Behavioural Brain Research 265 (2014) 155–162

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Prior exposure to repeated immobilization or chronic unpredictable stress protects from some negative sequels of an acute immobilization Jordi Pastor-Ciurana a,1 , Cristina Rabasa a,1,2 , Juan A. Ortega-Sánchez a , Maria Sanchís-Ollè a , Marina Gabriel-Salazar a , Marta Ginesta a , Xavier Belda a , Núria Daviu a , Roser Nadal b , Antonio Armario a,∗ a Institut de Neurociències and Red de Trastornos Adictivos (RTA), Unitat de Fisiologia Animal, Facultat de Biociències, Universitat Autònoma de Barcelona, Bellaterra, Spain b Unitat de Psicobiologia, Facultat de Psicologia, Universitat Autònoma de Barcelona, Bellaterra, Spain

h i g h l i g h t s • Chronic immobilization protects from the effects of an acute immobilization. • Chronic unpredictable stress partially protects from an acute immobilization. • There is evidence of cross-adaptation between different stressors.

a r t i c l e

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Article history: Received 22 November 2013 Received in revised form 18 February 2014 Accepted 19 February 2014 Available online 28 February 2014 Keywords: Chronic immobilization Chronic unpredictable stress Cross-adaptation Saccharin preference Food intake Open-field

a b s t r a c t Exposure to chronic unpredictable stress (CUS) is gaining acceptance as a putative animal model of depression. However, there is evidence that chronic exposure to stress can offer non-specific stress protection from some effects of acute superimposed stressors. We then compared in adult male rats the protection afforded by prior exposure to CUS with the one offered by repeated immobilization on boards (IMO) regarding some of the negative consequences of an acute exposure to IMO. Repeated exposure to IMO protected from the negative consequences of an acute IMO on activity in an open-field, saccharin intake and body weight gain. Active coping during IMO (struggling) was markedly reduced by repeated exposure to the same stressor, but it was not affected by a prior history of CUS, suggesting that our CUS protocol does not appear to impair active coping responses. CUS exposure itself caused a strong reduction of activity in the open-field but appeared to protect from the hypo-activity induced by acute IMO. Moreover, prior CUS offered partial protection from acute IMO-induced reduction of saccharin intake and body weight gain. It can be concluded that a prior history of CUS protects from some of the negative consequences of exposure to a novel severe stressor, suggesting the development of partial cross-adaptation whose precise mechanisms remain to be studied. © 2014 Elsevier B.V. All rights reserved.

1. Introduction

Abbreviations: CUS, chronic unpredictable stress; GEE, generalized estimating equations; GENLIN, generalized linear models; HPA, hypothalamic-pituitaryadrenal axis; IMO, immobilization on boards; IMOa, acute immobilization; IMOch, chronic intermittent immobilization; NS, non acutely stressed; SAM, sympatheticadreno-medullary axis. ∗ Corresponding author at: Institut de Neurociències and Unitat de Fisiologia Animal, Facultat de Biociències, Universitat Autònoma de Barcelona, 08193 Bellaterra, Barcelona, Spain. Tel.: +34 935811664; fax: +34 935812390. E-mail address: [email protected] (A. Armario). 1 These authors contributed equally to this work. 2 Present address: Department of Physiology, Institute of Neuroscience and Physiology, The Sahlgrenska Academy at the University of Gothenburg, Medicinaregatan 11, PO Box 432, SE-405 30 Gothenburg, Sweden. http://dx.doi.org/10.1016/j.bbr.2014.02.028 0166-4328/© 2014 Elsevier B.V. All rights reserved.

Exposure to purely or predominantly emotional stressors (herein emotional stressors) resulted in a wide range of physiological and behavioural changes. The best characterized physiological changes are the activation of the hypothalamic-pituitary-adrenal (HPA) and sympathetic-adreno-medullary (SAM) axes [1]. The activation of the HPA axis results in the release of ACTH and glucocorticoids (corticosterone in rats), whereas the activation of the latter increases plasma levels of adrenaline and noradrenaline. Physiological response to stress is accompanied by behavioural changes that typically include alterations of normal activity and exploratory behaviour in novel environments, enhanced anxiety, and interference with learning and memory processes [2–4].

