Influence of enrichment on behavioral and neurogenic effects of antidepressants in Wistar rats submitted to repeated forced swim test

Progress in Neuro-Psychopharmacology & Biological Psychiatry 58 (2015) 15–21

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Progress in Neuro-Psychopharmacology & Biological Psychiatry

Influence of enrichment on behavioral and neurogenic effects of antidepressants in Wistar rats submitted to repeated forced swim test Fernanda Possamai, Juliano dos Santos, Thais Walber, Juliana C. Marcon, Tiago Souza dos Santos, Cilene Lino de Oliveira ⁎ Departamento de Ciências Fisiológicas, Centro de Ciências Biológicas, Universidade Federal de Santa Catarina—UFSC, Campus Trindade, Florianópolis, 88040-900 SC, Brazil

a r t i c l e

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Article history: Received 3 June 2014 Received in revised form 17 October 2014 Accepted 22 October 2014 Available online 6 December 2014 Keywords: Animal model Antidepressant Behavior Environment Hippocampal neurogenesis

a b s t r a c t Repeated forced swimming test (rFST) may detect gradual effects of antidepressants in adult rats. Antidepressants, as enrichment, affected behavior and neurogenesis in rats. However, the influence of enrichment on behavioral and neurogenic effects of antidepressants is unknown. Here, effects of antidepressants on rFST and hippocampal neurogenesis were investigated in rats under enriched conditions. Behaviors of male Wistar rats, housed from weaning in standard (SE) or enriched environment (EE), were registered during rFST. The rFST consisted of 15 min of swimming (pretest) followed by 5 min of swimming in the first (test), seventh (retest 1) and fourteenth (retest 2) days after pretest. One hour before the test, rats received an intraperitoneal injection of saline (1 ml/kg), fluoxetine (2.5 mg/kg) or imipramine (2.5 or 5 mg/kg). These treatments were performed daily until the day of the retest 2. After retest 2, rats were euthanized for the identification of markers for neurogenesis in the hippocampus. Fluoxetine or imipramine decreased immobility in retests 1 and 2, as compared to saline. EE abolished these differences. In EE, fluoxetine or imipramine (5 mg/kg) reduced immobility time in retest 2, as compared to the test. Independent of the housing conditions, fluoxetine and imipramine (5 mg/kg) increased the ratio of immature neurons per progenitor cell in the hippocampus. In summary, antidepressants or enrichment counteracted the high immobility in rFST. Enrichment changed the effects of antidepressants in rFST depending on the type, and the dose of a substance but failed to change neurogenesis in control or antidepressant treated-rats. Effects of antidepressants and enrichment on rFST seemed neurogenesisindependent. © 2014 Published by Elsevier Inc.

1. Introduction A process to develop new substances to treat Major Depression requires innovative translational research and more predictive animal models (e.g., Belzung, 2014). Refinement of the current animal models may be a strategy to find innovative ones (e.g., Berton et al., 2012). The use of rat forced swimming test (FST, Porsolt et al., 1978) is frequent in the literature because it is considered uncomplicated, inexpensive, reliable across laboratories, sensitive and relatively selective for detecting substances with potential activity as antidepressants. Several modifications of the FST in rats were tried in order to keep the valuable characteristics of it while overcoming some of its negative aspects Abbreviations: DCX, doublecortin; DCX-ir, Doublecortin immunoreactive cell; EE, enrichedenvironment; FST, forcedswimming test; Ki-67-ir,Ki-67 immunoreactive nucleus; NRIs, noradrenaline reuptake inhibitors; NSRIs, non-selective inhibitors of monoamine reuptake; PND, postnatal day; rFST, repeated Forced swimming test; SE, standard environment; SSRIs, selective serotonin reuptake inhibitors. ⁎ Corresponding author at: Departamento de Ciências Fisiológicas, Centro de Ciências Biológicas, Universidade Federal de Santa Catarina, Campus Universitário Trindade, 88049-900 Florianopolis, SC, Brazil. Tel.: +55 48 3721 4689; fax: +55 48 3721 9672. E-mail address: [email protected] (C. Lino de Oliveira).

http://dx.doi.org/10.1016/j.pnpbp.2014.10.017 0278-5846/© 2014 Published by Elsevier Inc.

