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Susceptibility to subchronic unpredictable stress is related to individual reactivity to threat stimuli in mice
Behavioural Brain Research 155 (2004) 291–299
Research report
Susceptibility to subchronic unpredictable stress is related to individual reactivity to threat stimuli in mice C. Ducottet∗ , A. Aubert, C. Belzung EA3248 Psychobiologie des Emotions, UFR des Sciences et Techniques, Parc de Grandmont, 37 200 Tours, France Received 20 April 2004; accepted 30 April 2004 Available online 5 June 2004
Abstract As in many complex behavioral responses, inter-individual variability can be observed in the responses to a chronic mild stress. While some subjects exhibit more resilient behaviours, others appear more susceptible to stress. This study hypothesizes that this variability relies on the individual appraisal of the stressful event. To study this assumption, mice were first subjected to a conditioned task occurring in a circular arena. In this task, a mild air-puff (i.e. stressor) in a given quadrant of the arena was coupled with the presence or the absence of a light in the same quadrant. Half of mice were then submitted to a 15-day subchronic stress consisting in various environmental and social mild stressors randomly applied two or three times a day. At the end of this procedure, the occurrence of depressive-like behaviours in stressed mice was assessed using measures of the stress regime (i.e. physical state, choice test, grooming test). The physical state assessed the physical appearance of mice. The grooming test consisted in measuring the time spent in grooming after mice were sprayed upon with a viscous solution. The choice test consisted in measuring the time spent in an uncomfortable place (i.e. whose floor was covered with damp sawdust) versus a more comfortable one (i.e. with dry sawdust) to evaluate the reactivity to a negative stimulus previously encountered during the subchronic stress. Multiple regression analyses revealed a relationship between attention toward salient stressful stimuli in the conditioned task and susceptibility to the subchronic stress procedure. These results are discussed regarding their relevance for the understanding of aetiologies of depressive illnesses. © 2004 Elsevier B.V. All rights reserved. Keywords: Subchronic stress; Cognitive orientation; Conditioned task; Grooming
1. Introduction Chronic social stress has been widely used to assess depressive-like behaviours in animals like rodents [15,17,18,36], tree shrews [9,10,17] or birds [4]. Social stress represents a type of stressor that animals may encounter in their natural environment. Moreover, it constitutes a common negative stressor in humans. Such procedures exhibit therefore relevant face validity for the study of stress-related disorders. Many behavioural modifications have been observed in animals after a chronic social stress. Most of them report decrease in many activities like locomotor, grooming [7,8] or feeding behaviours, but also physiological changes such as alteration of the circadian body temperature rhythm or a decrease in body weight (for a review, see [20]). Similar modifications have been identi∗ Corresponding author. Tel.: +33 2 47 36 69 98; fax: +33 2 47 36 72 85. E-mail address: [email protected] (C. Ducottet).
0166-4328/$ – see front matter © 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.bbr.2004.04.020
fied in rodents submitted to an unpredictable chronic mild stress procedure. Indeed, these animals exhibit alterations in external physical appearance (i.e. dirty tarnished fur), decision-making [26] or sleep [22]. Recently, several authors have reported a sizeable interindividual variability in the physiological and behavioural responses observed after such a stress procedure [4,14,25,28,35]. This variability could reveal of primary importance for the investigation of the factors underlying susceptibility to stressful events. However, many of these studies use genetically-selected animals exhibiting extreme response patterns. This urges for the study of these inter-individual differences in a non selected population expressing the full range of behavioural patterns. The inter-individual variability has been interpreted as dependent on the coping style (to the stressors). A growing literature describes various coping styles used by animals [16], such as passive/active coping or, more recently, proactive/reactive coping. These styles seem related to the reactivity to subchronic stress. Indeed, reactive mice have been
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shown to be more susceptible to chronic stress and to recover less rapidly than proactive ones [2,28,36]. Previous study has considered the reactivity of mice to stressful events and therefore the susceptibility of an animal to chronic stressors in term of different individual appraisal of the situation [8]. Individuals that assess a situation as highly threatening will mobilize more resources to face or flight the stressor perceived. This can also be seen in anxious humans that exhibit attentional biases toward threatening cues of their environment [1,21]. Cognitive styles and coping styles are not necessarily opposed and a particular cognitive style can be a characteristic of a specific coping style. Cognitive and coping styles represent two different approaches but can be related to a general cognitive structure. The principal aim of this study is to examine the role of cognitive style in response to a subchronic mild stress. In this study, we investigated the relationship between the cognitive orientation mice exhibit toward a mild stressor, as revealed in a conditioned task, and the reactivity to a 2-week subchronic stress with both social and environmental stressors. A heterozygous mice population (Swiss) was chosen for this study in order to guarantee variability of responses. Cognitive orientations were assessed in a conditioned task in which animals had to associate the onset and the removal of a mild stressor with light switches (on and off respectively). The mild aversiveness of the stimulus allowed us to obtain a large variability of responses. After this first assessment, mice were confronted to a subchronic stress. This paradigm is based on social and environmental stressors. As mice are macrosmatic animals, odours play a major role in their social life. Therefore, social disturbing stimuli consisted in the daily reversal of cages between two randomly chosen counterparts (including soiled sawdust). Animals were thus kept under an unstable olfactive environment. This procedure is similar to the sensory contact model [18] but does not include physical contact and needs much less animals. Environmental stressors consisted in unpredictable uncomfortable events in the mice physical environment, i.e. known to induce stress in rodents [37]. At the end of the subchronic stress, the physical state of animals was assessed. This measure has been shown to be altered in stressed mice, this alteration being reversed by antidepressants [6,13,29]. Animals were then tested in two settings: a grooming test and a choice place test procedure. Grooming activity represents an important and multifunctional behaviour in many animals. Indeed, grooming serves to maintain the body hygiene of mice but has also been found to act as an antibacterial treatment in case of wound, as chemocommunication in a social context and as a displacement behavior in conflicting situations [32]. Previous studies have shown a decrease of grooming after a chronic stress [11,17] which is reversed by antidepressant treatment [29]. The choice place paradigm was used as a new proce-
dure to assess reaction to stress after the subchronic stress regime. Indeed, it allows detecting changes in the reactivity of stressed mice to a negative stimulus that was previously included in the subchronic stress procedure.
2. Material and methods 2.1. Animals Forty male mice of the outbred Swiss strain (Centre d’elevage Janvier, France), aged of 5 weeks at their arrival at the laboratory were used in this study. They were maintained under standard laboratory conditions, in a temperature-controlled room (24 ± 2 ◦ C), with food and water ad libitum and under a reversed 12/12 h light/dark cycle (light on at 8 pm). Mice were group-housed (10 mice per cages of 40 cm × 27 cm × 17 cm size) during all the experiment except for the mice of the stressed group that were singly-housed (in 26 cm × 23 cm × 14 cm cages) at the start of the subchronic stress until the end of the study. Behavioural testing started after a 3-week period of laboratory habituation. This allows to have adult mice (8 weeks old) when testing reactivity to the subchronic stress. This study was conducted in accordance with the Guide for Care and Use of Laboratory Animals established by the National Institute of Health of the United States of America and with applicable guidelines from the Ministère de l’Agriculture of France and European Community. 2.1.1. General procedure Since the protocol implies many manipulations of our animals, mice were first handled and exposed to an experimental apparatus (i.e. plus-maze) in order to reduce over-reactivity (habituation to experimental manipulation) and therefore biases. No data were recorded during this pre-test phase. Four days after this habituation period, mice were submitted to a conditioned task (see further). Three days after conditioned learning, subjects were submitted to a subchronic mild stress (see after). 2.2. Conditioned learning task 2.2.1. Apparatus The learning task took place in a circular arena (69 cm diameter and 31 cm-high walls) with four equidistant holes ( 4 cm diameter, 2 cm above the floor each) made in the wall (see Fig. 1). The floor was covered with familiar odour-saturated sawdust taken from home-cage. This allowed us to include some familiar cues in this apparatus to avoid extreme neophobic responses in mice. The arena was covered with a transparent plastic lid. A light (blue light, 50 lux) was placed above each hole. The arena was divided into five sectors: one central square (31 cm × 31 cm) and four lateral quadrants (one in front of each hole). An air puff administered through a small plastic pump
C. Ducottet et al. / Behavioural Brain Research 155 (2004) 291–299
Light Air puff
One sector
Hole
Fig. 1. The conditioned task apparatus used. Each sector contained a hole made in the wall. When a sector was illuminated, an air puff was sent by the hole each time the mouse entered this illuminated sector. Behaviour of the mouse were observed. Then, the light was switched off and the air puff removed. Behaviours of the mouse were observed again.