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After daily repeated exposure to the stressor, the impact of an acute session of the same (homotypic) stressor is very often reduced. This has been frequently reported regarding the HPA and the SMA axes [1,5]. As exposure to novel (heterotypic) stressors resulted in normal or enhanced HPA and SMA responses [1,5–7], it is assumed that the reduction of the response caused by daily repeated exposure to the same stressor is due to a lower emotional activation, consequence of the familiarity with the situation. Less is known about how repeated exposure to a particular stressor affects the behavioural response to an acute challenge with the same stressor. In a series of papers in the 60–70s some authors reported that acute exposure to severe stressors impaired performance of rats in some tasks requiring an important degree of motor activity, but such impairment progressively decreased after repeated exposure to the stressor [8–10]. Quite interestingly, protection offered by chronic stress was not limited to the homotypic stressor, demonstrating cross-adaptation between different stressors. This non-specific adaptation is likely to involve brain noradrenaline function as severe chronic stressors consistently increased noradrenaline synthesis capabilities (i.e. synthesis of tyrosine-hydroxylase and other enzymes, see [7]) and tyrosine supplementation prevented both noradrenergic depletion after severe stressors and behavioural inhibition [11,12]. The possibility of non-specific cross-adaptation is particularly important regarding the consequences of exposure to chronic unpredictable stress (CUS), also known as chronic variable or chronic mild stress. Exposure to CUS, a model developed by Katz et al. (1981) [13] and later developed by Willner and colleagues (see [14]), has been considered as a putative animal model of depression causing for instance reduced activity in novel environments, anxiety, anhedonia (mainly evaluated by reduced consumption of sucrose), and the development of passive coping strategies in the forced swim test [14,15]. However, there is also evidence that under certain conditions CUS not only did not induce anxiety, but can even reduce it [16–20]. Moreover, some recent studies in rats suggest that activation of the HPA axis and brain c-fos expression in response to a novel stressor may be reduced in animals by prior exposure to CUS [21,22], although results regarding the HPA axis are not consistent [21–24]. From all the above considerations we hypothesized that a prior history of chronic experience with unpredictable stressful situations may confer partial protection from the detrimental consequences of an acute severe stressor such as immobilization on boards (IMO). Then, in the present work we compared the protection offered by a prior history of chronic IMO stress and by a CUS procedure that did not include IMO as stressor, regarding the negative consequences of an acute session of IMO. This comparison can shed lights on the possible dual consequences (detrimental, protective) of a prior history of stress on the response to novel encountered stressful situations.

2. Methods 2.1. Animals Fifty-three male Sprague–Dawley rats obtained from the breeding centre of the Universitat Autònoma de Barcelona were used. Rats were 2-month-old at the beginning of the experiment. Animals were housed individually under standard conditions of temperature (22 ± 1 ◦ C) in a 12 h light/dark schedule (lights on at 7:00) with ad libitum access to food and water. Rats were allowed at least 1 week to acclimate themselves to the animal room before starting the experiment. Animals were handled at least 3 times on different days for approximately 2 min. The experimental procedures were always done in the morning, with exception of CUS (Fig. 1). This