(Borsini et al., 1989; Cryan et al., 2005; Dal-Zotto et al., 2000; Detke et al., 1997; Kitamura et al., 2004; Vieira et al., 2008). For example, a modified version of FST in rats (Cryan et al., 2005; Detke et al., 1995, 1997; Lucki, 1997) allowed for detection of substances such as selective serotonin reuptake inhibitors (SSRIs) that were ineffective in the classical protocol developed by Porsolt et al. (1978). In addition, modified FST in rats discriminated between the effects of SSRIs and noradrenaline reuptake inhibitors (NRIs) after subacute (Detke et al., 1995) or chronic treatment (Cryan et al., 2005; Detke et al., 1997). Subsequent modifications in FST provided conditions for the discrimination of antidepressants from substances with psychostimulant properties (such as caffeine, Vieira et al., 2008) or for detecting gradual effects of low doses of antidepressants over time (repeated FST, Mezadri et al., 2011). Similar to the modified FST (e.g. Detke et al., 1995), the repeated FST (Mezadri et al., 2011) consisted of placing the rat into a tank filled with water for 15-min on the first experimental day (pretest) followed by a subsequent 5-min of forced swimming session 24 h later (test). In addition, the test was then repeated on the seventh (retest 1) and fourteenth (retest 2) days after the pretest (Mezadri et al., 2011). In these sessions, rats adopted a typical posture of immobility (floating in the water, making only minimal movement necessary to keep the head above water)

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after vigorous struggle and alternated it with climbing and swimming movements (Lino-de-Oliveira et al., 2005; Mezadri et al., 2011; Porsolt et al., 1978). Repetition shortened latency to immobility and increased endurance of immobility in the retests as compared to the test (Mezadri et al., 2011). Antidepressant treatment counteracted the effects of repetition (Gutiérrez-García and Contreras, 2009; Mezadri et al., 2011). Since the pharmacological treatment began 1 h prior the test, and was repeated daily until retest 2, there was three different opportunities for the evaluation of drug effect in the same group of rats (Gutiérrez-García and Contreras, 2009; Mezadri et al., 2011). Therefore, the within-subject analysis allowed for using a smaller number of rats compared to the standard protocols because it might detect the effects of short and long-term treatments in a single group of rats. In addition, repeated FST provided an opportunity to compare the onset of action for different treatments. Despite the potential of repeated FST to detect new antidepressants, the mechanisms underlying antidepressant action in this test remain elusive. In other behavioral tests, hippocampal neurogenesis seemed a requirement to the effects of antidepressants (Santarelli et al., 2003). In addition, antidepressants increased hippocampal neurogenesis after long-term treatment (e.g. Keilhoff et al., 2006; Malberg et al., 2000; Pinnock et al., 2009). Therefore, the present work investigated the correlation between behavior in repeated FST and markers of proliferation and immature neurons in the hippocampus of adult rats. Hippocampal neurogenesis, as well as behavior of adult rats were regulated by changes in the environment (Bjørnebekk et al., 2006, 2008; Brenes et al., 2008; Brenes-Saenz et al., 2006; Gutiérrez-García and Contreras, 2009; Simpson et al., 2012). For that reason, in the present work pharmacological treatments were performed in rats housed in standard or enriched environment. Previously (Gutiérrez-García and Contreras, 2009; Mezadri et al., 2011), the low doses of fluoxetine or imipramine selected for this study reduced immobility in repeated FST. Factorial analysis suggested that active behaviors of rats in repeated FST could discriminate between distinct classes of antidepressants (Mezadri et al., 2011). Therefore, active behaviors were also scored in the present study. 2. Method 2.1. Animals All rats (n = 80) used in this study were supplied by the central vivarium facilities of the Federal University of Santa Catarina, and all procedures were previously approved by the local Committee for Ethics in Animal Research (CEUA—UFSC, 158/CEUA/PRPE/2011). Male Wistar rats, all 21 days old (post-weaning, postnatal day 21, PND21) were housed 4-per-cage, under standard conditions of temperature (21 ± 1 °C), on reversed 12 h–12 h light–dark cycle (lights on at 6 p.m.) and with ad libitum access to food (Nuvital®) and water during all experimental period. Reversed light–dark cycle allows for experimentation in rat's active period (e.g. Prager et al., 2011). Behavioral experiments (injections and swimming sessions) were performed between 8 a.m. and noon (i.e. during the dark phase of the reversed light–dark cycle). During the experimental period, rats were housed in standard (SE, cages with 41 cm length × 34 cm wide × and 16 cm height) or in enriched (EE) environments. EE consisted of two different cages of plexiglass (the large one with 55.5 cm length × 36.5 cm wide × 40.3 cm height and a small one with 45.7 cm length × 28 cm wide × 32.6 cm height) connected by a PVC tube (100 mm diameter). Cages were lined with sawdust and contained several toys such as plastic tubing, small balls, clappers, rope, ramps and toilet paper tubes to shred. The food and water devices were kept in a box and the toys in another. 2.2. Drugs and injections Fluoxetine (Sigma-Aldrich Inc., St. Louis, USA; dose 2.5 mg/kg) and imipramine (Sigma-Aldrich Inc., St. Louis, USA; dose 2.5 or 5 mg/kg)

