1 Pilot-study
Investigating the effects of chronic
unpredictable mild stress and repetition of
behavioural tests on the expression of depressive-like components in adult male Wistar rats
The effect of cage enrichments and fluoxetine on depressive-like
behaviour in adult male Wistar rats
Thiwanan Khamphong, 11994274, University of Amsterdam, 2020 Prof. Dr. Judith Homberg, MSc. Kari Bosch, Department of Cognitive Neuroscience, Donders Institute for Brain, Cognition and Behaviour, Radboudumc Prof. Dr. Paul Lucassen, University of AmsterdamAbstract
To investigate the effect of environmental enrichment in combination with antidepressants on the depressive-like behaviour in rats, it is essential to first determine a proper model to induce and evaluate depressive-like behaviour in animals. The most commonly used and validated animal models are chronic unpredictable mild stress (CUMS) models. However, the CUMS protocol has many methodological variations in terms of stressors duration and types, which contribute to the ambiguous CUMS results. Studies have also shown that two weeks CUMS can successfully evoke depressive-like behaviour in rats. In addition, natural animal’s behaviour such as the ability to adapt to environmental stimuli should be taken into consideration. However, it is not known whether repetition of the same behaviour test performances could also induce behavioural adaptation in rats. Furthermore, whether the CUMS-induced depressive-like behaviour will persist in rats until the second test series. Therefore, the current pilot-study aimed to assess 1) whether two weeks CUMS protocol induces depressive-like behaviour that perseveres until the second test series in adult male Wistar rats 2) whether repetition of the same two behavioural test series could lead to behavioural habituation. Results showed a significant effect of stress on sleep/wake rhythm, exploratory behaviour in open field and elevated plus maze in rats, while test amount had a significant effect on exploratory and anxious-like behaviour. In addition, a significant interaction effect of stress and test amount was observed on exploratory and anxious-like behaviour in the elevated plus maze. Rats that performed two test series appeared to be more anxious compared to one test series rats. In conclusion, results of the second test series suggest, that the effects of two weeks of CUMS were observed on sleep/wake rhythm, anxious-like and exploratory behaviour in rats, with no effect on the behavioural despair sucrose preference and sociability. Furthermore, repetition of the same behavioural test performances did not lead to behavioural habituation in rats.Introduction
Depression is one of the most prevalent form of mental illness that severely affects a person’s quality of life. Depressive disorder is characterized by a spectrum of symptoms, ranging from mild to severe. Main symptoms of depression include anhedonia, anxiety,
circadian rhythm disturbance, and cognitive impairments (Salgado-Delgado, R., 2011; Ménard, C., 2016). At worst, the condition could lead to death by suicide, which is the main cause of high mortality rate of the illness (WHO., 2015). Despite a large number of
2 depression-related studies, the etiology
of depression still remains elusive. Together with the complexity of the illness, the effectiveness of the current available treatments such as anti-depressants also remain limited (Bosaipo, N. B. et al., 2017). Therefore, it is important to understand the underlying mechanisms of the development of depression and the action of anti-depressants.
Current first-line treatment of depression, next to cognitive behavioural therapy, consists of antidepressants, with selective serotonin reuptake inhibitors (SSRIs) being one of the most utilized due to its low rate of undesired side-effects and relatively good tolerability. The mechanism of action of SRRIs is to enhance the neurotransmission of serotonin in the brain by inhibiting serotonin reuptake of neurons (Sivolap, Y. P., 2017). In addition, serotonin induces neuroplasticity by modulating the glutamatergic transmission and N-methyl D-aspartate (NMDA) receptor. Neuroplasticity is the adapting ability of the brain to the changes in environmental stimuli, with synaptic homeostasis being one of the mechanisms of neuroplasticity that is relevant to the study of psychopathology (Kraus, C., 2017). Dysfunction of neuroplasticity has been shown to correlate with depression (Duman, R. S., 2004; Hayley, S., 2005).
Apart from the utilization of antidepressants, other studies also suggest that enrichments of the
environment improves the
neuroplasticity in depressive rodents by stimulating neurogenesis (Schloesser, R. J., 2010). However, it is unknown whether environmental enrichments in combination with antidepressants such as SSRIs has a positive effect on depression. Since a variety of models
used to evoke depressive-like behaviour in rodents exist, it is essential to first determine a proper model to induce depressive-like behaviour.
