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Effects of acute fluoxetine, paroxetine and desipramine on rats tested on the elevated plus-maze
Behavioural Brain Research 176 (2007) 202–209
Research report
Effects of acute fluoxetine, paroxetine and desipramine on rats tested on the elevated plus-maze Dominique Drapier a , Dani`ele Bentu´e-Ferrer b,c , Bruno Laviolle d , Bruno Millet a , Herv´e Allain b,c , Michel Bourin c,∗ , Jean-Michel Reymann b,c a
Service Hospitalo-Universitaire de Psychiatrie, Centre Hospitalier Guillaume R´egnier, 108 avenue du G´en´eral Leclerc, 35703 Rennes Cedex, France b Laboratoire de Pharmacologie Exp´ erimentale et Clinique, Facult´e de M´edecine, CS 34317, 35043 Rennes Cedex, France c EA 3256 Neurobiologie de l’anxi´ et´e et de la d´epression, Laboratoire de Pharmacologie, Facult´e de M´edecine, BP 53508, 1 rue Gaston Veil, 44035 Nantes Cedex, France d Centre d’Investigation Clinique INSERM 0203, CHU Pontchaillou, 35033 Rennes, France Received 16 May 2006; received in revised form 28 September 2006; accepted 2 October 2006 Available online 13 November 2006
Abstract Antidepressants are usually prescribed for the treatment of depression but more recently have also been recommended for the treatment of anxiety disorders. The purpose of this study was to investigate the anxiogenic- or anxiolytic-like effects of an acute administration of antidepressants (serotonergic and noradrenergic compounds) in male Wistar rats submitted to the elevated plus-maze. Fluoxetine (2.5, 5, 10, 15 mg/kg), paroxetine (0.1, 0.5, 3, 12 mg/kg) and desipramine (2.5, 5, 10 mg/kg) or their vehicles were administered intraperitoneally 30 min prior to testing. Diazepam (0.5, 1.5, 2.5 mg/kg) was used as a positive comparator for anxiolytic effect. In comparison with control animals, the percentage of time the rats treated with fluoxetine (5 and 10 mg/kg) and paroxetine (3 and 12 mg/kg) spent in the open arms decreased. The percent of inactive time spent in the open arms also decreased in rats given fluoxetine (5 and 10 mg/kg) and paroxetine (12 mg/kg). Desipramine was inactive on all these parameters. In conclusion, acute treatment with fluoxetine and paroxetine, but not with desipramine, produced a pattern of anxiety behavior. Thus, the pharmacological mechanism appears to be due more to serotonergic than adrenergic neurotransmission. The elevated plus-maze exhibits good sensitivity for detecting anxiogenic effects of antidepressant drugs and the conventional parameters are sufficient and reliable for detecting such effects. © 2006 Elsevier B.V. All rights reserved. Keywords: Antidepressants; Anxiety; Rats; Plus-maze; Ethological parameters
1. Introduction Benzodiazepines (BZDs) have been the mainstay of pharmacological treatment of anxiety over the last four decades despite a high incidence of side effects. Tolerance, physical dependence and withdrawal symptoms, as well as sedation and risk of abuse can raise significant problems [45]. Antidepressants are usually prescribed for the treatment of depression, but more recently these compounds, in particular drugs belonging to the class of selective serotonin re-uptake inhibitors (SSRIs), were also recommended for the treatment of anxiety in adults and children [1,35]. Antidepressants are useful for the treatment of gener-
∗
Corresponding author. Tel.: +33 2 40 41 28 52; fax: +33 2 40 41 28 56. E-mail address: [email protected] (M. Bourin).
0166-4328/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.bbr.2006.10.002
alized anxiety disorders (GAD) [3,28,43] and panic disorders (PD) [13], but also for social anxiety disorder (SAD) [7], obsessive compulsive disorders (OCD) [18] and post-traumatic stress disorder (PTSD) [6,15], where BZDs fail to provide adequate symptom relief. In certain cases, SSRIs have been approved for these indications and may be more appropriate than BZDs for the treatment of anxiety disorders. On the other hand, it has been frequently reported that the initial effect of acute administration of serotonergic compounds in humans is increased anxiety [17,44]. Numerous animal studies have aimed at better understanding the neurobiological mechanisms implied in the etiology of anxiety [19,33]. It has been hypothesized that increased 5HT neurotransmission is associated with anxiogenesis, whereas a decrease could induce anxiolysis [2,12]. The role of 5-HT in anxiety is undoubtedly complex and may be dependent on a number of factors including 5-HT neurotransmission in multiple
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brain regions, the type of behavioral paradigms used to assess anxiety, and/or the emotional and cognitive contexts of the tests [22–24,31,40]. Several microdialysis studies have demonstrated that a single intraperitoneal (IP) injection of SSRIs increased the extracellular concentration of 5-HT: for example, paroxetine and citalopram in mice cortex [14]; paroxetine, citalopram and fluoxetine in rat diencephalon and frontal cortex [16]; citalopram and fluoxetine in rat hippocampus [8]. However, the data obtained in animal studies using antidepressants is contradictory and it still remains unclear whether these SSRIs truly display an anxiogenic or an anxiolytic effect or, to the contrary, have no effect as concluded by Borsini et al. [10], who analyzed 107 articles dealing with the effects of antidepressant treatment in animal models of anxiety. Moreover, little is known about pharmacological mechanism underlying the possible anxiolytic effect of SSRIs. Several factors could explain these inconsistent findings. It is noteworthy for example that the experimental procedures generally used to assess drug effects were developed and optimized initially for evaluating BZDs [4]. As a consequence, the predictive validity of the elevated plus-maze (EPM) might well be limited to BZD-related drugs. The aim of the present study was to compare under identical experimental conditions the effects of an acute administration of two serotonergic compounds, fluoxetine and paroxetine, and one noradrenergic compound, desipramine, by means of the same behavioral test widely validated for analyses of anxiety-like behavior [5,36], the elevated plus-maze (EPM). Diazepam was used as a positive comparator for anxiolytic effect. 2. Materials and methods 2.1. Animals Adult male Wistar rats (CERJ) weighing 200–300 g were kept in an approved animal house (authorization no. A 35006) at 22 ± 2 ◦ C with a 12-h light/dark cycle (light turned on at 0800 h). Tap water and food pellets were available ad libitum. The animals were housed in groups of four in polycarbonate cages (44 cm × 30 cm × 18 cm). During the 15 days previous the test, the rats were handled only for the changes of litter (three times a week) and to be weighed the day before the experimentation. The care provided to the animals, before, during and after the protocol was in compliance with the European Communities Council Directive. Researchers were authorized to conduct animal studies by the Veterinary Department of the French Ministry of Health (authorization no. 35-01).
2.2. Drugs, treatments and study protocol Tween-80, diazepam, fluoxetine and desipramine were provided by Sigma® (Sigma–Aldrich, 38297 St. Quentin Fallavier, France). Paroxetine was a gift of SmithKlineBeecham laboratory. Paroxetine and fluoxetine were dissolved in a saline solution. Desipramine and diazepam were suspended in a 1% Tween-80 saline solution. Thirty minutes prior to testing, the animals were given, after randomization, an intraperitoneal injection (5 ml/kg) of the relevant drug or vehicle. After the injection, the rats were put back into their home cage. All injections were administered between 0830 and 1030 h. Experiments were conducted blind with respect to treatment. Selected doses reflected usual doses point out in the literature for this kind of experiments, mentioned in particular in the review of Borsini et al. [10].
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Doses and number of animals by group were as follows: • Diazepam: 0.5 mg/kg, n = 12; 1.5 mg/kg, n = 11; 2.5 mg/kg, n = 11. The control group (n = 11) received a 1% Tween-80 saline solution, under the same conditions. • Fluoxetine: 2.5 mg/kg, n = 9; 5 mg/kg, n = 19; 10 mg/kg, n = 19; 15 mg/kg, n = 9. The control group (n = 18) received the saline solution, under the same conditions. • Paroxetine: 0.1 mg/kg, n = 12; 0.5 mg/kg, n = 12; 3 mg/kg, n = 13; 12 mg/kg, n = 13. The control group (n = 21) received the saline solution, under the same conditions. • Desipramine: 2.5 mg/kg, n = 15; 5 mg/kg, n = 15; 10 mg/kg, n = 14. The control group (n = 17 received a 1% Tween-80 saline solution, under the same conditions.
