Integrative systems 1151
Unpredictable chronic mild stress not chronic restraint stress induces depressive behaviours in mice Shenghua Zhua, Ruoyang Shib, Junhui Wangc,d, Jun-Feng Wanga and Xin-Min Lic The chronic stress model was developed on the basis of the stress–diathesis hypothesis of depression. However, these behavioural responses associated with different stress paradigms are quite complex. This study examined the effects of two chronic stress regimens on anxiety-like and depressive behaviours. C57BL/6 mice were subjected to unpredictable chronic mild stress or to chronic restraint stress for 4 weeks. Subsequently, both anxiety-like behaviours (open field, elevated plus maze and novelty suppressed feeding) and depression-like behaviours (tail suspension, forced swim and sucrose preference) were evaluated. Both chronic stress models generated anxietylike behaviours, whereas only unpredictable chronic mild stress could induce depressive behaviours such as increased immobility and decreased sucrose consumption. These results of the present study provide additional evidence on how chronic stress affects behavioural
Introduction Depression is characterized by affective, cognitive and physiological impairments, and is one of the most common psychiatric disorders [1]. Approximately 20% of the population is afflicted with depression [2]. Given that socioenvironmental chronic stressors influence the development of depression, stress-based animal models have been used to explore biological mechanisms of this disorder. Most of these animal models resemble some of the dysfunctions observed in human depression, including pronounced weight loss, increased anxiety, as well as anhedonia [3,4]. Prolonged exposure of experimental animals to a variety of stressors is associated with significant changes in animal behaviours. Animals subjected to chronic restraint stress (CRS), a model of chronic stress, show increased anxiety level and possible depressive behaviours [5]. However, in terms of changes in behaviour, it has been shown that predictable restraint stress has a less negative impact than unpredictable stressors as animals are habituated following repeated exposure to restraint [6]. Therefore, unpredictable chronic mild stress (UCMS) has been developed as an experimental model of depression because of their uncertainty [6]. Converging evidence has shown that UCMS induces anhedonia-like behaviour in mice [7]. The aim of the present study was to analyse both UCMS and CRS paradigms and to compare possible behavioural differences between these models of chronic stress. 0959-4965 © 2014 Wolters Kluwer Health | Lippincott Williams & Wilkins
responses and point to the importance of the validity of animal models of chronic stress in studying depression. NeuroReport 25:1151–1155 © 2014 Wolters Kluwer Health | Lippincott Williams & Wilkins. NeuroReport 2014, 25:1151–1155 Keywords: anxiety, chronic restraint stress, depression, unpredictable chronic mild stress Departments of aPharmacology and Therapeutics, bHuman Anatomy and Cell Science, Faculty of Medicine, University of Manitoba, Winnipeg, Manitoba, c Department of Psychiatry, Faculty of Medicine and Dentistry, University of Alberta, Edmonton, Alberta, Canada and dMental Health Center, Shantou University, Shantou, Guangdong, China Correspondence to Xin-Min Li, MD, PhD, Department of Psychiatry, 1E7.31 Walter C. Mackenzie Health Sciences Centre, Faculty of Medicine and Dentistry, University of Alberta, Edmonton, AB, Canada T6G 2B7 Tel: + 1 780 4076503; fax: + 1 780 4076804; e-mail: [email protected] Received 10 June 2014 accepted 9 July 2014
The changes in the anxiety-like and depressive behaviours were evaluated using the open-field test, elevated plus maze test, novelty suppressed feeding test, tailsuspension test (TST), forced-swim test (FST) and sucrose preference test.
Materials and methods Animals
Seven-week-old female C57BL/6 mice were purchased from Charles River Canada (Montreal, Canada). Upon arrival, the animals were housed four per cage, with free access to food and water under controlled laboratory conditions (a 12 : 12-h light/dark cycle, room temperature 21 ± 1°C, and 60% humidity). Animals were allowed to habituate to the novel environment and they were handled daily by the same individual for 7 days before commencing stress procedures. On the basis of this parameter and body weight, animals were matched and divided into stress and control groups. All behavioural testing was performed in the light phase between 09:00 and 17:00 h. All procedures with animals were performed in accordance with the guidelines established by the Canadian Council on Animal Care and were approved by the Animal Care Committee of the University of Manitoba. Stress models
The animals were divided into three groups: control, CRS and UCMS. Controls were kept undisturbed in DOI: 10.1097/WNR.0000000000000243
Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.