was used as a negative stimulus (S−). The mouthpiece of this pump was inserted in one of the four holes. 2.2.2. Procedure Mice were first familiarized with the arena during 30 min on days 1 and 2, by groups of five mice. During the last 10 min of this familiarisation period, one lateral quadrant (randomly chosen) was illuminated by the light. On days 3, mice were individually familiarized during 10 min with a lateral quadrant illuminated at random during the last 5 min. On days 4, mice were individually introduced in the central sector and allowed to explore the arena during 1 min. Then, when the mouse entered one of the two adjacent quadrants of the target quadrant, this later was illuminated during 1 min and an air puff was blown to the animal each time it entered this quadrant and until it left it. The light was then switched off, the air puff was removed and the mouse was observed during 1 min. Two trials were performed per day from days 4–6 in order to obtain a conditioned association between the illumination of a quadrant and the presence of the air puff considered as a negative stimulus (further called S−). The target quadrant changed semi-randomly between trials. On days 7, mice were confronted to a single probe trial without any air puff; it consisted of 1 min of exploration, 2 min with an illuminated quadrant (S− presence cue) and 2 min without any light (S− removal cue). The probe trial was video-taped and later analysed in slow-motion when necessary. During both periods (S− presence cue and S− removal cue periods) the latency to reach the target quadrant, the time spent in each quadrant (target, opposite, lateral left and lateral right ones), the time spent in the central sector, the number of nose-pokes inside holes, the number of rearings (vertical movements) and the number
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of oriented behaviours (i.e. when a mouse orients its head toward the target sector being immobile or walks toward the target and suddenly changes its direction) were recorded. Latency to reach the target quadrant allowed to assess level of attractiveness of a mouse toward the target sector when the light is switched on and then switched off. The time spent in each sector was recorded to detect whether mice tended to avoid or to approach the target sector. The number of nose pokes and rearings in the target sector were recorded to reveal the level of the mice exploration of both sectors and of the air puff source location (during both S− presence and S− removal cue periods). This provides information about the reactivity of mice toward the S−. The number of oriented behaviours revealed a possible behavioural orientation of the mice toward the target sector. 2.3. The subchronic stress procedure The original chronic stress procedure was proposed by Willner et al. [37] in rats. In this study, we used a modified version of the original procedure. Half of mice (n = 20) were confronted to a subchronic unpredictable mild stress. It included social stressors (a mouse was introduced in the cage of a congener, this last being itself transferred to another cage) and other unpredictable mild stressors during 2 weeks (see Table 1 for details). Stressed mice were singly housed at the start of the procedure while control mice were maintained group housed during all the experiment in a separate room. Despite the apparent unbalance, such a group-housed control group was preferred to a singly-housing condition as social isolation is highly stressful for mice and thus should per se contribute to the subchronic stress effects [5,23,31]. At the end of the procedure, mice were left one day without any stressor, except during the observation of their physical state test in order to avoid a direct effect of the last stressor to the subsequent observations. 2.4. Physical state evaluation Physical appearance evaluation was chosen as it represents a simple and quick method to assess stress state in mice. Various body areas (back, face, belly, tail, ears, eyes and noses) were observed and quoted “0”if clean or “1” if dirty or piloerected. Sum of these scores gave us an index of the general physical state of a mouse. It was evaluated the day before and on day 1 after the subchronic stress period. Physical state variation was calculated by subtracting the final from the initial value. Analyses were performed on this variation. 2.5. Grooming test On days 2 after the end of the subchronic stress, control mice were singly housed during 1 h in cages similar to the ones of stressed mice, in order to be familiarized with them. Then, both stressed and control mice received two
Light (3–5 p.m.) Dark (4–6 a.m.)
sprays of a sweet solution (10%) on their back. This solution acted as a releaser and a persisting factor of grooming due to its viscosity. The relative palatability of the solution prevented possible inhibition of self-grooming in case of aversive taste/smell. Total time spent in grooming behaviour and number of rearings (i.e. all vertical movement) were recorded during a 5 min session. This last behaviour was chosen as it was the main allocentered behaviour observed versus the egocentered grooming behaviour. Rearings provided a measure of the exploration of the animal (i.e. vigilance) during the test. Control mice were then again placed in their home cage.
Social stress (6 p.m.)
Social stress (4 p.m.) Light (7–8 p.m.)
2 sawdust changes (10 and 11 a.m.) Social stress (3 p.m.)
Light (3–5 p.m.) Dark (4–6 a.m.)
2.6. Choice place test
Social stress (13 p.m.) Cages tilt at 45 ◦ C (3–5 p.m.)
Social stress + no sawdust (11–12:30 a.m.) Cages tilt at 45 ◦ C (3–4 p.m.)
Succession of 4 light/dark every 30 min (9:30–11:30 a.m.) Succession of 4 light/dark every 30 min (9:30–11:30 a.m.) Dark (5–6 h) Damp sawdust (10 a.m.–13 p.m.) No sawdust (9:30–11 h) Week 2 End of the reversal cycle (10 a.m.)
Social stress (3 p.m.) No sawdust (3–5 p.m.)
Social stress (9:30 a.m.) Week 1 Social stress (12 a.m.)
3 sawdust changes (10:30–11:30 a.m.) Social stress (3 p.m.)
Cages tilt at 45 ◦ C (2–3 p.m. 30)
Social stress (12 a.m.)
Reversal of the light dark cycle Light on (2–6 p.m.)
Reversal of the light dark cycle Reversal of the light dark cycle
Damp sawdust (9:30–11 a.m.) Social stress (2 p.m.) Social stress (12 a.m.)
Day 7 Day 5 Day 2 Day 1
Day 3
Day 4
Day 6
C. Ducottet et al. / Behavioural Brain Research 155 (2004) 291–299
Table 1 Procedure used during the 2-week period of subchronic mild stress
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Mice reactivity to a mild stressor previously used in the subchronic stress (damp sawdust) was assessed in a large cage (40 cm × 27 cm × 17 cm) subdivided in two parts of equal size, separated by a wooden barrier (1.5 cm high). The floor was covered with sawdust. One part contained damp sawdust while the other contained dry one. The cage was covered by a grid. A mouse was introduced on the damp sawdust part. The time spent in each part, the latency to enter the dry part, the number of rearings, the number of grooming behaviours and the time spent in climbing the grid were recorded during 2 min. This test was performed on days 3 after the end of the stress. 2.7. Statistical analysis Classical non-parametric analyses were performed on measures obtained in the conditioned task and after the subchronic stress. We used the Friedman rank-sum test to compare the time spent in each sector of the conditioned task. Wilcoxon test was used for post-hoc pairwise comparisons. A ε risk was used to correct for multiple comparison errors, with ε = α/(k((k − 1)/2)), k being the number of groups, and α the risk that was not corrected for multiple comparisons [30]. In this study, k = 4 (i.e. 4 sectors): so, to satisfy a 5% significance threshold, probability must be lower than 0.008. Mann–Whitney tests were then performed to compare control and stressed groups on parameters recorded after the subchronic stress. Stepwise multiple regression analyses were carried out on stressed mice only (N = 20) to study the relationship between parameters of the conditioned task and responses to the subchronic stress. We used step up analyses with inclusion and exclusion criteria chosen respectively at 0.99 and 0.98. Behaviours observed in the conditioned task were used as predictive variables. Measures recorded after the subchronic stress was chosen as dependant variables. The multiple correlation coefficient (R2 ) and standardized regression coefficient (β) were calculated. Spearman correlations were also performed between conditioned task parameters and variables measuring sensitivity to the subchronic stress.