2.3. Elevated plus-maze and general procedure The EPM was made of wood and painted black. The apparatus consisted of two opposite open arms (45 cm × 10 cm) without sidewalls and two enclosed arms (45 cm × 10 cm × 30 cm) with sides and end walls, extending from a central square (10 cm × 10 cm). The maze was elevated to the height of 60 cm above the floor and placed in a moderately lighted room (30 lx as measured at the center of the maze). At the onset of test, the handling naive animals were placed at the center of the EPM, facing towards an open arm and their behavior was recorded during 5 min. The Video-Track® system, used for recording the animal’s behavior, was composed of a roof-hand-held camera connected to a computer. This apparatus allowed the measurement of activity (i.e. movement time spent) or inactivity (time spent at rest, in an observation mode or grooming), time and distances covered in each part of the maze during a 5-min period of time. The following parameters were analyzed in our study: total distance covered, percentage of distance and total time spent in each of the open arms, percentage of inactive time spent in the open arms compared to total inactive time spent in the EPM as a whole. Each rat’s behavior was also recorded on videotape and analyzed by a person not involved in the experimental procedure. The time devoted to the following ethological parameters were then recorded: risk assessment (RA), the rat exiting an enclosed arm with the forepaws and head only and investigating the surroundings, not necessarily accompanied by body stretching; protected head dips (PHD), the rat stretching to dip its head into the open space and observing the environment, the rear part of the body remaining in a closed arm with the forepaws in the center of the EPM; non-protected head dips (NPHD), the rat dipping its head into the open space and observing the environment, the body being in an open arm. The EPM was cleaned between experiments with a 5% alcohol solution.
2.4. Statistical analysis Statistical analysis was performed with SAS statistical software, version 9.1 (SAS Institute, Cary, NC). Data are presented as mean with the corresponding standard error of the mean (m ± S.E.M.). Comparisons between groups were performed using an ANOVA or the Kruskal–Wallis (KW) test if needed. The corresponding F or KW statistics are indicated. In case of significant differences between groups, each treatment dose was compared to the corresponding control group by use of the Dunnett’s two-tailed t-test to control the overall rate of type I error in multiple testing. For all analyses, a p-value < 0.05 was considered to be significant.
3. Results 3.1. Video-track analysis 3.1.1. Diazepam effects The total distance covered by the treated rats during the 5-min test was not significantly different from controls (Fig. 1a).
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cantly increased in animals given diazepam 2.5 mg/kg (+113%) (Fig. 1d). 3.1.2. Fluoxetine effects The total distance covered by the rats during the 5-min test was significantly different between groups (KW = 12.54; p = 0.0137). The distance decreased with increasing doses and the difference became statistically significant with the highest dose (15 mg/kg; −33%) (Fig. 2a). The percentage of distance covered in the open arms tended to be significantly different between groups (KW = 9.28;
Fig. 1. Effects of acute diazepam, 0.5 mg/kg (n = 12), 1.5 mg/kg (n = 11), 2.5 mg/kg (n = 11) administered IP, 30 min before the test, on rats tested in the elevated plus-maze (mean ± S.E.M.). The control group (n = 11) received a 1% Tween-80 in saline solution, under the same conditions. (a) Total distance, (b) percentage of distance covered in the open arms, (c) total time spent in the open arms, and (d) percentage of inactivity time spent in the open arms (* p < 0.05).
The percentage of distance covered in the open arms tended to be significantly different between groups (F = 2.69; p = 0.0585); the percentage of distance in the open arms in the group given diazepam 2.5 mg/kg was higher (89%) than control group (Fig. 1b). The total time spent in the open arms showed a significant difference between groups (F = 3.18; p = 0.0339). The total time spent in the open arms was longer in all treated groups than in the control group but the difference was only significant in the group given diazepam 2.5 mg/kg (+98%) (Fig. 1c). The percentage of inactive time spent in the open arms showed a significant difference between groups (F = 3.27; p = 0.0308). Inactivity time in the open arms was signifi-
Fig. 2. Effects of acute fluoxetine, 2.5 mg/kg (n = 9), 5 mg/kg (n = 19), 10 mg/kg (n = 19), 15 mg/kg (n = 9) administered IP, 30 min before the test, on rats tested in the elevated plus-maze (mean ± S.E.M.). The control group (n = 18) received a saline solution, under the same conditions. (a) Total distance, (b) percentage of distance covered in the open arms, (c) total time spent in the open arms, and (d) percentage of inactivity time spent in the open arms (* p < 0.05).
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p = 0.0545); the percentage of distance in the open arms in the group given fluoxetine (5 mg/kg) was lower (−45%) (Fig. 2b). The total time spent in the open arms showed a significant difference between groups (KW = 13.58; p = 0.0088). The total time spent in the open arms was significantly shorter in groups given doses of 5 and 10 mg/kg (−45% and −43%, respectively). At the dose of 15 mg/kg the decrease was similar to that observed for the other doses (−37%), but the difference did not reach statistical significance (Fig. 2c). The percentage of inactive time spent in the open arms showed a significant difference between groups (KW = 16.04; p = 0.0030). Inactivity time in the open arms was significantly decreased in animals given fluoxetine 5 and 10 mg/kg (−47% and −52%, respectively). At the dose of 15 mg/kg, the decrease (−47%) did not reach significance (Fig. 2d). The 2.5 mg/kg fluoxetine dose had no observable effect on any of the parameters considered. 3.1.3. Paroxetine effects The total distance covered by the treated rats during the 5-min test was not significantly different from controls (Fig. 3a). The percentage of distances covered in the open arms showed no significant difference between groups. However, with the three doses 0.5, 3 and 12 mg/kg, the percentage of distance covered in the open arms decreased between 33% and 37% (Fig. 3b). The total time spent in the open arms showed a significant difference between groups (KW = 12.72; p = 0.0127); the total time spent in the open arms decreased significantly in animals given 3 and 12 mg/kg (−41% and −45%, respectively) (Fig. 3c). The percentage of inactivity time spent in the open arms showed a significant difference between groups (KW = 14.61; p = 0.0056); inactivity time in open arms decreased significantly in animals given 12 mg/kg (−55%) (Fig. 3d). 3.1.4. Desipramine effects The total distance covered showed significant differences between groups (KW = 13.87; p = 0.0039). The distance decreased with increasing doses, but this difference was only statistically significant at 5 mg/kg (−21%) and 10 mg/kg (−32%) (Fig. 4a). None of the other parameters was significantly different when compared to controls (Figs. 4b–d).
Fig. 3. Effect of acute paroxetine, 0.1 mg/kg (n = 12), 0.5 mg/kg (n = 12), 3 mg/kg (n = 13), 12 mg/kg (n = 13), administered IP, 30 min before the test, on rats tested in the elevated plus-maze (mean ± S.E.M.). The control group (n = 20) received a saline solution, under the same conditions. (a) Total distance, (b) percentage of distance covered in the open arms, (c) total time spent in the open arms, and (d) percentage of inactivity time spent in the open arms (* p < 0.05).
4. Discussion 3.2. Videotape analysis For diazepam, among the ethological parameters (NPHD, PHD, RA), only RA showed a significant difference between groups (KW = 15.35; p = 0.0015), but compared to the control group, the RA time decreased in all groups (−59%, −78%, and −96%, respectively) for diazepam (Fig. 5a–c). For fluoxetine and desipramine no difference between groups was observed for the ethological parameters. For paroxetine, time spent for RA and PHD was significantly different between groups (respectively KW = 13.99, p = 0.0073 and KW = 15.76, p = 0.0033). In comparison with the group of control, RA time increased at the doses of 3 and 12 mg/kg and PHD decreased with the dose of 0.5 mg/kg (Fig. 6a–c).
The results of our study clearly show that acute treatment with a single IP injection of fluoxetine (see [38] for review on the molecule) at the dose of 5 or 10 mg/kg decreased the time the rats spent in the open arms of the EPM. This effect was not induced by changes in motor activity, for theses doses, since the total distance covered by the rats was not modified. When exposed to an EPM, rats tend to avoid the open arms and prefer to stay in the enclosed arms. So, drugs that elicit a decrease of the time spent in the open arms are considered as anxiogenic [36]. At the higher 15 mg/kg dose, fluoxetine probably had a sedative effect since the total distance covered in the maze was shorter [9]. The 2.5 mg/kg fluoxetine dose had no observable effect on any of the parameters considered.
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Fig. 5. Effect of acute diazepam, 0.5 mg/kg (n = 12), 1.5 mg/kg (n = 11), 2.5 mg/kg (n = 11), administered IP, 30 min before the test on ethological parameters. (a) Risk assessment, (b) protected head dips, and (c) non-protected head dips, on rats tested in the elevated plus-maze (mean ± S.E.M.). The control groups (n = 11), received 1% Tween-80 in saline solution, under the same conditions.