1152 NeuroReport 2014, Vol 25 No 14
their home cages during the entire period of treatment, receiving only ordinary facility care with daily support of food and water. CRS was applied by placing the animal in well-ventilated polypropylene restrainers. The restraint procedure was performed during the morning between 09:00 and 11:00 h. The animals were stressed for 1 h/day for 4 weeks. The UCMS mice were subjected to different kinds of stressors such as restraint in a plastic tube, cage tilting, placement in an empty cage with no nesting, placement in crowded cages, lights on for a short period of time during the dark phase and white noises [8]. On average, two of these stressors were applied daily at different times, as shown in Table 1, with a semirandomized schedule. The stress procedure lasted for 4 weeks before the behavioural testing. Anxiety-like behaviour evaluation Open-field test
The open-field test was performed in a bare square box (36 × 36 inches) and it was partitioned into outer and inner zones. Mice were allowed to explore the maze for 5 min, after which they were returned to their home cage. The maze was cleaned with 75% ethanol wipes before commencing testing with the next mouse. The total distance was analysed as measures of motor ability. The time in the inner zone and total ambulation (m) were taken as measures of anxiety. Elevated plus maze test
The elevated plus maze consisted of two open and two closed arms (50 × 10 cm). Closed arms had 45 cm high walls at the sides and end. The entire maze was elevated to a height of 50 cm above the floor. Each mouse was Time and length of stressors used in 1 week of the unpredictable chronic mild stress procedure (following weeks will be different presentation)
Table 1
Restraint in a plastic tube a.m. Monday p.m. a.m. Tuesday p.m. a.m. Wednesday p.m. a.m. Thursday p.m. a.m. Friday p.m. a.m. Saturday p.m. a.m. Sunday p.m.
Cage tilt (45°)
Empty cage/ no nesting
13:00 ↓ 15:00
Crowded cage
Illumination in the dark phase
9:30 ↓ 12:30 11:00 ↓ 21:00
9:00 ↓ 13:00 12:00 ↓ 20:00
11:00 ↓ 13:00
White noise
18:00 ↓ 20:00
15:00 ↓ 17:00 18:00 ↓ 20:00
9:00 ↓ 11:00
16:00 ↓ 19:00 12:00 ↓ 20:00
9:00 ↓ 13:00
placed in the central square (10 × 10 cm) facing an open arm and allowed to explore the maze for 5 min [9]. One valid entry into any of the four arms was measured as all four paws of a mouse crossed from the central region into an arm, and the number of total open arm entries and the amount of time spent in the open arms were recorded. Novelty suppressed feeding
The test was performed as described previously [10]. The floor of the box (40 × 40 × 23 cm) was covered with 2 cm of bedding. Before the test, mice were deprived from food for 24 h. During the test, a single pellet of chow was placed on a white paper in the centre of the box. Animals were placed at one corner of the box. The latency to eat was recorded. The valid eat was defined as the mouse sitting on its haunches and biting the chow using its forepaws. Immediately after the bite, the mouse was moved back to its home cage with enough food supply. The amount of food consumed in the subsequent 5 min was measured. For this test, there was a cutoff time of 10 min. Depressive behaviour evaluation Tail-suspension test
Mice were suspended in the air using a paper clip fixed by tape wrapped around their tails about 20 mm from the extremity of the tail. The duration of immobility was recorded during the last 4 min of the 6-min test, which reflected the depressive state of the animal [11]. Forced-swim test
Mice were placed into plastic buckets (19 cm diameter, 23 cm deep, filled with 23–25°C water). As described previously by Porsolt et al. [12], only the last 4 min were scored for mobility duration. Immediately after the test, mice were covered by a dry towel and then placed under a heating lamp until it was dry. Sucrose preference test
Mice were first trained with both bottles while grouphoused before the behaviour test. The animals were exposed to a palatable sucrose solution (1%; Sigma, St Louis, Missouri, USA), followed by 4 h of water deprivation and a 1-h exposure to two identical bottles: one filled with sucrose solution and the other with plain water [13]. Liquid consumption was determined by measuring the change in the weight of fluid consumed. Sucrose preference was defined as the ratio of the volume of sucrose liquid compared with the total water consumed during the 1-h testing period. Food was withdrawn during the test. Statistical analysis
18:00 ↓ 20:00
All results were expressed as mean ± SEM. The significance of differences was determined by one-way analysis of variance (ANOVA), followed by a Tukey
Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.
Unpredictable stress induces depression Zhu et al. 1153
post-hoc test for multiple comparisons. A P value of less than 0.05 was considered statistically significant.
sucrose solution compared with the controls [one-way ANOVA, F(2,34) = 4.47, followed by Tukey’s multiple comparison test, P < 0.05]. There was no difference among the groups in the total fluid intake during the test sessions [one-way ANOVA, F(1,29) = 0.66, P > 0.5; Fig. 2d].
Results To evaluate anxiety-like behaviours of stressed mice, the open-field test, elevated plus maze and novelty suppressed feeding test were applied. Mice subjected to both UCMS and CRS showed a significant reduction in the time spent in the inner zone [one-way ANOVA, F(2,35) = 4.56, followed by Tukey’s multiple comparison test, P < 0.05; Fig. 1a]. In addition, mice subjected to CRS showed an increase in the total distance moved in comparison with the controls [one-way ANOVA, F(2,35) = 9.74, followed by Tukey’s multiple comparison test, P < 0.05; Fig. 1b]. Consistently, in the elevated plus maze test, the time spent in the open arms was significantly decreased in both CRS and UCMS groups [one-way ANOVA, F(2,34) = 7.4, followed by Tukey’s multiple comparison test, P < 0.05; Fig. 1c], whereas the number of entries to open arms was not affected (Fig. 1d). In the novelty suppressed feeding test, UCMStreated but not CRS-treated mice had significantly longer latencies before the first bite than the controls [one-way ANOVA, F(2,34) = 3.17, followed by Tukey’s multiple comparison test, P < 0.05; Fig. 1e]. However, the total amount of food consumed in CRS was slightly higher than that in the controls (Fig. 1f), suggesting that appetite may be likely to confound this test.