were dissolved in 0.9% NaCl solution (injected in the control group as well) and administered intraperitoneally (IP, 1 ml/kg). These substances and doses were selected based on the literature (Gutiérrez-García and Contreras, 2009; Mezadri et al., 2011). 2.3. Experimental design and forced swimming procedures On the first two days in the laboratory (from PND21 to PND23), all rats were kept in SE for adaptation to laboratory conditions. From PND23 on, half of the rats (n = 40) was randomly assigned to the EE and transferred to the EE cages. Both groups, SE and EE, were maintained in their particular environments for 40 days, and after this time, were submitted to the repeated FST (Mezadri et al., 2011, Experimental Design at Fig. S1). On the 41st day of differential lodging, the rats (PND64) were exposed to the pretest session of forced swimming (15 min, in the first experimental day) and then randomly assigned to a particular group of pharmacological treatment: fluoxetine, imipramine 2.5 mg/kg, imipramine 5 mg/kg or saline. Twenty-four hours later, rats were treated with an IP injection of the selected treatment and, 1 h later, presented to the test session (5 min, second experimental day). In the following 13 days, rats were kept in their particular environmental condition and treated daily with an IP injection according to their experimental group initially assigned. The test session (5 min) was repeated on the 7th day (retest 1) and the 14th day (retest 2). In these days, pharmacological treatment was completed 1 h before the sessions of swimming. After retest 2, rats (PND 78) were anesthetized and perfused transcardially. The repeated FST consisted of individually placing the rats into a cylindrical tank (50 cm height × 25 cm diameter) containing clean water at 25 °C (25 cm deep). These conditions of the test were already described previously (Detke et al., 1995; Lino-de-Oliveira et al., 2005; Mezadri et al., 2011). After each session rats were taken out of the water and allowed to dry under a lamp (40 W, 10 min) before being returned to their home cages. All test sessions were recorded by a webcam (Logitech QuickCam) positioned 70 cm above the tank, to enable posterior evaluation. Behavioral categories scored were immobility, swimming, climbing and diving (definitions in Table S1). An experimenter blind to the treatment performed the behavioral analyses. The parameters evaluated for each category were: 1—latency (time elapsed between placing the animal in the tank and the first bout of each behavior observed), 2—frequency (number of bouts), and 3—duration (summary of the time spent in all bouts). The behavioral parameters were scored by the software Ethowatcher® (developed by the Laboratory of Comparative Neurophysiology of the Federal University of Santa Catarina, freely available on www.ethowatcher.ufsc.br, IEB-UFSC, Crispim-Junior et al., 2012). 2.4. Immunohistochemistry procedures and cellular quantification After the retest 2, rats were anesthetized (Urethane 35%) and perfused transcardially with a sucrose solution (9.25% in 0.02 M phosphate buffer (PB), pH 7.2, with 0.3 ml of heparin, at 37 °C), followed by 4% paraformaldehyde in PB. The brains were removed, blocked and postfixed for 4 h in the same fixative, transferred to a 0.1 M phosphatebuffered saline solution (PBS, pH 7.2) and then cut on a vibratome at 40 μm (250 μm apart throughout hippocampus). Sections were stored in a cryoprotectant at −20 °C, until required for the immunohistochemical reactions to detect Ki-67 (Ki-67-ir) or doublecortin (DCX-ir) (according to Schiavon et al., 2010). Briefly, all washings and incubations steps were performed under free-floating (gentle shaking) and room temperature (RT), unless otherwise stated. Washing steps (5 min each) consisted of three changes of 0.1 M PBS plus 0.25% Triton X-100 (PBST) between incubations. Endogenous peroxidase was blocked by incubation (30 min) with 100% methanol plus 0.3% H2O2 solution. Unspecific sites were blocked by incubation (90 min) in a solution containing 1% bovine serum albumin (BSA) in PBST followed by an overnight incubation (4 °C) with the primary antibody [rabbit anti-