The most validated and commonly used animal model to evoke depressive behaviour in animals is the chronic unpredictable mild stress (CUMS) model. This model uses long-term or chronic stress exposure to induce depressive-like behaviour in rodents that to some extent, mimics aspects of human clinical psychopathology. Depressive behaviour in rodents is evaluated by performing behavioural tests (Willner P., et al 2017). Chronic stress is associated with physiological changes comprising neuroendocrine pathway mediated by stress system such as the hypothalamic-pituitary-adrenal (HPA) axis, which in turn leads to atrophy of neurons in the prefrontal cortex and hippocampus. The HPA axis is normally involved in the regulation of stress hormones such as cortisol (in human, and corticosterone in rodents) and is essential for survival and maintaining bodily equilibrium. Short-term repeated stress can be beneficial, because stress promotes adaptation to daily life challenges and stressors. However, long-term repeated stress can cause HPA axis imbalance, which can lead to impairments in mood, cognition and memory. HPA axis abnormalities is one of the most consistent biological validation to study depressive features (Gregus et al., 2005; Duman, R. S., & Aghajanian, G. K., 2012).
Nonetheless, the HPA axis imbalance can be recovered by the animals’ adapting ability to the repeated stressors (Babb, J. A. et al., 2014). Due to the adapting ability or habituation in animals, some chronic paradigms often fail to induce depressive behaviour. The CUMS paradigms have overcome this obstacle by using unpredictable repeated, instead of homotypic repeated stressors (Grissom,
3 N., & Bhatnagar, S. 2009), preventing
animal adaptation to the used stressors. This makes the CUMS paradigm an effective method and essential to the development of novel antidepressants. However, CUMS model suffers from a major problem, which is the low rates of cross-laboratory reproducibility. In general, most CUMS protocol are reproducible within laboratories, but cross-laboratories appear to be more difficult due to changes in environmental factors leading to ambiguous results, which are hard to interpret. Most studies that fail to reproduce cross-lab evidence often remain unpublished (Planchez, B., 2019). Therefore, to improve the reliability of the CUMS model, it is important to identify the experimental variables that cause the ambiguous CUMS results.
Previous studies have shown that the inconsistency of results can be caused by the variation in the stressor duration and type (Belovicova et al. 2017; Antoniuk et al., 2019), with the evidence of recent studies showing that two weeks CUMS can successfully evoke depressive-like behaviour in rats (Guedri, K., 2017; Koprdova, R., 2016).
Considering the adapting ability observed in animals, their susceptibility to stressors should also be taken into account (Salomons, A. R. et al., 2010). In addition, since two behavioural test series will be carried out during the planned follow-up study, it is important to investigate whether this repetition of the same behavioural test could lead to behavioural adaptation in rats. Furthermore, whether the CUMS-induced depressive-like behaviour will persist in rats until the second test series. Therefore, the goal of the full pilot-study is to assess which components of depressive-like behaviour are induced by the proposed two weeks CUMS, and which behavioural tests can be used to evaluate the induced depressive-like
behaviour in rats. However, due to the time frame of the internship, here we address only parts of these objectives. We set out to investigate the following: 1) whether two weeks CUMS protocol induces depressive-like behaviour that perseveres until the second test series in adult male Wistar rats, and 2) whether repetition of the same behavioural tests could lead to behavioural habituation.
Protocol
AnimalsMale Wistar rats (N=72) were obtained from Charles River, the Netherlands. All experiments were performed according to the Dutch federal regulations for animal protection, and approved by the Veterinary Authority of the Radboud University Medical Center, Nijmegen, the Netherlands. Throughout the whole experiment, rats were housed in a standard cage under an artificial light/dark cycle (12:12 h; lights on at 8:00 p.m.) and room temperature was maintained at 21°C ±1°C. Rats were housed in pairs per cage, cages were changed weekly. The habituation period endured for at least eight days. Five days prior to initiating experimental stress procedure, rats were handled each day for two minutes to habituate the animals to get picked up and to minimize stress responses when approached. Food (rat/mouse maintenance, Ssniff, v1534, Spezialdiäten GmbH, Germany) and saccharine water were available ad
libitum.