Discussion Consistent with previous studies, we confirmed that both UCMS and CRS induced anxiety-like behaviours in the open-field test, elevated plus maze and novelty suppressed feeding test. However, an increase in the immobility time in the TST and FST was only observed in animals exposed to UCMS. In addition, the UCMS group also showed reduced sucrose preference at the end of the UCMS regimen, suggesting decreased responsiveness to rewarding stimuli, the core characteristic of depressive states in animal models. The current study, by comparing the effects of two different models of chronic stress that were carried out at the same time, may help to show the validity of animal models of chronic stress in studying depression. Exposure to chronic stress is known to alter behavioural responses including increased anxiety [14], impaired working memory [15] and some depressive behaviours [16]. In this study, our results show that 4 weeks of CRS and UCMS is sufficient to induce anxiety in female C57BL/6 mice as measured by the decrease in centre measures in the open-field test and the decreased time in open arms in the elevated plus maze test (Fig. 1). This elevated anxiety in stressed mice of both groups may be a direct consequence of chronic stress. In addition, hyperlocomotion is considered a nonspecific consequence of prolonged exposure to restraint in rodents and it could be a confounding factor in anxiety and depression models in mice [17]. However, heightened locomotor activity was only observed in mice exposed to
Mice subjected to UCMS showed depressive behaviours, evaluated as the time of immobility in both the TST [one-way ANOVA, F(2,34) = 4.55, followed by Tukey’s multiple comparison test, P < 0.05; Fig. 2a] and the FST [one-way ANOVA, F(2,34) = 9.40, followed by Tukey’s multiple comparison test, P < 0.05; Fig. 2b]. However, there was no significant difference between the CRS and the control group (P > 0.05). As shown in Fig. 2c, UCMS mice but not CRS mice consumed significantly less Fig. 1
20 10 0
CON CRS UCMS
∗
30 20 10 0
CON CRS UCMS
40 30
∗ ∗
20 10 0
(e) 10
CON CRS UCMS
(f) 15
500 ∗
8 6 4 2 0
CON CRS UCMS
400 300 200 100 0
CON CRS UCMS
Food consumption (mg/g)
∗
40
(d) 50
Time in open arms (s)
∗
Total ambulation (m)
Time spent in centre (s)
40 30
(c) 50
Latency to feed (s)
(b) 50
Total open arm entries
(a)
10
5
0
CON CRS UCMS
Effects of exposure to CRS or UCMS for 4 weeks on anxiety-like behaviours. In the open-field test, decreased time spent in the inner zone in both CRS-exposed and UCMS-exposed mice (a) and increased locomotor activity in mice subjected to CRS (b). In the elevated plus maze test, less time was spent in the open arms by both CRS-exposed and UCMS-exposed mice, whereas the total number of entries to the open arms was not changed (d). In the novelty suppressed feeding test, the latency to feed was increased in mice subjected to UCMS compared with the control (e), whereas food consumption was not altered in all groups (f). All values are represented as mean ± SEM, n = 10–15 per group (*P < 0.05 vs. control mice). CON, control; CRS, chronic restraint stress; UPCS, unpredictable chronic stress.
Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.
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NeuroReport 2014, Vol 25 No 14
Fig. 2
90
∗
60 30
120
∗#
90 60 30
80
CON CRS UCMS
∗
60 40 20 0
0
0
(d)
100
Total liquid consumption (g)
120
(c)
150
Preference index (%)
(b)
150
Immobility duration (s)
Immobility duration (s)
(a)
CON CRS UCMS
4 3 2 1 0
CON CRS UCMS
CON CRS UCMS
Effects of exposure to CRS or UCMS for 4 weeks on depressive behaviours. The immobility time was significantly increased in the UCMS group, but not the CRS group, in comparison with the control mice in both the tail-suspension test (a) and the forced-swim test (b). The UCMS group also showed reduced sucrose preference (c), whereas the total liquid consumption was not affected in all groups (d). All values are presented as mean ± SEM, n = 10–15 per group (*P < 0.05 vs. control; #P < 0.05 vs. CRS). CON, control; CRS, chronic restraint stress; UPCS, unpredictable chronic stress.
CRS not UCMS in the open-field test, but not the elevated plus maze and novelty suppressed feeding test, suggesting that the level of locomotion did not confound the measurement of anxiety-like behaviours in mice subjected to CRS.