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DCX (ab18723-100, Abcam, Eugene, OR, USA), 1:2000 or rabbit anti-Ki67 (DRM 004, Acris, Germany), 1:1000 diluted in PBST plus 1% BSA]. Incubation (2 h) with secondary biotinylated antibodies (goat anti-rabbit, Vector Laboratories, Burlingame, CA, USA; 1:1000) were followed by incubation (1.5 h) with avidin–biotin complex (Vector Laboratories, 1:500). Immunoreactions were visualized using 0.05% DAB (3.3-diaminobenzidine, Sigma-Aldrich, St. Louis, MO, USA) plus 0.015% H2O2. Sections were mounted on gelatin-coated glass slides, air-dried, protected from light for at least 24 h and then dehydrated in a graded series of alcohols and xylene before being coverslipped with DPX (Sigma-Aldrich, St. Louis, MO, USA). Typical staining of Ki-67-ir (round, dark brown dots) and DCX-ir (perikarya and processes) were seen in the subgranular zone of the dentate gyrus of hippocampus (DG, Fig. S2). For each animal, a section placed in the interval between the bregma (−) 3.3 to (−) 4.7 mm (Paxinos and Watson, 1982) was randomly selected for the quantification of Ki-67-ir. From the same interval, a section was randomly selected for the quantification of DCX-ir. For random selection, every section of hippocampus of a given animal was labeled with a number from 1 to n (n = maximal number of sections available for that animal) and then selected through generation of random numbers (Min = 1, Max = n, http://www.random.org/). Therefore, present data represent number of Ki-67-ir nuclei or DCX-ir cells per section of DG. All measurements were performed at high magnification (400×, optic microscope Olympus, BH-2; digital camera attached PixeLINK, Ontario, Canada) to enable distinction of single nuclei. Quantification was performed with the aid of the software Image J (www.rsweb.nih.gov/ij/). For detailed description of cell quantification see Fig. S3. Pharmacological treatments affected Ki-67-ir more intensely than DCX-ir scores, to highlight these differences the ratio DCX-ir/Ki-67-ir was also calculated. All data were expressed as percentage (%) of the control group. 2.5. Statistical analysis Statistical analyses were performed with the aid of Statistica 8 (Stasoft, Tulsa, OK, USA). Normality and homoscedacity were evaluated with Kolmogorov–Smirnov and Levene tests, respectively. Unpaired Student t-test was used to verify statistical differences between SE and EE during the pretest session. For each environmental condition (SE or EE), repeated measures two-way ANOVA (Factors: Treatment, Repetition), followed by Duncan's test, was performed to compare behavioral parameters within test, retest 1 and retest 2. Kruskal–Wallis ANOVA by Ranks followed by Mann–Whitney U test was applied to verify statistical differences in the gain of body weight and immunohistochemical data. Correlations between immunohistochemical data with behavioral parameters were evaluated using Spearman Rank Order. Values of P b 0.05 were accepted as being significant in all statistical tests performed. 3. Results 3.1. General remarks All rats remained healthy (eyes, fur, weight) during the whole period of the experiment and no external or internal injuries were seen in the area of the IP injections. In all cages with enrichment, rats seem to interact with the objects because their positions were altered from day to day. All rats gained weight over the period of the research, independent of the lodging condition or pharmacological treatment (Table S2). However, over the course of the experiment the rats treated with fluoxetine housed in EE gained significantly (t-Test, p b 0.05) less weight (9.8 + 1 g) than those housed in SE (15.6 ± 1.3 g).