Experimental design
Male Wistar rats were randomly assigned to CUMS-exposed or no stress exposed (control) groups. After fourteen days of CUMS-exposure or no stress exposure, behavioural tests were performed on the animals to assess depressive-like behaviour. Both CUMS exposed and control groups were divided into two groups, each performing either
4 one or two behavioural tests series. This
resulted in total of four experimental groups (n=18, N=72) (Table 1, Figure 1). Only one behavioural test was performed per day, with minimal one day inter-tests sessions to minimize interactions between tests. In addition, behavioural tests were performed from least to most stressful (figure 1). In our
analysis we will focus on the readouts from the behavioural tests at test point II. Behavioural test consisted of phenotyping, sucrose test, open field maze, elevated plus maze, social Y maze and learned helplessness. Table 1: Experiment design of behavioural testing
Week 1 - 2 Week 3 - 4 Week 5 - 6 Week 7 - 8 Control-2 Regular housing Test point I Regular housing Test point II Control-1 Regular housing Regular housing Regular housing Test point II CUMS-2 CUMS Test point I Regular housing Test point II CUMS-1 CUMS Regular housing Regular housing Test point II
Chronic unpredictable mild stress
Rats assigned to the CUMS groups were exposed to stressors for fourteen consecutive days, immediately after their habituation period. Stressors that were used included social isolation, wet bedding, cage tilt (45°), light/dark reversal, strobe light, white noise (90 dB), and deprivation of water in the first three hours of the active period (dark phase). Stressors were provided for eight hours, except for the stroboscopic light, noise and water deprivation, which were provided three times one hour per day. In addition, light/dark reversal endured for 24 hours (Table 2).
Table 2: CUMS paradigm
Day Stressor Duration 1 Social isolation 8 h. 2 Wet bedding 8 h. 3 Cage tilt (45°) 8 h. 4 Light dark reversal 8 h. 5 Cage tilt (45°) 8 h. 6 Social isolation 8 h. 7 Strobe light 3x 60 min. 8 Wet bedding 8 h. 9 Noise 3x 60 min. 10 Water deprivation 3 h. 11 Light dark reversal 24 h. 12 Noise 3x 60 min. h. = hour; min. = minutes Behavioural tests
The first behavioural test series commenced on the fifteen day of the
experiment, after the CUMS exposure or no stress exposure housing. Only rats that were assigned to perform two test series underwent the first test point, while rats allocated to perform one test series were excluded and housed regularly during this time. The second test point occurred two weeks after the first test point, all experimental groups were included (Table 1). All behavioural tests were recorded with a camera and analysed using EthoVision XT (version 3.1, Noldus), except for sucrose preference test. Housing in PhenoTyper: activity patterns measurement Figure 1 The order of the behavioural test Each behavioural test was performed separately per day, with minimal one day inter-tests sessions to minimize interactions between tests. In addition, behavioural tests were performed from least to most stressful tests
5 Activity patterns of rats were assessed
using PhenoTyper cages (PhenoTyper 4500, Noldus) to determine the total distance moved (cm). Rats were single housed and allowed to habituate for one day. After habituation period, activities of rats were recorded for two consecutive days, each day from 8:00 am till 8:00 pm (24 h). During Phenotyper housing, food and saccharine water were available ad libitum, with an extra drinking bottle of drink water were added to familiarize the animals with the presence of two bottles for the sucrose preference test.
Sucrose preference test
Anhedonic behaviour in rats, characterized by sensitivity to reward(sucrose) were assessed using
the sucrose preference test. Rats were transferred from PhenoTyper housing to a standard housing, with one day period to habituate the regular cage. Rats were single housed and their sucrose preferences were determined for the next two consecutive days. During the sucrose preference test, two drinking bottles were provided with one being filled with sucrose in saccharine water, while the other was filled with saccharine water bottles were switched daily to prevent side preference bias. Subsequently, the amount of sucrose intake was determined by the weight of bottles and the percentage of sucrose preference was calculated as follow: 𝑆𝑢𝑐𝑟𝑜𝑠𝑒 𝑤𝑎𝑡𝑒𝑟 (𝑔)
𝑥 100
𝑆𝑢𝑐𝑟𝑜𝑠𝑒 𝑤𝑎𝑡𝑒𝑟 (𝑔) + 𝑠𝑎𝑐𝑐ℎ𝑎𝑟𝑖𝑛𝑒 𝑤𝑎𝑡𝑒𝑟 (𝑔)
Open field maze
Anxiety-like behaviour was assessed using the open field maze (OFM). Rats performed this test under 5 lux and were placed in a square shaped OFM (100x100x40 cm) with their head faced to one of the corners. Animals were able to freely explore the OFM for 5 minutes. Rat’s movements were recorded using a camera to determine the time spent in the center area of the open field (63 x 63), latency to enter the center area and the distance moved (cm). The maze was cleaned using 70% ethanol after each animal session.