Acknowledgements
Contradictory results have been found when evaluating the effects of chronic stress on these depressive behavioural alternations [18]. One recent study has shown that predictable chronic stress even actually decreases depressive behaviours [19]. These inconsistent behavioural responses to CRS can be related to adaptations of the hypothalamic–pituitary–adrenal axis, which might desensitize or stabilize hypothalamus–pituitary–adrenal to these physical stressors [20]. However, converging evidence has shown that some models of unpredictable stress, such as the models used here, could consistently produce depression-like behaviours in animal studies [7,21]. The results of this study showed that UCMS but not CRS predictably produces an overall increase in immobility duration in both the TST and the FST (Fig. 2). Prolonged immobility in the FST and TST was used as a proxy for the symptoms of behavioural despair [11,12]. This despair behaviour was further validated by the sucrose preference test, in which animals in the UCMS group showed a decreased preference rate towards sucrose solution, indicating that UCMS induces anhedonia in mice. Together, both behavioural despair and reduced response to rewarding stimuli induced by UCMS are characterized as a depressive-like behaviour.
There are no conflicts of interest.
Shenghua Zhu is a recipient of the graduate studentships from the Manitoba Health Research Council. Conflicts of interest
References 1
2 3
4 5
6
7
8 9
10
11
Conclusion Both chronic stress models generated anxiety-like behaviours, whereas only UCMS could induce depressive behaviours including increased immobility and decreased sucrose consumption. These data provide new evidence showing that the behavioural responses to different stress paradigms are quite complex.
12
13
Moussavi S, Chatterji S, Verdes E, Tandon A, Patel V, Ustun B. Depression, chronic diseases, and decrements in health: results from the World Health Surveys. Lancet 2007; 370:851–858. Berton O, Nestler EJ. New approaches to antidepressant drug discovery: beyond monoamines. Nat Rev Neurosci 2006; 7:137–151. Cryan JF, Markou A, Lucki I. Assessing antidepressant activity in rodents: recent developments and future needs. Trends Pharmacol Sci 2002; 23:238–245. Krishnan V, Nestler EJ. The molecular neurobiology of depression. Nature 2008; 455:894–902. Zhao X, Seese RR, Yun K, Peng T, Wang Z. The role of galanin system in modulating depression, anxiety, and addiction-like behaviors after chronic restraint stress. Neuroscience 2013; 246:82–93. Bondi CO, Rodriguez G, Gould GG, Frazer A, Morilak DA. Chronic unpredictable stress induces a cognitive deficit and anxiety-like behavior in rats that is prevented by chronic antidepressant drug treatment. Neuropsychopharmacology 2008; 33:320–331. Mineur YS, Belzung C, Crusio WE. Effects of unpredictable chronic mild stress on anxiety and depression-like behavior in mice. Behav Brain Res 2006; 175:43–50. Mineur YS, Prasol DJ, Belzung C, Crusio WE. Agonistic behavior and unpredictable chronic mild stress in mice. Behav Genet 2003; 33:513–519. He J, Xu H, Yang Y, Zhang X, Li XM. Chronic administration of quetiapine alleviates the anxiety-like behavioural changes induced by a neurotoxic regimen of DL-amphetamine in rats. Behav Brain Res 2005; 160:178–187. David DJ, Samuels BA, Rainer Q, Wang JW, Marsteller D, Mendez I, et al. Neurogenesis-dependent and -independent effects of fluoxetine in an animal model of anxiety/depression. Neuron 2009; 62:479–493. Yan B, He J, Xu H, Zhang Y, Bi X, Thakur S, et al. Quetiapine attenuates the depressive and anxiolytic-like behavioural changes induced by global cerebral ischemia in mice. Behav Brain Res 2007; 182:36–41. Porsolt RD, Bertin A, Jalfre M. Behavioral despair in mice: a primary screening test for antidepressants. Arch Int Pharmacodyn Ther 1977; 229:327–336. Banasr M, Chowdhury GM, Terwilliger R, Newton SS, Duman RS, Behar KL, Sanacora G. Glial pathology in an animal model of depression: reversal of stress-induced cellular, metabolic and behavioral deficits by the glutamatemodulating drug riluzole. Mol Psychiatry 2010; 15:501–511.
Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.
Unpredictable stress induces depression Zhu et al. 1155
14
Campos AC, Fogaça MV, Aguiar DC, Guimarães FS. Animal models of anxiety disorders and stress. Rev Bras Psiquiatr 2013; 35 (Suppl 2): S101–S111. 15 Shansky RM, Rubinow K, Brennan A, Arnsten AF. The effects of sex and hormonal status on restraint-stress-induced working memory impairment. Behav Brain Funct 2006; 2:8. 16 Naert G, Ixart G, Maurice T, Tapia-Arancibia L, Givalois L. Brain-derived neurotrophic factor and hypothalamic–pituitary–adrenal axis adaptation processes in a depressive-like state induced by chronic restraint stress. Mol Cell Neurosci 2011; 46:55–66. 17 Strekalova T, Spanagel R, Dolgov O, Bartsch D. Stress-induced hyperlocomotion as a confounding factor in anxiety and depression models in mice. Behav Pharmacol 2005; 16:171–180.