Fig. 4. Effect of acute desipramine, 2.5 mg/kg (n = 15), 5 mg/kg (n = 15), 10 mg/kg (n = 14) administered IP, 30 min before the test, on rats tested in the elevated plus-maze (mean ± S.E.M.). The control group (n = 17) received 1% Tween-80 in saline solution, under the same conditions. (a) Total distance), (b) percentage of distance covered in the open arms, (c) total time spent in the open arms, and (d) percentage of inactivity time spent in the open arms (* p < 0.05).
We also found that paroxetine (see [11] for review on the molecule) induced an anxiogenic effect after acute IP administration for both the 3 and 12 mg/kg doses, as demonstrated by the total time and/or the percentage of inactivity spent in the open arms. At the smallest dose (0.1 mg/kg) paroxetine was inactive as none of the measured parameters were affected. Earlier EMP results with SSRIs are still a subject of debat. Several authors have observed an acute anxiogenic-like effect. Kurt et al. [30] shown in mice that acute sertraline (10 mg/kg IP) and fluoxetine (20 mg/kg IP) treatment significantly decreased the time spent in open arms and fluoxetine also decreased the total number of enclosed arm entries. Silva et al. [41]
demonstrated that acute fluoxetine (5 mg/kg IP) induced the same effect in rats. For Holmes and Rodgers [25], fluoxetine (5–20 mg/kg IP) produced non-significant trends for increased anxiety-like behavior in maze-naive mice, but significantly and dose-dependently increased anxiety-like behavior and suppressed locomotor activity in maze-experienced mice. Kocks et al. [29] using a smaller dose of paroxetine (0.5 mg/kg IP) than those of the present study, observed a similar anxiogeniclike behaviors in rats adapted to handling, but not in handling naive animals. But, Griebel et al. [20] demonstrated, on the contrary, an anxiolytic-like effect with fluoxetine (20 mg/kg IP) in Wistar–Kyoto rats, both on conventional and ethological parameters, like Varty et al. [47] with fluoxetine (1–30 mg/kg PO) and paroxetine (0.3–10 mg/kg PO) in gerbils. Other authors were unable to demonstrate any effect at all, for example, RodriguesFilho and Takahashi [37] with fluoxetine (5–20 mg/kg IP) in mice or Troelsen et al. [46] with citalopram (5–40 mg/kg IP), fluoxetine (1–30 mg/kg IP) and paroxetine (1–30 mg/kg IP) after acute treatment, in the mouse zero-maze, a modified elevated plus-maze. Taken together, these results highlight the importance of experimental conditions (animal species, han-
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Fig. 6. Effect of acute fluoxetine, 2.5 mg/kg (n = 9), 5 mg/kg (n = 19), 10 mg/kg (n = 19), 15 mg/kg (n = 9), acute paroxetine, 0.1 mg/kg (n = 12), 0.5 mg/kg (n = 12), 3 mg/kg (n = 13), 12 mg/kg (n = 13), and acute desipramine, 2.5 mg/kg (n = 8), 5 mg/kg (n = 8), 10 mg/kg (n = 7), administered IP, 30 min before the test on ethological parameters. (a) Risk assessment, (b) protected head dips, and (c) non-protected head dips, on rats tested in the elevated plus-maze (mean ± S.E.M.). The control groups (n = 18, n = 20, n = 17, respectively) received saline or Tween-80 in saline solution, under the same conditions (* p < 0.05).
dling of the animals or not, etc.) on the interpretation of results. Conversely, our procedures failed to detect any significant effect of desipramine (see [27] for review on the molecule) on anxiety behavior. The only effect noted was an anxiogenic trend at the 2.5 and 10 mg/kg doses and an anxiolytic one at the 5 mg/kg dose, but none were statistically significant. At our knowledge, only one study looked at the effect of acute desipramine in EPM and also failed to demonstrate any desipramine (5–20 mg/kg IP) effect in EPM, in mice [37]. As attempted, the reference benzodiazepine diazepam display a clear and consistent anxiolytic-like behavioral profile, the total distance and time spent in the open arms being increased dose-dependently. These results validated the sensibility of this model in our laboratory. All these results reinforce previously published data in favor of an anxiogenic effect of a single administration of SSRIs, as assessed by the EPM. We decided to study fluoxetine and paroxetine among the currently available SSRIs because fluoxetine
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is considered as the leading drug in this therapeutic class and because paroxetine has been awarded fully marketing approval for its anxiolytic properties in humans. Moreover, among all the currently available SSRI antidepressants, paroxetine is the most potent inhibitor of serotonin re-uptake [26]. The dose range used has been validated in a large number of preclinical trials (see [10] for review). So far, the mechanistic explanation of the antidepressant anxiogenic effect remains unclear. The fact that we clearly demonstrated an anxiogenic effect with two drugs acting selectively on the 5-HT system, but not with desipramine, which preferentially acts on the noradrenergic system, suggests that serotonergic transmission is more implicated in acute anxiety than noradrenergic transmission. However, desipramine is not a specific compound and also binds to the cholinergic and histaminergic receptors. These properties might explain the sedative effects that we observed with the highest doses. This result is highly consistent with clinical reports of jitteriness in humans after acute administration of serotonergic drugs [40]. This conclusion is comforted by the studies looking at the role of 5-HT receptors in mediating SSRIs-induced anxiogenesis. For example, Bagdy et al. [2] have shown that anxiety-like effects induced by acute fluoxetine (2.5–10 mg/kg IP) and sertraline (15 mg/kg IP) are reversed by a 5-HT2C receptor antagonist, but not by a 5-HT1A one. According to Millan [34], at the onset of treatment by SSRIs, indirect activation of 5-HT2C receptors participates in their anxiogenic effects and conversely, progressive down-regulation of 5-HT2C receptors parallels the gradual onset of clinical efficacy of SSRIs. The validity and appropriateness of the EPM to predict anxiolytic or anxiogenic pharmacological properties of antidepressants also merits discussion since the EPM test was not initially constructed for that purpose. An anxiolytic or anxiogenic drug effect may entail a combination of anxiolytic, sedative, amnesic, anticonvulsant or myorelaxant elements. This is why some authors have introduced ethological parameters to more specifically examine the anxiety component [32,39]. One important ethological parameter, RA, a concept that has emerged from work on antipredator defense in rodents, refers to a behavioral pattern observed in animals exposed to a dangerous situation. Variability in this pattern is particularly well exposed by the EPM test, as has been reported for animals given 5-HT agents [21]. The coherence of our results in terms of the observed dose–effect relationship is an argument in favor of the EPM as a valid model for testing antidepressants. Our conclusions result from a conventional analysis of the time the rats spent in the open or closed arms of the maze. The fact that the ethological parameters did significantly vary adds further credibility to the conclusion that paroxetine has an anxiogenic effect at two different active doses (3 and 12 mg/kg). These results are comparable with those of Griebel et al. [21] who showed that RA responses are sensitive to the action of 5-HT1A receptor ligands, but that their modulation by drugs targeting 5-HT2A , 5-HT2C , or 5-HT3 receptors such as SSRIs has not been convincingly established. On the other hand, we reported any effect with fluoxetine, whereas Silva and Brandao [42] demonstrated that acute fluoxetine (5.6, 10 mg/kg) reduced unprotected head dipping indicated an anxiogenic profile.
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In conclusion, our results demonstrate that acute administration of SSRIs has an anxiogenic effect in rats, and that serotonergic transmission is preferentially involved. Our findings also favor the position that EPM conventional measurements are sufficient and reliable for detecting anxiogenic-like effects of serotonergic drugs. Acknowledgements
[17] [18]
[19]
We thank Dr. Gerald Pope and Mr. Bill Bowen for assistance with the English version of this manuscript and Mrs. Julie Querzerho for technical assistance.