C. Ducottet et al. / Behavioural Brain Research 155 (2004) 291–299
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Table 2 Behaviours observed in the conditioned task
3. Results 3.1. Conditioned task Friedman test revealed no significant differences between the time spent in the different quadrants during the S− presence cue (Friedman’s χ2 = 2.5; d.f. = 3; P = 0.5) while it showed significant differences during the S− removal cue (χ2 = 10.98; d.f. = 3; P < 0.05). As to this last period, mice exhibited a trend to avoid the target sector as they spent more time in the opposite quadrant than in the target one (P < 0.01). Significant differences were observed between the number of oriented behaviours made during the S− presence cue and the S− removal cue period (P < 0.01). Number of nose pokes and rearing in the target sector tended to vary between the two periods (P = 0.08 for both). Interestingly, a large individual variability was observed in the responses to this probe trial, as revealed by the high interquartiles values obtained for all measures. Indeed, for all variables observed during the conditioned task, values spread on a continuum which indicates that the individuals exhibited various types of responses. Such variability is favourable to multiple regression analyses. Data are shown in Table 2.
S− presence cue
S− removal cue
Median
IQ
Median
IQ
Time in quadrant (sec)
20.8
13.9
Time in opposite quadrant (sec) Time in right adjacent quadrant (sec) Time in left adjacent quadrant (sec)
28.3
−9 +12.9 −9.3 +5.9 −6.3 +5.8 −5.9
−3.9 +17.9 −19.1 +13.1 −5.4 +17.1 −6.7
20 28.8
Time in centre (sec)
2.9
Number of nose pokes
0
Number of rearings
2
Number of oriented behaviours
4
+6.6 −2.2 +4.9 −0 +1 −1 +2 −1.3 +1
38.8a 16.3 30.6
4.8 1 1 2
+9.9 −2.6 +2.2 −1 +1 −0 +1.3 −0 +1
3.2. Reactivity to the subchronic stress
Medians and interquartiles (IQ) of the time spent in each quadrant of the circular arena, the time spent in the centre, the number of nose pokes in the hole of the target quadrant, the number of rearings in the target quadrant and the number of oriented behaviours toward the target quadrant. sec: Seconds a Significant difference (P = 0.01 level) between the time spent in the opposite vs. the target quadrant.
Stressed mice did not differ from the control ones for the physical state test (Mann–Whitney’s U = 174; P = 0.7; data not shown). Indeed, they did not show a significant physical external deterioration after the subchronic stress. However, the Mann–Whitney test revealed a significant decrease of the time spent in grooming behaviours in the stressed mice compared to the controls (U = 67; P < 0.01). This corresponds to the reduction of an important and multifunctional behaviour for rodents. Stressed mice also exhibited an increase of an exploratory behaviour (rearings) that may represent an interfering behaviour to grooming (U = 110; P <
0.05; Fig. 2). Confronted to the choice place test, stressed mice spent more time in the negative damp sawdust part than control mice (U = 19; P < 0.01) and spent less time in climbing the grid than controls (U = 60; P < 0.01). This may reveal a decrease of the most demanding items in term of effort (see Fig. 3). Latency to enter the more comfortable dry part was weakly increased in stressed mice (U = 237; P = 0.19; see Fig. 3). The number of rearing, the number of grooming behaviours and the time spent in the comfortable dry part of the cage did not vary between the two groups (U > 152; P > 0.3; data not shown).
Control Stressed
120
* 160
**
120
100 80 60
80 40
40
Number of rearing
Time spent in grooming (sec)
200
20
0
0
(a)
(b)
Fig. 2. Grooming behaviours (a) and rearings (b) during the grooming test. Mice were sprayed with a sweet solution that acted as a releasing and persisting factor of grooming behaviours. (a) Shows total time spent in grooming behaviours and (b) shows number of rearings during the test. Data shown represent medians and quartiles. ∗ P < 0.05 and ∗∗ P < 0.01 when compared to control group. Sec: seconds.
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**
60
Time (sec)
20
16
12
40 8
20 4
* 0
0
Dry part
Damp part
Latency to enter the dry sector( sec)
Control Stressed
80
Climbing
(b)
(a)
Fig. 3. Time spent in each half of the choice place arena (a) and latency to enter the dry part (b) in the choice place test. The mild stressor was damp sawdust. The two parts were separated by a small barrier (1.5 cm) and covered by a grid. Climbing occurred on this grid. Data shown represent medians and quartiles. ∗ P < 0.05 and ∗∗ P < 0.01 when compared to control group. Sec: seconds. Table 3 Stepwise multiple regression analyses predicting behaviours in the grooming test Conditioned task: S− presence cue (air puff)
Time spent grooming (sec) β R2
Time in opposite quadrant (sec) Time in centre (sec) Nose pokes in target quadrant
S.E.
Number of rearings P
= 0.6; P < 0.01
0.51 −0.37 0.36
β R2
±0.16 ±0.16 ±0.16
0.005 0.033 0.04
S.E.
P
= 0.46; P < 0.01
−0.44 – −0.5
±0.18 – ±0.18
0.02 – 0.01
Behaviours observed in the conditioned task were used as predictor variables. Time spent in grooming and number of rearings were used as dependant variables. R2 : multiple correlation coefficient, β: standardized regression coefficient, S.E.: standard error, sec: seconds.
3.3. Relationship between the conditioned task and reactivity to the subchronic stress Stepwise multiple regression analyses indicated a relationship between some behaviours observed after the subchronic stress (the time spent in grooming and the number of rearings in the grooming test as well as the time spent in climbing the grid during the choice place test) with parameters observed during the S− presence cue period of the initial conditioned task. Indeed, grooming duration was associated with the time spent both in the opposite and in the central quadrant and with the number of nose pokes in the target quadrant. The number of rearings was associated with the time spent in the opposite quadrant and with the number of nose pokes in the target (Table 3). As to the choice place test, climbings were related to both the number of rearings in the target quadrant and the latency to reach the target (Table 4). Behaviours observed in the conditioned task during the S− removal cue period failed to be significantly associated with any variable measuring reactivity to the subchronic stress. Physical state test was not considered in these analyses since no evolution of this index was observed (i.e. the index was constantly equal to zero). Significant correlations were observed between behaviour observed after the
subchronic stress and behaviour recorded in the initial conditioned task during the S− presence cue period (ρ’s > 0.41, P’s < 0.04). These Spearman correlations are summarized in Table 5. Behaviours observed in the conditioned task during the S− removal cue period failed to correlate with any variable recorded after the subchronic stress while physical state variation did not show significant correlation with any behaviour observed during the two periods of the conditioned task (i.e. during the S− presence and S− removal cue periods). Table 4 Stepwise multiple regression analyses predicting the time of climbing in the choice place test Conditioned task: S− presence cue (air puff) (R2 = 0.38; P < 0.001)
Time spent in climbing β
S.E.