18
19
20
21
Pijlman FT, Wolterink G, Van Ree JM. Physical and emotional stress have differential effects on preference for saccharine and open field behaviour in rats. Behav Brain Res 2003; 139:131–138. Parihar VK, Hattiangady B, Kuruba R, Shuai B, Shetty AK. Predictable chronic mild stress improves mood, hippocampal neurogenesis and memory. Mol Psychiatry 2011; 16:171–183. Marin MT, Cruz FC, Planeta CS. Chronic restraint or variable stresses differently affect the behavior, corticosterone secretion and body weight in rats. Physiol Behav 2007; 90:29–35. Pucilowski O, Overstreet DH, Rezvani AH, Janowsky DS. Chronic mild stress-induced anhedonia: greater effect in a genetic rat model of depression. Physiol Behav 1993; 54:1215–1220.
Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.
Unpredictable chronic mild stress not chronic restraint stress induces depressive behaviours in mice Shenghua Zhua, Ruoyang Shib, Junhui Wangc,d, Jun-Feng Wanga and Xin-Min Lic The chronic stress model was developed on the basis of the stress–diathesis hypothesis of depression. However, these behavioural responses associated with different stress paradigms are quite complex. This study examined the effects of two chronic stress regimens on anxiety-like and depressive behaviours. C57BL/6 mice were subjected to unpredictable chronic mild stress or to chronic restraint stress for 4 weeks. Subsequently, both anxiety-like behaviours (open field, elevated plus maze and novelty suppressed feeding) and depression-like behaviours (tail suspension, forced swim and sucrose preference) were evaluated. Both chronic stress models generated anxietylike behaviours, whereas only unpredictable chronic mild stress could induce depressive behaviours such as increased immobility and decreased sucrose consumption. These results of the present study provide additional evidence on how chronic stress affects behavioural
Introduction Depression is characterized by affective, cognitive and physiological impairments, and is one of the most common psychiatric disorders [1]. Approximately 20% of the population is afflicted with depression [2]. Given that socioenvironmental chronic stressors influence the development of depression, stress-based animal models have been used to explore biological mechanisms of this disorder. Most of these animal models resemble some of the dysfunctions observed in human depression, including pronounced weight loss, increased anxiety, as well as anhedonia [3,4]. Prolonged exposure of experimental animals to a variety of stressors is associated with significant changes in animal behaviours. Animals subjected to chronic restraint stress (CRS), a model of chronic stress, show increased anxiety level and possible depressive behaviours [5]. However, in terms of changes in behaviour, it has been shown that predictable restraint stress has a less negative impact than unpredictable stressors as animals are habituated following repeated exposure to restraint [6]. Therefore, unpredictable chronic mild stress (UCMS) has been developed as an experimental model of depression because of their uncertainty [6]. Converging evidence has shown that UCMS induces anhedonia-like behaviour in mice [7]. The aim of the present study was to analyse both UCMS and CRS paradigms and to compare possible behavioural differences between these models of chronic stress. 0959-4965 © 2014 Wolters Kluwer Health | Lippincott Williams & Wilkins
responses and point to the importance of the validity of animal models of chronic stress in studying depression. NeuroReport 25:1151–1155 © 2014 Wolters Kluwer Health | Lippincott Williams & Wilkins. NeuroReport 2014, 25:1151–1155 Keywords: anxiety, chronic restraint stress, depression, unpredictable chronic mild stress Departments of aPharmacology and Therapeutics, bHuman Anatomy and Cell Science, Faculty of Medicine, University of Manitoba, Winnipeg, Manitoba, c Department of Psychiatry, Faculty of Medicine and Dentistry, University of Alberta, Edmonton, Alberta, Canada and dMental Health Center, Shantou University, Shantou, Guangdong, China Correspondence to Xin-Min Li, MD, PhD, Department of Psychiatry, 1E7.31 Walter C. Mackenzie Health Sciences Centre, Faculty of Medicine and Dentistry, University of Alberta, Edmonton, AB, Canada T6G 2B7 Tel: + 1 780 4076503; fax: + 1 780 4076804; e-mail: [email protected] Received 10 June 2014 accepted 9 July 2014
The changes in the anxiety-like and depressive behaviours were evaluated using the open-field test, elevated plus maze test, novelty suppressed feeding test, tailsuspension test (TST), forced-swim test (FST) and sucrose preference test.
Materials and methods Animals
Seven-week-old female C57BL/6 mice were purchased from Charles River Canada (Montreal, Canada). Upon arrival, the animals were housed four per cage, with free access to food and water under controlled laboratory conditions (a 12 : 12-h light/dark cycle, room temperature 21 ± 1°C, and 60% humidity). Animals were allowed to habituate to the novel environment and they were handled daily by the same individual for 7 days before commencing stress procedures. On the basis of this parameter and body weight, animals were matched and divided into stress and control groups. All behavioural testing was performed in the light phase between 09:00 and 17:00 h. All procedures with animals were performed in accordance with the guidelines established by the Canadian Council on Animal Care and were approved by the Animal Care Committee of the University of Manitoba. Stress models
The animals were divided into three groups: control, CRS and UCMS. Controls were kept undisturbed in DOI: 10.1097/WNR.0000000000000243
Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.
