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The differential effects of chronic imipramine or citalopram administration on physiological and behavioral outcomes in naïve mice
Behavioural Brain Research 245 (2013) 101–106
Contents lists available at SciVerse ScienceDirect
Behavioural Brain Research journal homepage: www.elsevier.com/locate/bbr
Short communication
The differential effects of chronic imipramine or citalopram administration on physiological and behavioral outcomes in naïve mice Tatyana Strekalova a,b,∗,1 , Daniel C. Anthony c,1 , Oleg Dolgov d,e , Konstantin Anokhin e , Aslan Kubatiev f , Harry M.W. Steinbusch b , Careen Schroeter a a
Maastricht Medical Center in Annadal, Department of Preventive Medicine, Becanusstraat 17 A0, 6216 BX Maastricht, Netherlands Department of Neuroscience, Maastricht University, Universiteitssingel 40, NL 6229 ER Maastricht, Netherlands c Department of Pharmacology, Oxford University, Mansfield Road, OX1 3QT Oxford, UK d Department of Molecular Cell Biology, Max-Planck Institute of Biophysical Chemistry, Am Fassberg 11, D-37077 Göttingen, Germany e Department of Neurobiology of Learning and Memory, Institute of Normal Physiology, P.K. Anokhin Institute of Normal Physiology and Department of Neurophysiology and Cognitive Sciences, National Research Center “Kurchatov Institute”, Baltyiskaia 8, 125315 Moscow, Russia f Institute of General Pathology and Pathophysiology, Baltyiskaia 8, 125315 Moscow, Russia b
h i g h l i g h t s I I I I I
Citalopram and imipramine alter behavior in naïve mice in the normal therapeutic range. Imipramine increases sucrose and water intake, and home cage activity in naïve mice. Imipramine impairs learning in the object recognition task in naïve mice. In naïve mice, citalopram increases body mass and disrupts object exploration. Both drugs need to be used with caution as reference standards in depression models.
a r t i c l e
i n f o
Article history: Received 30 January 2013 Received in revised form 8 February 2013 Accepted 12 February 2013 Available online 19 February 2013 Keywords: Animal models of depression Imipramine Citalopram Reference antidepressant Pharmacological validation Mouse
a b s t r a c t Tricyclics and selective serotonin reuptake inhibitors (SSRIs) are probably the most widely employed reference antidepressants in animal studies on depression. Using imipramine and citalopram, we sought to assess which drug would be more appropriate as pharmacological reference in paradigms of depression in C57BL6N mice by measuring their effect on liquid consumption, home cage activity, body weight and long-term memory in naïve animals treated with each compound at generally used dose of 15 mg/kg/day. Continuous logging of home cage movement, weekly monitoring of vertical activity in a novel cage, and body weight was recorded during four-week treatment period and for four weeks after discontinuation of the antidepressant; sucrose preference was evaluated at weekly intervals during drug administration. A novel object recognition memory test was performed in mice treated the antidepressants for two weeks. Compared to control, imipramine-treated mice displayed increased sucrose and water intake, as well as enhanced home-cage and novelty exploration activities, and reduced body weight. Imipramine also impaired learning in the object recognition task, but citalopram diminished object exploration sufficiently to invalidate the test. Citalopram-treated animals demonstrated no changes in a sucrose test and had elevated body mass. Thus basic physiological and behavioral outcomes in naïve mice were significantly altered by the chronic administration of imipramine and, to a lesser extent, citalopram. As altered variables are crucial for the evaluation of antidepressant-like effects in mice, our data suggest that, at commonly used doses, both drugs must be applied in mouse models of depression with caution. © 2013 Elsevier B.V. All rights reserved.