P
Latency to target (s) Number of rearings Time in centre (s)
0.54 −0.24 −0.17
±0.13 ±0.13 ±0.14
0.0003 0.07 ns
Behaviours observed in the conditioned task were used as predictor variables while the time spent in climbing the grid in the choice place test was used as dependant variable. R2 : multiple correlation coefficient, β: standardized regression coefficient, S.E.: standard error, sec: seconds, and ns: non significant (i.e. P > 0.05).
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Table 5 Correlations between variables assessing reactivity to a subchronic stress and variables measured in the conditioned task Conditioned task with the S− presence cue (illumination) Time in opposite quadrant (sec)
Time in central quadrant (sec)
Number of nose pokes (sec)
Total time in lateral quadrant (sec)
Latency to the target quadrant (sec)
Number of oriented behaviours
Grooming test Time spent in grooming (sec) Number of rearings
0.58∗∗ −0.42#
−0.52∗∗ 0.54∗∗
ns −0.41#
ns ns
ns ns
ns 0.55∗∗
Choice place test Time in dry sector (sec) Time in damp sector (sec) Time spent in climbing Number grooming
ns ns ns ns
−0.42# ns ns 0.47∗
ns ns ns −0.49∗
ns ns 0.62∗∗ ns
ns −0.47∗∗ 0.70∗∗ ns
ns ns ns 0.47∗
Spearman correlations were made between behaviours observed in both the grooming test and the choice place test and the variables recorded in the conditioned task when the S− was supposed to be present (S− presence cue); ns: non significant (i.e. P >.05), and sec: seconds. ∗ P <.05. ∗∗ P < 0.01. # P < 0.07
4. Discussion The purpose of this study was to investigate the relationship between the reactivity of mice toward an acute mild stressor and their subsequent reactivity to a subchronic mild stress. Reactivity to threat was observed during a conditioned task, both during the S− presence cue (light switched on) and during the S− removal cue (light switched off). As expected, a great variability of response was observed in the parameters measured in this task during both periods, some mice spending more time in the target quadrant and others exhibiting few time in this quadrant. After 2 weeks of subchronic stress, mice exhibited a decrease in the time spent in grooming behaviours after they were sprayed with a sweet solution. Grooming is an important and multifunctional behaviour in rodents that act to maintain body hygiene, protection of wounds against bacteria, social communication (i.e. chemosignal), displacement behaviors and relaxation in stressful situations [32]. It was proposed that this reduction in grooming activities may be due to a retraction of mice toward a highly demanding situation [12,24]. However, during this test, stressed mice exhibited more rearings than controls, which may reflect a long distance exploration (i.e. vigilance). This increase in vigilance may be related to the fact that stressed mice may display increased apprehension toward the stressful spray (i.e. due to the stressful regime of the subchronic stress) which may interfere with grooming in reason of a competition between motivational systems. Indeed, as proposed by Bolles [3], stressed mice may select the most appropriate behaviors (i.e. vigilance versus hygiene/body maintenance) to this situation, in accordance with the behavioural hierarchies defined by the previous experience of subjects (i.e. repetition of stressors). Interestingly, Zalaquett and Thiessen [38] observed more vigilant behaviours in mice confronted to the odour of a stressed conspecific.
Unexpectedly, physical appearance did not degrade after the subchronic stress. This does not confirm data of previous studies that have reported a physical deterioration of the stressed mice [6–8,13,29]. The lack of physical changes may be explained by the use of a different strain of mice in this study. Indeed, an outbred mice population may include high responder as well as low responder and this may mask the stress effect on this measure. The previous studies were mainly undertaken with the high responding BALB/c strain [6–8,13,34]. In the choice place test, stressed mice spent more time in the negative part of the test (damp sawdust) than the control ones but did not differ from control animals for the time spent in the dry part. Stressed mice decreased their climbing time but did not change their locomotion (data not shown). This reduction may be due to a decrease of the most demanding behaviors in term of effort. One may also propose that they exhibited an alteration of their exploratory behaviours. Some authors have observed such decrease of exploration in mice after a social stress [18,19,27]. Different explanations have been given as increase of emotionality, motor retardation or sensitization of vegetative processes. Stepwise multiple regression analyses revealed relationships between some behavioural responses indicating sensitivity to the subchronic stress and some variables observed in the conditioned task, especially with parameters recorded when the cue signalling the presence of the S− was presented (first period of the probe task). For example, low level of grooming behaviours and high number of rearings (i.e. high sensitivity to the subchronic stress) were associated with some variables indicating a high reactivity toward the S− (few exploration of the target quadrant, attention toward the S−). Further, low levels of climbing in the choice place task (another variable indicating high sensitivity to the subchronic stress) were related to rapid behavioural orientation toward the S− when light was switched-on and
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to low exploration of the target quadrant. These data are confirmed by Spearman correlations indicating higher emotional reactivity to the S− in mice exhibiting the highest behavioural changes after the subchronic stress. Indeed, in stressed mice, a large decrease in grooming or increase of rearings in the “grooming test” was correlated with a longer time spent in the central quadrant and less time spent in the opposite quadrant of the conditioned task, with few nose-pokes in the target and many oriented behaviors. Moreover, regarding the choice test, decrease in climbing behaviors or a long time in the damp sector were correlated with a small latency to reach the target quadrant. Similar findings regarding the relationship between high emotional reactivity and high susceptibility to chronic stress have been obtained by some authors with different pre and post-stress tests [34,35]. Moreover, many authors have stated about the higher susceptibility to stress of reactive/passive animals [2,28,36]. Some authors have observed a high emotional reactivity associated to this coping style [8,33] that corresponds to behaviourally inhibited animals [16]. These results indicate an association between higher susceptibility to the subchronic stress and higher initial reactivity to threat stimuli associated with cognitive orientation of mice toward the stressor. However, it remains unclear if such cognitive orientations are directed toward the S− or toward each salient cues of the environment (e.g. positive or negative). Further studies are needed in this way. Interestingly, no relationship is found between the reaction to the S− removal and variables indicating sensitivity to the subchronic stress, indicating that prediction of the chronic stress susceptibility concerned the mice reactivity to a stimulus that is directly perceived. One may hypothesize that mice displaying such high level of reactivity toward an aversive stimulus may exhibit similar reactivity to each repeated stressor during the subchronic stress procedure, leading to a greater impact of these threats on these mice. So, emotional reactivity to a negative stimulus seems to represent a factor of susceptibility to repeated threats and play a role in the variability observed in the responses to the stress procedure.
Acknowledgements This study was supported by the Post-Genome Grant from French Ministere de l’Industrie. Authors also greatly acknowledge Raymond Jegat who built the experimental apparatus and Samuel Leman for helping in English text.