1152 NeuroReport 2014, Vol 25 No 14
their home cages during the entire period of treatment, receiving only ordinary facility care with daily support of food and water. CRS was applied by placing the animal in well-ventilated polypropylene restrainers. The restraint procedure was performed during the morning between 09:00 and 11:00 h. The animals were stressed for 1 h/day for 4 weeks. The UCMS mice were subjected to different kinds of stressors such as restraint in a plastic tube, cage tilting, placement in an empty cage with no nesting, placement in crowded cages, lights on for a short period of time during the dark phase and white noises [8]. On average, two of these stressors were applied daily at different times, as shown in Table 1, with a semirandomized schedule. The stress procedure lasted for 4 weeks before the behavioural testing. Anxiety-like behaviour evaluation Open-field test
The open-field test was performed in a bare square box (36 × 36 inches) and it was partitioned into outer and inner zones. Mice were allowed to explore the maze for 5 min, after which they were returned to their home cage. The maze was cleaned with 75% ethanol wipes before commencing testing with the next mouse. The total distance was analysed as measures of motor ability. The time in the inner zone and total ambulation (m) were taken as measures of anxiety. Elevated plus maze test
The elevated plus maze consisted of two open and two closed arms (50 × 10 cm). Closed arms had 45 cm high walls at the sides and end. The entire maze was elevated to a height of 50 cm above the floor. Each mouse was Time and length of stressors used in 1 week of the unpredictable chronic mild stress procedure (following weeks will be different presentation)
Table 1
Restraint in a plastic tube a.m. Monday p.m. a.m. Tuesday p.m. a.m. Wednesday p.m. a.m. Thursday p.m. a.m. Friday p.m. a.m. Saturday p.m. a.m. Sunday p.m.
Cage tilt (45°)
Empty cage/ no nesting
13:00 ↓ 15:00
Crowded cage
Illumination in the dark phase
9:30 ↓ 12:30 11:00 ↓ 21:00
9:00 ↓ 13:00 12:00 ↓ 20:00
11:00 ↓ 13:00
White noise
18:00 ↓ 20:00
15:00 ↓ 17:00 18:00 ↓ 20:00
9:00 ↓ 11:00
16:00 ↓ 19:00 12:00 ↓ 20:00
9:00 ↓ 13:00
placed in the central square (10 × 10 cm) facing an open arm and allowed to explore the maze for 5 min [9]. One valid entry into any of the four arms was measured as all four paws of a mouse crossed from the central region into an arm, and the number of total open arm entries and the amount of time spent in the open arms were recorded. Novelty suppressed feeding
The test was performed as described previously [10]. The floor of the box (40 × 40 × 23 cm) was covered with 2 cm of bedding. Before the test, mice were deprived from food for 24 h. During the test, a single pellet of chow was placed on a white paper in the centre of the box. Animals were placed at one corner of the box. The latency to eat was recorded. The valid eat was defined as the mouse sitting on its haunches and biting the chow using its forepaws. Immediately after the bite, the mouse was moved back to its home cage with enough food supply. The amount of food consumed in the subsequent 5 min was measured. For this test, there was a cutoff time of 10 min. Depressive behaviour evaluation Tail-suspension test
Mice were suspended in the air using a paper clip fixed by tape wrapped around their tails about 20 mm from the extremity of the tail. The duration of immobility was recorded during the last 4 min of the 6-min test, which reflected the depressive state of the animal [11]. Forced-swim test
Mice were placed into plastic buckets (19 cm diameter, 23 cm deep, filled with 23–25°C water). As described previously by Porsolt et al. [12], only the last 4 min were scored for mobility duration. Immediately after the test, mice were covered by a dry towel and then placed under a heating lamp until it was dry. Sucrose preference test
Mice were first trained with both bottles while grouphoused before the behaviour test. The animals were exposed to a palatable sucrose solution (1%; Sigma, St Louis, Missouri, USA), followed by 4 h of water deprivation and a 1-h exposure to two identical bottles: one filled with sucrose solution and the other with plain water [13]. Liquid consumption was determined by measuring the change in the weight of fluid consumed. Sucrose preference was defined as the ratio of the volume of sucrose liquid compared with the total water consumed during the 1-h testing period. Food was withdrawn during the test. Statistical analysis
18:00 ↓ 20:00
All results were expressed as mean ± SEM. The significance of differences was determined by one-way analysis of variance (ANOVA), followed by a Tukey
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Unpredictable stress induces depression Zhu et al. 1153
post-hoc test for multiple comparisons. A P value of less than 0.05 was considered statistically significant.
sucrose solution compared with the controls [one-way ANOVA, F(2,34) = 4.47, followed by Tukey’s multiple comparison test, P < 0.05]. There was no difference among the groups in the total fluid intake during the test sessions [one-way ANOVA, F(1,29) = 0.66, P > 0.5; Fig. 2d].