work has been carried out in accordance with the Guide for the Care and Use of Laboratory Animals (National Institutes of Health) and approved by the Ethical Committee for Animal Experimentation of the Universitat Autònoma de Barcelona and by the Generalitat de Catalunya. 2.2. Experimental procedures Animals were assigned to three experimental groups: (i) controls (n = 18), undisturbed from day 1 to 9, (ii) chronic intermittent IMO (IMOch, n = 18), daily exposed to 1 h of IMO from day 1 to 9, and (iii) chronic unpredictable stress (CUS, n = 17), animals exposed to the CUS paradigm for 9 days (Fig. 1). On day 10, 9 animals from the two former groups and 8 for the last group were exposed to 1 h IMO (IMOa) and the others remained undisturbed, with no additional exposure to stress (NS). On day 11 (24 h after the last IMO) all animals were exposed to an open-field for 15 min. After that, three animals from each NS group were sacrificed for other purposes. In the remaining animals, food intake and body weight were daily measured for 4 days. Saccharin intake was only measured in those animals exposed to the acute IMO on day 10 because preliminary results indicated that neither chronic IMO nor CUS affected significantly saccharin intake measured at the end of the chronic stress period. These latter values could then be used as a baseline to study the impact of the acute IMO. The CUS consisted of the exposure to 3 different stressors (restraint, footshock and forced swim) following the schedule indicated in Fig. 1. Animals were always transported to the stress room in their home-cage. For restraint, animals were placed during 30, 60 or 90 min into cylindrical PVC tubes measuring 6 cm diameter and 21.5 cm length. The rear top of the apparatus was closed by a cork letting the tail to protrude from the tube. Several holes (0.5 cm in diameter) in the walls of the cylinder provided fresh air. For the footshock, rats received repeatedly a 6 s shock (1.5 mA) each min for 30, 60 or 90 min. Rats were put into individual clear Plexiglas® boxes (19.7 cm × 11.8 cm × 20.0 cm) provided with a metal removable grid floor of 15 stainless steel rods (0.4 cm diameter and spaced 0.9 cm centre to centre) connected to a shocker that delivered scrambled AC current (Cibertec, Madrid, Spain). Shock chambers were carefully cleaned with ethanol (5%, v/v) before introducing the animals. For forced swim, animals were allocated in transparent cylindrical plastic tanks (height = 40 cm, internal diameter = 19 cm) containing water (25 ◦ C) to a level of 24 cm where they remained for 20 min [25]. Afterwards, they were withdrawn from water and kindly dried with a towel before being returned to their home-cages. After 1 h of rest, they underwent 10 additional min of forced swim. Water was always changed before introducing the animals into the tanks. All the CUS procedures were done in a room with white walls illuminated by a white fluorescent light. The chronic IMO procedure consisted of immobilizing the animals for 1 h by taping their four limbs to metal mounts attached to a board [26]. Head movements were restricted with two plastic pieces (7 cm × 6 cm) and the body was subjected to the board by means of a piece of plastic cloth (10 cm-wide) attached with Velcro® , which surrounded all the trunk. Animals remained immobilized in a room provided with white fluorescent light. 2.3. Behavioural assessment 2.3.1. Activity/exploration The open-field was a rectangular grey plastic box opened at the top (56 cm × 36.5 cm × 31 cm) with dim illumination provided by a white 25 W bulb placed 1.20 m above the centre of the surface of the box. Animals were placed in a corner of the open-field facing the wall. The box was cleaned between animals with ethanol solution

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Fig. 1. CUS schedule schematic representation. Type of stressor, stress length and daily starting time are indicated. 20 + 10 min means 20 min of forced swim followed by 1 h of rest in the home-cage and 10 additional min of exposure to the same stressor.

(5%, v/v in tap water). The animals were transported from the vivarium to the adjacent rooms inside their home-cages. Behaviour was recorded by one video camera (Sony SSC-M388 CE, BW) suspended from the ceiling (1.20 m above the surface of the open-fields, two per camera). An experimenter blind to the treatment measured exploratory and motor behaviour by counting the number of rearings and ambulations in 5 min blocks. The open-field exposure was carried out in the morning (09–13 h) and 4 animals were simultaneously exposed in two different rooms. Central and peripheral activity was measured. 2.3.2. Struggling to IMO To measure struggling behaviour in response to IMO, immobilized animals were placed against a black background and separated from each other by a black wood wall. For behavioural recording, a video camera was positioned just behind the immobilized rats (two per camera). It was considered as struggling the display of strong mobility, exaggerated rear back movements and attempts to escape. An experimenter blind to the treatment measured total time spent struggling under IMO for the first 10 min on days 1, 2 and 10.