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[30] Siegel S, Castellan NJ. Nonparametric statistics for the behavioural sciences. 2nd ed. New York: McGraw-Hill; 1988. p. 399. [31] Spani D, Arras M, Konig B, Rulicke T. Higher heart rate of laboratory mice housed individually vs in pairs. Lab Anim 2003;37:54– 62. [32] Spruijt BM, Van Hooff JA, Gispen WH. Ethology and neurobiology of grooming behaviour. Physiol R 1992;72:825–52. [33] Steimer T. Neuroendocrine correlates of emotional reactivity and coping in male rats from the Roman high (RHA/Verh)- and low (RLA/Verh)-avoidance lines. Behav Genet 1997;27:503–12. [34] Tannenbaum B, Anisman H. Impact of chronic intermittent challenges in stressor-susceptible and resilient strains of mice. Biol Psychiatry 2003;53:292–303. [35] Touyarot K, Venero C, Sandi C. Spatial learning impairment induced by chronic stress is related to individual differences in novelty reactivity: search for neurobiological correlates. Psychoneuroendocrinology 2004;29:290–305. [36] Veenema AH, Meijer OC, de Kloet ER, Koolhaas JM. Genetic selection for coping style predicts stressor susceptibility. J Neuroendocrinol 2003;15:256–67. [37] Willner P, Towell A, Sampson D, Sophokleous S, Muscat R. Reduction of sucrose preference by chronic unpredictable mild stress, and its restoration by a tricyclic antidepressant. Psychopharmacology 1987;93:358–64. [38] Zalaquett C, Thiessen D. The effects of odors from stressed mice on conspecific behaviour. Physiol Behav 1991;50:221–7.
Research report
Susceptibility to subchronic unpredictable stress is related to individual reactivity to threat stimuli in mice C. Ducottet∗ , A. Aubert, C. Belzung EA3248 Psychobiologie des Emotions, UFR des Sciences et Techniques, Parc de Grandmont, 37 200 Tours, France Received 20 April 2004; accepted 30 April 2004 Available online 5 June 2004
Abstract As in many complex behavioral responses, inter-individual variability can be observed in the responses to a chronic mild stress. While some subjects exhibit more resilient behaviours, others appear more susceptible to stress. This study hypothesizes that this variability relies on the individual appraisal of the stressful event. To study this assumption, mice were first subjected to a conditioned task occurring in a circular arena. In this task, a mild air-puff (i.e. stressor) in a given quadrant of the arena was coupled with the presence or the absence of a light in the same quadrant. Half of mice were then submitted to a 15-day subchronic stress consisting in various environmental and social mild stressors randomly applied two or three times a day. At the end of this procedure, the occurrence of depressive-like behaviours in stressed mice was assessed using measures of the stress regime (i.e. physical state, choice test, grooming test). The physical state assessed the physical appearance of mice. The grooming test consisted in measuring the time spent in grooming after mice were sprayed upon with a viscous solution. The choice test consisted in measuring the time spent in an uncomfortable place (i.e. whose floor was covered with damp sawdust) versus a more comfortable one (i.e. with dry sawdust) to evaluate the reactivity to a negative stimulus previously encountered during the subchronic stress. Multiple regression analyses revealed a relationship between attention toward salient stressful stimuli in the conditioned task and susceptibility to the subchronic stress procedure. These results are discussed regarding their relevance for the understanding of aetiologies of depressive illnesses. © 2004 Elsevier B.V. All rights reserved. Keywords: Subchronic stress; Cognitive orientation; Conditioned task; Grooming
1. Introduction Chronic social stress has been widely used to assess depressive-like behaviours in animals like rodents [15,17,18,36], tree shrews [9,10,17] or birds [4]. Social stress represents a type of stressor that animals may encounter in their natural environment. Moreover, it constitutes a common negative stressor in humans. Such procedures exhibit therefore relevant face validity for the study of stress-related disorders. Many behavioural modifications have been observed in animals after a chronic social stress. Most of them report decrease in many activities like locomotor, grooming [7,8] or feeding behaviours, but also physiological changes such as alteration of the circadian body temperature rhythm or a decrease in body weight (for a review, see [20]). Similar modifications have been identi∗ Corresponding author. Tel.: +33 2 47 36 69 98; fax: +33 2 47 36 72 85. E-mail address: [email protected] (C. Ducottet).
0166-4328/$ – see front matter © 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.bbr.2004.04.020
fied in rodents submitted to an unpredictable chronic mild stress procedure. Indeed, these animals exhibit alterations in external physical appearance (i.e. dirty tarnished fur), decision-making [26] or sleep [22]. Recently, several authors have reported a sizeable interindividual variability in the physiological and behavioural responses observed after such a stress procedure [4,14,25,28,35]. This variability could reveal of primary importance for the investigation of the factors underlying susceptibility to stressful events. However, many of these studies use genetically-selected animals exhibiting extreme response patterns. This urges for the study of these inter-individual differences in a non selected population expressing the full range of behavioural patterns. The inter-individual variability has been interpreted as dependent on the coping style (to the stressors). A growing literature describes various coping styles used by animals [16], such as passive/active coping or, more recently, proactive/reactive coping. These styles seem related to the reactivity to subchronic stress. Indeed, reactive mice have been
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shown to be more susceptible to chronic stress and to recover less rapidly than proactive ones [2,28,36]. Previous study has considered the reactivity of mice to stressful events and therefore the susceptibility of an animal to chronic stressors in term of different individual appraisal of the situation [8]. Individuals that assess a situation as highly threatening will mobilize more resources to face or flight the stressor perceived. This can also be seen in anxious humans that exhibit attentional biases toward threatening cues of their environment [1,21]. Cognitive styles and coping styles are not necessarily opposed and a particular cognitive style can be a characteristic of a specific coping style. Cognitive and coping styles represent two different approaches but can be related to a general cognitive structure. The principal aim of this study is to examine the role of cognitive style in response to a subchronic mild stress. In this study, we investigated the relationship between the cognitive orientation mice exhibit toward a mild stressor, as revealed in a conditioned task, and the reactivity to a 2-week subchronic stress with both social and environmental stressors. A heterozygous mice population (Swiss) was chosen for this study in order to guarantee variability of responses. Cognitive orientations were assessed in a conditioned task in which animals had to associate the onset and the removal of a mild stressor with light switches (on and off respectively). The mild aversiveness of the stimulus allowed us to obtain a large variability of responses. After this first assessment, mice were confronted to a subchronic stress. This paradigm is based on social and environmental stressors. As mice are macrosmatic animals, odours play a major role in their social life. Therefore, social disturbing stimuli consisted in the daily reversal of cages between two randomly chosen counterparts (including soiled sawdust). Animals were thus kept under an unstable olfactive environment. This procedure is similar to the sensory contact model [18] but does not include physical contact and needs much less animals. Environmental stressors consisted in unpredictable uncomfortable events in the mice physical environment, i.e. known to induce stress in rodents [37]. At the end of the subchronic stress, the physical state of animals was assessed. This measure has been shown to be altered in stressed mice, this alteration being reversed by antidepressants [6,13,29]. Animals were then tested in two settings: a grooming test and a choice place test procedure. Grooming activity represents an important and multifunctional behaviour in many animals. Indeed, grooming serves to maintain the body hygiene of mice but has also been found to act as an antibacterial treatment in case of wound, as chemocommunication in a social context and as a displacement behavior in conflicting situations [32]. Previous studies have shown a decrease of grooming after a chronic stress [11,17] which is reversed by antidepressant treatment [29]. The choice place paradigm was used as a new proce-
dure to assess reaction to stress after the subchronic stress regime. Indeed, it allows detecting changes in the reactivity of stressed mice to a negative stimulus that was previously included in the subchronic stress procedure.