[20]
References
[22]
[1] Argyropoulos SV, Sandford JJ, Nutt DJ. The psychobiology of anxiolytic drugs. Part 2. Pharmacological treatments of anxiety. Pharmacol Ther 2000;88:213–27. [2] Bagdy G, Graf M, Anheuer ZE, Modos EA, Kantor S. Anxiety-like effects induced by acute fluoxetine, sertraline or m-CPP treatment are reversed by pretreatment with the 5-HT2C receptor antagonist SB-242084 but not the 5-HT1A receptor antagonist WAY-100635. Int J Neuropsychopharmacol 2001;4:399–408. [3] Ball SG, Kuhn A, Wall D, Shekhar A, Goddard AW. Selective serotonin reuptake inhibitor treatment for generalized anxiety disorder: a doubleblind, prospective comparison between paroxetine and sertraline. J Clin Psychiatry 2005;66:94–9. [4] Belzung C. Rodent models of anxiety-like behaviors: are they predictive for compounds acting via non-benzodiazepine mechanisms? Curr Opin Invest Drugs 2001;2:1108–11. [5] Carobrez AP, Bertoglio LJ. Ethological and temporal analyses of anxietylike behavior: the elevated plus-maze model 20 years on. Neurosci Biobehav Rev 2005;29:1193–205. [6] Berlant J. New drug development for post-traumatic stress disorder. Curr Opin Invest Drugs 2003;4:37–41. [7] Blanco C, Raza MS, Schneier FR, Liebowitz MR. The evidence-based pharmacological treatment of social anxiety disorder. Int J Neuropsychopharmacol 2003;6:427–42. [8] Boothman LJ, Mitchell SN, Sharp T. Investigation of the SSRI augmentation properties of 5-HT(2) receptor antagonists using in vivo microdialysis. Neuropharmacology 2006;50:726–32. [9] Borelli KG, Nobre MJ, Brandao ML, Coimbra NC. Effects of acute and chronic fluoxetine and diazepam on freezing behavior induced by electrical stimulation of dorsolateral and lateral columns of the periaqueductal gray matter. Pharmacol Biochem Behav 2004;77:557–66. [10] Borsini F, Podhorna J, Marazziti D. Do animal models of anxiety predict anxiolytic-like effects of antidepressants? Psychopharmacology (Berlin) 2002;163:121–41. [11] Bourin M, Chue P, Guillon Y. Paroxetine: a review. CNS Drug Rev 2001;7:25–47. [12] Briley M, Chopin P, Moret C. Effect of serotonergic lesion on “anxious” behaviour measured in the elevated plus-maze test in the rat. Psychopharmacology (Berlin) 1990;101:187–9. [13] Bruce SE, Vasile RG, Goisman RM, Salzman C, Spencer M, Machan JT, et al. Are benzodiazepines still the medication of choice for patients with panic disorder with or without agoraphobia? Am J Psychiatry 2003;160:1432–8. [14] David DJ, Bourin M, Jego G, Przybylski C, Jolliet P, Gardier AM. 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 2003;140:1128–36. [15] Donnelly CL. Pharmacologic treatment approaches for children and adolescents with posttraumatic stress disorder. Child Adolesc Psychiatr Clin N Am 2003;12:251–69. [16] Felton TM, Kang TB, Hjorth S, Auerbach SB. Effects of selective serotonin and serotonin/noradrenaline reuptake inhibitors on extracellular serotonin
[21]
[23] [24]
[25]
[26] [27] [28] [29]
[30]
[31]
[32] [33] [34]
[35] [36]
[37]
[38] [39]
[40]
[41]
in rat diencephalon and frontal cortex. Naunyn Schmiedebergs Arch Pharmacol 2003;367:297–305. Gordon JA, Hen R. The serotonergic system and anxiety. Neuromol Med 2004;5:27–40. Greist JH, Bandelow B, Hollander E, Marazziti D, Montgomery SA, Nutt DJ, et al. World Council of Anxiety. WCA recommendations for the longterm treatment of obsessive-compulsive disorder in adults. CNS Spectrosc 2003;8(8 Suppl 1):7–16. Griebel G. 5-Hydroxytryptamine interacting drugs in animal models of anxiety disorders: more than 30 years of research. Pharmacol Ther 1995;65:319–95. Griebel G, Cohen C, Perrault G, Sanger DJ. Behavioral effects of acute and chronic fluoxetine in Wistar–Kyoto rats. Physiol Behav 1999;67:315–20. Griebel G, Rodgers RJ, Perrault G, Sanger DJ. Risk assessment behavior: evaluation of utility in the study of 5-HT related drugs in the rat elevated plus-maze test. Pharmacol Biochem Behav 1997;57:817–27. Handley S. Serotonin in animal model of anxiety: the importance of stimulus and response. In: Cowen P, Idzikowski C, editors. Serotonin, sleep and mental disorder. Petersfield: Wrighston; 1991. p. 89–115. Handley S, McBlane JW. 5-HT drugs in animal models of anxiety. Psychopharmacology (Berlin) 1993;112:13–20. Handley SL, Mc Blane JW, Critchley MA, Njung’e K. Multiple serotonin mechanisms in animal models of anxiety: environmental, emotional and cognitive factors. Behav Brain Res 1993;58:203–10. Holmes A, Rodgers RJ. Prior exposure to the elevated plus-maze sensitizes mice to the acute behavioral effects of fluoxetine and phenelzine. Eur J Pharmacol 2003;459:221–30. Hyttel J. Pharmacological characterization of selective serotonin reuptake inhibitors (SRRIs). Int Clin Psychopharmacol 1994;9(Suppl 1):19–26. Janowsky DS, Byerley B. Desipramine: an overview. J Clin Psychiatry 1984;45:3–9. Kapczinski F, Lima MS, Souza JS, Schmitt R. Antidepressants for generalized anxiety disorder. Cochrane Database Syst Rev 2003;2:CD003592. Koks S, Beljajev S, Koovit I, Abramov U, Bourin M, Vasar E. 8-OH-DPAT, but not deramiclane, antagonizes the anxiogenic-like action of paroxetine in an elevated plus-maze. Psychopharmacology (Berlin) 2001;153:365–72. Kurt M, Arik AC, Celik S. The effects of sertraline and fluoxetine on anxiety in the elevated plus-maze test in mice. J Basic Physiol Pharmacol 2000;11:173–80. Lin D, Parsons LH. Anxiogenic-like effect of serotonin (1B) receptor stimulation in the rat elevated plus-maze. Pharmacol Biochem Behav 2002;71:581–7. Lister R. Ethologically-based animal models of anxiety disorders. Pharmacol Ther 1990;46:321–40. Millan MJ. The neurobiology and control of anxious states. Prog Neurobiol 2003;70:83–244. Millan MJ. Serotonin 5-HT2C receptors as a target for the treatment of depressive and anxious states: focus on novel therapeutic strategies. Therapie 2005;60:441–60. Nash LT, Hack S. The pharmacological treatment of anxiety disorders in children and adolescents. Expert Opin Pharmacother 2002;3:555–71. Pellow S, Chopin P, File SE, Briley M. Validation of open:closed arm entries in an elevated plus-maze as a measure of anxiety in the rat. J Neurosci Meth 1985;14:149–67. Rodrigues-Filho R, Takahashi RN. Antinociceptive effects induced by desipramine and fluoxetine are dissociated from their antidepressant or anxiolytic action in mice. Int J Neuropsychopharmacol 1999;2:263–9. Rossi A, Barraco A, Donda P. Fluoxetine: a review on evidence based medicine. Ann Gen Hosp Psychiatry 2004;3:2. Roy V, Chapillon P. Further evidences that risk assessment and object exploration behaviours are useful to evaluate emotional reactivity in rodents. Behav Brain Res 2004;154:439–48. Salchner P, Singewald N. Neuroanatomical substrates involved in the anxiogenic-like effect of acute fluoxetine treatment. Neuropharmacology 2002;43:1238–48. Silva MT, Alves CR, Santarem EM. Anxiogenic-like effect of acute and chronic fluoxetine on rats tested on the elevated plus-maze. Braz J Med Biol Res 1999;32:333–9.
D. Drapier et al. / Behavioural Brain Research 176 (2007) 202–209 [42] Silva RC, Brandao ML. Acute and chronic effects of gepirone and fluoxetine in rats tested in the elevated plus-maze: an ethological analysis. Pharmacol Biochem Behav 2000;65:209–16. [43] Sramek JJ, Zarotsky V, Cutler NR. Generalised anxiety disorder: treatment options. Drugs 2002;62:1635–48. [44] Stahl SM. Basic psychopharmacology of antidepressants. Part 1. Antidepressants have seven distinct mechanisms of action. J Clin Psychiatry 1998;59(Suppl 4):5–14.
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[45] Stewart SH, Westra HA. Benzodiazepine side-effects: from the bench to the clinic. Curr Pharm Des 2002;8:1–3. [46] Troelsen KB, Nielsen EO, Mirza NR. Chronic treatment with duloxetine is necessary for an anxiolytic-like response in the mouse zero maze: the role of the serotonin transporter. Psychopharmacology (Berlin) 2005;181:741–50. [47] Varty GB, Morgan CA, Cohen-Williams ME, Coffin VL, Carey GJ. The gerbil elevated plus-maze. I. Behavioral characterization and pharmacological validation. Neuropsychopharmacology 2002;27:357–70.