∗ Corresponding author at: School for Mental Health and Neuroscience, Department of Neuroscience, Maastricht University Universiteitssingel 40, NL 6229 ER Maastricht, Netherlands. Tel.: +31 43 38 84 110; fax: +31 43 36 71 096. E-mail address: [email protected] (T. Strekalova). 1 These authors equally contributed to this work. 0166-4328/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.bbr.2013.02.015
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The failure to discover new powerful antidepressants has raised important questions regarding the limitations of preclinical studies [1,2]. The failures may stem from a challenge to select the most appropriate reference drug and its dosage in Phase I trials with new pharmacotherapies [3,4]. Species-specific differences might be the primary reason for the distinct effects of investigational drugs in experimental animals compared to man [5]. For instance, studies in a transgenic rat that solely expresses human SERT have revealed species-specific selectivity of selective serotonin reuptake inhibitors (SSRIs) and tricyclics [6]. Furthermore, it is known that imipramine displays higher affinity for rat SERT than human SERT [7]. Despite this known phenomenon, tricyclics and SSRIs are often employed as reference treatment at a range of clinical doses in rodent studies in pre-clinical models of depression, and the results are commonly extrapolated to human conditions. Imipramine and citalopram are important and widely prescribed compounds as therapy for depression [2]. They are typically used in rodent models of depression at daily doses of 10–20 mg/kg for 2–8 weeks, which is meant to simulate a human equivalent therapeutic dosing regime [8–11]. However, the optimal dosing regime in human subjects can result in differential effects in animals. This issue has not been explored systematically, and this is especially true where prolonged dosing in mouse models of neuropsychiatric disorders is involved and lasting effects of treatment might be anticipated after discontinuation. Consequently, we sought to study the effects of chronic treatment of either imipramine or citalopram at the dose 15 mg/kg/day in naïve C57BL6N mice, and assess possible treatment effects after the termination of dosing. As ‘no effect on controls’ is a prerequisite for a reference drug, our goal was to determine whether tricyclics or SSRIs affect basic physiological and behavioral parameters in naïve animals. Three-month-old male C57BL/6N mice were used (for details on animals, housing and study design see Supplementary data). First, we investigated the effects of a 4-week-dosing regime; mice were treated, via drinking water, with either with imipramine (Sigma–Aldrich, Cambridge Soft Corporation, Cambridge, MA, USA) or citalopram (Lundbeck, Copenhagen, Denmark) as described elsewhere [12,13]; control mice were left untreated (n = 10 per group; see Supplementary data). From the 5th day of dosing, a sucrose preference test [13] was performed at weekly intervals for 4 weeks; mice had free choice, for 8 h, of bottles containing either a 1% sucrose solution in tap water or tap water (see Supplementary data). The consumption of both solutions and total intake of liquids was estimated by weighing the bottles. Preference for sucrose was calculated as [Volume of sucrose solution/Volume of sucrose solution + Volume of water] × 100%. During the 4-week treatment period, and a subsequent 4-week period of washout with no dosing, mean velocity of horizontal home cage movement was registered and calculated each hour using the infrared beam system and software of SAMAB [14]; it was averaged over 24 h and over a week. Weekly, exploratory rears were scored under red light during a 5-min period in the novel cage test, where a standard plastic cage (21 cm × 21 cm × 15 cm) was used [13] and body weight were measured weekly (see Supplementary data). Separately, we addressed the question of whether two-week antidepressant treatment interferes with learning in a two-day object recognition task (see Supplementary data). In this model, animals were exposed simultaneously to a familiar and a new object. The new object was placed in the “non-preferred” area of the observation cylinder to contrast behavioral manifestations of novelty exploration with an opposing competing motivator. Preference for the object was calculated as a percentage time of exploration for each object over the total duration of exploration: [Object exploration/Total exploration of both objects] × 100%. An increased preference in the exploration of a new object that has replaced the
former one in the “non-preferred” area from Day 1 to Day 2, was taken as recognition of a novel object and memory for the former one. Data were analyzed with GraphPad Prism version 5.00 for Windows (San Diego, CA, USA) using unpaired and paired t-test, one-way ANOVA, which was followed by a Tukey test and repeated measures ANOVA, where appropriate. The level of confidence was set at 95% (p < 0.05). There were significant differences between the groups in the overall comparison of absolute amounts of intake of sucrose solution on weeks one to four of dosing (week 1: p = 0.0255, F = 4.748, R2 = 0.3880; week 2: p = 0.0247, F = 4.759, R2 = 0.3891; week 3: p = 0.006, F = 7.022, R2 = 0.4903; week 4: p = 0.0025, F = 8.332, R2 = 0.5648, ANOVA; Fig. 1A). Post hoc testing revealed significant increase in this measure in imipramine-treated animals when compared to control mice on week two, three and four (p < 0.05, q = 5.447; p < 0.05, q = 6.181 and p < 0.05, q = 5.695, Tukey’s test, respectively; Fig. 1A). Citalopram-treated animals exhibited no change in sucrose solution intake at any time point during the experiment (p > 0.05, Tukey’s test). Multiple