. (1997). In the present study, 10 mg/kg selegiline caused decreases in motor activities in the initial phase of active exploration, and weak increases in activities in the phase after initial exploration periods, but did not affect cumulative motor activities. Thus, the analyses at detail revealed that selegiline hardly stimulated motor activities, comparing with a psychostimulant like (+)-methamphetamine (Figs. 2, 4, and 6). It is noteworthy that selegiline, at a single dose which lacked stimulant effects, had an antidepressant effect, indicating that its effect is not merely due to a stimulation of motor activity. On the other hand, repeated administration of a selective noradrenaline reuptake inhibitor nortriptyline at 5 mg/kg caused a reduction in immobility time and an increase in active path, but the single administration was ineffective.
Table 1 The effects of receptor antagonists on selegiline-induced behavioral effects in FST in mice Drug
FST Immobility (s)
Sal Sel Sal Sel Sal Sel Sal Sel Sal Sel Sal Sel Sal Sel Sal Sel Sal Sel Sal Sel Sal Sel Sal Sel Sal Sel Sal Sel
Sal Sal SCH SCH Sal Sal Eti Eti Sal Sal WAY WAY Sal Sal Ket Ket Sal Sal Pra Pra Sal Sal Yoh Yoh Sal Sal Pro Pro
167.2 F 9.0 115.0 F 10.5*** 179.6 F 9.8 160.3 F 7.7## 181.9 F 10.7 105.9 F 26.9* 134.1 F 22.7 86.1 F 22.7** 162.9 F 11.7 80.8 F 15.1*** 148.2 F 17.0 97.1 F13.0** 119.1 F16.4 54.9 F 15.5* 135.9 F 11.9 58.4 F 17.2* 118.5 F 13.6 64.9 F 13.6** 144.2 F 12.6 76.0 F 14.2* 137.8 F 15.7 80.3 F 12.9* 113.8 F 10.6 76.8 F 11.1** 154.1 F18.8 76.2 F 21.1* 127.2 F 26.4 84.7 F 12.5*
Active path (cm) a
df = 3, P b 0.0001
df = 3, P = 0.0207
df = 3, P = 0.0017
df = 3, P = 0.0019
df = 3, P = 0.0005
df = 3, P = 0.0082
df = 3, P = 0.0363
543.0 F 70.6 1032.6 F 106.6*** 476.2 F 87.7 685.0 F 67.8# 272.6 F 49.5 740.6 F 176.2* 468.3 F 129.7 964.7 F 197.1** 341.4 F 52.7 925.3 F 109.9*** 384.1 F 71.7 795.7 F 105.2*** 646.2 F 124.2 1147.9 F 127.2* 597.7 F 77.5 1256.8 F 186.0* 564.4 F 73.4 994.2 F 102.4** 589.0 F 97.8 1178.9 F 159.6** 497.2 F 83.7 934.8 F 99.9** 533.2 F 45.3 726.8 F 51.7* 398.0 F 89.4 977.9 F 196.4* 562.8 F 166.8 825.7 F 83.1*
df = 3, P b 0.0001
df = 3, P = 0.0062
df = 3, P b 0.0001
df = 3, P = 0.0011
df = 3, P = 0.0005
df = 3, P = 0.0017
df = 3, P = 0.0349
Selegiline (Sel; 10 mg/kg) or saline (Sal) were given 1 h before the test. SCH 23390 (SCH; 0.03 mg/kg), eticlopride (Eti; 0.01 mg/kg), WAY 100,635 (WAY; 0.1 mg/kg), ketanserin (Ket; 5 mg/kg), prazosin (Pra; 2 mg/kg), yohimbine (Yoh; 5 mg/kg) and propranolol (Pro; 5 mg/kg) were administered simultaneously with selegiline or saline. Each value represents as mean F S.E.M. (n = 7–28). Post hoc Wilcoxon’s test: *P b 0.05, **P b 0.01 and ***P b 0.001 vs. saline-treated group. #P b 0.05 and ##P b 0.01 vs. selegiline-treated group. a Kruskal–Wallis’ test.
S. Shimazu et al. / European Neuropsychopharmacology 15 (2005) 563–571
569
Fig. 7. Effects of selegiline on MAO activities in whole brains. Panels (A) and (B) show total-MAO and MAO-B inhibition 2 h after a single injection of selegiline (1–10 mg/kg), nortriptyline (5 mg/kg) or saline, respectively. Panels (C) and (D) show total-MAO and MAO-B inhibition after repeated injections (3 times) of drugs, respectively. Selegiline, nortriptyline or saline was given 25, 6, and 2 h before the decapitation. MAO activities were measured using fluorometric assay. Each value represents as mean F S.E.M. (n = 10 for single saline injection group and n = 5 for other groups). **P b 0.01 and ***P b 0.001 vs. saline-treated control.