2.3.3. Saccharin intake Rats have strong preference for sweet solutions such as those containing sucrose or saccharin and the intake of these compounds are considered to be due to their hedonic properties [14]. However, saccharin, in contrast to sucrose, has not caloric value. As IMO results in reduced food intake [27], we wanted to avoid the problems of interpreting changes in consumption of a sweet solution having caloric properties. Then we chose to measure saccharin intake. Animals were allowed to drink a saccharin sodium salt (Sigma, S1002-500G) solution (0.1%, w/v in tap water) ad libitum in non-drip bottles (250 ml). A bottle of the same type filled with tap water was added as well. The two bottles were separated and were refilled and side-switched each day to avoid place preference. Animals from the three chronic stress groups to be exposed to acute IMO on day 10 were allowed to habituate to the bottles before starting the chronic stress protocol. At the beginning of the chronic stress period saccharin bottles were withdrawn and they were re-introduced three days before finishing the chronic treatment. This was done to prevent rebound intake of saccharin on the first day of saccharin availability after a period of withdrawal. Saccharin was then daily measured until day 4 post-acute IMO.

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Fig. 2. Effects of chronic stress on food intake (A) and body weight gain (B) over all the experimental period. Mean and S.E.M. are represented (each group n = 17–18). Always: 1 symbol p < 0.05; 2 symbols p < 0.01; and 3 symbols p < 0.001. * vs control; # vs IMOch.

Animals always had free-access to food. Four animals that did not show clear preference for saccharin before treatment were excluded from the analysis. 2.4. Data analysis The statistical analysis was performed using the “Statistical Package for Social Sciences” (SPSS, version 18). Overall changes in body weight and food intake throughout the chronic stress period were analyzed with generalized linear models (GENLIN) [28], with chronic stress as the between-subjects factor (three levels: control, IMOch and CUS). The same analysis was performed regarding struggling on day 10 and baseline levels of food intake, body weight and saccharin intake at the end of chronic treatment. In order to identify the influence of prior chronic stress history on the consequences of an acute exposure to IMO over the next days we studied the changes respect to corresponding baseline levels using generalized lineal models with repeated measures (generalized estimating equations, GEE) [29]. The analysis of changes in body weight and food intake included two between-subjects factors [chronic stress (3 levels: control, IMOch and CUS) and acute IMO (2 levels: NS and IMOa)] and day as the within-subjects factor (4 levels corresponding to 4 days post-stress: PS1, PS2, PS3 and PS4). The same type of analysis was used to assess activity and exploratory behaviour in the open-field, but in this case “block” was the within-subjects factor (3 levels of 5 min). GEE analysis was also used to study changes in saccharin intake, with chronic stress as the only between-subjects factor (3 levels: control, IMOch and CUS) and day (4 levels) as the within-subjects factor. Finally, to assess the changes in IMO-induced struggling behaviour over the days in the IMOch group, a GEE analysis was used, with one within-subjects factor (day: 3 levels). Significance level was set at p < 0.05. 3. Results The evaluation of food intake and body weight gain throughout the 9 days of chronic stress showed a significant chronic stress effect [Wald 2 (2) = 46.2; p < 0.001 and Wald 2 (2) = 154.7; p < 0.001, respectively]. Further comparisons revealed a significant reduction of food intake (Fig. 2A) in the two chronic stress groups (IMOch and CUS) compared with controls (p < 0.001 in the two cases). The effects were more pronounced after IMOch than CUS (p < 0.001). The comparison of body weight gain yielded similar results as food intake (Fig. 2B). Therefore, these data suggest that the CUS procedure was less severe than IMOch. The GEE analysis revealed that struggling behaviour during IMO (Fig. 3A) declined over days [Wald 2 (2) = 31.1; p < 0.001]. The reduction was not significant after second IMO exposure (day 2), but it was after repeated experience with the stressor (day 10 vs day 1, p < 0.001). Comparison of the three chronic stress groups on day 10 (Fig. 3B) showed a significant chronic stress effect