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remained similar between the groups (Table S3). From the test to retest 2, rats of the control group housed in SE displayed reduced latency to immobility while increased duration and frequency of immobility (Table S4). Retesting also increased the frequency of swimming of control rats (Table S5). Scores of climbing were not affected by retesting. Housing in EE prevented the effects of retesting on immobility and swimming of control rats (Tables S4 and S5). 3.3. Behaviors of male rats in the repeated FST: effects of the antidepressants in standard housing Data of statistical analyses are Tables S4–S6. In SE, pharmacological treatments failed to affect any parameters of climbing (Fig. 1 and Table S6) while affected the parameters of immobility (Fig. 1 and Table S4) and swimming (Fig. 1 and Table S5). In fact, imipramine (2.5 mg/kg) reduced immobility time in retest 1 and 2 as compared to saline (Fig. 1 and Table S4). Imipramine (5 mg/kg), as well as fluoxetine, increased latency to immobility and reduced immobility time and frequency in retests 1 and 2, as compared to saline (Fig. 1 and Table S4). Imipramine (5 mg/kg) also reduced the frequency of swimming in the test and retest 2 (Table S5). 3.4. Behaviors of male rats in the repeated FST: effects of the antidepressants in enriched housing Data of statistical analyses are in Tables S4–S6. In rats housed in EE, there were no significant differences between pharmacological treatments and saline (Fig. 1 and Tables S4–S6). However, there were significant differences between the scores in the test and retests within a pharmacological treatment. Indeed, treatment with imipramine (5 mg/kg), as well as fluoxetine, decreased significantly immobility time in retest 2 when compared to the test (Fig. 1 and Table S4). In addition, fluoxetine increased significantly swimming time and frequency in retests 1 and 2 as compared to the test (Fig. 1 and Table S5). Treatment with fluoxetine also increased the frequency of climbing in retest 2 when compare to the test (Table S6). 3.5. Neurogenesis in the hippocampus of male rats submitted to the repeated FST: effects of pharmacological treatment in different housing conditions In SE conditions, pharmacological treatment failed to affect significantly the percentage of Ki-67-ir (Kruskal–Wallis, H (2, N = 15) = 1.01, p = 0.6, Fig. 2 A) or DCX-ir (Kruskal–Wallis test: H (2, N = 12) = 2.9, p = 0.2, Fig. 2 B) in relation to control. In EE conditions, the percentage of Ki-67-ir in the DG of rats submitted to repeated FST was significantly lowered after pharmacological treatment (Kruskal–Wallis, H (2, 15) = 9.8, p = 0.007, Fig. 2 A) while percentage of DCX-ir remained unchanged (Kruskal–Wallis, H (2, N = 12) = 4.6, p = 0.09, Fig. 2 B). Indeed, the percentage of Ki-67-ir nuclei in the DG of rats housed in EE and treated with imipramine (5 mg/kg) or fluoxetine was significantly smaller than in those treated with saline or imipramine (2.5 mg/kg) (Mann–Whitney, p b 0.05, Fig. 2 A). Raw data can be found in Table S7. Pharmacological treatment affected significantly the ratio DCX-ir/Ki67-ir independent of the housing condition (Kruskal–Wallis, H (3, N = 34) = 14.3, p = 0.002, Fig. 2 C). Post hoc analysis indicated that imipramine (5 mg/kg) and fluoxetine increased the ratio DCX-ir/Ki-67-ir, as compared to saline (Mann–Whitney, p b 0.05, Fig. 2 C). There was no significant correlation between the number of Ki-67-ir nuclei and DCX-ir cells in the DG neither between these markers and behaviors scored by rats in the repeated FST (data not shown).

3.2. Behaviors of male rats in the repeated FST: effects of housing conditions on the control group

4. Discussion

Data of statistical analyses are in Tables S3–S6. In the pretest, the latency to climbing was shorter in EE than in SE while other parameters

This study replicated the findings showing that repeated FST in rats could detect the gradual effects of low doses of imipramine and

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Fig. 1. Duration (s) of immobility (upper panel), swimming (middle panel) and climbing (lower panel) recorded for 5 min during the test (T), retest 1 (R1), and retest 2 (R2) in male rats. Bars (white = SAL, n = 15; light gray = IMI2.5, n = 8, dark gray = IMI5, n = 6, black = FLX, n = 6) represent mean ± S.E.M. from SE (left) and EE (right) groups. * = significantly different from respective SAL group, p b 0.05 (Duncan's test); # = significantly different from the respective test, p b 0.05 (Duncan's test). Abbreviations: SAL = saline, IMI2.5 = imipramine (2.5 mg/kg, IP), IMI5 = imipramine (5 mg/kg, IP), FLX = fluoxetine (2.5 mg/kg, IP), SE = standard environment, EE = enriched environment.

fluoxetine on behavior (Gutiérrez-García and Contreras, 2009; Mezadri et al., 2011). In addition, it revealed that the enriched environment per se prevented the effects of retesting on parameters of immobility, similarly to the treatment with the small doses of imipramine or fluoxetine. However, the enriched environment changed the effects of the pharmacological treatments on behavior in substance and dose-related fashion. Moreover, the treatment with imipramine (5 mg/kg) or fluoxetine for fourteen days increased the ratio between proliferating cells and new neurons in the hippocampus indicating that antidepressants favored differentiation of progenitors. These last effects of the antidepressant seemed independent of the environmental conditions. Current procedures were consistent with those previously published (Dal-Zotto et al., 2000; Gutiérrez-García and Contreras, 2009; Mezadri et al., 2011), rats housed in standard conditions shown in the pretest a

typical repertoire for the first contact with the forced swimming situation. Over retesting, the latency to immobility decreased while other parameters of immobility increased as expected for male rats treated with saline and housed in a standard environment (Dal-Zotto et al., 2000; Gutiérrez-García and Contreras, 2009; Mezadri et al., 2011). In those rats treated with fluoxetine (2.5 mg/kg), anti-immobility effect was visible earlier (retest 1) than previously reported (retest 2, Mezadri et al., 2011). The reversed light cycle (lights on at 6 pm) used in the present study might be a factor relevant for this contrasting finding. Reversed light cycle may affect the activity of rats in general (Prager et al., 2011) and in the traditional FST (Kelliher et al., 2000; Verma et al., 2010). On the other hand, reversed light cycle seemed not to alter the onset of imipramine action in repeated FST because current data were similar to those obtained in rats maintained in regular light cycle