Onodera et al. (2000) demonstrated that another selective noradrenaline reuptake inhibitor desipramine had no effect in the FST in a single injection, but decreased immobility time in repeated administration. These phenomena suggest that the antidepressant effects cannot be explained solely by the change in monoaminergic tone (Nestler et al., 2002) and may involve adaptive changes in intracellular signal transduction and synaptic connectivity (Manji et al., 2001; Nestler et al., 2002). Porsolt et al. (1978) reported that antidepressants reduce open field activities. In the present study, a single treatment of nortriptyline showed a significant decrease in locomotor activities in initial phase of active exploration, though repeated administrations of nortriptyline showed no effect. Whereas, single and repeated treatments of 10 mg/kg selegiline significantly reduced motor activities in initial phase of active exploration, which may suggests its antianxious effect to unfamiliar environment. Selegiline orally treated is mainly metabolized by the liver cytochrome P-450 system and the metabolites identified are desmethylselegiline, ( )-methamphetamine and ( )-amphetamine in rats (Heinonen et al., 1994). The ratio of l-( )-methamphetamine, which is metabolized from selegiline in in vitro liver microsomal system of mice, are similar to that of rats (Levai et al., 2004). In the present study, 10 mg/kg s.c. ( )-methamphetamine, but not 1 and 3 mg/kg s.c., resulted in a significant decrease in immobility time and an increase in active path, without affecting locomotor activity. Using rats, Melega et al. (1999) have shown that the area under the curve of metabolically
generated ( )-methamphetamine after administration of 10 mg/kg s.c. selegiline was approximately 15% of that observed after administration of 2.5 mg/kg s.c. ( )methamphetamine, in rats. Taken together, ( )-methamphetamine generated after the administration of 10 mg/kg s.c. selegiline might not play an important role in the antidepressant-like effects of selegiline. Furthermore, a psychostimulant (+)-methamphetamine resulted in a marked decrease in immobility time and an increase in locomotor activity. The reduction of immobility induced by (+)methamphetamine seems to be caused by increase in general motor activity (Kitada et al., 1981). Thus, effects of selegiline and its metabolite ( )-methamphetamine on the immobility time could be clearly distinguished from those of psychostimulants such as (+)-methamphetamine. In the present study, selegiline inhibited total-MAO and MAO-B activities. Fozard et al. (1985) showed that a MAOB inhibitor [(E)-2-(3,4-dimethoxyphenyl)-3-fluoroallylamine hydrochloride, MDL 72145] failed to decrease immobility time, although its inhibitory effect of MAO-B is equivalent to that of selegiline. Gordon et al. (1999) suggested that antidepressant-like effects of selegiline were mediated by MAO-A inhibition rather than MAO-B inhibition. It has been reported that selegiline at 8 mg/kg i.p. and 10 mg/kg s.c. inhibited 20% MAO-A activities in rat striatum, and about a half of MAO-A activities in rat brains, respectively (Paterson et al., 1991, Tipton et al., 1976). The inhibition in MAO-A activities by the administration of 10 mg/kg s.c. selegiline may be contribute to antidepressant-like effects of selegiline. Repeated treatment
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of nortriptyline caused a significant inhibition of 18% in MAO-B activities, which may not contribute to its antidepressant effect. Moreover, the antidepressant-like effects of selegiline were completely inhibited by a dopamine D1 receptor antagonist SCH 23390. Nikulina et al. (1991) demonstrated a dopamine D1 receptor agonist SKF 38393 increased swimming activity and antagonist SCH 23390 reduced. Selegiline, as a bcatecholaminergic activity enhancer,Q increased the release of dopamine by 30 to 90% in striatum, substantia nigra, and tuberculum olfactorium (Knoll et al., 1996). Anti-immobility effects of selegiline may be through dopamine D1 receptor activation due to increases in dopamine level by bcatecholaminergic activity enhancerQ effects and MAO-B inhibitory effect. Electroconvulsive therapy, MAO inhibitors or an atypical depressant bupropion were applied for refractory depression that was resistance to selective serotonin reuptake inhibitor or serotonin–noradrenaline reuptake inhibitor (Stern et al., 1983; Sackeim et al., 1990; Thase et al., 1995). Elevation of extracellular dopamine level in frontal cortex, striatum and nucleus accumbens is considered as a possible mechanism for the effect of electroconvulsive therapy, MAO inhibitors and bupropion (Nomikos et al., 1989, 1991a,b; Segal et al., 1992; Yoshida et al., 1997, 1998; Curet et al., 1998; Li et al., 2002). As selegiline facilitates the activities of nigrostriatal and mesolimbic dopaminergic system (Knoll, 1987, 1996), it may be useful in the treatment of refractory depression. In conclusion, selegiline exerts the antidepressant-like effects in the FST by prolonging escape-directed behavior, rather than by a generalized motor stimulant effect observed in mice treated with (+)-methamphetamine. The antidepressant-like effects of selegiline are related to the dopamine D1 receptor activation subsequent to elevation of dopamine level. In addition, the effects of selegiline could not be explained solely by a metabolite of selegiline ( )-methamphetamine, or its inhibitory effects of MAO-B. Selegiline could be clinically useful for depression. References Birkmayer, W., Riederer, P., Ambrozi, L., Youdim, M.B.H., 1977. Implications of combined treatment with dMadoparT and l-deprenil in Parkinson’s disease. A long-term study. Lancet 26, 439 – 443. Birkmayer, W., Riederer, P., Linauer, P., Knoll, J., 1984. l-Deprenyl plus l-phenylalanine in the treatment of depression. J. Neural Transm. 59, 81 – 87. Curet, O., Damoiseau-Ovens, G., Sauvage, C., Sontag, N., Avenet, P., Depoortere, H., Caille, D., Bergis, O., Scatton, B., 1998. Preclinical profile of befloxatone, a new reversible MAO-A inhibitor. J. Affect. Disord. 51, 287 – 303. Ferigolo, M., Barros, H.M.T., Marquardt, A.R., Tannhauser, M., 1998. Comparison of behavioral effects of moclobemide and deprenyl during forced swimming. Pharmacol. Biochem. Behav. 60, 431 – 437. Fozard, J.R., Zreika, M., Robin, M., Palfreyman, M.G., 1985. The functional consequences of inhibition of monoamine oxidase type B: comparison of the pharmacological properties of l-deprenyl and MDL 72145, Naunyn. Schmiedebergs. Arch. Pharmacol. 331, 186 – 193.
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