Fig. 3. Effect of chronic stress on struggling behaviour during the first 10 min of exposure to IMO. Mean and S.E.M are represented (n = 8–9 per group). (A) Time spent struggling in response to IMO after repeated exposure to the stressor. ***p < 0.001 vs day 1. (B) Effect of prior chronic stress experience on struggling response to an acute IMO on day 10. **p < 0.01 vs control; ### p < 0.001 vs IMOch.

(Wald 2 (2) = 14.4; p < 0.001). Further comparisons revealed that the IMOch group showed reduced struggling compared with the stress-naive control group and the CUS group (p < 0.01 and p < 0.001, respectively), whereas prior CUS exposure did not affect struggling response to acute IMO. The food intake over the 24 h preceding acute IMO exposure was used as the baseline to study the response of the three chronic stress groups to an acute IMO. The GEE analysis of baseline levels revealed significant group differences (Wald 2 (2) = 54.5; p < 0.001). Both IMOch and CUS groups showed reduced food intake as compared with controls (p < 0.001 in the two cases), with no differences between them (Fig. 4A1). The GEE analysis of changes in food intake after the last acute IMO revealed significant effects for chronic stress (Wald 2 (2) = 40.8; p < 0.001), acute IMO (Wald 2 (1) = 16.5; p < 0.001), day (Wald 2 (3) = 291.1; p < 0.001) and the interactions chronic stress × day (Wald 2 (6) = 22.9; p < 0.001) and acute IMO × day (Wald 2 (4) = 16.247; p < 0.001). To better illustrate the differential impact of the acute IMO in the three chronic stress groups, we compared for each group and post-IMO day the changes in food intake in NS and acute IMO groups (see details in Fig. 4A2). In controls, acute exposure to IMO reduced food intake over the four post-IMO days studied, whereas in the IMOch group, there was not additional impact of the last IMO and the animals progressively recovered normal food intake over the four postIMO days. Interestingly, CUS animals did show an impact of the acute IMO, but this was lower than in controls, indicating a partial protection. The body weight gain over the chronic stress period was considered as the baseline to study the impact of the superimposed acute IMO. Chronic stress differences in baseline body weight gain during the chronic stress period were observed (Wald 2 (2) = 102.7; p < 0.001). Further comparisons showed (Fig. 4B1) lower body weight gain in IMOch compared with CUS and in the two chronic stress groups compared with controls (always p < 0.001). The GEE analysis of changes in body weight gain respect to their corresponding baseline values revealed significant effect for chronic stress (Wald 2 (2) = 19.8; p < 0.001), acute IMO (Wald 2 (1) = 7.3; p < 0.01), day (Wald 2 (3) = 454.8; p < 0.001) and the interactions chronic stress × acute IMO (Wald 2 (2) = 15.7; p < 0.001) and chronic stress × acute IMO × day (Wald 2 (6) = 13.1; p < 0.05) (Fig. 4B2). As in the case of food intake, we compared for each chronic group and post-IMO day the changes in body weight between NS and acute IMO groups. Control animals were negatively affected by the acute IMO and the impact was observed on all days studied (p < 0.001). In contrast, the acute IMO did not alter body weight gain either in IMOch or CUS groups. No baseline group differences were observed in saccharin intake (Fig. 5A1). The GEE analysis of changes in saccharin intake respect to baseline after the acute IMO showed significant effect for chronic stress [Wald 2 (2) = 20.7; p < 0.001], day [Wald

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Fig. 4. Influence of prior chronic stress exposure on food intake (A) and body weight change (B) after an acute IMO. It is represented means and S.E.M. of food intake in the 24 h preceding acute IMO (baseline, A1), changes in food intake respect to baseline levels (A2), cumulative body weight gain during all the period of chronic stress (baseline, B1) and cumulative body weight changes respect to baseline (B2). The number of animals not exposed to IMO was 6 per group and the number of animals exposed to acute IMO was 8–9 per group. Always, 1 symbol means p < 0.05, 2 symbols p < 0.01 and 3 symbols p < 0.001. PS indicates the day post-stress. * vs controls, # vs chronic IMO, • vs corresponding non-acute IMO group.