comparison tests revealed significant differences between experimental groups in the overall consumption of water intake in week two to four of treatment (week 2: p = 0.0071, F = 7.328, R2 = 0.4637; week 3: p = 0.0065, F = 7.435, R2 = 0.4701; week 4: p = 0.0073, F = 7.2569, R2 = 0.4602, ANOVA; Fig. 1B). Significant increases of water intake were found in imipramine-treated vs. control mice in weeks two, three and four (p < 0.05, q = 7.384; p < 0.05, q = 8.076 and p < 0.05, q = 7.523, Tukey’s test, respectively; Fig. 1B). Citalopram-treated animals displayed no change in this variable (p > 0.05, Tukey’s test). Overall comparison of sucrose preference revealed no significant differences between the groups suggesting a lack of the effect of the antidepressants on this parameter (p > 0.05, ANOVA). Repeated measures ANOVA test revealed significant differences in the sucrose preference between the weekly measurements in all three groups of mice which demonstrated that preference increased as the assays were repeated from a base line to week four of the experiment (p < 0.05 for each group; Fig. 1C). ANOVA revealed significant differences between the groups on the averaged speed of home cage horizontal activity and body weight that was registered during the treatment period (week 1: p = 0.0251, F = 4.759, R2 = 0.3882; week 2: p = 0.0036, F = 8.375, R2 = 0.5276; week 3: p = 0.004, F = 8.137, R2 = 0.5204; week 4: p = 0.0161, F = 5.505, R2 = 0.4233; Fig. 2A). Imipramine-treated mice had elevated speed of the home cage activity when compared to control mice on weeks two to four (p < 0.05, q = 4.823; p < 0.05, q = 4.663; p < 0.05, q = 3.760, Tukey’s test, respectively). Citalopramtreated mice exhibited no difference in home cage locomotion throughout the treatment (p > 0.05, Tukey’s test; Fig. 2A). After discontinuation of the drug delivery, the stimulatory effects of imipramine on home cage locomotion remained significant for two the first weeks (week 5: p = 0.0041, F = 8.101, R2 = 0.5193; week 6: p = 0.0182, F = 5.298, R2 = 0.4140; Fig. 2A), but not for subsequent weeks (p > 0.05, ANOVA). Imipraminetreated mice had elevated speed of the home cage activity, as compared to control mice at these time points (p < 0.05, q = 4.622 and p < 0.05, q = 3.821, Tukey’s test, respectively). Citalopramtreated mice exhibited no difference in the home cage locomotion compared with the non-treated control group during the aftertreatment period (p > 0.05, Tukey’s test; Fig. 2A). In the course of the treatment, the three groups exhibited significant differences in the number of exploratory rears (week 1: p = 0.0304, F = 4.055, R2 = 0.2526; week 2: p < 0.001, F = 19.12, R2 = 0.6144; week 3: p = 0.005, F = 10.63, R2 = 0.4697; week 4: p < 0.001, F = 17.35, R2 = 0.5911, ANOVA; Fig. 2B). Imipraminetreated mice had elevated novelty exploration scores at these time
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Fig. 1. Effects of imipramine and citalopram on sucrose preference. Compared to the non-treatment control, on weeks two to four of the drug administration, imipraminetreated mice significantly increased the intake of (A) the sucrose solution (*p < 0.05 vs. control group; Tukey’s test) and (B) water (*p < 0.05 vs. control group; Tukey’s test). However, (C) over the entire treatment period, there were no significant differences in sucrose preference between antidepressant and non-treated control groups (p > 0.05, Tukey’s test). There was a significant increase in sucrose preference from the baseline to week four in all groups of mice (p < 0.05 for each group; repeated measures ANOVA).
points, as compared to control mice (p < 0.05, q = 3.766; p < 0.05, q = 8.143; p < 0.05, q = 5.868; p < 0.05, q = 7.068, Tukey’s test, respectively). Citalopram-treated mice showed no differences in this measurement (p > 0.05, Tukey’s test; Fig. 2B). Repeated Measures ANOVA test revealed a significant decline in the number of
exploratory rears between weekly measurements (from baseline to week four) in non-treated and citalopram-treated mice (p < 0.05; Fig. 2B), but not in imipramine-treated animals (p > 0.05). After termination of dosing, the differences in exploratory rearing activity between the groups remained significant for the
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Fig. 2. Effects of imipramine and citalopram on home cage activity, novelty exploration, and body weight during and after dosing. (A) Imipramine treatment significantly elevated speed in the home cage activity, as compared to control mice, on weeks 2–4 of dosing and during the first two weeks after its termination; citalopram treatment animals did not affect this parameter at any time point (*p < 0.05 vs. control group, Tukey’s test). (B) Imipramine-treated mice displayed significantly increased exploratory activity in the novel cage, compared to control mice, on weeks 1–4 of dosing and during the first two weeks after its termination; this behavior was unaltered in the citalopram-treated animals (*p < 0.05 vs. control group, Tukey’s test). There was a significant decrease of the number of exploratory rears from baseline to week eight in non-treated and citalopram-treated mice (p < 0.05 for each group; repeated measures ANOVA), but not in animals that received imipramine (p > 0.05; repeated measures ANOVA). (C) Compared to control mice, imipramine treatment reduced body weight in weeks 2–4 of dosing, and citalopram treatment significantly increased body weight in weeks 3 and 4 of administration (*p < 0.05 vs. control group, Tukey’s test). After a discontinuation of the treatment, there were no significant changes in body weight in either antidepressant treatment group in comparison to control mice (p > 0.05 vs. control group, Tukey’s test).
