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Adolescent social housing conditions and resilience to adult social stress

Westgeest, Y.

Abstract

This research focused on finding indications of increased resilience to social stress by social housing conditions during adolescence. During this period, low aggressive Wistar rats were housed in pairs, in a colony with other Wistar rats or in a mixed colony with socially more skillful Wild-Type Groningen (WTG) rat for 25 days (PND 40 -75). After these different housing conditions, rats were housed individually and subjected to a social stressful event: a defeat in the resident-intruder paradigm. Severity of anxiety and stress was measured by an open field test, the elevated plus maze, serotonin 1A receptor sensitivity, changes in circadian body temperature, locomotor activity in the home cage and indicators of anhedonia such as sucrose consumption and behavioural and physiological anticipation to a reward.

Test results, however, show no direct indications of changes in resilient behaviour. For future research it is recommended that in mixed strain social colonies WTG rats are used that exceed Wistar rats in age.

This age difference will stimulate defensive behaviour of the Wistar rats that might result in an increased resilience towards social stress in later life.

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Introduction

In general, humans and many animal species grow up in complex social structures1. These structures support development of social skills which are vital to behavioural adaptation and survival of individuals belonging to this species.

Social stress in this structure potentially leads to (psycho) pathologies in individuals who are the victims of aggressive behaviour. Anxiety and depression appear as common forms of psychopathologies at individuals concerned2, 3. In our human society this negative social behaviour amongst others is clearly reflected in social bullying. Bullying, a major issue in today’s society, is presented as repetitive negative behaviour which results in physical injury and/or psychological suffering of the victim4. These victims are often individuals behaving slightly different compared to the rest of the individuals in the environment. They tend not to defend or stand up for themselves5. Bullying behaviour is frequently observed in juveniles and adolescents although bullying is also observed in adults6, 7. In a later stage of their lives victims of social bullying behaviour may show serious disorders like a general bad health2, psychological complaints and emotional disorders8. Victims (re)-act significantly more anxious and insecure than non-victimized individuals of the peer group and show defects in their emotional system9, a process facilitating coping with provocative situations resulting in decreased amounts of stress of negative emotions10. They regard themselves as being unattractive, less intelligent, and feel ashamed within their social environment. This frequently results in forms of sensitivity towards others and quiet behaviour combined with a negative view on situations and their own performances.

Victims most of the time feel lonely and are non-aggressive4, sometimes they show indicators of a post-traumatic stress disorder11.

Bullying can be described as a phenomenon with repeated social conflicts in which the victim continuously loses these conflicts4. By studying social loss or defeat experiences in animal models, insight into the possible neuronal mechanisms involved in the development of

psychopathology in victims of bullying behaviour can be gained12.

Research models

This social stress experience is demonstrated in rat and mice experiments using a resident- intruder model. According this model, a male rat or mouse is put in the home cage of a male conspecific, the resident. The resident is usually older, heavier and trained for attacking any intruders in his territory, resulting in submission of the intruder. The intruder will experience this a type of social stress resulting in long- lasting changes in brain, behaviour and physiology when defeated animals are housed individually8. These changes can be regarded as attempts to adapt to the situation but in some cases also as indicators of stress pathology.

Animal social defeat is therefore applicable for understanding the changes that human victims show13. Results of experiences with rats confronted with social defeat show a delayed body growth, reduced mobility, reduced activity in the open field, increased anxiety in the elevated plus maze and an increase in HPA activity8.

Most commonly, laboratory strains like the Wistar rat are used as an experimental model for research purposes on bullying behaviour14. This rat strain has been bred to be extremely docile when handling these animals. In contrast to the wild rat, its common ancestor, the Wistar rat shows very little aggressive behaviour due to this selection breeding of the strain. In addition to differences in levels of aggression, research also shows considerably less social skills for the Wistar rat when compared to the so-called Wild-type Groningen (WTG) strain. This WTG rat strain is known for its higher developed social behaviour skills when compared to the Wistar strain14, 15.