2. Material and methods 2.1. Animals Forty male mice of the outbred Swiss strain (Centre d’elevage Janvier, France), aged of 5 weeks at their arrival at the laboratory were used in this study. They were maintained under standard laboratory conditions, in a temperature-controlled room (24 ± 2 ◦ C), with food and water ad libitum and under a reversed 12/12 h light/dark cycle (light on at 8 pm). Mice were group-housed (10 mice per cages of 40 cm × 27 cm × 17 cm size) during all the experiment except for the mice of the stressed group that were singly-housed (in 26 cm × 23 cm × 14 cm cages) at the start of the subchronic stress until the end of the study. Behavioural testing started after a 3-week period of laboratory habituation. This allows to have adult mice (8 weeks old) when testing reactivity to the subchronic stress. This study was conducted in accordance with the Guide for Care and Use of Laboratory Animals established by the National Institute of Health of the United States of America and with applicable guidelines from the Ministère de l’Agriculture of France and European Community. 2.1.1. General procedure Since the protocol implies many manipulations of our animals, mice were first handled and exposed to an experimental apparatus (i.e. plus-maze) in order to reduce over-reactivity (habituation to experimental manipulation) and therefore biases. No data were recorded during this pre-test phase. Four days after this habituation period, mice were submitted to a conditioned task (see further). Three days after conditioned learning, subjects were submitted to a subchronic mild stress (see after). 2.2. Conditioned learning task 2.2.1. Apparatus The learning task took place in a circular arena (69 cm diameter and 31 cm-high walls) with four equidistant holes ( 4 cm diameter, 2 cm above the floor each) made in the wall (see Fig. 1). The floor was covered with familiar odour-saturated sawdust taken from home-cage. This allowed us to include some familiar cues in this apparatus to avoid extreme neophobic responses in mice. The arena was covered with a transparent plastic lid. A light (blue light, 50 lux) was placed above each hole. The arena was divided into five sectors: one central square (31 cm × 31 cm) and four lateral quadrants (one in front of each hole). An air puff administered through a small plastic pump
C. Ducottet et al. / Behavioural Brain Research 155 (2004) 291–299
Light Air puff
One sector
Hole
Fig. 1. The conditioned task apparatus used. Each sector contained a hole made in the wall. When a sector was illuminated, an air puff was sent by the hole each time the mouse entered this illuminated sector. Behaviour of the mouse were observed. Then, the light was switched off and the air puff removed. Behaviours of the mouse were observed again.
was used as a negative stimulus (S−). The mouthpiece of this pump was inserted in one of the four holes. 2.2.2. Procedure Mice were first familiarized with the arena during 30 min on days 1 and 2, by groups of five mice. During the last 10 min of this familiarisation period, one lateral quadrant (randomly chosen) was illuminated by the light. On days 3, mice were individually familiarized during 10 min with a lateral quadrant illuminated at random during the last 5 min. On days 4, mice were individually introduced in the central sector and allowed to explore the arena during 1 min. Then, when the mouse entered one of the two adjacent quadrants of the target quadrant, this later was illuminated during 1 min and an air puff was blown to the animal each time it entered this quadrant and until it left it. The light was then switched off, the air puff was removed and the mouse was observed during 1 min. Two trials were performed per day from days 4–6 in order to obtain a conditioned association between the illumination of a quadrant and the presence of the air puff considered as a negative stimulus (further called S−). The target quadrant changed semi-randomly between trials. On days 7, mice were confronted to a single probe trial without any air puff; it consisted of 1 min of exploration, 2 min with an illuminated quadrant (S− presence cue) and 2 min without any light (S− removal cue). The probe trial was video-taped and later analysed in slow-motion when necessary. During both periods (S− presence cue and S− removal cue periods) the latency to reach the target quadrant, the time spent in each quadrant (target, opposite, lateral left and lateral right ones), the time spent in the central sector, the number of nose-pokes inside holes, the number of rearings (vertical movements) and the number
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of oriented behaviours (i.e. when a mouse orients its head toward the target sector being immobile or walks toward the target and suddenly changes its direction) were recorded. Latency to reach the target quadrant allowed to assess level of attractiveness of a mouse toward the target sector when the light is switched on and then switched off. The time spent in each sector was recorded to detect whether mice tended to avoid or to approach the target sector. The number of nose pokes and rearings in the target sector were recorded to reveal the level of the mice exploration of both sectors and of the air puff source location (during both S− presence and S− removal cue periods). This provides information about the reactivity of mice toward the S−. The number of oriented behaviours revealed a possible behavioural orientation of the mice toward the target sector. 2.3. The subchronic stress procedure The original chronic stress procedure was proposed by Willner et al. [37] in rats. In this study, we used a modified version of the original procedure. Half of mice (n = 20) were confronted to a subchronic unpredictable mild stress. It included social stressors (a mouse was introduced in the cage of a congener, this last being itself transferred to another cage) and other unpredictable mild stressors during 2 weeks (see Table 1 for details). Stressed mice were singly housed at the start of the procedure while control mice were maintained group housed during all the experiment in a separate room. Despite the apparent unbalance, such a group-housed control group was preferred to a singly-housing condition as social isolation is highly stressful for mice and thus should per se contribute to the subchronic stress effects [5,23,31]. At the end of the procedure, mice were left one day without any stressor, except during the observation of their physical state test in order to avoid a direct effect of the last stressor to the subsequent observations. 2.4. Physical state evaluation Physical appearance evaluation was chosen as it represents a simple and quick method to assess stress state in mice. Various body areas (back, face, belly, tail, ears, eyes and noses) were observed and quoted “0”if clean or “1” if dirty or piloerected. Sum of these scores gave us an index of the general physical state of a mouse. It was evaluated the day before and on day 1 after the subchronic stress period. Physical state variation was calculated by subtracting the final from the initial value. Analyses were performed on this variation. 2.5. Grooming test On days 2 after the end of the subchronic stress, control mice were singly housed during 1 h in cages similar to the ones of stressed mice, in order to be familiarized with them. Then, both stressed and control mice received two
Light (3–5 p.m.) Dark (4–6 a.m.)
sprays of a sweet solution (10%) on their back. This solution acted as a releaser and a persisting factor of grooming due to its viscosity. The relative palatability of the solution prevented possible inhibition of self-grooming in case of aversive taste/smell. Total time spent in grooming behaviour and number of rearings (i.e. all vertical movement) were recorded during a 5 min session. This last behaviour was chosen as it was the main allocentered behaviour observed versus the egocentered grooming behaviour. Rearings provided a measure of the exploration of the animal (i.e. vigilance) during the test. Control mice were then again placed in their home cage.
Social stress (6 p.m.)
Social stress (4 p.m.) Light (7–8 p.m.)
2 sawdust changes (10 and 11 a.m.) Social stress (3 p.m.)
Light (3–5 p.m.) Dark (4–6 a.m.)
2.6. Choice place test
Social stress (13 p.m.) Cages tilt at 45 ◦ C (3–5 p.m.)
Social stress + no sawdust (11–12:30 a.m.) Cages tilt at 45 ◦ C (3–4 p.m.)
Succession of 4 light/dark every 30 min (9:30–11:30 a.m.) Succession of 4 light/dark every 30 min (9:30–11:30 a.m.) Dark (5–6 h) Damp sawdust (10 a.m.–13 p.m.) No sawdust (9:30–11 h) Week 2 End of the reversal cycle (10 a.m.)
Social stress (3 p.m.) No sawdust (3–5 p.m.)
Social stress (9:30 a.m.) Week 1 Social stress (12 a.m.)
3 sawdust changes (10:30–11:30 a.m.) Social stress (3 p.m.)
Cages tilt at 45 ◦ C (2–3 p.m. 30)
Social stress (12 a.m.)
Reversal of the light dark cycle Light on (2–6 p.m.)
Reversal of the light dark cycle Reversal of the light dark cycle
Damp sawdust (9:30–11 a.m.) Social stress (2 p.m.) Social stress (12 a.m.)