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two first weeks (week 5: p < 0.001, F = 14.50, R2 = 0.5472; week 6: p = 0.029, F = 4.120, R2 = 0.2556, ANOVA; Fig. 2B), but not during the subsequent weeks of the experiment (p > 0.05, ANOVA). Novel cage exploratory scores were elevated in imipramine-treated mice, as compared to control mice at these time points (p < 0.05, q = 6.435 and p < 0.05, q = 4.019, Tukey’s test, respectively). Novel cage exploration in citalopram-treated mice was unchanged compared to controls (p > 0.05, Tukey’s test, Fig. 2B). During the treatment period, there were significant differences in body weight between experimental groups (week 1: p = 0.005, F = 7.205, R2 = 0.446; week 2: p = 0.004, F = 12.78, R2 = 0.601; week 3: p < 0.001, F = 13.15, R2 = 0.65; week 4: p = 0.0002, F = 13.776, R2 = 0.605; ANOVA, Fig. 2B). Imipramine-treated mice had reduced body weight at weeks two, three and four, as compared to control mice (p < 0.05, q = 4.83; p < 0.05, q = 4.204; p < 0.05, q = 3.724, Tukey’s test, respectively). Citalopram-treated mice showed significant gain of body mass in week three and four (p < 0.05, q = 4.078 and p < 0.05, q = 3.697, respectively, Tukey’s test; Fig. 2B). During the post-treatment period, overall group differences in body weight lasted for all four weeks (p < 0.05, ANOVA), while post hoc analysis revealed no difference between the treatment groups compared to control mice (p > 0.05; Tukey’s test). As differences in exploration might interfere with the evaluation of mouse performance in an object recognition memory task, the potential effects of treatment on its total duration was assessed on Day 1 of the test. ANOVA revealed a significant difference between the three groups (p = 0.00691, F = 6.08, R2 = 0.308). A 2-week treatment with imipramine did not change exploratory behavior (p > 0.05, Tukey’s test; Fig. 3A), compared to the control group, but the administration of citalopram resulted in a significant reduction in the total duration of object exploration (p < 0.05, q = 4.204, Tukey’s test), therefore, this group of mice was excluded from a subsequent experiment as its outcome could not be interpreted in this learning paradigm. On day 2 of the object recognition memory task, preference for the exploration of the “non-preferred” area, where the new object is placed, was significantly elevated in control mice (p = 0.0045, t = 3.332, df = 9, R = 0.5508, paired t-test; Fig. 3B), indicating memory of the object in the preferred area. On Day 2, the imipraminetreated group of animals showed a significantly lower preference for the new object compared to the control group (p = 0.0037, t = 3.339, df = 18, unpaired t-test). Importantly, initial preference to the exploration in a “non-preferred” area evaluated on Day 1 was unchanged in imipramine-treated mice (p > 0.05, t-test; Fig. 3B). Thus the difference observed between the groups on Day 2 cannot be attributed to general effects on exploratory behavior, and is most likely to be due to a memory impairment in this group. Together, our study has revealed that profound changes in a number of physiological and behavioral outcomes are induced in naïve C57BL6N mice by an antidepressant treatment that is typically used as a reference in pre-clinical studies on depression. It should be noted that the C57BL6 mouse is the most common line used in psychopharmacological research [4,8,10,21]. Because imipramine-treated mice displayed an increase in sucrose and water intake, lasting enhancement of the home-cage activity, decreased body weight and disrupted long-term memory in the object recognition test, this drug cannot be regarded as an optimal pharmacological reference in some common mouse models of depression, e.g., chronic mild stress and sucrose test. Citalopram did not affect drinking and locomotor outcomes, but increased body mass and affected object exploration in a novel situation. However, it might be considered a more suitable antidepressant standard than imipramine for many, but not all, depression paradigms in C57BL6 mice. The use of imipramine, but not citalopram, in mouse tests, which are reliant on the drinking of palatable solutions as a measure of hedonic sensitivity, can be compromised by its
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Fig. 3. Effects of citalopram and imipramine on object exploration and recognition. (A) Compared to the non-treated control animals, citalopram treatment reduced total exploration time compared to control (*p < 0.05 vs. non-treated control group; Tukey’s test); imipramine had no effect of this measure (p > 0.05; Tukey’s test). (B) On Day 1, the preference to explore an object at the “non-preferred” site was similar in control and imipramine-treated mice (p > 0.05; unpaired t-test). On Day 2, non-treated controls demonstrated a significant increase in preference to explore a new object in the “non-preferred” site compared to baseline (*p < 0.05; paired ttest). Imipramine-treated mice had significantly reduced time spent exploring the novel object as compared to control animals (# p < 0.05 vs. non-treated control group; unpaired t-test). Each column represents the mean ± SEM. Con: non-treated control group; Imi: imipramine-treated group; Cit: citalopram-treated group.
non-specific stimulatory effect on liquid intake. The effects of two antidepressants on locomotion and body weight in mice are similar to results obtained on rats and other small rodents [15–20]. Hitherto no data was available describing the lasting behavioral effects of imipramine and citalopram during the post-treatment period in small rodents, and, as our results show, the effects of the antidepressants persist. This must be taken into account during experimental design where might be expected that the changes observed in naïve animals will also occur in animals with an experimentally induced depression-like syndrome and, thus, confound the evaluation of their phenotype [3,9,22]. Together, our findings establish the limitations for the application of these two perhaps most commonly used antidepressants in pre-clinical models of depression, where the accuracy is determined by the elements sensitive to the treatment employed. Our results also suggest that an elevated metabolic rate might be expected in imipramine-treated mice. Chronic administration of imipramine was reported to increase oxidative energy metabolism and stimulate activity of the cytochrome proteins, while the effects of SSRIs were opposite [11,23,24]. Hence, alteration of oxidative energy metabolism may underpin some of the actions of imipramine and citalopram reported in this study in naïve mice. In a wider context, the current study further demonstrates how important it is to consider species-specific differences in the response antidepressant dosing regimes between humans and small rodents, in order to avoid potential misleading conclusions that might arise in subsequent clinical trials.
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We acknowledge the important contribution of Prof. Peter Gruss, Dr. Barbara I. Mayer, technical support of Joao Nunes and Diogo Borros, as well as financial support of NARSAD, USA (17611 to T.S.), DFG STR 699–1/2 to T.S. and RFBR 11-04-01411.