Based on these differences in social behaviour a study5 let adolescent male Wistar rats either grew up with either a female partner of the same strain or with slightly older WTG males and females. They expected that the Wistar males would be subjected to a kind of social

“bullying” in the mixed strain colonies because of their socially impaired behaviour. Yet, when exposed to social defeat in the previously described resident-intruder test, the Wistar rats

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seemed less affected by this defeat when compared to Wistar male rats that grew up with a female. It appeared that Wistar rats reared with WTG rats experience some kind of resilience training5.

Resilience is regarded the ability of individuals to avoid the effects of stress16. There appears to be large variation in resilience to social stress14. WTG rats actually seem to be less sensitive to social stress compared to Wistar rats17.

During both the prenatal18 and this adolescent19 stage of life the brain of an individual is most subject to changes and therefore social stimuli20,21. Changes in brain structures involve reproductive behaviour and the regulation of behaviour crucial to survive independently and without protection of parents and family. The changes can be either neurochemical or structural, caused by all kind of stimuli; both positive and negative (stress12,22). Main brain areas involved in social stress are the prefrontal cortex and the hippocampus17. The changes in the brain during this young adolescent phase in life are characterized as developmental plasticity2. This plasticity can be an explanation for the presence of a learning component in resilience against social stress. Rats demonstrate this learning process in play fights, starting with one rat soliciting another rat. This social play fight experience is stress reducing and crucial for behavioural flexibility, development of social skill, cognitive capacities and group cohesion37. An explanation for the resilient behaviour of Wistar rats in mixed strain colonies might be that they learn defensive-like behaviour during their interaction in play fights with WTG rats, using that experience to more effectively deal with the aggressive behaviour of the resident rat.

In this way rats might become more socially resilient rather than, as initially expected in the study of Buwalda et al.2, more stressed as victims of adolescent bullying. These findings are largely consistent with the match-mismatch theory23; individuals experiencing stress at a young age know how to cope with similar stressful situations at a later age. Individuals growing up without stress during childhood cannot handle stressful situations later in life effectively. Therefore, being able to successfully adapt to new social environments in a later stage

in life is strongly related to one’s early life experiences in social environments23.

The study’s first aim is to examine effects of social housing of adolescent rats on levels of resilience against social aggression in adulthood.

The second aim is on examination of optional stimulation of resilience by housing socially less skillful Wistar rats with socially more skillful WTG rats. Changes in physiology, behaviour and epigenetics are key targets of interest. By letting young adolescent Wistar rats grow up together with socially skillful WTG rats, these Wistar rats are expected to behave more resilient compared to Wistars growing up with similarly socially impaired Wistar rats in a colony. Wistar rats from the mixed strain colony setups are expected to defend themselves more efficient during the resident intruder test compared to the other Wistar rats.

In a study38 it is showed that social housing conditions during puberty do have an effect on the physiology of the victims after defeat. Pair housed male guinea pigs were compared to colony-housed males at an age of 4 months.

Social housing seems to have pervasive effects on social behaviour and HPA activity. Because social housing has different effects compared to individual housing, all rats are individually housed8. For reasons of test liability all resident intruder tests are recorded. This allows later analysis of adapted behaviour (e.g. ways of (upright) fighting per individual rat5). The stress of the resident intruder tests can be measured by different experiments such as serotonin receptor 1A sensitivity 24, locomotor activity, circadian temperature patterns, and anhedonia.

Serotonin sensitivity

Serotonin (5-HT) receptor sensitivity can be used as a measure of sensitivity to stress.

Previous research24 shows that ligands of the 5- HT1a receptor induce hypothermia in humans.

Depressed patients show less steep decline in temperature decrease compared to control healthy humans injected with a 5-HT1a agonist, indicating less 5-HT1a receptor sensitivity.