Elevated plus maze
Anxiety-like behaviour of rats was also evaluated using the elevated plus maze (EPM). The maze consisted of two open arms (48.5x10 cm) and two closed arms (49x10 cm) arranged in opposite directions as a ‘plus sign’ around a center
platform. The maze was located 46.5 cm above the floor and animals performed the test under red light. Rats were place in the middle of the center area with their heads faced toward one of the open arms and were able to explore the maze freely for 5 minutes. Rats’ movements were recorded using a camera to determine the time spent in both open and closed arms, frequency to enter arms and distance moved(cm). The maze was cleaned with 70% ethanol after each animal session.
Social Y maze
The sociability and preference for social novelty of rats were assessed using the social Y maze and consisted of three arms (50x15x40 cm each), each at an angle of 120°, with a chamber at the end of each arm. Two of the three chambers were closed with a retractable wire door. Four
6 hours prior to the social Y maze, rats
were isolated and housed individually. The social test was performed under red lights. Before interacting with other rats, each rat was allowed to habituate the maze and explore freely for 15 minutes. After all animals were habituated to the maze, the rats performed the social test for 10 minutes. During the test, the cage-mate was present in one of the chambers, while an unfamiliar rat from the same experimental group was present in the other chamber. Rats were able to interact with one of the two rats in other chambers. The time spent with either its cage-mate or unfamiliar rat (i.e. nose point directed to one of the chambers at a close distance (<5 cm)) and the latency and frequency to enter one of the arms were assessed. The maze was cleaned with 70% ethanol after each session.
Learned helplessness
Behavioural despair or helplessness was assessed by the learned helplessness test using a dual channel shuttle box was used (ENV-010MD, Med Associates, St. Albans, VT, USA). The box consisted of two chambers setting divided by a closed retractable door in the center and was placed in a dark, sound attenuated environment. The floor of the chambers consisted of a grid enabling to conduct foot shocks. The test was performed for two consecutive days. On the first day, rats were able to habituate the novel environment for 5 minutes before they were exposed to 60 electric shocks of
0.60 mA for 6 to 15 seconds with inter-trial time of 24 seconds. Though, the center door was open and rats were able to move freely to the other chamber, the electric shocks were unescapable (i.e. presented in both chambers). On the second day, 24 hours after the unescapable shock exposure, rats were exposed to 10 trials of escapable electric shocks of 0.60 mA. The electric shocks were present in one of the chambers. Rats were able to escape the shocks by translocating through an open center door to the chamber with no shocks. The time it took for the animal to escape the shock, as well as the number of escaped trials was assessed. All chambers were cleaned with 70% ethanol after each session.
Statistical analysis
All data were analysed using Excel (version 16.37) and R-studio (version R 3.6.2 GUI 1.70 El Capitan build). The effect of stress and/or test amount on the behavioural test performances were analysed using two-way ANOVA, in case the data were normal distributed. Otherwise, Kruskal Wallis rank test was used. T-test was used as a comparisons test to determine the significant difference between experimental groups in case the data were normal distributed, in any other cases Wilcox rank test was used. Normality of data was determined using Shapiro-Wilk normality test. The significance level was set at p ≤ 0.05 for all statistical comparisons. Graphs were made using GraphPad Prism (version 8.4.3. (417).
7
Results
Activity patterns measurements Sleep/wake rhythms of rats were assessed by their activity patterns, determined by measuring the total distance moved of rats during two consecutive days in dark and light phase (Figure 2). A significant main effect of stress was observed on total distance moved during the light phases on day 1(p= 0.031) and day 2(p=0.01262). No further significant effects of stress or test amount were found. The total distance moved of CUMS-2 rats during the first day in light phase was significant higher compared to control-2 rats (p<0.001). Although, both CUMS-1 and 2 rats appeared to be less active during the dark phase compared to control rats, these differences were not significant.