[12]
[13]
Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.bbr.2013.02.015.
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Contents lists available at SciVerse ScienceDirect
Behavioural Brain Research journal homepage: www.elsevier.com/locate/bbr
Short communication
The differential effects of chronic imipramine or citalopram administration on physiological and behavioral outcomes in naïve mice Tatyana Strekalova a,b,∗,1 , Daniel C. Anthony c,1 , Oleg Dolgov d,e , Konstantin Anokhin e , Aslan Kubatiev f , Harry M.W. Steinbusch b , Careen Schroeter a a
Maastricht Medical Center in Annadal, Department of Preventive Medicine, Becanusstraat 17 A0, 6216 BX Maastricht, Netherlands Department of Neuroscience, Maastricht University, Universiteitssingel 40, NL 6229 ER Maastricht, Netherlands c Department of Pharmacology, Oxford University, Mansfield Road, OX1 3QT Oxford, UK d Department of Molecular Cell Biology, Max-Planck Institute of Biophysical Chemistry, Am Fassberg 11, D-37077 Göttingen, Germany e Department of Neurobiology of Learning and Memory, Institute of Normal Physiology, P.K. Anokhin Institute of Normal Physiology and Department of Neurophysiology and Cognitive Sciences, National Research Center “Kurchatov Institute”, Baltyiskaia 8, 125315 Moscow, Russia f Institute of General Pathology and Pathophysiology, Baltyiskaia 8, 125315 Moscow, Russia b
h i g h l i g h t s I I I I I
Citalopram and imipramine alter behavior in naïve mice in the normal therapeutic range. Imipramine increases sucrose and water intake, and home cage activity in naïve mice. Imipramine impairs learning in the object recognition task in naïve mice. In naïve mice, citalopram increases body mass and disrupts object exploration. Both drugs need to be used with caution as reference standards in depression models.
a r t i c l e
i n f o
Article history: Received 30 January 2013 Received in revised form 8 February 2013 Accepted 12 February 2013 Available online 19 February 2013 Keywords: Animal models of depression Imipramine Citalopram Reference antidepressant Pharmacological validation Mouse
a b s t r a c t Tricyclics and selective serotonin reuptake inhibitors (SSRIs) are probably the most widely employed reference antidepressants in animal studies on depression. Using imipramine and citalopram, we sought to assess which drug would be more appropriate as pharmacological reference in paradigms of depression in C57BL6N mice by measuring their effect on liquid consumption, home cage activity, body weight and long-term memory in naïve animals treated with each compound at generally used dose of 15 mg/kg/day. Continuous logging of home cage movement, weekly monitoring of vertical activity in a novel cage, and body weight was recorded during four-week treatment period and for four weeks after discontinuation of the antidepressant; sucrose preference was evaluated at weekly intervals during drug administration. A novel object recognition memory test was performed in mice treated the antidepressants for two weeks. Compared to control, imipramine-treated mice displayed increased sucrose and water intake, as well as enhanced home-cage and novelty exploration activities, and reduced body weight. Imipramine also impaired learning in the object recognition task, but citalopram diminished object exploration sufficiently to invalidate the test. Citalopram-treated animals demonstrated no changes in a sucrose test and had elevated body mass. Thus basic physiological and behavioral outcomes in naïve mice were significantly altered by the chronic administration of imipramine and, to a lesser extent, citalopram. As altered variables are crucial for the evaluation of antidepressant-like effects in mice, our data suggest that, at commonly used doses, both drugs must be applied in mouse models of depression with caution. © 2013 Elsevier B.V. All rights reserved.
∗ Corresponding author at: School for Mental Health and Neuroscience, Department of Neuroscience, Maastricht University Universiteitssingel 40, NL 6229 ER Maastricht, Netherlands. Tel.: +31 43 38 84 110; fax: +31 43 36 71 096. E-mail address: [email protected] (T. Strekalova). 1 These authors equally contributed to this work. 0166-4328/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.bbr.2013.02.015
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The failure to discover new powerful antidepressants has raised important questions regarding the limitations of preclinical studies [1,2]. The failures may stem from a challenge to select the most appropriate reference drug and its dosage in Phase I trials with new pharmacotherapies [3,4]. Species-specific differences might be the primary reason for the distinct effects of investigational drugs in experimental animals compared to man [5]. For instance, studies in a transgenic rat that solely expresses human SERT have revealed species-specific selectivity of selective serotonin reuptake inhibitors (SSRIs) and tricyclics [6]. Furthermore, it is known that imipramine displays higher affinity for rat SERT than human SERT [7]. Despite this known phenomenon, tricyclics and SSRIs are often employed