Often stress results in forms of depression25. Consequently, the postsynaptic receptor sensitivity provides an indication of mood reflecting perhaps the amount of stress individuals perceived24. 5-HT is involved in the

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control of somatic processes which are disturbed in affective disorders: mood, motor activity, function, circadian rhythms and body temperature24. In this research, rats from the mixed colonies are expected to show less decrease in temperature after a 5-HT1a receptor agonist.

Rhythms and body temperature

Circadian rhythms and body temperature can also function as indicators for the amount of stress experienced. Previous research outcomes26 show effects at Wistar rats’ activity and temperature after stressful periods. Directly after the stressful event a decrease in the amplitude of body temperature was found. At rest, the average body temperature proved to be higher. Even after 10 days, the body temperature was still not recovered from the stressful experience. Another effect is that locomotor activity in the home cage immediately decreases after the stressful event.

A reduction in activity and a decreased amplitude of body temperature fluctuation are regarded parameters to read out the amount of stress experienced26. In this research the rats of the Wistar colonies are expected to show a bigger decrease in body temperature and locomotor activity after the resident intruder test.

Anhedonia

Sucrose consumption

An important symptom of human depression is anhedonia, the inability to experience pleasure from factors which usually have a positive effect at mood. In stressed animals anhedonia is reflected as the impaired capability to anticipate for a reward27. In this study sucrose consumption and a pavlovian conditioning test are used as parameters for anhedonia.

Research17 shows the preference for a 1%

sucrose solution versus normal tap water.

Previous research indicates that Wistar rats already appear to have stronger preference for sucrose water than WTG rats5. In that study, pair-housed Wistar rats showed a significant decrease in sucrose intake after a social defeat, while WTG rats did not show this decrease and neither did Wistar males growing up with WTG conspecifics. In our experiments we therefore expect that the decrease in

consumption will not be observed in Wistar males housed in a colony setup together with WTG conspecifics. These rats also are expected to be less affected after the resident intruder test and consequently deal with less behavioural and physiological disturbances after defeat stress.

Pavlovian conditioning test

Based on Wistar rats’ preference for sucrose, research27 performed a classical Pavlovian conditioning test rewarding rats with sucrose pellets. Male Wistar rats received a visual light stimulus combined with an auditory stimulus.

This conditioning took place with an increasing time interval prior to reward supplies. Through an increased number of behavioural changes, rats showed conditioned anticipatory behaviour for the time period which starts with supplying the stimuli and ends providing sucrose rewards.

This increased number of behavioural changes could be found at rats subjected to a social defeat, however appeared to be lower in individually housed rats after their defeat. This was hypothesized to reflect the lowered sensitivity to rewards in individually housed rats after defeat indicative of anhedonia28. In the present study we expect rats that are less affected by stress to show increased hyperactivity.

Epigenetics

Finally, chronic social stress can lead to epigenetic DNA changes22,29. The negative development of bullied human victims appears to be related to epigenetic changes in the brain during puberty31. Epigenetic changes can occur by less strict bonding of DNA, allowing this DNA to (de-) activate different genes. HDAC6, an enzyme regulating the strictness of the DNA binding, seems to have an association with adaptation to the chronic stress that bullying entails (see appendix 1)30. A reduction in HDAC6 expression is a hallmark for more resilient animals31. Wistar rats in the mixed colonies are expected to experience less stress during the resident intruder test compared to Wistar rats growing up with other Wistar rats.

This increased resilience to social defeat stress might be reflected in Wistar rats that grow up in the mixed colonies having less HDAC6 in the raphe nucleus (see appendix 1). This will lead

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to less translocation of the glucocorticoid receptor to the core, resulting in a reduced signaling of raphe serotonergic neurons by glucocorticoids released during stress.

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Methods

Animals

Wistar rats (male n=40, female n=20) were obtained from the Harlan Laboratories (Horst, The Netherlands) at PND 24. Upon their arrival, the female animals were housed together, the male animals in groups of 5 respectively 6 in transparent plastic cages (60x35x18cm). Wild Type Groningen (WTG) rats (male n=20, female n=20) were obtained from the University of Groningen at PND 24.