Research report
Effects of acute fluoxetine, paroxetine and desipramine on rats tested on the elevated plus-maze Dominique Drapier a , Dani`ele Bentu´e-Ferrer b,c , Bruno Laviolle d , Bruno Millet a , Herv´e Allain b,c , Michel Bourin c,∗ , Jean-Michel Reymann b,c a
Service Hospitalo-Universitaire de Psychiatrie, Centre Hospitalier Guillaume R´egnier, 108 avenue du G´en´eral Leclerc, 35703 Rennes Cedex, France b Laboratoire de Pharmacologie Exp´ erimentale et Clinique, Facult´e de M´edecine, CS 34317, 35043 Rennes Cedex, France c EA 3256 Neurobiologie de l’anxi´ et´e et de la d´epression, Laboratoire de Pharmacologie, Facult´e de M´edecine, BP 53508, 1 rue Gaston Veil, 44035 Nantes Cedex, France d Centre d’Investigation Clinique INSERM 0203, CHU Pontchaillou, 35033 Rennes, France Received 16 May 2006; received in revised form 28 September 2006; accepted 2 October 2006 Available online 13 November 2006
Abstract Antidepressants are usually prescribed for the treatment of depression but more recently have also been recommended for the treatment of anxiety disorders. The purpose of this study was to investigate the anxiogenic- or anxiolytic-like effects of an acute administration of antidepressants (serotonergic and noradrenergic compounds) in male Wistar rats submitted to the elevated plus-maze. Fluoxetine (2.5, 5, 10, 15 mg/kg), paroxetine (0.1, 0.5, 3, 12 mg/kg) and desipramine (2.5, 5, 10 mg/kg) or their vehicles were administered intraperitoneally 30 min prior to testing. Diazepam (0.5, 1.5, 2.5 mg/kg) was used as a positive comparator for anxiolytic effect. In comparison with control animals, the percentage of time the rats treated with fluoxetine (5 and 10 mg/kg) and paroxetine (3 and 12 mg/kg) spent in the open arms decreased. The percent of inactive time spent in the open arms also decreased in rats given fluoxetine (5 and 10 mg/kg) and paroxetine (12 mg/kg). Desipramine was inactive on all these parameters. In conclusion, acute treatment with fluoxetine and paroxetine, but not with desipramine, produced a pattern of anxiety behavior. Thus, the pharmacological mechanism appears to be due more to serotonergic than adrenergic neurotransmission. The elevated plus-maze exhibits good sensitivity for detecting anxiogenic effects of antidepressant drugs and the conventional parameters are sufficient and reliable for detecting such effects. © 2006 Elsevier B.V. All rights reserved. Keywords: Antidepressants; Anxiety; Rats; Plus-maze; Ethological parameters
1. Introduction Benzodiazepines (BZDs) have been the mainstay of pharmacological treatment of anxiety over the last four decades despite a high incidence of side effects. Tolerance, physical dependence and withdrawal symptoms, as well as sedation and risk of abuse can raise significant problems [45]. Antidepressants are usually prescribed for the treatment of depression, but more recently these compounds, in particular drugs belonging to the class of selective serotonin re-uptake inhibitors (SSRIs), were also recommended for the treatment of anxiety in adults and children [1,35]. Antidepressants are useful for the treatment of gener-
∗
Corresponding author. Tel.: +33 2 40 41 28 52; fax: +33 2 40 41 28 56. E-mail address: [email protected] (M. Bourin).
0166-4328/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.bbr.2006.10.002
alized anxiety disorders (GAD) [3,28,43] and panic disorders (PD) [13], but also for social anxiety disorder (SAD) [7], obsessive compulsive disorders (OCD) [18] and post-traumatic stress disorder (PTSD) [6,15], where BZDs fail to provide adequate symptom relief. In certain cases, SSRIs have been approved for these indications and may be more appropriate than BZDs for the treatment of anxiety disorders. On the other hand, it has been frequently reported that the initial effect of acute administration of serotonergic compounds in humans is increased anxiety [17,44]. Numerous animal studies have aimed at better understanding the neurobiological mechanisms implied in the etiology of anxiety [19,33]. It has been hypothesized that increased 5HT neurotransmission is associated with anxiogenesis, whereas a decrease could induce anxiolysis [2,12]. The role of 5-HT in anxiety is undoubtedly complex and may be dependent on a number of factors including 5-HT neurotransmission in multiple
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brain regions, the type of behavioral paradigms used to assess anxiety, and/or the emotional and cognitive contexts of the tests [22–24,31,40]. Several microdialysis studies have demonstrated that a single intraperitoneal (IP) injection of SSRIs increased the extracellular concentration of 5-HT: for example, paroxetine and citalopram in mice cortex [14]; paroxetine, citalopram and fluoxetine in rat diencephalon and frontal cortex [16]; citalopram and fluoxetine in rat hippocampus [8]. However, the data obtained in animal studies using antidepressants is contradictory and it still remains unclear whether these SSRIs truly display an anxiogenic or an anxiolytic effect or, to the contrary, have no effect as concluded by Borsini et al. [10], who analyzed 107 articles dealing with the effects of antidepressant treatment in animal models of anxiety. Moreover, little is known about pharmacological mechanism underlying the possible anxiolytic effect of SSRIs. Several factors could explain these inconsistent findings. It is noteworthy for example that the experimental procedures generally used to assess drug effects were developed and optimized initially for evaluating BZDs [4]. As a consequence, the predictive validity of the elevated plus-maze (EPM) might well be limited to BZD-related drugs. The aim of the present study was to compare under identical experimental conditions the effects of an acute administration of two serotonergic compounds, fluoxetine and paroxetine, and one noradrenergic compound, desipramine, by means of the same behavioral test widely validated for analyses of anxiety-like behavior [5,36], the elevated plus-maze (EPM). Diazepam was used as a positive comparator for anxiolytic effect. 2. Materials and methods 2.1. Animals Adult male Wistar rats (CERJ) weighing 200–300 g were kept in an approved animal house (authorization no. A 35006) at 22 ± 2 ◦ C with a 12-h light/dark cycle (light turned on at 0800 h). Tap water and food pellets were available ad libitum. The animals were housed in groups of four in polycarbonate cages (44 cm × 30 cm × 18 cm). During the 15 days previous the test, the rats were handled only for the changes of litter (three times a week) and to be weighed the day before the experimentation. The care provided to the animals, before, during and after the protocol was in compliance with the European Communities Council Directive. Researchers were authorized to conduct animal studies by the Veterinary Department of the French Ministry of Health (authorization no. 35-01).
2.2. Drugs, treatments and study protocol Tween-80, diazepam, fluoxetine and desipramine were provided by Sigma® (Sigma–Aldrich, 38297 St. Quentin Fallavier, France). Paroxetine was a gift of SmithKlineBeecham laboratory. Paroxetine and fluoxetine were dissolved in a saline solution. Desipramine and diazepam were suspended in a 1% Tween-80 saline solution. Thirty minutes prior to testing, the animals were given, after randomization, an intraperitoneal injection (5 ml/kg) of the relevant drug or vehicle. After the injection, the rats were put back into their home cage. All injections were administered between 0830 and 1030 h. Experiments were conducted blind with respect to treatment. Selected doses reflected usual doses point out in the literature for this kind of experiments, mentioned in particular in the review of Borsini et al. [10].
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Doses and number of animals by group were as follows: • Diazepam: 0.5 mg/kg, n = 12; 1.5 mg/kg, n = 11; 2.5 mg/kg, n = 11. The control group (n = 11) received a 1% Tween-80 saline solution, under the same conditions. • Fluoxetine: 2.5 mg/kg, n = 9; 5 mg/kg, n = 19; 10 mg/kg, n = 19; 15 mg/kg, n = 9. The control group (n = 18) received the saline solution, under the same conditions. • Paroxetine: 0.1 mg/kg, n = 12; 0.5 mg/kg, n = 12; 3 mg/kg, n = 13; 12 mg/kg, n = 13. The control group (n = 21) received the saline solution, under the same conditions. • Desipramine: 2.5 mg/kg, n = 15; 5 mg/kg, n = 15; 10 mg/kg, n = 14. The control group (n = 17 received a 1% Tween-80 saline solution, under the same conditions.