as reference treatment at a range of clinical doses in rodent studies in pre-clinical models of depression, and the results are commonly extrapolated to human conditions. Imipramine and citalopram are important and widely prescribed compounds as therapy for depression [2]. They are typically used in rodent models of depression at daily doses of 10–20 mg/kg for 2–8 weeks, which is meant to simulate a human equivalent therapeutic dosing regime [8–11]. However, the optimal dosing regime in human subjects can result in differential effects in animals. This issue has not been explored systematically, and this is especially true where prolonged dosing in mouse models of neuropsychiatric disorders is involved and lasting effects of treatment might be anticipated after discontinuation. Consequently, we sought to study the effects of chronic treatment of either imipramine or citalopram at the dose 15 mg/kg/day in naïve C57BL6N mice, and assess possible treatment effects after the termination of dosing. As ‘no effect on controls’ is a prerequisite for a reference drug, our goal was to determine whether tricyclics or SSRIs affect basic physiological and behavioral parameters in naïve animals. Three-month-old male C57BL/6N mice were used (for details on animals, housing and study design see Supplementary data). First, we investigated the effects of a 4-week-dosing regime; mice were treated, via drinking water, with either with imipramine (Sigma–Aldrich, Cambridge Soft Corporation, Cambridge, MA, USA) or citalopram (Lundbeck, Copenhagen, Denmark) as described elsewhere [12,13]; control mice were left untreated (n = 10 per group; see Supplementary data). From the 5th day of dosing, a sucrose preference test [13] was performed at weekly intervals for 4 weeks; mice had free choice, for 8 h, of bottles containing either a 1% sucrose solution in tap water or tap water (see Supplementary data). The consumption of both solutions and total intake of liquids was estimated by weighing the bottles. Preference for sucrose was calculated as [Volume of sucrose solution/Volume of sucrose solution + Volume of water] × 100%. During the 4-week treatment period, and a subsequent 4-week period of washout with no dosing, mean velocity of horizontal home cage movement was registered and calculated each hour using the infrared beam system and software of SAMAB [14]; it was averaged over 24 h and over a week. Weekly, exploratory rears were scored under red light during a 5-min period in the novel cage test, where a standard plastic cage (21 cm × 21 cm × 15 cm) was used [13] and body weight were measured weekly (see Supplementary data). Separately, we addressed the question of whether two-week antidepressant treatment interferes with learning in a two-day object recognition task (see Supplementary data). In this model, animals were exposed simultaneously to a familiar and a new object. The new object was placed in the “non-preferred” area of the observation cylinder to contrast behavioral manifestations of novelty exploration with an opposing competing motivator. Preference for the object was calculated as a percentage time of exploration for each object over the total duration of exploration: [Object exploration/Total exploration of both objects] × 100%. An increased preference in the exploration of a new object that has replaced the
former one in the “non-preferred” area from Day 1 to Day 2, was taken as recognition of a novel object and memory for the former one. Data were analyzed with GraphPad Prism version 5.00 for Windows (San Diego, CA, USA) using unpaired and paired t-test, one-way ANOVA, which was followed by a Tukey test and repeated measures ANOVA, where appropriate. The level of confidence was set at 95% (p < 0.05). There were significant differences between the groups in the overall comparison of absolute amounts of intake of sucrose solution on weeks one to four of dosing (week 1: p = 0.0255, F = 4.748, R2 = 0.3880; week 2: p = 0.0247, F = 4.759, R2 = 0.3891; week 3: p = 0.006, F = 7.022, R2 = 0.4903; week 4: p = 0.0025, F = 8.332, R2 = 0.5648, ANOVA; Fig. 1A). Post hoc testing revealed significant increase in this measure in imipramine-treated animals when compared to control mice on week two, three and four (p < 0.05, q = 5.447; p < 0.05, q = 6.181 and p < 0.05, q = 5.695, Tukey’s test, respectively; Fig. 1A). Citalopram-treated animals exhibited no change in sucrose solution intake at any time point during the experiment (p > 0.05, Tukey’s test). Multiple comparison tests revealed significant differences between experimental groups in the overall consumption of water intake in week two to four of treatment (week 2: p = 0.0071, F = 7.328, R2 = 0.4637; week 3: p = 0.0065, F = 7.435, R2 = 0.4701; week 4: p = 0.0073, F = 7.2569, R2 = 0.4602, ANOVA; Fig. 1B). Significant increases of water intake were found in imipramine-treated vs. control mice in weeks two, three and four (p < 0.05, q = 7.384; p < 0.05, q = 8.076 and p < 0.05, q = 7.523, Tukey’s test, respectively; Fig. 1B). Citalopram-treated animals displayed no change in this variable (p > 0.05, Tukey’s test). Overall comparison