Day 7 Day 5 Day 2 Day 1
Day 3
Day 4
Day 6
C. Ducottet et al. / Behavioural Brain Research 155 (2004) 291–299
Table 1 Procedure used during the 2-week period of subchronic mild stress
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Mice reactivity to a mild stressor previously used in the subchronic stress (damp sawdust) was assessed in a large cage (40 cm × 27 cm × 17 cm) subdivided in two parts of equal size, separated by a wooden barrier (1.5 cm high). The floor was covered with sawdust. One part contained damp sawdust while the other contained dry one. The cage was covered by a grid. A mouse was introduced on the damp sawdust part. The time spent in each part, the latency to enter the dry part, the number of rearings, the number of grooming behaviours and the time spent in climbing the grid were recorded during 2 min. This test was performed on days 3 after the end of the stress. 2.7. Statistical analysis Classical non-parametric analyses were performed on measures obtained in the conditioned task and after the subchronic stress. We used the Friedman rank-sum test to compare the time spent in each sector of the conditioned task. Wilcoxon test was used for post-hoc pairwise comparisons. A ε risk was used to correct for multiple comparison errors, with ε = α/(k((k − 1)/2)), k being the number of groups, and α the risk that was not corrected for multiple comparisons [30]. In this study, k = 4 (i.e. 4 sectors): so, to satisfy a 5% significance threshold, probability must be lower than 0.008. Mann–Whitney tests were then performed to compare control and stressed groups on parameters recorded after the subchronic stress. Stepwise multiple regression analyses were carried out on stressed mice only (N = 20) to study the relationship between parameters of the conditioned task and responses to the subchronic stress. We used step up analyses with inclusion and exclusion criteria chosen respectively at 0.99 and 0.98. Behaviours observed in the conditioned task were used as predictive variables. Measures recorded after the subchronic stress was chosen as dependant variables. The multiple correlation coefficient (R2 ) and standardized regression coefficient (β) were calculated. Spearman correlations were also performed between conditioned task parameters and variables measuring sensitivity to the subchronic stress.
C. Ducottet et al. / Behavioural Brain Research 155 (2004) 291–299
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Table 2 Behaviours observed in the conditioned task
3. Results 3.1. Conditioned task Friedman test revealed no significant differences between the time spent in the different quadrants during the S− presence cue (Friedman’s χ2 = 2.5; d.f. = 3; P = 0.5) while it showed significant differences during the S− removal cue (χ2 = 10.98; d.f. = 3; P < 0.05). As to this last period, mice exhibited a trend to avoid the target sector as they spent more time in the opposite quadrant than in the target one (P < 0.01). Significant differences were observed between the number of oriented behaviours made during the S− presence cue and the S− removal cue period (P < 0.01). Number of nose pokes and rearing in the target sector tended to vary between the two periods (P = 0.08 for both). Interestingly, a large individual variability was observed in the responses to this probe trial, as revealed by the high interquartiles values obtained for all measures. Indeed, for all variables observed during the conditioned task, values spread on a continuum which indicates that the individuals exhibited various types of responses. Such variability is favourable to multiple regression analyses. Data are shown in Table 2.
S− presence cue
S− removal cue
Median
IQ
Median
IQ
Time in quadrant (sec)
20.8
13.9
Time in opposite quadrant (sec) Time in right adjacent quadrant (sec) Time in left adjacent quadrant (sec)
28.3
−9 +12.9 −9.3 +5.9 −6.3 +5.8 −5.9
−3.9 +17.9 −19.1 +13.1 −5.4 +17.1 −6.7
20 28.8
Time in centre (sec)
2.9
Number of nose pokes
0
Number of rearings
2
Number of oriented behaviours
4
+6.6 −2.2 +4.9 −0 +1 −1 +2 −1.3 +1
38.8a 16.3 30.6
4.8 1 1 2
+9.9 −2.6 +2.2 −1 +1 −0 +1.3 −0 +1
3.2. Reactivity to the subchronic stress
Medians and interquartiles (IQ) of the time spent in each quadrant of the circular arena, the time spent in the centre, the number of nose pokes in the hole of the target quadrant, the number of rearings in the target quadrant and the number of oriented behaviours toward the target quadrant. sec: Seconds a Significant difference (P = 0.01 level) between the time spent in the opposite vs. the target quadrant.
Stressed mice did not differ from the control ones for the physical state test (Mann–Whitney’s U = 174; P = 0.7; data not shown). Indeed, they did not show a significant physical external deterioration after the subchronic stress. However, the Mann–Whitney test revealed a significant decrease of the time spent in grooming behaviours in the stressed mice compared to the controls (U = 67; P < 0.01). This corresponds to the reduction of an important and multifunctional behaviour for rodents. Stressed mice also exhibited an increase of an exploratory behaviour (rearings) that may represent an interfering behaviour to grooming (U = 110; P <
0.05; Fig. 2). Confronted to the choice place test, stressed mice spent more time in the negative damp sawdust part than control mice (U = 19; P < 0.01) and spent less time in climbing the grid than controls (U = 60; P < 0.01). This may reveal a decrease of the most demanding items in term of effort (see Fig. 3). Latency to enter the more comfortable dry part was weakly increased in stressed mice (U = 237; P = 0.19; see Fig. 3). The number of rearing, the number of grooming behaviours and the time spent in the comfortable dry part of the cage did not vary between the two groups (U > 152; P > 0.3; data not shown).
Control Stressed
120
* 160
**
120
100 80 60
80 40
40
Number of rearing
Time spent in grooming (sec)
200
20
0
0
(a)
(b)
Fig. 2. Grooming behaviours (a) and rearings (b) during the grooming test. Mice were sprayed with a sweet solution that acted as a releasing and persisting factor of grooming behaviours. (a) Shows total time spent in grooming behaviours and (b) shows number of rearings during the test. Data shown represent medians and quartiles. ∗ P < 0.05 and ∗∗ P < 0.01 when compared to control group. Sec: seconds.
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**
60
Time (sec)
20
16
12
40 8
20 4
* 0
0
Dry part
Damp part
Latency to enter the dry sector( sec)
Control Stressed
80
Climbing
(b)
(a)
Fig. 3. Time spent in each half of the choice place arena (a) and latency to enter the dry part (b) in the choice place test. The mild stressor was damp sawdust. The two parts were separated by a small barrier (1.5 cm) and covered by a grid. Climbing occurred on this grid. Data shown represent medians and quartiles. ∗ P < 0.05 and ∗∗ P < 0.01 when compared to control group. Sec: seconds. Table 3 Stepwise multiple regression analyses predicting behaviours in the grooming test Conditioned task: S− presence cue (air puff)
Time spent grooming (sec) β R2
Time in opposite quadrant (sec) Time in centre (sec) Nose pokes in target quadrant
S.E.
Number of rearings P
= 0.6; P < 0.01
0.51 −0.37 0.36
β R2
±0.16 ±0.16 ±0.16
0.005 0.033 0.04
S.E.
P
= 0.46; P < 0.01
−0.44 – −0.5
±0.18 – ±0.18
0.02 – 0.01
Behaviours observed in the conditioned task were used as predictor variables. Time spent in grooming and number of rearings were used as dependant variables. R2 : multiple correlation coefficient, β: standardized regression coefficient, S.E.: standard error, sec: seconds.