2.3. Elevated plus-maze and general procedure The EPM was made of wood and painted black. The apparatus consisted of two opposite open arms (45 cm × 10 cm) without sidewalls and two enclosed arms (45 cm × 10 cm × 30 cm) with sides and end walls, extending from a central square (10 cm × 10 cm). The maze was elevated to the height of 60 cm above the floor and placed in a moderately lighted room (30 lx as measured at the center of the maze). At the onset of test, the handling naive animals were placed at the center of the EPM, facing towards an open arm and their behavior was recorded during 5 min. The Video-Track® system, used for recording the animal’s behavior, was composed of a roof-hand-held camera connected to a computer. This apparatus allowed the measurement of activity (i.e. movement time spent) or inactivity (time spent at rest, in an observation mode or grooming), time and distances covered in each part of the maze during a 5-min period of time. The following parameters were analyzed in our study: total distance covered, percentage of distance and total time spent in each of the open arms, percentage of inactive time spent in the open arms compared to total inactive time spent in the EPM as a whole. Each rat’s behavior was also recorded on videotape and analyzed by a person not involved in the experimental procedure. The time devoted to the following ethological parameters were then recorded: risk assessment (RA), the rat exiting an enclosed arm with the forepaws and head only and investigating the surroundings, not necessarily accompanied by body stretching; protected head dips (PHD), the rat stretching to dip its head into the open space and observing the environment, the rear part of the body remaining in a closed arm with the forepaws in the center of the EPM; non-protected head dips (NPHD), the rat dipping its head into the open space and observing the environment, the body being in an open arm. The EPM was cleaned between experiments with a 5% alcohol solution.
2.4. Statistical analysis Statistical analysis was performed with SAS statistical software, version 9.1 (SAS Institute, Cary, NC). Data are presented as mean with the corresponding standard error of the mean (m ± S.E.M.). Comparisons between groups were performed using an ANOVA or the Kruskal–Wallis (KW) test if needed. The corresponding F or KW statistics are indicated. In case of significant differences between groups, each treatment dose was compared to the corresponding control group by use of the Dunnett’s two-tailed t-test to control the overall rate of type I error in multiple testing. For all analyses, a p-value < 0.05 was considered to be significant.
3. Results 3.1. Video-track analysis 3.1.1. Diazepam effects The total distance covered by the treated rats during the 5-min test was not significantly different from controls (Fig. 1a).
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cantly increased in animals given diazepam 2.5 mg/kg (+113%) (Fig. 1d). 3.1.2. Fluoxetine effects The total distance covered by the rats during the 5-min test was significantly different between groups (KW = 12.54; p = 0.0137). The distance decreased with increasing doses and the difference became statistically significant with the highest dose (15 mg/kg; −33%) (Fig. 2a). The percentage of distance covered in the open arms tended to be significantly different between groups (KW = 9.28;
Fig. 1. Effects of acute diazepam, 0.5 mg/kg (n = 12), 1.5 mg/kg (n = 11), 2.5 mg/kg (n = 11) administered IP, 30 min before the test, on rats tested in the elevated plus-maze (mean ± S.E.M.). The control group (n = 11) received a 1% Tween-80 in saline solution, under the same conditions. (a) Total distance, (b) percentage of distance covered in the open arms, (c) total time spent in the open arms, and (d) percentage of inactivity time spent in the open arms (* p < 0.05).
The percentage of distance covered in the open arms tended to be significantly different between groups (F = 2.69; p = 0.0585); the percentage of distance in the open arms in the group given diazepam 2.5 mg/kg was higher (89%) than control group (Fig. 1b). The total time spent in the open arms showed a significant difference between groups (F = 3.18; p = 0.0339). The total time spent in the open arms was longer in all treated groups than in the control group but the difference was only significant in the group given diazepam 2.5 mg/kg (+98%) (Fig. 1c). The percentage of inactive time spent in the open arms showed a significant difference between groups (F = 3.27; p = 0.0308). Inactivity time in the open arms was signifi-
Fig. 2. Effects of acute fluoxetine, 2.5 mg/kg (n = 9), 5 mg/kg (n = 19), 10 mg/kg (n = 19), 15 mg/kg (n = 9) administered IP, 30 min before the test, on rats tested in the elevated plus-maze (mean ± S.E.M.). The control group (n = 18) received a saline solution, under the same conditions. (a) Total distance, (b) percentage of distance covered in the open arms, (c) total time spent in the open arms, and (d) percentage of inactivity time spent in the open arms (* p < 0.05).
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p = 0.0545); the percentage of distance in the open arms in the group given fluoxetine (5 mg/kg) was lower (−45%) (Fig. 2b). The total time spent in the open arms showed a significant difference between groups (KW = 13.58; p = 0.0088). The total time spent in the open arms was significantly shorter in groups given doses of 5 and 10 mg/kg (−45% and −43%, respectively). At the dose of 15 mg/kg the decrease was similar to that observed for the other doses (−37%), but the difference did not reach statistical significance (Fig. 2c). The percentage of inactive time spent in the open arms showed a significant difference between groups (KW = 16.04; p = 0.0030). Inactivity time in the open arms was significantly decreased in animals given fluoxetine 5 and 10 mg/kg (−47% and −52%, respectively). At the dose of 15 mg/kg, the decrease (−47%) did not reach significance (Fig. 2d). The 2.5 mg/kg fluoxetine dose had no observable effect on any of the parameters considered. 3.1.3. Paroxetine effects The total distance covered by the treated rats during the 5-min test was not significantly different from controls (Fig. 3a). The percentage of distances covered in the open arms showed no significant difference between groups. However, with the three doses 0.5, 3 and 12 mg/kg, the percentage of distance covered in the open arms decreased between 33% and 37% (Fig. 3b). The total time spent in the open arms showed a significant difference between groups (KW = 12.72; p = 0.0127); the total time spent in the open arms decreased significantly in animals given 3 and 12 mg/kg (−41% and −45%, respectively) (Fig. 3c). The percentage of inactivity time spent in the open arms showed a significant difference between groups (KW = 14.61; p = 0.0056); inactivity time in open arms decreased significantly in animals given 12 mg/kg (−55%) (Fig. 3d). 3.1.4. Desipramine effects The total distance covered showed significant differences between groups (KW = 13.87; p = 0.0039). The distance decreased with increasing doses, but this difference was only statistically significant at 5 mg/kg (−21%) and 10 mg/kg (−32%) (Fig. 4a). None of the other parameters was significantly different when compared to controls (Figs. 4b–d).
Fig. 3. Effect of acute paroxetine, 0.1 mg/kg (n = 12), 0.5 mg/kg (n = 12), 3 mg/kg (n = 13), 12 mg/kg (n = 13), administered IP, 30 min before the test, on rats tested in the elevated plus-maze (mean ± S.E.M.). The control group (n = 20) received a saline solution, under the same conditions. (a) Total distance, (b) percentage of distance covered in the open arms, (c) total time spent in the open arms, and (d) percentage of inactivity time spent in the open arms (* p < 0.05).
4. Discussion 3.2. Videotape analysis For diazepam, among the ethological parameters (NPHD, PHD, RA), only RA showed a significant difference between groups (KW = 15.35; p = 0.0015), but compared to the control group, the RA time decreased in all groups (−59%, −78%, and −96%, respectively) for diazepam (Fig. 5a–c). For fluoxetine and desipramine no difference between groups was observed for the ethological parameters. For paroxetine, time spent for RA and PHD was significantly different between groups (respectively KW = 13.99, p = 0.0073 and KW = 15.76, p = 0.0033). In comparison with the group of control, RA time increased at the doses of 3 and 12 mg/kg and PHD decreased with the dose of 0.5 mg/kg (Fig. 6a–c).
The results of our study clearly show that acute treatment with a single IP injection of fluoxetine (see [38] for review on the molecule) at the dose of 5 or 10 mg/kg decreased the time the rats spent in the open arms of the EPM. This effect was not induced by changes in motor activity, for theses doses, since the total distance covered by the rats was not modified. When exposed to an EPM, rats tend to avoid the open arms and prefer to stay in the enclosed arms. So, drugs that elicit a decrease of the time spent in the open arms are considered as anxiogenic [36]. At the higher 15 mg/kg dose, fluoxetine probably had a sedative effect since the total distance covered in the maze was shorter [9]. The 2.5 mg/kg fluoxetine dose had no observable effect on any of the parameters considered.
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Fig. 5. Effect of acute diazepam, 0.5 mg/kg (n = 12), 1.5 mg/kg (n = 11), 2.5 mg/kg (n = 11), administered IP, 30 min before the test on ethological parameters. (a) Risk assessment, (b) protected head dips, and (c) non-protected head dips, on rats tested in the elevated plus-maze (mean ± S.E.M.). The control groups (n = 11), received 1% Tween-80 in saline solution, under the same conditions.