Sucrose preference test
Anhedonic-like behaviour was assessed using sucrose preference test. Results showed no significant effect of both stress and test amount on the percentage and mean sucrose preference on the first and second day. Furthermore, no significance differences were observed of sucrose preferences between experimental groups (figure 3). Figure 2 Sleep/wake rhythms of rats assessed by determining the total distance moved during two consecutive days in dark and light phases A) Stress appeared to have a significant effect on the total distance moved of
rats during the first day, only in the light phase. With CUMS-2 rats being significantly more active compared to control-2 rats. During the dark phase of the first day, no significant effect of stress and test amount, and differences was observed between the experimental groups. (B) On the second day, no significant effect of stress and test amount, and differences of total distance moved were found between the experimental groups.
8 Open field maze Exploratory behaviour and anxiety were evaluated using the OFM. Stress (p= 0.01049) and an interaction effect of stress and test amount (p= 0.00454) appeared to have a significant effect on the total distance moved in open field. No further significant effect of test amount was observed on the total distance moved. The total distance moved of CUMS-1 rats were significantly higher than control-1 rats (p= 0.001) (figure 4A). In addition, no significant effects of both stress and test amount were observed on latency to first enter the center area, frequency of actually
entering the center area and total time spent in center area. Despite non-significant effects of stress and test amount on the total time spent in center area, CUMS-1 rats appeared to spend significantly more time in center area compared to control-1 (p= 0.029) and CUMS-2 rats (p= 0.032) (figure 4D). CUMS-1 rats also displayed a non-significant higher latency and frequency to actually entering the center area compared to control-1 rats. While CUMS-2 rats displayed a non-significant higher latency to first entering center area, they also showed a lower frequency of actually entering and spending time in center area (figure 4B, C).
Figure 3 Anhedonic behaviour of rats assessed by sucrose preference test
(A)/(B) no significant effect of both stress and
test amount on the percentage and mean 1% sucrose preference on the first and second day. Furthermore, no significance differences were observed of sucrose preferences between the experimental groups.
9
Elevated plus maze
Exploratory and anxiety-like behaviour were assessed using EPM. Results showed a significant main effect of test amount on the total time spent in open arms (p= 0.0006725), but no significant effect of stress. However, a significant effect of stress (p=0.036) and interaction-effect of stress and test amount (p<0.001) were observed on the total time spent in closed arms with no significant effect of test amount. CUMS-1 rats spent significantly less time on the closed arms compared to CUMS-2 rats (p<0.001), but they spent more time in open arms compared to CUMS-2 rats (p=0.022) and control-1 rats (p<0.001)
(figure 5A). Furthermore, the total distance moved in both open and closed arms were determined. Test amount appeared to have a significant effect on the total distance moved in both open (p<0.001) and closed (p<0.001) arms. While stress (p= 0.0042) and interaction-effect (p=0.004) significantly affected the total distance moved in only closed arms, with no further significant effect of stress found in open arms. The total distance moved in open arms of CUMS-1 rats was higher compared to CUMS-2 (p<0.001) and control-1 (p=0.026). Control-1 rats also had a significant higher total distance moved in closed arms compared to control-2 (p=0.026) (figure 5B). Figure 4 Exploratory and anxiety-like behaviour of rats were evaluated by performing the open field test (A) Stress (p= 0.01049) and an interaction effect of stress and test amount (p= 0.00454) appeared to have a significant effect on the total distance moved. CUMS-1 rats were significantly more active compared to control-1 rats (p= 0.001). (B)/(C)/(D) No significant effect or differences between experimental groups were found on frequency, latency and total time spent in center area. (D) CUMS-1 rats spent significantly more time in the open field compared to control-1 (p= 0.029) and CUMS-2 rats (p= 0.032).