Results To evaluate anxiety-like behaviours of stressed mice, the open-field test, elevated plus maze and novelty suppressed feeding test were applied. Mice subjected to both UCMS and CRS showed a significant reduction in the time spent in the inner zone [one-way ANOVA, F(2,35) = 4.56, followed by Tukey’s multiple comparison test, P < 0.05; Fig. 1a]. In addition, mice subjected to CRS showed an increase in the total distance moved in comparison with the controls [one-way ANOVA, F(2,35) = 9.74, followed by Tukey’s multiple comparison test, P < 0.05; Fig. 1b]. Consistently, in the elevated plus maze test, the time spent in the open arms was significantly decreased in both CRS and UCMS groups [one-way ANOVA, F(2,34) = 7.4, followed by Tukey’s multiple comparison test, P < 0.05; Fig. 1c], whereas the number of entries to open arms was not affected (Fig. 1d). In the novelty suppressed feeding test, UCMStreated but not CRS-treated mice had significantly longer latencies before the first bite than the controls [one-way ANOVA, F(2,34) = 3.17, followed by Tukey’s multiple comparison test, P < 0.05; Fig. 1e]. However, the total amount of food consumed in CRS was slightly higher than that in the controls (Fig. 1f), suggesting that appetite may be likely to confound this test.
Discussion Consistent with previous studies, we confirmed that both UCMS and CRS induced anxiety-like behaviours in the open-field test, elevated plus maze and novelty suppressed feeding test. However, an increase in the immobility time in the TST and FST was only observed in animals exposed to UCMS. In addition, the UCMS group also showed reduced sucrose preference at the end of the UCMS regimen, suggesting decreased responsiveness to rewarding stimuli, the core characteristic of depressive states in animal models. The current study, by comparing the effects of two different models of chronic stress that were carried out at the same time, may help to show the validity of animal models of chronic stress in studying depression. Exposure to chronic stress is known to alter behavioural responses including increased anxiety [14], impaired working memory [15] and some depressive behaviours [16]. In this study, our results show that 4 weeks of CRS and UCMS is sufficient to induce anxiety in female C57BL/6 mice as measured by the decrease in centre measures in the open-field test and the decreased time in open arms in the elevated plus maze test (Fig. 1). This elevated anxiety in stressed mice of both groups may be a direct consequence of chronic stress. In addition, hyperlocomotion is considered a nonspecific consequence of prolonged exposure to restraint in rodents and it could be a confounding factor in anxiety and depression models in mice [17]. However, heightened locomotor activity was only observed in mice exposed to
Mice subjected to UCMS showed depressive behaviours, evaluated as the time of immobility in both the TST [one-way ANOVA, F(2,34) = 4.55, followed by Tukey’s multiple comparison test, P < 0.05; Fig. 2a] and the FST [one-way ANOVA, F(2,34) = 9.40, followed by Tukey’s multiple comparison test, P < 0.05; Fig. 2b]. However, there was no significant difference between the CRS and the control group (P > 0.05). As shown in Fig. 2c, UCMS mice but not CRS mice consumed significantly less Fig. 1
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Effects of exposure to CRS or UCMS for 4 weeks on anxiety-like behaviours. In the open-field test, decreased time spent in the inner zone in both CRS-exposed and UCMS-exposed mice (a) and increased locomotor activity in mice subjected to CRS (b). In the elevated plus maze test, less time was spent in the open arms by both CRS-exposed and UCMS-exposed mice, whereas the total number of entries to the open arms was not changed (d). In the novelty suppressed feeding test, the latency to feed was increased in mice subjected to UCMS compared with the control (e), whereas food consumption was not altered in all groups (f). All values are represented as mean ± SEM, n = 10–15 per group (*P < 0.05 vs. control mice). CON, control; CRS, chronic restraint stress; UPCS, unpredictable chronic stress.
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Effects of exposure to CRS or UCMS for 4 weeks on depressive behaviours. The immobility time was significantly increased in the UCMS group, but not the CRS group, in comparison with the control mice in both the tail-suspension test (a) and the forced-swim test (b). The UCMS group also showed reduced sucrose preference (c), whereas the total liquid consumption was not affected in all groups (d). All values are presented as mean ± SEM, n = 10–15 per group (*P < 0.05 vs. control; #P < 0.05 vs. CRS). CON, control; CRS, chronic restraint stress; UPCS, unpredictable chronic stress.
CRS not UCMS in the open-field test, but not the elevated plus maze and novelty suppressed feeding test, suggesting that the level of locomotion did not confound the measurement of anxiety-like behaviours in mice subjected to CRS.
Acknowledgements
Contradictory results have been found when evaluating the effects of chronic stress on these depressive behavioural alternations [18]. One recent study has shown that predictable chronic stress even actually decreases depressive behaviours [19]. These inconsistent behavioural responses to CRS can be related to adaptations of the hypothalamic–pituitary–adrenal axis, which might desensitize or stabilize hypothalamus–pituitary–adrenal to these physical stressors [20]. However, converging evidence has shown that some models of unpredictable stress, such as the models used here, could consistently produce depression-like behaviours in animal studies [7,21]. The results of this study showed that UCMS but not CRS predictably produces an overall increase in immobility duration in both the TST and the FST (Fig. 2). Prolonged immobility in the FST and TST was used as a proxy for the symptoms of behavioural despair [11,12]. This despair behaviour was further validated by the sucrose preference test, in which animals in the UCMS group showed a decreased preference rate towards sucrose solution, indicating that UCMS induces anhedonia in mice. Together, both behavioural despair and reduced response to rewarding stimuli induced by UCMS are characterized as a depressive-like behaviour.