Fig. 4. Effect of acute desipramine, 2.5 mg/kg (n = 15), 5 mg/kg (n = 15), 10 mg/kg (n = 14) administered IP, 30 min before the test, on rats tested in the elevated plus-maze (mean ± S.E.M.). The control group (n = 17) received 1% Tween-80 in saline solution, under the same conditions. (a) Total distance), (b) percentage of distance covered in the open arms, (c) total time spent in the open arms, and (d) percentage of inactivity time spent in the open arms (* p < 0.05).
We also found that paroxetine (see [11] for review on the molecule) induced an anxiogenic effect after acute IP administration for both the 3 and 12 mg/kg doses, as demonstrated by the total time and/or the percentage of inactivity spent in the open arms. At the smallest dose (0.1 mg/kg) paroxetine was inactive as none of the measured parameters were affected. Earlier EMP results with SSRIs are still a subject of debat. Several authors have observed an acute anxiogenic-like effect. Kurt et al. [30] shown in mice that acute sertraline (10 mg/kg IP) and fluoxetine (20 mg/kg IP) treatment significantly decreased the time spent in open arms and fluoxetine also decreased the total number of enclosed arm entries. Silva et al. [41]
demonstrated that acute fluoxetine (5 mg/kg IP) induced the same effect in rats. For Holmes and Rodgers [25], fluoxetine (5–20 mg/kg IP) produced non-significant trends for increased anxiety-like behavior in maze-naive mice, but significantly and dose-dependently increased anxiety-like behavior and suppressed locomotor activity in maze-experienced mice. Kocks et al. [29] using a smaller dose of paroxetine (0.5 mg/kg IP) than those of the present study, observed a similar anxiogeniclike behaviors in rats adapted to handling, but not in handling naive animals. But, Griebel et al. [20] demonstrated, on the contrary, an anxiolytic-like effect with fluoxetine (20 mg/kg IP) in Wistar–Kyoto rats, both on conventional and ethological parameters, like Varty et al. [47] with fluoxetine (1–30 mg/kg PO) and paroxetine (0.3–10 mg/kg PO) in gerbils. Other authors were unable to demonstrate any effect at all, for example, RodriguesFilho and Takahashi [37] with fluoxetine (5–20 mg/kg IP) in mice or Troelsen et al. [46] with citalopram (5–40 mg/kg IP), fluoxetine (1–30 mg/kg IP) and paroxetine (1–30 mg/kg IP) after acute treatment, in the mouse zero-maze, a modified elevated plus-maze. Taken together, these results highlight the importance of experimental conditions (animal species, han-
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Fig. 6. Effect of acute fluoxetine, 2.5 mg/kg (n = 9), 5 mg/kg (n = 19), 10 mg/kg (n = 19), 15 mg/kg (n = 9), acute paroxetine, 0.1 mg/kg (n = 12), 0.5 mg/kg (n = 12), 3 mg/kg (n = 13), 12 mg/kg (n = 13), and acute desipramine, 2.5 mg/kg (n = 8), 5 mg/kg (n = 8), 10 mg/kg (n = 7), administered IP, 30 min before the test on ethological parameters. (a) Risk assessment, (b) protected head dips, and (c) non-protected head dips, on rats tested in the elevated plus-maze (mean ± S.E.M.). The control groups (n = 18, n = 20, n = 17, respectively) received saline or Tween-80 in saline solution, under the same conditions (* p < 0.05).
dling of the animals or not, etc.) on the interpretation of results. Conversely, our procedures failed to detect any significant effect of desipramine (see [27] for review on the molecule) on anxiety behavior. The only effect noted was an anxiogenic trend at the 2.5 and 10 mg/kg doses and an anxiolytic one at the 5 mg/kg dose, but none were statistically significant. At our knowledge, only one study looked at the effect of acute desipramine in EPM and also failed to demonstrate any desipramine (5–20 mg/kg IP) effect in EPM, in mice [37]. As attempted, the reference benzodiazepine diazepam display a clear and consistent anxiolytic-like behavioral profile, the total distance and time spent in the open arms being increased dose-dependently. These results validated the sensibility of this model in our laboratory. All these results reinforce previously published data in favor of an anxiogenic effect of a single administration of SSRIs, as assessed by the EPM. We decided to study fluoxetine and paroxetine among the currently available SSRIs because fluoxetine
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is considered as the leading drug in this therapeutic class and because paroxetine has been awarded fully marketing approval for its anxiolytic properties in humans. Moreover, among all the currently available SSRI antidepressants, paroxetine is the most potent inhibitor of serotonin re-uptake [26]. The dose range used has been validated in a large number of preclinical trials (see [10] for review). So far, the mechanistic explanation of the antidepressant anxiogenic effect remains unclear. The fact that we clearly demonstrated an anxiogenic effect with two drugs acting selectively on the 5-HT system, but not with desipramine, which preferentially acts on the noradrenergic system, suggests that serotonergic transmission is more implicated in acute anxiety than noradrenergic transmission. However, desipramine is not a specific compound and also binds to the cholinergic and histaminergic receptors. These properties might explain the sedative effects that we observed with the highest doses. This result is highly consistent with clinical reports of jitteriness in humans after acute administration of serotonergic drugs [40]. This conclusion is comforted by the studies looking at the role of 5-HT receptors in mediating SSRIs-induced anxiogenesis. For example, Bagdy et al. [2] have shown that anxiety-like effects induced by acute fluoxetine (2.5–10 mg/kg IP) and sertraline (15 mg/kg IP) are reversed by a 5-HT2C receptor antagonist, but not by a 5-HT1A one. According to Millan [34], at the onset of treatment by SSRIs, indirect activation of 5-HT2C receptors participates in their anxiogenic effects and conversely, progressive down-regulation of 5-HT2C receptors parallels the gradual onset of clinical efficacy of SSRIs. The validity and appropriateness of the EPM to predict anxiolytic or anxiogenic pharmacological properties of antidepressants also merits discussion since the EPM test was not initially constructed for that purpose. An anxiolytic or anxiogenic drug effect may entail a combination of anxiolytic, sedative, amnesic, anticonvulsant or myorelaxant elements. This is why some authors have introduced ethological parameters to more specifically examine the anxiety component [32,39]. One important ethological parameter, RA, a concept that has emerged from work on antipredator defense in rodents, refers to a behavioral pattern observed in animals exposed to a dangerous situation. Variability in this pattern is particularly well exposed by the EPM test, as has been reported for animals given 5-HT agents [21]. The coherence of our results in terms of the observed dose–effect relationship is an argument in favor of the EPM as a valid model for testing antidepressants. Our conclusions result from a conventional analysis of the time the rats spent in the open or closed arms of the maze. The fact that the ethological parameters did significantly vary adds further credibility to the conclusion that paroxetine has an anxiogenic effect at two different active doses (3 and 12 mg/kg). These results are comparable with those of Griebel et al. [21] who showed that RA responses are sensitive to the action of 5-HT1A receptor ligands, but that their modulation by drugs targeting 5-HT2A , 5-HT2C , or 5-HT3 receptors such as SSRIs has not been convincingly established. On the other hand, we reported any effect with fluoxetine, whereas Silva and Brandao [42] demonstrated that acute fluoxetine (5.6, 10 mg/kg) reduced unprotected head dipping indicated an anxiogenic profile.
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In conclusion, our results demonstrate that acute administration of SSRIs has an anxiogenic effect in rats, and that serotonergic transmission is preferentially involved. Our findings also favor the position that EPM conventional measurements are sufficient and reliable for detecting anxiogenic-like effects of serotonergic drugs. Acknowledgements
[17] [18]
[19]
We thank Dr. Gerald Pope and Mr. Bill Bowen for assistance with the English version of this manuscript and Mrs. Julie Querzerho for technical assistance.