10 Likewise, a significant effect of test
amount was found on the frequency entering the both open (p<0.001) and closed arms (p<0.001), with a significant interaction-effect observed only in closed arms (p=0.027). No further significant effect of stress was observed. CUMS-1 rats entered the open arms significantly more frequent compared to CUMS-2 (p<0.001), control-1 (p=0.019) and control-2 rats (p=0.032). CUMS-1 rats also entered the closed arms significant more frequently compared to control-2 rats (p=0.049). Whereas CUMS-2 rats entered closed arm
significantly less frequent compared to control-1 rats (p=0.010) (figure 5C). In addition, results showed a significant effect of test amount on the latency to first enter open (p<0.001) and closed arms (p=0.006). No further significant effect of stress was found. CUMS-1 rats had a significant lower latency rate to first enter the open arms (p<0.001), but higher latency rate to first enter the closed arms compared to CUMS-2 rats (p=0.007). Furthermore, CUMS-1 rats had a significant lower latency rate to first enter the open arms compared to control-2 rats (p=0.0073) (figure 5D). Figure 5 Anxiety-like and exploratory behaviour in rats assessed using elevate plus maze (EPM) (A) CUMS-1 rats spent significantly less time on the closed arms compared to CUMS-2 rats (p<0.001), while they spent more time in open arms compared to CUMS-2 rats (p=0.022) and control-1 rats (p<0.001). (B) The total distance moved in open arms of CUMS-1 rats was higher compared to CUMS-2 (p<0.001) and control-1 (p=0.026). While control-1 rats had a significantly higher total distance moved in closed arms compared to control-2 (p=0.026). (C) CUMS-1 rats entered the open arms significantly more frequent compared to CUMS-2 (p<0.001), control-1 (p=0.019) and control-2 rats (p=0.032). CUMS-1 rats also entered the closed arms significant more frequently compared to control-2 rats (p=0.049). Whereas CUMS-2 rats entered closed arm significantly less frequent compared to control-1 rats (p=0.010) (D) CUMS-1 rats had a significant lower latency rate to first enter the open arms (p<0.001), but higher latency rate to first enter the closed arms compared to CUMS-2 rats (p=0.007). Furthermore, CUMS-1 rats had a significant lower latency rate to first enter the open arms compared to control-2 rats (p=0.0073).
11 Social Y maze
The sociability and preference for social novelty of rats were assessed by determining the total time spent with unknown or familiar rats. No significant effects of both stress and test amount were found on the total time spent with unknown or familiar rats (figure 6A). In addition, no significant differences were
observed of the total time spent with unknown or familiar rats between the experimental groups. Similarly, no significant effects of stress and test amount, and no differences in the frequency of interacting with unknown or familiar rats were found (figure 6B). Learned Helplessness Behavioural despair of rats was assessed by determining the average escaping latency. Test amount appeared to have a significant effect on the average escaping latency only on the first trial (p= 0.0012). No further effect of test amount was observed in other trials. Stress appeared to have a significant effect on the average escaping latency, only on the sixth trial (p = 0.03253), with no further effect of stress observed in other trials. Both CUMS-1(p=0.021) and control-1 rats (p=0.014) had a significant higher
average escaping latency compared to control-2 rats in the first trial (figure 7A). Moreover, the number of rats that successfully escaped was determined. No significant effect of both stress and test amount was observed on the total number of rats that successfully escaped. However, an interaction effect of stress and test amount was observed in the seventh trial (p=0.021). CUMS-2 rats had a significant lower rate of escaping compared to control-2 rats in the fifth (p= 0.03527), seventh (p= 0.03527) and eighth trial (p=0.01335). Furthermore, Figure 6 The sociability and preference for social novelty of rats were assessed using social Y maze (A)/(B) No significant effects
of both stress and test amount were found on the total time spent with unknown or familiar rats. In addition, no significant differences were observed of the total time spent with unknown or familiar rats between the experimental groups. Similarly, no significant effects of stress and test amount, and differences in the frequency of interacting with unknown or familiar rats were found.
12 CUMS-2 rats appeared to have a
significant lower rate of escaping compared to control-1 rats in the fifth (p=0.03527), sixth (p= 0.03527), seventh (p= 0.03527), eighth trial (p=0.01335). Control-1 rats had a significant lower rat of successfully escaping compared to control-2 rats in the third (p=0.03527), seventh (p= 0.00395) and tenth trial (p=0.01335) (figure 7B, C).
Discussion
In this pilot-study, the effects of two weeks CUMS and repetition of behavioural tests series on the expression of depressive-like components were assessed in adult male Wistar rats. Results showed a significant effect of stress on sleep/wake rhythm, exploratory behaviour in OFM and EPM in rats, while test amount had a significant effect on exploratory and anxious-like behaviour. In addition, a significant interaction effect of stress and test amount were observed on exploratory and anxious-like behaviour in the EPM. With CUMS-1 rats being significantly more explorative and less anxious compared to control-1 and CUMS-2 rats, while CUMS-2 rats appeared to be non-significant less explorative and more anxious compared to control-2 and CUMS-1 rats.
Furthermore, increased behavioural despair was non-significant, but more obvious in CUMS-2 rats compared to control-2 and CUMS-1 rats, with no further significant differences or effects
of stress and test amount on behavioural despair in most trial during learned helplessness test.