There are no conflicts of interest.
Shenghua Zhu is a recipient of the graduate studentships from the Manitoba Health Research Council. Conflicts of interest
References 1
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Conclusion Both chronic stress models generated anxiety-like behaviours, whereas only UCMS could induce depressive behaviours including increased immobility and decreased sucrose consumption. These data provide new evidence showing that the behavioural responses to different stress paradigms are quite complex.
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Moussavi S, Chatterji S, Verdes E, Tandon A, Patel V, Ustun B. Depression, chronic diseases, and decrements in health: results from the World Health Surveys. Lancet 2007; 370:851–858. Berton O, Nestler EJ. New approaches to antidepressant drug discovery: beyond monoamines. Nat Rev Neurosci 2006; 7:137–151. Cryan JF, Markou A, Lucki I. Assessing antidepressant activity in rodents: recent developments and future needs. Trends Pharmacol Sci 2002; 23:238–245. Krishnan V, Nestler EJ. The molecular neurobiology of depression. Nature 2008; 455:894–902. Zhao X, Seese RR, Yun K, Peng T, Wang Z. The role of galanin system in modulating depression, anxiety, and addiction-like behaviors after chronic restraint stress. Neuroscience 2013; 246:82–93. Bondi CO, Rodriguez G, Gould GG, Frazer A, Morilak DA. Chronic unpredictable stress induces a cognitive deficit and anxiety-like behavior in rats that is prevented by chronic antidepressant drug treatment. Neuropsychopharmacology 2008; 33:320–331. Mineur YS, Belzung C, Crusio WE. Effects of unpredictable chronic mild stress on anxiety and depression-like behavior in mice. Behav Brain Res 2006; 175:43–50. Mineur YS, Prasol DJ, Belzung C, Crusio WE. Agonistic behavior and unpredictable chronic mild stress in mice. Behav Genet 2003; 33:513–519. He J, Xu H, Yang Y, Zhang X, Li XM. Chronic administration of quetiapine alleviates the anxiety-like behavioural changes induced by a neurotoxic regimen of DL-amphetamine in rats. Behav Brain Res 2005; 160:178–187. David DJ, Samuels BA, Rainer Q, Wang JW, Marsteller D, Mendez I, et al. Neurogenesis-dependent and -independent effects of fluoxetine in an animal model of anxiety/depression. Neuron 2009; 62:479–493. Yan B, He J, Xu H, Zhang Y, Bi X, Thakur S, et al. Quetiapine attenuates the depressive and anxiolytic-like behavioural changes induced by global cerebral ischemia in mice. Behav Brain Res 2007; 182:36–41. Porsolt RD, Bertin A, Jalfre M. Behavioral despair in mice: a primary screening test for antidepressants. Arch Int Pharmacodyn Ther 1977; 229:327–336. Banasr M, Chowdhury GM, Terwilliger R, Newton SS, Duman RS, Behar KL, Sanacora G. Glial pathology in an animal model of depression: reversal of stress-induced cellular, metabolic and behavioral deficits by the glutamatemodulating drug riluzole. Mol Psychiatry 2010; 15:501–511.
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Unpredictable stress induces depression Zhu et al. 1155
14
Campos AC, Fogaça MV, Aguiar DC, Guimarães FS. Animal models of anxiety disorders and stress. Rev Bras Psiquiatr 2013; 35 (Suppl 2): S101–S111. 15 Shansky RM, Rubinow K, Brennan A, Arnsten AF. The effects of sex and hormonal status on restraint-stress-induced working memory impairment. Behav Brain Funct 2006; 2:8. 16 Naert G, Ixart G, Maurice T, Tapia-Arancibia L, Givalois L. Brain-derived neurotrophic factor and hypothalamic–pituitary–adrenal axis adaptation processes in a depressive-like state induced by chronic restraint stress. Mol Cell Neurosci 2011; 46:55–66. 17 Strekalova T, Spanagel R, Dolgov O, Bartsch D. Stress-induced hyperlocomotion as a confounding factor in anxiety and depression models in mice. Behav Pharmacol 2005; 16:171–180.
18
19
20
21
Pijlman FT, Wolterink G, Van Ree JM. Physical and emotional stress have differential effects on preference for saccharine and open field behaviour in rats. Behav Brain Res 2003; 139:131–138. Parihar VK, Hattiangady B, Kuruba R, Shuai B, Shetty AK. Predictable chronic mild stress improves mood, hippocampal neurogenesis and memory. Mol Psychiatry 2011; 16:171–183. Marin MT, Cruz FC, Planeta CS. Chronic restraint or variable stresses differently affect the behavior, corticosterone secretion and body weight in rats. Physiol Behav 2007; 90:29–35. Pucilowski O, Overstreet DH, Rezvani AH, Janowsky DS. Chronic mild stress-induced anhedonia: greater effect in a genetic rat model of depression. Physiol Behav 1993; 54:1215–1220.
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