Upon their arrival, the female animals were also housed together, the male animals in groups of 2 respectively 3 assuring that offspring of one litter stays together. These animals were also housed in transparent plastic cages (60x35x18cm). Food and water were supplied ad libitum. Temperatures were approximately 22⁰C during a 12:12 h day-night regime (lights on at 22:00, lights off at 10:00 to enable testing at the active phase of the animals). At PND 30 the female rats were anesthetized with Isoflurane-O2 and oviducts were cut. 1 mg flunixin-meglumin per kg weight was used as analgesia. At PND=40 rats were placed in the experimental setup, divided over group A, B, or E/F, see figure 1.

Male rats in group A and B were marked with black, red or white hair dye on their back to allow a quick discrimination between animals during behavioural observations. Male rats received a transmitter (Data Sciences International (St. Paul, MN, USA) model TA10TA-F40) at PND=60. Rats stayed in the experimental setup until PND 75, after that they are individually housed in cages (43x26,5x15,5cm) until PND=104 (see appendix 2 for an overview).

Hierarchy determination

The hierarchy of rats in the experimental setup cages A and B was determined by observing the first 10 minutes of the dark phase (subjective day) and checking for playing behaviour by scoring the amount of introducing fights, winning fights and excessive grooming other rats. Each day a hierarchy was calculated by observation, wherefrom the most dominant rat was given 1 point, the second most dominant rat 2 points etc. The total amount of points per rat after the period in the experimental setup was used as the dominance within the hierarchy.

Social defeat stress

Social stress was caused by social defeats by highly aggressive adult (approximately six months) male WTG rats, selected on their aggression. These males were selected on high offensive aggression when an intruder entered the cage. These resident rats were housed in a separate room in cages (45x50x30cm) with a female, to stimulate territorial aggression. Thirty minutes prior to the resident-intruder test the females were removed from the cage. On PND=80 and 81 the experimental animals were transported to the cage of the resident rats. The resident rats react with territorial, threatening behaviour and attacks, which stimulates the experimental rat to show submissive behaviour.

The experimental rats stayed in the cage for 15 minutes. After this time, the experimental rats were placed in a wire mesh cage (23x13x13 cm) and placed back in the resident cage until 60 minutes have passed. After this hour, experimental rats returned to the home cage.

Behaviour was subdivided in: upright behaviour (standing on hind legs), rearing (standing on hind legs against the resident, attacked (total time being attacked by the

Figure 1. Group A and B cage size (83x55x40cm), group E and F cage size (60x35x18cm).

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resident), cage exploration, social exploration (total time that the intruder explores the resident), freezing, washing, being explored (total time being explored by the resident), being chased by the resident and keep an eye on the resident (total time that the intruder actively watched what the resident is doing, and adapted to this situation by walking away or towards the resident).

Telemetry

Surgical implantation of the transmitter took place at PND = 60. For the experimental cage A, the two most submissive rats receive a transmitter, for the experimental cage B both the Wistar and the WTG rats received transmitters. All the control males in group E and F received transmitters as well. After PND=75 experimental rats were individually housed which enabled measurements of home cage activity and body temperature.

Measurements were recorded every 5 minutes with sensor plates underneath the home cage and processed by the LabPro computer program until the end of the experiment.

Before and after the defeat from the resident intruder test the biotelemetry provided information to compare.

Open field test

At PND = 76 rats were tested for anxiety levels and exploratory behaviour as a baseline prior to the resident intruder test. Rats were placed in the corner of a square (100 x 100cm) and observed (with Ethovision XT, Noldus Information Technology, Wageningen, Netherlands) for five minutes. In the middle of the square a small Lego block was placed.

Behaviour was tested at frequency in different zones (figure 2), the total duration in these zones and the time it took to enter the zone in the middle where the object was.