of sucrose preference revealed no significant differences between the groups suggesting a lack of the effect of the antidepressants on this parameter (p > 0.05, ANOVA). Repeated measures ANOVA test revealed significant differences in the sucrose preference between the weekly measurements in all three groups of mice which demonstrated that preference increased as the assays were repeated from a base line to week four of the experiment (p < 0.05 for each group; Fig. 1C). ANOVA revealed significant differences between the groups on the averaged speed of home cage horizontal activity and body weight that was registered during the treatment period (week 1: p = 0.0251, F = 4.759, R2 = 0.3882; week 2: p = 0.0036, F = 8.375, R2 = 0.5276; week 3: p = 0.004, F = 8.137, R2 = 0.5204; week 4: p = 0.0161, F = 5.505, R2 = 0.4233; Fig. 2A). Imipramine-treated mice had elevated speed of the home cage activity when compared to control mice on weeks two to four (p < 0.05, q = 4.823; p < 0.05, q = 4.663; p < 0.05, q = 3.760, Tukey’s test, respectively). Citalopramtreated mice exhibited no difference in home cage locomotion throughout the treatment (p > 0.05, Tukey’s test; Fig. 2A). After discontinuation of the drug delivery, the stimulatory effects of imipramine on home cage locomotion remained significant for two the first weeks (week 5: p = 0.0041, F = 8.101, R2 = 0.5193; week 6: p = 0.0182, F = 5.298, R2 = 0.4140; Fig. 2A), but not for subsequent weeks (p > 0.05, ANOVA). Imipraminetreated mice had elevated speed of the home cage activity, as compared to control mice at these time points (p < 0.05, q = 4.622 and p < 0.05, q = 3.821, Tukey’s test, respectively). Citalopramtreated mice exhibited no difference in the home cage locomotion compared with the non-treated control group during the aftertreatment period (p > 0.05, Tukey’s test; Fig. 2A). In the course of the treatment, the three groups exhibited significant differences in the number of exploratory rears (week 1: p = 0.0304, F = 4.055, R2 = 0.2526; week 2: p < 0.001, F = 19.12, R2 = 0.6144; week 3: p = 0.005, F = 10.63, R2 = 0.4697; week 4: p < 0.001, F = 17.35, R2 = 0.5911, ANOVA; Fig. 2B). Imipraminetreated mice had elevated novelty exploration scores at these time
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Fig. 1. Effects of imipramine and citalopram on sucrose preference. Compared to the non-treatment control, on weeks two to four of the drug administration, imipraminetreated mice significantly increased the intake of (A) the sucrose solution (*p < 0.05 vs. control group; Tukey’s test) and (B) water (*p < 0.05 vs. control group; Tukey’s test). However, (C) over the entire treatment period, there were no significant differences in sucrose preference between antidepressant and non-treated control groups (p > 0.05, Tukey’s test). There was a significant increase in sucrose preference from the baseline to week four in all groups of mice (p < 0.05 for each group; repeated measures ANOVA).
points, as compared to control mice (p < 0.05, q = 3.766; p < 0.05, q = 8.143; p < 0.05, q = 5.868; p < 0.05, q = 7.068, Tukey’s test, respectively). Citalopram-treated mice showed no differences in this measurement (p > 0.05, Tukey’s test; Fig. 2B). Repeated Measures ANOVA test revealed a significant decline in the number of
exploratory rears between weekly measurements (from baseline to week four) in non-treated and citalopram-treated mice (p < 0.05; Fig. 2B), but not in imipramine-treated animals (p > 0.05). After termination of dosing, the differences in exploratory rearing activity between the groups remained significant for the
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Fig. 2. Effects of imipramine and citalopram on home cage activity, novelty exploration, and body weight during and after dosing. (A) Imipramine treatment significantly elevated speed in the home cage activity, as compared to control mice, on weeks 2–4 of dosing and during the first two weeks after its termination; citalopram treatment animals did not affect this parameter at any time point (*p < 0.05 vs. control group, Tukey’s test). (B) Imipramine-treated mice displayed significantly increased exploratory activity in the novel cage, compared to control mice, on weeks 1–4 of dosing and during the first two weeks after its termination; this behavior was unaltered in the citalopram-treated animals (*p < 0.05 vs. control group, Tukey’s test). There was a significant decrease of the number of exploratory rears from baseline to week eight in non-treated and citalopram-treated mice (p < 0.05 for each group; repeated measures ANOVA), but not in animals that received imipramine (p > 0.05; repeated measures ANOVA). (C) Compared to control mice, imipramine treatment reduced body weight in weeks 2–4 of dosing, and citalopram treatment significantly increased body weight in weeks 3 and 4 of administration (*p < 0.05 vs. control group, Tukey’s test). After a discontinuation of the treatment, there were no significant changes in body weight in either antidepressant treatment group in comparison to control mice (p > 0.05 vs. control group, Tukey’s test).