Fig. 5. Influence of prior chronic stress exposure on baseline and acute-IMO induced changes in saccharin intake. It is represented means and S.E.M. (n = 7–8 per group) of saccharin intake in the 24 h preceding acute IMO (baseline, A1) and the changes in saccharin intake respect to baseline levels caused by an acute IMO (A2). The number of animals not exposed to IMO was 6 per group and the number of animals exposed to acute IMO was 8–9 per group. One symbol p < 0.05, 2 symbols p < 0.01 and 3 symbols p < 0.001. PS indicates day post-stress,  vs baseline, * vs controls, # vs IMOch.

2 (3) = 188.6; p < 0.001] and the interaction chronic stress × day [Wald 2 (6) = 27.0; p < 0.001]. Decomposition of the interaction showed a marked acute IMO-induced reduction of saccharin intake in controls that progressively recovered over the days (Fig. 5A2). CUS animals showed a similar but less marked pattern, whereas no reduction was observed in IMOch animals. In fact, CUS animals needed 3 days to return to baseline consumption whereas controls were still below baseline levels on PS4. Regarding the open-field, the ambulation and rearing number analyses were divided in 3 blocks of 5 min. Central and peripheral ambulations were separately measured, but no differential group effect was found among the two and we present total ambulations. The GEE analysis showed significant effects for chronic stress (Wald 2 (2) = 108.8; p < 0.001), acute IMO (Wald 2 (1) = 4.4; p < 0.05), block (Wald 2 (2) = 10.3; p < 0.01) and the interactions chronic stress × block (Wald 2 (4) = 174.1; p < 0.001) and chronic stress × acute IMO × block (Wald 2 (4) = 21.5; p < 0.001) (Fig. 6A). Further comparisons showed that in controls, an acute IMO reduced the number of ambulations in blocks 1 (p < 0.001) and block 3

(p < 0.05). Chronic IMO animals were not affected by acute IMO. CUS itself caused a marked hypo-activity, which was not longer evident in the last block (p < 0.001 in blocks 1 and 2 vs controls), whereas no additional effect of acute IMO was observed. The GEE analysis of the number of rearings revealed significant effects for chronic stress (Wald 2 (2) = 83.7; p < 0.001), block (Wald 2 (2) = 76.5; p < 0.05) and the interaction chronic stress × block (Wald 2 (4) = 227.2; p < 0.001 (Fig. 6B), with no effect of acute IMO. Further comparisons revealed that IMOch group showed greater number of rearings than controls (p < 0.05 in all blocks), whereas the CUS group showed less rearings than the controls in blocks 1 and 2 (p < 0.05 in the two cases). 4. Discussion It has been repeatedly reported that daily exposure to the same (homotypic) stressor causes a reduction of certain physiological changes elicited by an acute exposure to the situation (adaptation), including the activation of the HPA and SAM axes. In contrast,

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Fig. 6. Influence of prior chronic stress exposure on open-field performance 24 h after exposure to an acute IMO. Means and S.E.M. of the number of ambulations (A) and rearings (B) are represented (n = 8–9 per group). Activity was measured in three blocks of 5 min each. One symbol p < 0.05, 2 symbols p < 0.01 and 3 symbols p < 0.001. * vs controls within the same block, • vs corresponding non-acute IMO group within the same block.