In addition, only CUMS-2 rats were significantly more active during the light phase compared to control rats. Figure 7 Behavioural despair was assessed using learned helplessness test (A) The average escaping latency was significantly affected by test amount only on the first trial (p= 0.0012), and by stress only on sixth trial (p = 0.03253). Both CUMS-1 (p=0.021) and control-1 rats (p=0.014) had a significant higher average escaping latency compared to control-2 rats in the first trial (B)/(C) The total escaping rate was only significantly affected by the interaction effect of stress and test amount in the seventh trial (p=0.021). CUMS-2 rats had significantly lower rate of escaping compared to control-2 rats in the fifth (p= 0.03527), seventh (p= 0.03527) and eighth trial (p=0.01335). Furthermore, CUMS-2 rats appeared to have a significant lower rate of escaping compared to control-1 rats in the fifth (p=0.03527), sixth (p= 0.03527), seventh (p= 0.03527), eighth trial (p=0.01335). Control-1 rats had a significant lower rat of successfully escaping compared to control-2 rats in the third (p=0.03527), seventh (p= 0.00395) and tenth trial (p=0.01335).
13 Interestingly, neither stress nor test
amount appeared to have a significant effect on the sucrose preference and sociability in rats.
Thus, these results of the second test series suggest that the effects of two weeks CUMS were still observed on sleep/wake rhythm, anxious-like and exploratory behaviour in male Wistar rats, but not on the behavioural despair, sucrose preference and sociability. Furthermore, repetition of the same behavioural test performances did not lead to behavioural habituation in rats.
Considering the adapting ability of the animals to its environment, behavioural habituation to the repetition of the same behavioural tests is expected in rats. However, rats with two test series performances do not display behavioural habituation, but rather express more depressive-like components compared to one test series rats. Presumably, the non-habituation effect could be explained by the interfering effect of the first test series, i.e. electric foot shocks, which could possibly induce the stressful experiences in rats. This could contribute to the higher level of anxious-like behaviour in CUMS-2 compared to CUMS-1 rats, debunking behavioural habituation to behavioural test in CUMS-2 rats.
Anxiety, together with exploratory behaviour, are important components of depressive-like behaviour, which can be determined by comparing animals’ natural behaviours with their behaviours after stress exposure. Based on the natural habitats of most rodents, closed off areas are more preferred by rodents rather than open areas. At the same time, rodents display a tendency to explore a new environment. These natural behaviours of rodents were used to determine anxiety-like and exploratory behaviour, with avoidance of open areas, i.e. center area in open field or open arms
in EPM, and less exploratory behaviour indicating depressive-like state in rats (Belzung, C.,1999; Horii, Y., McTaggart, I., & Kawaguchi, M. 2018). Results show that CUMS-1 rats preferred open areas and displayed more exploratory behaviour compared to control-1 and CUMS-2 rats, while CUMS-2 rats preferred closed areas and were less explorative compared to control-2 and CUMS-1 rats. This indicates higher levels of anxiety-like and less exploratory behaviour in CUMS-2 compared to CUMS-1 rats.
However, the lowered exploratory behaviour in CUMS-2 could also be explained by behavioural habituation, since CUMS-2 rats could become familiarized by the OFM and EPM, leading to less interest in exploring the environment. Nevertheless, due to a higher level of anxious-like behaviour observed in CUMS-2 compared to CUMS-1 rats, indicating non-habituation to behavioural test by CUMS-2 rats.
Other components of depressive-like behaviour in animal is the disturbance of sleep/wake rhythm. In general, rodents are naturally more active during the dark phase and less active during the light phase, changes in these activity pattern insinuates the sleep/wake rhythm disturbance (Christiansen, S. L.,2016; Frank, M. G., 2017; Tapia-Osorio, A., 2013). Results show that only CUMS-2 rats appear to be significantly more active during the light phase, while both CUMS-1 and 2 rats were non-significantly less active during the dark phase. Together with the evidences of a significant effect of stress on activity pattern during the light phase, these results suggest a sleep/wake rhythm disturbance, which appear to be more prominent in CUMS-2 rats compared to CUMS-1 rats.