Elevated plus maze

Rats (PND = 82) were tested for anxiety with an elevated plus maze (height 55 cm, arm-length 45 cm). Experimental rats were placed in the maze for five minutes while a camera was recording the movement of the rat between the open and the closed arms.

Figure 2: A schematic overview of the different zones in the open field test.

The amount of anxiety in a new surrounding was measured depending on the time spent in the open and closed arms, which are safer. The platform at the crossing was not taken into account. The anxiety rate (The higher this rate, the less anxiety an animal is) was measured using the following formula:

Time (%) at the open arms / Time (%) at open and closed arms

Anhedonia - Sucrose preference test

Anhedonia is based on having less interest in rewarding stimuli after social stress. In this research, anhedonia was tested by a sucrose preference test and Pavlovian conditioning test.

For the sucrose test, daily measurements calculated the intake of a 1% sucrose solution versus tap water. The baseline consisted of the days PND = 76 to 79, prior to the resident intruder test. The bottles were weighed and switched every day to exclude place preference or conditioning. Sucrose water intake is calculated in comparison to the total amount of fluid intake in %. Previous research showed a decrease in sucrose consumption after repeated social defeat, calculated by the ratio sucrose intake versus the total amount of fluid intake in

% 5. In this study we checked if the intake of sucrose water was elevated due to social stress of the resident intruder test and whether this elevation returned to baseline.

Anhedonia – Pavlovian conditioning test

For the Pavlov conditioning test white chocolate was used. Rats received daily chocolate for 10 days in a row (PND= 94-103) after a stimuli of red light combined with an alarm for 10 seconds. The first day rats received chocolate

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Figure 3. Growth of the different experimental groups in grams throughout the experiment.

two minutes after the stimuli, the second day four minutes, the third day six minutes, the fourth day 10 minutes and the fifth and following days 15 minutes after the stimuli.

Research 27 indicates that rats that anticipated on a reward constantly switched the kind of behaviour. The amount of different kinds of behaviour can therefore be used as an indication of anticipation on a reward. With the telemetry system of this research, the amount of behavioural patterns could not be recorded.

Changes in behaviour might be reflected in the amount of activity hits read by the telemetry system, which was used as a measure of anhedonia.

5-HT1A sensitivity

Three weeks (PND=102) after social defeat the rats were injected with a 5-HT1A agonist (F15599 ultrapure AD (0,4mg/ml/kg)) to measure the potential decrease in 5-HT1A sensitivity by telemetry, reflecting the impact of social defeat stress.

Statistic analysis

Most tests were analysed by using an analysis of variance (ANOVA). Before the analysis of variance could take place the requirements should be satisfied (normal distribution and homogeneity of the variance). To fulfil these conditions a transformation of the model sometimes was required. Not every rat showed all the classified behaviour, resulting in some zero’s in the data. A normal distribution could not be obtained, therefore a binary binominal model was used for the analysis. For all the tests the program R (The R Project for Statistical Computing, Bell Laboratories, Murray Hill, New Jersey, USA) was used, with the packages

‘car’ and ‘multcomp’.

Results

Growth

In figure 3 the growth curves of the different groups of rats are shown. There appears to be a relation between dominance and body mass.

Dominant rats are heavier. In figure 4 the last ten days of the growth curves are zoomed in (A, B, C and D), to visualise the effects. Visually, the growth curves form 3 pairs each include two groups of rats. Figure 4a shows an overview of

the weight of all rats. The two groups with the lowest weight (figure 4b) contain the WTG rats of colony B and the WTG of F (controls). The middle groups (figure 4c) contain the Wistar rats of B and the subordinate Wistar rats of A.

The two groups with the highest weight (figure 4d) contain the Wistar rats of E and the dominant Wistar rats of A. At post natal day 40 differences between groups in body weight are present (figure 5). The differences in weight between the Wistar rats and the WTG rats are increasing throughout the experiment.