exposure to a different (heterotypic) stressor results in a normal or enhanced response. However, there is some evidence for crossadaptation between different stressors when other variables (e.g. active behaviour) were studied. In the present work we compared protection conferred by prior daily exposure to the same stressor with that conferred by prior exposure to CUS. In response to a single session of IMO, stress-naïve rats showed reduced food and saccharin intake as well as impaired body weight gain for some days after initial exposure. In addition, reduced activity in a novel environment was observed on the day after IMO. This is the expected pattern of changes based on previous reports [30–33]. Although the effect of IMO on saccharin intake has not been previously reported, other types of stressors (restraint or unpredictable tail-shocks) have been reported to reduce saccharin or sucrose intake in the 24 h following the stressor [34,35]. The latter reported effects of stress in saccharin intake were transient as they were only observed the day after stress. Nevertheless, the long-lasting effect of IMO on saccharin intake observed in the present work is not surprising considering the greater IMO-induced activation of physiological markers of stress compared with other stressors (i.e. [36]) and its long-lasting impact on food intake and body weight [36,37]. Daily repeated exposure to IMO for 9 days markedly reduced body weight gain and food intake, in accordance with previous reports [38,39]. Although both chronic IMO and CUS reduced food intake and body weight gain over the experimental period, the effects were stronger after chronic IMO, suggesting an overall greater impact of the latter paradigm. After the last acute IMO challenge on day 10, chronic IMO rats showed no additional reduction of body weight or food intake, in striking contrast to impact of the acute IMO in stress-naïve rats. It might be argued that the reduction of food intake was nearly at the maximum for emotional stressors on the first 2 days after the termination of IMO or CUS and there was no room for further decrease after acute IMO. However, this does not appear to be the case in the following days. Therefore, it is reasonable to consider that the negative consequences of an acute episode of IMO were blunted by prior experience with the stressor. The weight gain of IMOch rats was similar to that observed in stress-naïve rats despite the fact that their baseline food intake was lower. These data can be explained by a higher food efficiency of chronic IMO rats, probably related to metabolic adaptations to reduced food intake. In addition to the above protective effect of prior chronic IMO, this procedure completely protected from the marked reduction of saccharin intake observed in stress-naïve rats after acute IMO. Adaptation of saccharin intake to a daily repeated stressor has been previously reported [34]. Chronic IMO rats were also protected from the immediate or delayed (24 h) inhibitory effects of an acute IMO on activity of animals in a novel environment, in accordance with previous data [30,31,40,41]. Therefore, both anhedonic and behavioural inhibitory effects of severe stressors appear

to be progressively reduced after repeated experience with the stressor, likely reflecting a process of adaptation (habituation) typically observed with neuroendocrine markers [1]. Saccharin intake was not significantly reduced in CUS animals when measured on the last days of the chronic treatment. Importantly, a prior history of CUS partially protected from the reduction of saccharin intake caused by an acute IMO as the impact was of lower magnitude and the recovery was faster than in stress-naïve rats. It thus appears that repeated exposure to IMO offers complete protection from acute IMO-induced anhedonia, whereas a partial cross-protection was offered by CUS. The finding that the overall inhibition of food intake caused by the two chronic stressors was not associated with a reduction of saccharin intake and that the impact of an acute superimposed IMO followed a different pattern with both parameters strongly supports dissociated regulatory mechanisms. Most studies on stress-induced anhedonia have been done using sucrose intake [14]. To our knowledge, only one previous study has measured the impact of chronic repeated exposure to the same stressor (3 h of daily restraint) on sucrose intake and they reported an enhanced rather than reduced inhibitory effect over time [42], in contrast to the present results with saccharin. The discrepancies could be explained by the use of sucrose instead of saccharin as sucrose, in contrast to saccharin, has caloric value. For instance, exposure to CUS has failed to decrease saccharin preference [43], and when both sucrose and saccharin consumptions were measured in the same experiment CUS reduced sucrose but not saccharin consumption in rats [44]. Moreover, in the Naert et al.’s study [42] access to sucrose was allowed after 17 h of prior water deprivation, whereas in the present study rats were not deprived and had free simultaneous access to saccharin and water intake. Considering the extensive use of sucrose intake/preference to evaluate anhedonia, more attention should be paid to the influence of caloric properties of sweet solutions and the deprivation protocols before testing. In this regard, it would be of interest to compare in further studies the impact of stress on both saccharin and sucrose intakes. Repeatedly immobilized rats showed a reduction of struggling behaviour compared with rats exposed to IMO for the first time, in accordance with previous results after repeated restraint stress [45–47]. In contrast, a prior history of CUS did not modify the struggling behaviour displayed by rats during the initial exposure to an acute IMO. These data strongly suggest that our CUS protocol is not inducing a generalized reduction of active coping behaviour when facing novel stressful situations. Nevertheless, it cannot be ruled out that a more prolonged period of CUS exposure, by increasing the experience of lack of control over the stressful situations, could result in the development of depression-like behaviour reflected in less attempts to escape from the IMO board or in anhedonia. In contrast to chronic IMO, prior exposure to CUS caused a marked hypo-activity in the novel environment (open-field)