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Fig. 2. Number of proliferative cells (Ki-67-ir-nuclei, upper panel) and immature cells (DCX-ir-perikarya, middle panel) per section of the dentate gyrus of male rats submitted to the repeated FST (n = 3–7 per group). The ratio proliferative/immature cells (DCX-ir/ Ki-67-ir) per section of dentate gyrus (n = 3–5). Data are expressed as percentage of the respective SAL group (% Control, dashed horizontal line). Bars (light gray = IMI2.5, dark gray = IMI5, black = FLX) represent mean ± S.E.M. from SE (left) and EE (right) groups. * = significantly different from respective SAL group, p b 0.05 (Mann–Whitney test). Abbreviations: see Fig. 1. Means ± S.E.M. of raw data available in Table S7.

(Gutiérrez-García and Contreras, 2009). In the present standard conditions, the onset of effect was similar to imipramine and fluoxetine. The model of repeated FST, based on the factorial analysis of rats housed in SE conditions (Mezadri et al., 2011), predicted that small doses of antidepressants would not change any parameter in the test. However, would prevent an increase of immobility in retests 1 and 2 (putative index for antidepressant detection in repeated FST, Mezadri et al., 2011). In addition, low doses of SSRIs would increase swimming time (or the frequency of swimming, or frequency of immobility, or all of them) whereas other antidepressant substances would increase climbing time (or latency to immobility, or frequency of climbing, or all of them) in retests 1 and 2 (Mezadri et al., 2011). In the present study the small doses of fluoxetine (an SSRI) or imipramine (an NSRI) failed to change any parameter in the test while prevented the increase of immobility time over retesting, according to the model. Differing from the model, parameters of swimming and climbing in repeated FST were similarly affected by both antidepressants. Therefore, differing from the previous expectation, the scores of active behaviors were not suitable to discriminate SSRI-type drugs from other antidepressant substances (e.g. non-selective inhibitors of monoamine reuptake, NSRIs) in the standard condition.

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Continuous housing in enrichment from weaning decreased parameters of immobility of rats submitted to the traditional FST in the adulthood (Brenes et al., 2008; Brenes-Saenz et al., 2006; Porsolt et al., 1978). However, current data showed that rats in enriched or standard housing behaved similarly in the pretest and test (similar to previously published data Cui et al., 2006; Simpson et al., 2012). Several factors could account for the disparities between present (and Cui et al., 2006; Simpson et al., 2012) and other studies (Brenes et al., 2008; Brenes-Saenz et al., 2006; Porsolt et al., 1978). Strain, sex and age of rats, as well as the length of the exposure to the enrichment, the size of the cage and the objects available, may change the aspects of enrichment and its consequences (Simpson and Kelly, 2011). For example, Brenes et al. (2008) found decreased immobility in male Sprague– Dawley rats tested in the traditional FST after 11 weeks of enrichment in cages (120 cm length × 70 cm width × 100 cm height) containing a running wheel. Studying the same strain of male rats, Simpson et al. (2012) or Simpson and Kelly (2012) did not observe any modification of behavior in the traditional FST after nine weeks of enrichment (cage: 54 cm length × 38 cm width × 20 cm height) without a running wheel. In the present study, the conditions of enrichment and behavioral output were similar to Simpson et al. (2012) or Simpson and Kelly (2012). Indeed, the running wheel seemed to be an important element in the enriched environment for rats (Bjørnebekk et al., 2006, 2008). The absence of a running wheel could explain why the enrichment failed to reduce immobility time in the pretest and test sessions of the repeated FST. Despite the initial lack of effect in the pretest and test, enrichment prevented the behavioral effects of retesting in the repeated FST. Indeed, enrichment lowered the values of immobility in the saline's group during retests 1 and 2 producing a putative “floor effect” eliminating significant differences between saline and antidepressants seen in the standard conditions. Absence of antidepressant action on rats housed in enriched environment was seen previously (Simpson and Kelly, 2012; Simpson et al., 2012). Despite the “floor effect”, imipramine (5 mg/kg) and fluoxetine progressively reduced immobility from test to retest 2 indicating that the enrichment preserved the within effects. The within effects of imipramine observed in enrichment seem dosedependent once that the within effects of the dose 2.5 mg/kg were absent in enriched-housed group. In addition, enrichment appeared to influence the within effects of imipramine and fluoxetine on behavior in repeated FST in different ways: 1—affected only anti-immobility effects of imipramine while 2—potentiated the proactive effects of fluoxetine. Together these data indicate that features of environments may change, favor or impair the response of the subjects to antidepressants. The interference of the environmental conditions on the effects of antidepressants may help to explain some incoherent findings in preclinical and clinical trials (e.g. Belzung, 2014). Enrichment and antidepressants may affect several mutual aspects of brain function such as hippocampal neurogenesis (for review e.g. Tanti and Belzung, 2013). In this work, eight weeks housed in enrichment failed to affect the markers of proliferation or immature neurons in the DG of adult Wistar rats submitted to the repeated FST. The lack of effects of the environment on marker of neurogenesis could be related to several different methodological factors (for review see e.g. Simpson and Kelly, 2011). Besides the absence or a running wheel (Bjørnebekk et al., 2006), the length of enrichment and the strain of rats may be relevant to the influence of enrichment on neurogenesis (Birch et al., 2013). Indeed, in adult Wistar rats the proliferation in the hippocampus remained unchanged after six weeks of housing in an enriched environment (Birch et al., 2013). Concerning to immature neurons, six weeks of an enrichment favored the short term survival (within two weeks after birth) without affecting long-term one (six weeks after birth) of new cells in the DG of Wistar rats (Birch et al., 2013). These last data could help to explain the lack of significant differences between the numbers of immature cells in the DG of control rats in standard or enriched housing for eight weeks.