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Figure 4A Growth of all groups in grams from PND=94 to PND=104. Figure 4B shows the curves for the WTG rats exclusively, figure 4C for the subordinate Wistar rats and the Wistar rats from the mixed colonies as figure 4D shows the growth of the dominant Wistar rats and the control Wistar rats.

Weight PND 40

A Domi nant Wista

r

A Subordinate Wista r

B WT G

B Wista r

E Wista r

F WT G 0

50 100 150 200

*

**

* * *

**

Groups

Weight (in gram)

Figure 5. Weight on PND 40. * significant difference (p<0,05) compared to WTG rats of colony B. ** significant difference (p<0,05) compared to the controls WTG of F.

Open field prior to defeat Total distance moved

In figure 6 the total distance moved is reflected.

WTG rats, independent of the experimental setup, seem to move less compared to all the Wistar rat groups. The Wistar rats from the mixed setup, colony B, do not seem to behave differently from the other Wistar groups.

OF - Total Distance Moved

A Domi nant Wista

r

A Subordinate Wista r

B WT G

B Wista r

E Wista r

F WT G 0

1000 2000 3000 4000

5000 A Dominant Wistar

A Subordinate Wistar B WTG

B Wistar E Wistar F WTG

*

*

**

Groups

Total Distance Moved (cm)

Figure 6. Total distance moved during the open field in the arena (in cm). * significant difference (p<0.05) compared to WTG rats of colony B. ** significant difference (p<0.05) compared to the controls WTG of F.

Total duration zone 3

The total percentage of time spent in the middle zone corresponds with the previous finding; the middle zone is the location where the novel object is placed. WTG rats spent less time in this middle zone compared to the Wistar groups (figure 7). A significant difference is found between the WTG rats and Wistar rats, both of the mixed colonies.

OF - Zone 3 - Total Duration

A Domi nant Wista

r

A Subordinate Wista r

B WT G

B Wista r

E Wista r

F WT G 0

20 40 60

80 A Dominant Wistar

A Subordinate Wistar B WTG

B Wistar E Wistar F WTG

*

Groups

Total Duration (s)

Figure 7.Total duration spent in the middle zone during the open field in the arena (in sec).

* significant difference (p<0,05) compared to WTG rats of colony B. ** significant difference (p<0,05) compared to the controls WTG of F.

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Latency of first occurrence zone 3

The third parameter measured is latency time (figure 8); latency time is reflected as the time it takes to enter the middle zone from the start of the test. WTG rats take more time to enter this zone compared to Wistar rats. Significant differences are found between the WTG rats from the mixed colonies with the dominant and subordinate Wistar rats from setup A, and with Wistar rats of E, the controls. As for the WTG control rats, significant differences are found compared to all the Wistar groups. For all the parameters calculated in the open field it appears there are purely line differences between the Wistar and WTG strains.

OF - Zone 3 - Latency of first occurence

A Domi nant Wista

r

A Subordinate Wista r

B WT G

B Wista r

E Wista r

F WT G 0

50 100

150 A Dominant Wistar

A Subordinate Wistar B WTG

B Wistar E Wistar F WTG

* * *

** ** ** **

Groups

Latency of first occurence (s)

Figure 8. Time it takes to reach the middle zone during for the first time in the open field (in sec). * significant difference (p<0,05) compared to WTG rats of colony B. **

significant difference (p<0,05) compared to the controls WTG of F.

Resident intruder test

During the resident intruder tests the behaviour of the intruder was scored. Upright defensive behaviour, rearing behaviour, being attacked, exploration of the cage, social exploration, washing behaviour, freezing behaviour, being explored by the resident, being chased by the resident and the percentage of time that the intruder kept an eye on the resident were measured. In figure 9 the percentage of time spent on the different behaviours is shown.