[20]
References
[22]
[1] Argyropoulos SV, Sandford JJ, Nutt DJ. The psychobiology of anxiolytic drugs. Part 2. Pharmacological treatments of anxiety. Pharmacol Ther 2000;88:213–27. [2] Bagdy G, Graf M, Anheuer ZE, Modos EA, Kantor S. Anxiety-like effects induced by acute fluoxetine, sertraline or m-CPP treatment are reversed by pretreatment with the 5-HT2C receptor antagonist SB-242084 but not the 5-HT1A receptor antagonist WAY-100635. Int J Neuropsychopharmacol 2001;4:399–408. [3] Ball SG, Kuhn A, Wall D, Shekhar A, Goddard AW. Selective serotonin reuptake inhibitor treatment for generalized anxiety disorder: a doubleblind, prospective comparison between paroxetine and sertraline. J Clin Psychiatry 2005;66:94–9. [4] Belzung C. Rodent models of anxiety-like behaviors: are they predictive for compounds acting via non-benzodiazepine mechanisms? Curr Opin Invest Drugs 2001;2:1108–11. [5] Carobrez AP, Bertoglio LJ. Ethological and temporal analyses of anxietylike behavior: the elevated plus-maze model 20 years on. Neurosci Biobehav Rev 2005;29:1193–205. [6] Berlant J. New drug development for post-traumatic stress disorder. Curr Opin Invest Drugs 2003;4:37–41. [7] Blanco C, Raza MS, Schneier FR, Liebowitz MR. The evidence-based pharmacological treatment of social anxiety disorder. Int J Neuropsychopharmacol 2003;6:427–42. [8] Boothman LJ, Mitchell SN, Sharp T. Investigation of the SSRI augmentation properties of 5-HT(2) receptor antagonists using in vivo microdialysis. Neuropharmacology 2006;50:726–32. [9] Borelli KG, Nobre MJ, Brandao ML, Coimbra NC. Effects of acute and chronic fluoxetine and diazepam on freezing behavior induced by electrical stimulation of dorsolateral and lateral columns of the periaqueductal gray matter. Pharmacol Biochem Behav 2004;77:557–66. [10] Borsini F, Podhorna J, Marazziti D. Do animal models of anxiety predict anxiolytic-like effects of antidepressants? Psychopharmacology (Berlin) 2002;163:121–41. [11] Bourin M, Chue P, Guillon Y. Paroxetine: a review. CNS Drug Rev 2001;7:25–47. [12] Briley M, Chopin P, Moret C. Effect of serotonergic lesion on “anxious” behaviour measured in the elevated plus-maze test in the rat. Psychopharmacology (Berlin) 1990;101:187–9. [13] Bruce SE, Vasile RG, Goisman RM, Salzman C, Spencer M, Machan JT, et al. Are benzodiazepines still the medication of choice for patients with panic disorder with or without agoraphobia? Am J Psychiatry 2003;160:1432–8. [14] David DJ, Bourin M, Jego G, Przybylski C, Jolliet P, Gardier AM. 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 2003;140:1128–36. [15] Donnelly CL. Pharmacologic treatment approaches for children and adolescents with posttraumatic stress disorder. Child Adolesc Psychiatr Clin N Am 2003;12:251–69. [16] Felton TM, Kang TB, Hjorth S, Auerbach SB. Effects of selective serotonin and serotonin/noradrenaline reuptake inhibitors on extracellular serotonin
[21]
[23] [24]
[25]
[26] [27] [28] [29]
[30]
[31]
[32] [33] [34]
[35] [36]
[37]
[38] [39]
[40]
[41]
in rat diencephalon and frontal cortex. Naunyn Schmiedebergs Arch Pharmacol 2003;367:297–305. Gordon JA, Hen R. The serotonergic system and anxiety. Neuromol Med 2004;5:27–40. Greist JH, Bandelow B, Hollander E, Marazziti D, Montgomery SA, Nutt DJ, et al. World Council of Anxiety. WCA recommendations for the longterm treatment of obsessive-compulsive disorder in adults. CNS Spectrosc 2003;8(8 Suppl 1):7–16. Griebel G. 5-Hydroxytryptamine interacting drugs in animal models of anxiety disorders: more than 30 years of research. Pharmacol Ther 1995;65:319–95. Griebel G, Cohen C, Perrault G, Sanger DJ. Behavioral effects of acute and chronic fluoxetine in Wistar–Kyoto rats. Physiol Behav 1999;67:315–20. Griebel G, Rodgers RJ, Perrault G, Sanger DJ. Risk assessment behavior: evaluation of utility in the study of 5-HT related drugs in the rat elevated plus-maze test. Pharmacol Biochem Behav 1997;57:817–27. Handley S. Serotonin in animal model of anxiety: the importance of stimulus and response. In: Cowen P, Idzikowski C, editors. Serotonin, sleep and mental disorder. Petersfield: Wrighston; 1991. p. 89–115. Handley S, McBlane JW. 5-HT drugs in animal models of anxiety. Psychopharmacology (Berlin) 1993;112:13–20. Handley SL, Mc Blane JW, Critchley MA, Njung’e K. Multiple serotonin mechanisms in animal models of anxiety: environmental, emotional and cognitive factors. Behav Brain Res 1993;58:203–10. Holmes A, Rodgers RJ. Prior exposure to the elevated plus-maze sensitizes mice to the acute behavioral effects of fluoxetine and phenelzine. Eur J Pharmacol 2003;459:221–30. Hyttel J. Pharmacological characterization of selective serotonin reuptake inhibitors (SRRIs). Int Clin Psychopharmacol 1994;9(Suppl 1):19–26. Janowsky DS, Byerley B. Desipramine: an overview. J Clin Psychiatry 1984;45:3–9. Kapczinski F, Lima MS, Souza JS, Schmitt R. Antidepressants for generalized anxiety disorder. Cochrane Database Syst Rev 2003;2:CD003592. Koks S, Beljajev S, Koovit I, Abramov U, Bourin M, Vasar E. 8-OH-DPAT, but not deramiclane, antagonizes the anxiogenic-like action of paroxetine in an elevated plus-maze. Psychopharmacology (Berlin) 2001;153:365–72. Kurt M, Arik AC, Celik S. The effects of sertraline and fluoxetine on anxiety in the elevated plus-maze test in mice. J Basic Physiol Pharmacol 2000;11:173–80. Lin D, Parsons LH. Anxiogenic-like effect of serotonin (1B) receptor stimulation in the rat elevated plus-maze. Pharmacol Biochem Behav 2002;71:581–7. Lister R. Ethologically-based animal models of anxiety disorders. Pharmacol Ther 1990;46:321–40. Millan MJ. The neurobiology and control of anxious states. Prog Neurobiol 2003;70:83–244. Millan MJ. Serotonin 5-HT2C receptors as a target for the treatment of depressive and anxious states: focus on novel therapeutic strategies. Therapie 2005;60:441–60. Nash LT, Hack S. The pharmacological treatment of anxiety disorders in children and adolescents. Expert Opin Pharmacother 2002;3:555–71. Pellow S, Chopin P, File SE, Briley M. Validation of open:closed arm entries in an elevated plus-maze as a measure of anxiety in the rat. J Neurosci Meth 1985;14:149–67. Rodrigues-Filho R, Takahashi RN. Antinociceptive effects induced by desipramine and fluoxetine are dissociated from their antidepressant or anxiolytic action in mice. Int J Neuropsychopharmacol 1999;2:263–9. Rossi A, Barraco A, Donda P. Fluoxetine: a review on evidence based medicine. Ann Gen Hosp Psychiatry 2004;3:2. Roy V, Chapillon P. Further evidences that risk assessment and object exploration behaviours are useful to evaluate emotional reactivity in rodents. Behav Brain Res 2004;154:439–48. Salchner P, Singewald N. Neuroanatomical substrates involved in the anxiogenic-like effect of acute fluoxetine treatment. Neuropharmacology 2002;43:1238–48. Silva MT, Alves CR, Santarem EM. Anxiogenic-like effect of acute and chronic fluoxetine on rats tested on the elevated plus-maze. Braz J Med Biol Res 1999;32:333–9.
D. Drapier et al. / Behavioural Brain Research 176 (2007) 202–209 [42] Silva RC, Brandao ML. Acute and chronic effects of gepirone and fluoxetine in rats tested in the elevated plus-maze: an ethological analysis. Pharmacol Biochem Behav 2000;65:209–16. [43] Sramek JJ, Zarotsky V, Cutler NR. Generalised anxiety disorder: treatment options. Drugs 2002;62:1635–48. [44] Stahl SM. Basic psychopharmacology of antidepressants. Part 1. Antidepressants have seven distinct mechanisms of action. J Clin Psychiatry 1998;59(Suppl 4):5–14.
209
[45] Stewart SH, Westra HA. Benzodiazepine side-effects: from the bench to the clinic. Curr Pharm Des 2002;8:1–3. [46] Troelsen KB, Nielsen EO, Mirza NR. Chronic treatment with duloxetine is necessary for an anxiolytic-like response in the mouse zero maze: the role of the serotonin transporter. Psychopharmacology (Berlin) 2005;181:741–50. [47] Varty GB, Morgan CA, Cohen-Williams ME, Coffin VL, Carey GJ. The gerbil elevated plus-maze. I. Behavioral characterization and pharmacological validation. Neuropsychopharmacology 2002;27:357–70.