Additional factor that could contribute to the differences in the expression of depressive-like behaviour between
14 CUMS-exposed rats is stress resilience.
Some rodents are capable of coping with stress, leading to no development of depressive-like behaviour. Evidence has shown that 30% of CUMS induced rodents can become stress resilience, while remaining 70% are stress sensitive (Bergström, A., Jayatissa, M. N., Mørk, A., & Wiborg, O. 2008). Furthermore, the time interval between the last stressor and the initiation of the second test series could contribute to differences in the expression of depressive-like phenotype between CUMS-1 and 2 rats. Since the time interval between the last stressor and the second test series of CUMS-1 was four weeks, while CUMS-2 performed the first test series 24h after CUMS exposure, following a time interval of two weeks until the second test series. These differences in time frame and activity, i.e. one or two test series, between CUMS exposure and the anxiety measurements, possibly contribute to differences in the expression of depressive-like behaviour between CUMS-1 and CUMS2 rats. This is supported by other study, in which the delayed effect of CUMS on anxiety measurements in rats is shown, insinuating the importance of the time frame between the CUMS and behavioural tests (Matuszewich, L., 2007).
Despite CUMS paradigms being one of the most validated protocol used to evoke depressive-like behaviour in rodents, certain limitations can be identified in current pilot-study. One of which is the limited CUMS exposure duration to two weeks. This is considered a short period compared to other studies where CUMS exposure could endured up to 6 weeks (Sequeira-Cordero, A., 2019). The short CUMS duration could possibly contribute to the lowered to non-significant expression of the depressive-like components such as the behavioural despair, sucrose
preference and sociability by CUMS-exposed rats.
However, current experimental design with two weeks of CUMS exposure is necessary to coincide with the experimental design of the planned follow-up study, in which two weeks CUMS will also be applied. Despite the limited CUMS duration, a wide range of CUMS paradigm using two weeks of CUMS exposure are shown to effectively evoking depressive-like behaviour in rodents (Guedri, K., 2017; Koprdova, R., 2016). Therefore, the differences in the expression of depressive-like components in CUMS-exposed rats, as earlier mentioned could rather be due to stress resilience or differences in time frame and activity between the CUMS and behavioural tests. This showcases the importance of the current pilot-study by emphasising a possible factor that contributes to ambiguous chronic paradigm results.
As mentioned in the previous studies, natural animal’s behaviour, specifically the ability to adapt to changing environmental stimuli and animal’s susceptibility to stressors should be taken into consideration. Current pilot-study have found a non-habituation effect to the behavioural tests in rats, which presumably could be explained by the interfering effect of the first test series, i.e. electric foot shock. This insinuates the possibility of the interfering effect of the behavioural tests to the effect of CUMS. This knowledge could be useful during the methodological steps to measure the effect of CUMS paradigms, which are relevant when using animal model of depression.
Future research should be conducted to investigate the effect of ‘resting time’, which refers to the time frame between the last stressors exposure and the behavioural test, on the depressive-like behaviour in rats. Moreover, further
15 research should be done to investigate the
possible interfering effect of the behavioural tests to the effect of CUMS paradigms. Since rats with two test series performances did not display habituation, but rather expressed more depressive-like components compared to one test series rats.
To Summarize, the effects of two weeks CUMS were observed only on sleep/wake rhythm, anxious-like and exploratory behaviour in rats with no evidence of behavioural habituation in rats to repetition of the same behavioural test.
References
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18
Supplementals
19 Figure 8 Distribution of data Normality of data was observed on total distance moved during dark phase on day 2 (B), Sucrose preference on day 2 (C), the total distance moved in open field (D) and in closed arms of EPM (H), time spent and frequency to enter closed arms in EPM (G, I) and frequency to interact with known rats (M).
20 Figure 9 Main effect of stress and test amount on the total distance moved in PhenoTyper (sleep/wake rhythm assessment) Main effect of only stress was observed on the total distance moved only during the light phase. No further significant effect of stress and test amount was observed on the total distance moved of rats. Figure 10 Main effect of stress and test amount on the exploratory behaviour of rats in open field Main effect of stress and interaction effect of stress and test amount were found on the total distance moved in open field. No further effect of stress and test amount was found during open field test.
21 A B C D
22 E F G H Figure 12 Main effect of stress and test amount on anxious-like and exploratory behaviour during EPM Main effect of test amount was observed on total time spent in open arms (A), while main effect of stress and interaction effect of stress and test amount were observed on total time spent and distant moved in closed arms (B, D). Moreover, a significant effect of test amount was also observed on total distance moved (C, D), frequency (E, F) and latency (G, H) to enter both open and closed arms. In addition, an interaction effect of was found on frequency to enter closed arms (F).