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that was more dramatic during the first 5 min and then progressively recovered during the session. These data deserves further comments. Our CUS procedure includes exposure to footshock and there are several previous reports demonstrating footshock-induced hypo-activity in novel environment [48,49]. More importantly, we and others have demonstrated that prior exposure to a single footshock is enough to induce a long-lasting (more than 1 week) hypo-activity in novel environments that is not merely due to protracted effects of footshock, but rather dependent on the establishment of contextual fear conditioning [50,51]. The present data give support to all these previous results and suggest some kind of cognitive fear generalization after footshock. Exposure of CUS rats to an acute IMO did not further reduce such hypoactivity. A floor effect could explain the lack of effect of acute IMO on activity during the first 5 min, but not later when hypo-activity vanished, thus precluding a floor effect that would mask the effects of acute IMO. It is then possible that a prior history of CUS, while inducing per se an inhibition of activity, prevented from the effect of a severe stressor such as IMO. This hypothesis may be compatible with prior studies demonstrating that inhibition of motor activity/performance observed during the first few hours after exposure to severe stressors were reduced by chronic exposure to the same situation, but also by prior exposure to other types of stressors [8–10], suggesting cross-adaptation. Severe stressor-induced inhibition of activity/performance appears to be linked to the impossibility to maintain enough levels of noradrenaline in certain brain areas and cross-adaptation can be explained by the enhanced potential for noradrenaline synthesis after a prior history of chronic stress [8–10]. However, other neurotransmitters might be involved. Thus, daily repeated exposure to restraint not only protected from the anxiogenic effects of acute restraint but also of a different stressor (acute footshock), and this protective effect is apparently mediated by endogenous opioids [41]. 5. Conclusions The present results indicate that a prior history of CUS is able to induce some protection from the negative consequences of a novel severe acute stressor that shares qualitative similarities with the protection offered by chronic exposure to the same stressor. Interestingly, a clear dissociation between the two chronic stress protocols was observed with active coping behaviour on the board (struggling), suggesting that the CUS animals perfectly identify IMO as a novel stressor. The study of factors and mechanisms influencing either specific or non-specific protective effects of the two models of chronic stress may shed light about brain processing of stressors. Moreover, the present results are important regarding the question as to whether repeated experience with different type of stressors can inevitably result in sensitization to the negative consequences of further stressful situations or can, under some circumstances, confer protective effects. Acknowledgements This work was supported by grants from the Ministerio de Economía y Competitividad (SAF2011-28313), Instituto de Salud Carlos III (RD12/0001/0015, Redes Temáticas de Investigación Cooperativa en Salud, Ministerio de Sanidad y Consumo) and Generalitat de Catalunya (SGR2009-16). Cristina Rabasa and Núria Daviu were recipients of a predoctoral fellowship (Spanish Ministry of Education). Jordi Pastor-Ciurana and Maria Sanchís-Ollè are recipients of a predoctoral fellowship (Universitat Autònoma de Barcelona). Authors would like to thank Joan C. Balasch for generously designing Fig. 1.

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