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The variables changing the effects of enrichment may also influence the actions of the antidepressant treatment on markers for neurogenesis in the DG. The treatment with imipramine (5 mg/kg) or fluoxetine during the two weeks of the repeated FST decreased the number of proliferating cells in the DG, which was significant in enrichment. Regarding immature neurons, treatment with antidepressants failed to affect consistently the number of DCX-ir cells in the DG of Wistar rats housed in standard or enriched environment. These results were surprising because the literature is plenty of evidence showing pro-proliferative and pro-neurogenic effects of antidepressants (e.g. Keilhoff et al., 2006; Klomp et al., 2014; Malberg et al., 2000; Marcussen et al., 2008; Santarelli et al., 2003). However, factors such as the characteristics of the rat (strain, sex and age), the presence of the intermittent stress (repeated FST), and the doses and length of the antidepressant may help to explain these disparities. Indeed, antidepressants increased proliferation and neurogenesis in the hippocampus of male Wistar rats only when administered for longer than two weeks in rats younger than 65 days (Klomp et al., 2014; Marcussen et al., 2008; Mateus-Pinheiro et al., 2013). Moreover, fluoxetine in the doses of 10 mg/kg increased proliferation in the DG of Sprague–Dawley rats after fourteen days of treatment while the doses of 2.5 and 5 mg/kg failed to affect it (Pinnock et al., 2009). Therefore, in the present work the doses and the length of the treatment were sufficient to produce behavioral effects, but not enough to reveal increased neurogenesis in the DG. In addition, repeated FST could have affected hippocampal neurogenesis (Vega-Rivera et al., 2014) impairing the actions of the antidepressants on the number of newborn neurons. Two weeks of imipramine (5 mg/kg) or fluoxetine (2.5 mg/kg) were the most effective treatments to reduce immobility in repeated FST. These treatments reduced Ki-67-ir while failed to change DCX-ir in the DG increasing the ratio Ki-67-ir/DCX-ir in standard or enriched housing conditions. The increased ratio between proliferating and immature neurons markers may indicate an increase in that differentiation of progenitors into newborn neurons or, alternatively, an increase in newborn neurons survival. This last possibility seemed feasible once that the treatment with imipramine or fluoxetine (10 mg/kg for two weeks) increased the number of cells still alive and mature six weeks after their birth (Mateus-Pinheiro et al., 2013). Therefore, more appropriated experiments (for example, using BrdU) should be performed in order to verify these hypotheses. The lack of correlation between the numbers of proliferating or immature neurons with behavior in repeated FST indicated absence of a linear relationship between these both consequences of the treatments with antidepressants. In fact, antidepressant-like effects in FST may be seen even after the inhibition of the hippocampal neurogenesis (Zhang et al., 2012). 5. Conclusion In summary, behavioral outcome in repeated FST in rats depended on the housing conditions. Housing in enrichment impaired increased immobility over the repetition of the swimming sessions. In addition, enrichment affected the behavioral outcome of antidepressant treatment. However, the influence of enrichment on behavioral effects of antidepressants was not general but depended on the substance and the doses administered to the rats. In enriched conditions, two weeks treatment with antidepressants decreased the number of proliferating cells and failed to change the number of immature neurons in the DG. Conversely, fourteen days of treatment with antidepressants increased the ratio between the number of proliferating cells and immature neurons in the DG independent of housing conditions. As far as known, the present study is the first investigation of the effects of antidepressants associated to enrichment on parameters of neurogenesis in the DG. Future experiments should be done in order to understand why the association of the two pro-neurogenic stimuli reduced the number of proliferating cells and failed to change the number of immature neurons in the hippocampus of rats subjected to the repeated FST.