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two first weeks (week 5: p < 0.001, F = 14.50, R2 = 0.5472; week 6: p = 0.029, F = 4.120, R2 = 0.2556, ANOVA; Fig. 2B), but not during the subsequent weeks of the experiment (p > 0.05, ANOVA). Novel cage exploratory scores were elevated in imipramine-treated mice, as compared to control mice at these time points (p < 0.05, q = 6.435 and p < 0.05, q = 4.019, Tukey’s test, respectively). Novel cage exploration in citalopram-treated mice was unchanged compared to controls (p > 0.05, Tukey’s test, Fig. 2B). During the treatment period, there were significant differences in body weight between experimental groups (week 1: p = 0.005, F = 7.205, R2 = 0.446; week 2: p = 0.004, F = 12.78, R2 = 0.601; week 3: p < 0.001, F = 13.15, R2 = 0.65; week 4: p = 0.0002, F = 13.776, R2 = 0.605; ANOVA, Fig. 2B). Imipramine-treated mice had reduced body weight at weeks two, three and four, as compared to control mice (p < 0.05, q = 4.83; p < 0.05, q = 4.204; p < 0.05, q = 3.724, Tukey’s test, respectively). Citalopram-treated mice showed significant gain of body mass in week three and four (p < 0.05, q = 4.078 and p < 0.05, q = 3.697, respectively, Tukey’s test; Fig. 2B). During the post-treatment period, overall group differences in body weight lasted for all four weeks (p < 0.05, ANOVA), while post hoc analysis revealed no difference between the treatment groups compared to control mice (p > 0.05; Tukey’s test). As differences in exploration might interfere with the evaluation of mouse performance in an object recognition memory task, the potential effects of treatment on its total duration was assessed on Day 1 of the test. ANOVA revealed a significant difference between the three groups (p = 0.00691, F = 6.08, R2 = 0.308). A 2-week treatment with imipramine did not change exploratory behavior (p > 0.05, Tukey’s test; Fig. 3A), compared to the control group, but the administration of citalopram resulted in a significant reduction in the total duration of object exploration (p < 0.05, q = 4.204, Tukey’s test), therefore, this group of mice was excluded from a subsequent experiment as its outcome could not be interpreted in this learning paradigm. On day 2 of the object recognition memory task, preference for the exploration of the “non-preferred” area, where the new object is placed, was significantly elevated in control mice (p = 0.0045, t = 3.332, df = 9, R = 0.5508, paired t-test; Fig. 3B), indicating memory of the object in the preferred area. On Day 2, the imipraminetreated group of animals showed a significantly lower preference for the new object compared to the control group (p = 0.0037, t = 3.339, df = 18, unpaired t-test). Importantly, initial preference to the exploration in a “non-preferred” area evaluated on Day 1 was unchanged in imipramine-treated mice (p > 0.05, t-test; Fig. 3B). Thus the difference observed between the groups on Day 2 cannot be attributed to general effects on exploratory behavior, and is most likely to be due to a memory impairment in this group. Together, our study has revealed that profound changes in a number of physiological and behavioral outcomes are induced in naïve C57BL6N mice by an antidepressant treatment that is typically used as a reference in pre-clinical studies on depression. It should be noted that the C57BL6 mouse is the most common line used in psychopharmacological research [4,8,10,21]. Because imipramine-treated mice displayed an increase in sucrose and water intake, lasting enhancement of the home-cage activity, decreased body weight and disrupted long-term memory in the object recognition test, this drug cannot be regarded as an optimal pharmacological reference in some common mouse models of depression, e.g., chronic mild stress and sucrose test. Citalopram did not affect drinking and locomotor outcomes, but increased body mass and affected object exploration in a novel situation. However, it might be considered a more suitable antidepressant standard than imipramine for many, but not all, depression paradigms in C57BL6 mice. The use of imipramine, but not citalopram, in mouse tests, which are reliant on the drinking of palatable solutions as a measure of hedonic sensitivity, can be compromised by its
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Fig. 3. Effects of citalopram and imipramine on object exploration and recognition. (A) Compared to the non-treated control animals, citalopram treatment reduced total exploration time compared to control (*p < 0.05 vs. non-treated control group; Tukey’s test); imipramine had no effect of this measure (p > 0.05; Tukey’s test). (B) On Day 1, the preference to explore an object at the “non-preferred” site was similar in control and imipramine-treated mice (p > 0.05; unpaired t-test). On Day 2, non-treated controls demonstrated a significant increase in preference to explore a new object in the “non-preferred” site compared to baseline (*p < 0.05; paired ttest). Imipramine-treated mice had significantly reduced time spent exploring the novel object as compared to control animals (# p < 0.05 vs. non-treated control group; unpaired t-test). Each column represents the mean ± SEM. Con: non-treated control group; Imi: imipramine-treated group; Cit: citalopram-treated group.
non-specific stimulatory effect on liquid intake. The effects of two antidepressants on locomotion and body weight in mice are similar to results obtained on rats and other small rodents [15–20]. Hitherto no data was available describing the lasting behavioral effects of imipramine and citalopram during the post-treatment period in small rodents, and, as our results show, the effects of the antidepressants persist. This must be taken into account during experimental design where might be expected that the changes observed in naïve animals will also occur in animals with an experimentally induced depression-like syndrome and, thus, confound the evaluation of their phenotype [3,9,22]. Together, our findings establish the limitations for the application of these two perhaps most commonly used antidepressants in pre-clinical models of depression, where the accuracy is determined by the elements sensitive to the treatment employed. Our results also suggest that an elevated metabolic rate might be expected in imipramine-treated mice. Chronic administration of imipramine was reported to increase oxidative energy metabolism and stimulate activity of the cytochrome proteins, while the effects of SSRIs were opposite [11,23,24]. Hence, alteration of oxidative energy metabolism may underpin some of the actions of imipramine and citalopram reported in this study in naïve mice. In a wider context, the current study further demonstrates how important it is to consider species-specific differences in the response antidepressant dosing regimes between humans and small rodents, in order to avoid potential misleading conclusions that might arise in subsequent clinical trials.
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We acknowledge the important contribution of Prof. Peter Gruss, Dr. Barbara I. Mayer, technical support of Joao Nunes and Diogo Borros, as well as financial support of NARSAD, USA (17611 to T.S.), DFG STR 699–1/2 to T.S. and RFBR 11-04-01411.
[12]
[13]
Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.bbr.2013.02.015.
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