3.3. Relationship between the conditioned task and reactivity to the subchronic stress Stepwise multiple regression analyses indicated a relationship between some behaviours observed after the subchronic stress (the time spent in grooming and the number of rearings in the grooming test as well as the time spent in climbing the grid during the choice place test) with parameters observed during the S− presence cue period of the initial conditioned task. Indeed, grooming duration was associated with the time spent both in the opposite and in the central quadrant and with the number of nose pokes in the target quadrant. The number of rearings was associated with the time spent in the opposite quadrant and with the number of nose pokes in the target (Table 3). As to the choice place test, climbings were related to both the number of rearings in the target quadrant and the latency to reach the target (Table 4). Behaviours observed in the conditioned task during the S− removal cue period failed to be significantly associated with any variable measuring reactivity to the subchronic stress. Physical state test was not considered in these analyses since no evolution of this index was observed (i.e. the index was constantly equal to zero). Significant correlations were observed between behaviour observed after the
subchronic stress and behaviour recorded in the initial conditioned task during the S− presence cue period (ρ’s > 0.41, P’s < 0.04). These Spearman correlations are summarized in Table 5. Behaviours observed in the conditioned task during the S− removal cue period failed to correlate with any variable recorded after the subchronic stress while physical state variation did not show significant correlation with any behaviour observed during the two periods of the conditioned task (i.e. during the S− presence and S− removal cue periods). Table 4 Stepwise multiple regression analyses predicting the time of climbing in the choice place test Conditioned task: S− presence cue (air puff) (R2 = 0.38; P < 0.001)
Time spent in climbing β
S.E.
P
Latency to target (s) Number of rearings Time in centre (s)
0.54 −0.24 −0.17
±0.13 ±0.13 ±0.14
0.0003 0.07 ns
Behaviours observed in the conditioned task were used as predictor variables while the time spent in climbing the grid in the choice place test was used as dependant variable. R2 : multiple correlation coefficient, β: standardized regression coefficient, S.E.: standard error, sec: seconds, and ns: non significant (i.e. P > 0.05).
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Table 5 Correlations between variables assessing reactivity to a subchronic stress and variables measured in the conditioned task Conditioned task with the S− presence cue (illumination) Time in opposite quadrant (sec)
Time in central quadrant (sec)
Number of nose pokes (sec)
Total time in lateral quadrant (sec)
Latency to the target quadrant (sec)
Number of oriented behaviours
Grooming test Time spent in grooming (sec) Number of rearings
0.58∗∗ −0.42#
−0.52∗∗ 0.54∗∗
ns −0.41#
ns ns
ns ns
ns 0.55∗∗
Choice place test Time in dry sector (sec) Time in damp sector (sec) Time spent in climbing Number grooming
ns ns ns ns
−0.42# ns ns 0.47∗
ns ns ns −0.49∗
ns ns 0.62∗∗ ns
ns −0.47∗∗ 0.70∗∗ ns
ns ns ns 0.47∗
Spearman correlations were made between behaviours observed in both the grooming test and the choice place test and the variables recorded in the conditioned task when the S− was supposed to be present (S− presence cue); ns: non significant (i.e. P >.05), and sec: seconds. ∗ P <.05. ∗∗ P < 0.01. # P < 0.07
4. Discussion The purpose of this study was to investigate the relationship between the reactivity of mice toward an acute mild stressor and their subsequent reactivity to a subchronic mild stress. Reactivity to threat was observed during a conditioned task, both during the S− presence cue (light switched on) and during the S− removal cue (light switched off). As expected, a great variability of response was observed in the parameters measured in this task during both periods, some mice spending more time in the target quadrant and others exhibiting few time in this quadrant. After 2 weeks of subchronic stress, mice exhibited a decrease in the time spent in grooming behaviours after they were sprayed with a sweet solution. Grooming is an important and multifunctional behaviour in rodents that act to maintain body hygiene, protection of wounds against bacteria, social communication (i.e. chemosignal), displacement behaviors and relaxation in stressful situations [32]. It was proposed that this reduction in grooming activities may be due to a retraction of mice toward a highly demanding situation [12,24]. However, during this test, stressed mice exhibited more rearings than controls, which may reflect a long distance exploration (i.e. vigilance). This increase in vigilance may be related to the fact that stressed mice may display increased apprehension toward the stressful spray (i.e. due to the stressful regime of the subchronic stress) which may interfere with grooming in reason of a competition between motivational systems. Indeed, as proposed by Bolles [3], stressed mice may select the most appropriate behaviors (i.e. vigilance versus hygiene/body maintenance) to this situation, in accordance with the behavioural hierarchies defined by the previous experience of subjects (i.e. repetition of stressors). Interestingly, Zalaquett and Thiessen [38] observed more vigilant behaviours in mice confronted to the odour of a stressed conspecific.
Unexpectedly, physical appearance did not degrade after the subchronic stress. This does not confirm data of previous studies that have reported a physical deterioration of the stressed mice [6–8,13,29]. The lack of physical changes may be explained by the use of a different strain of mice in this study. Indeed, an outbred mice population may include high responder as well as low responder and this may mask the stress effect on this measure. The previous studies were mainly undertaken with the high responding BALB/c strain [6–8,13,34]. In the choice place test, stressed mice spent more time in the negative part of the test (damp sawdust) than the control ones but did not differ from control animals for the time spent in the dry part. Stressed mice decreased their climbing time but did not change their locomotion (data not shown). This reduction may be due to a decrease of the most demanding behaviors in term of effort. One may also propose that they exhibited an alteration of their exploratory behaviours. Some authors have observed such decrease of exploration in mice after a social stress [18,19,27]. Different explanations have been given as increase of emotionality, motor retardation or sensitization of vegetative processes. Stepwise multiple regression analyses revealed relationships between some behavioural responses indicating sensitivity to the subchronic stress and some variables observed in the conditioned task, especially with parameters recorded when the cue signalling the presence of the S− was presented (first period of the probe task). For example, low level of grooming behaviours and high number of rearings (i.e. high sensitivity to the subchronic stress) were associated with some variables indicating a high reactivity toward the S− (few exploration of the target quadrant, attention toward the S−). Further, low levels of climbing in the choice place task (another variable indicating high sensitivity to the subchronic stress) were related to rapid behavioural orientation toward the S− when light was switched-on and
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to low exploration of the target quadrant. These data are confirmed by Spearman correlations indicating higher emotional reactivity to the S− in mice exhibiting the highest behavioural changes after the subchronic stress. Indeed, in stressed mice, a large decrease in grooming or increase of rearings in the “grooming test” was correlated with a longer time spent in the central quadrant and less time spent in the opposite quadrant of the conditioned task, with few nose-pokes in the target and many oriented behaviors. Moreover, regarding the choice test, decrease in climbing behaviors or a long time in the damp sector were correlated with a small latency to reach the target quadrant. Similar findings regarding the relationship between high emotional reactivity and high susceptibility to chronic stress have been obtained by some authors with different pre and post-stress tests [34,35]. Moreover, many authors have stated about the higher susceptibility to stress of reactive/passive animals [2,28,36]. Some authors have observed a high emotional reactivity associated to this coping style [8,33] that corresponds to behaviourally inhibited animals [16]. These results indicate an association between higher susceptibility to the subchronic stress and higher initial reactivity to threat stimuli associated with cognitive orientation of mice toward the stressor. However, it remains unclear if such cognitive orientations are directed toward the S− or toward each salient cues of the environment (e.g. positive or negative). Further studies are needed in this way. Interestingly, no relationship is found between the reaction to the S− removal and variables indicating sensitivity to the subchronic stress, indicating that prediction of the chronic stress susceptibility concerned the mice reactivity to a stimulus that is directly perceived. One may hypothesize that mice displaying such high level of reactivity toward an aversive stimulus may exhibit similar reactivity to each repeated stressor during the subchronic stress procedure, leading to a greater impact of these threats on these mice. So, emotional reactivity to a negative stimulus seems to represent a factor of susceptibility to repeated threats and play a role in the variability observed in the responses to the stress procedure.
Acknowledgements This study was supported by the Post-Genome Grant from French Ministere de l’Industrie. Authors also greatly acknowledge Raymond Jegat who built the experimental apparatus and Samuel Leman for helping in English text.
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