Authors' contribution – Possamai, F.—performed experiments, collected and analyzed data, wrote the manuscript and approved the manuscript. – Santos, J.—performed experiments, collected data and approved the manuscript. – Walber, T.—performed experiments and approved the manuscript. – Marcon, J.C.—performed experiments and approved the manuscript. – Santos, T. S.—performed experiments and approved the manuscript. – Lino-de-Oliveira, C.—selected research theme, provided facilities and materials, interpreted and discussed data, wrote the manuscript and approved the final version of the manuscript.

Acknowledgments We are thankful to Dr. José Marino Neto and the students in his laboratory (CFS-CCB-UFSC and IEB-CTC-UFSC) for the constant support in several aspects of this study, mainly providing facilities to immunohistochemistry essays. F.P. was recipient of master fellowship from Capes through the “Programa Multicêntrico de Pós-Graduação em Ciências Fisiológicas, CFS-CCB-UFSC, Campus Trindade, Florianópolis, 88040-900 SC, Brazil”. T.S.S. was recipient of Ph.D. fellowship from Capes. J. S. and J.C.M. were recipients of PIBIC fellowships from CNPq. T. W. was recipient of undergraduate fellowship from UFSC (Monitoria). This work received financial support from Alexander von Humboldt Foundation (“Equipment Grants”) and CNPq (Proc. 472446/2012-6). All procedures carried out in the present study complied with the current laws in Brazil. Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.pnpbp.2014.10.017. References Belzung C. Innovative drugs to treat depression: did animal models fail to be predictive or did clinical trials fail to detect effects? Neuropsychopharmacology 2014;39(5): 1041–51. Berton O, Hahn CG, Thase ME. Are we getting closer to valid translational models for major depression? Science 2012;338(6103):75–9. Birch AM, McGarry NB, Kelly AM. Short-term environmental enrichment, in the absence of exercise, improves memory, and increases NGF concentration, early neuronal survival, and synaptogenesis in the dentate gyrus in a time-dependent manner. Hippocampus 2013;23(6):437–50. Bjørnebekk A, Mathé AA, Brené S. Running has differential effects on NPY, opiates, and cell proliferation in an animal model of depression and controls. Neuropsychopharmacology 2006;31(2):256–64. Bjørnebekk A, Mathé AA, Gruber SH, Brené S. Housing conditions modulate escitalopram effects on antidepressive-like behaviour and brain neurochemistry. Int J Neuropsychopharmacol 2008;11(8):1135–47. Borsini F, Lecci A, Sessarego A, Frassine R, Meli A. Discovery of antidepressant activity by forced swimming test may depend on pre-exposure of rats to a stressful situation. Psychopharmacology (Berl) 1989;97:183–8. Brenes JC, Rodrígues O, Fornaguera J. Differential effect of environment enrichment and social isolation on depressive-like behavior, spontaneous activity and serotonin and norepinephrine concentration in prefrontal cortex and ventral striatum. Pharmacol Biochem Behav 2008;89:85–93. Brenes-Saenz JC, Rodriguez-Villagra O, Fornaguera-Trias J. Factor analysis of forced swimming test, sucrose preference test and open field test on enriched, social and isolated reared rats. Behav Brain Res 2006;169:57–65. Crispim-Junior CF, Pederiva CN, Bose RC, García VA, Lino-de-Oliveira C, Marino-Neto J. Ethowatcher: validation of a tool for behavioral and video-tracking analysis in laboratory animals. Comput Biol Med 2012;42:257–64. Cryan JF, Page ME, Lucki I. Differential behavioral effects of the antidepressants reboxetine, fluoxetine, and moclobemide in a modified forced swim test following chronic treatment. Psychopharmacology (Berl) 2005;182:335–44. Cui M, Yang Y, Yang J, Zhang J, Han H, Ma W, Li H, Mao R, Xu L, Hao W, Cao J. Enriched environment experience overcomes the memory deficits and depressive-like behavior induced by early life stress. Neurosci Lett 2006;404(1–2):208–12. Dal-Zotto S, Marti O, Armario A. Influence of single or repeated experience of rats with forced swimming on behavioural and physiological responses to the stressor. Behav Brain Res 2000;114:175–81.

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