WTG rats perform more upright defensive behaviour compared to all the Wistar rat groups. The Wistar rats of the mixed colonies do not show copying behaviour represented by more upright behaviour. Similar results apply to rearing. WTG rats, both control and colony,

perform more rearing behaviour compared to the Wistar groups. No indications of copying behaviour of Wistar rats from the mixed colonies are shown. WTG rats are attacked for a longer period compared to Wistar rats. This period consists of the sum of all attacks without the period of threatening the intruder by the resident. There is a slight increase in time being attacked by the resident for the Wistar rats from the mixed colonies, compared to the other Wistar rat groups. Time spent on cage exploration seems to have no clear relation to strain. Whereas WTG rats and the subordinate Wistar rats from the Wistar colony perform more cage exploration, the Wistar rats from the mixed colonies show almost no cage exploration. The dominant Wistar rats from the Wistar colony and the control Wistar rats are positioned between these extremes. There is a clear strain difference for social exploration;

WTG rats spent more time exploring the intruder compared to all the Wistar rat groups.

The increase in time spent on social exploration of the Wistar rats from the mixed colonies is too small to be a significant result. Wistar rats show a very high percentage of time spent on freezing behaviour. There is a clear strain difference in this parameter, WTG rats show almost no freezing behaviour and Wistar rats from mixed colonies do not show any copying behaviour towards the WTG rats. The number of rats that spent time on washing is too small to see clear results. The explorative behaviours of the resident towards the intruder are variable.

Dominant Wistar rats from the Wistar colony are explored the least, whereas the control Wistar rats and WTG rats are explored the most. The percentage time being chased by the resident shows a strain difference between WTG and Wistar. WTG rats are chased more, but the difference in percentage is very small.

WTG rats keep an eye on the resident for a longer period compared to all the Wistar groups. The Wistar rats of the mixed colonies do not perform copying behaviour towards the WTG rat.

Because Wistar rats grow up with WTG rats, it could be expected that Wistar rats copy the (defensive) behaviour of these WTG rats.

Besides copying behaviour, it could also occur

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that Wistar rats simply learn how to react in fight situations. In that case, Wistar rats should behave differently compared to the other

Wistar groups. This hypothesis is not reflected in figure 9.

Upright

A Dom inant

Wistar

A Subordi nate Wistar

B WTG B Wistar

E Wistar F WTG 0

2 4 6 8

Groups

Percentage Time

Rearing

A Dom inant

Wistar

A Subordi nate Wistar

B WTG B Wistar

E Wistar F WTG 0

5 10 15

Groups

Percentage Time

Total duration of attacks

A Dom inant

Wistar

A Subordi nate Wistar

B WTG B Wistar

E Wistar F WTG 0

10 20 30

Groups

Percentage Time

Cage exploration

A Dom inant

Wistar

A Subordi nate Wistar

B WTG B Wistar

E Wistar F WTG 0

5 10 15

Groups

Percentage Time

Social exploration

A Dom inant

Wistar

A Subordi nate Wistar

B WTG B Wistar

E Wistar F WTG 0

5 10 15 20

Groups

Percentage Time

Freeze

A Dom inant

Wistar

A Subordi nate Wistar

B WTG B Wistar

E Wistar F WTG 0

20 40 60 80 100

Groups

Percentage Time

Washing

A Dom inant

Wistar

A Subordi nate Wistar

B WTG B Wistar

E Wistar F WTG 0.0

0.2 0.4 0.6 0.8 1.0

Groups

Percentage Time

Being explored

A Dom inant

Wistar

A Subordi nate Wistar

B WTG B Wistar

E Wistar F WTG 0

2 4 6 8

Groups

Percentage Time

Being chased

A Dom inant

Wistar

A Subordi nate Wistar

B WTG B Wistar

E Wistar F WTG 0.0

0.5 1.0 1.5

Groups

Percentage Time

Keep an eye on the resident

A Dom inant

Wistar

A Subordi nate Wistar

B WTG B Wistar

E Wistar F WTG 0

10 20 30 40 50

Groups

Percentage Time

Figure 9. Average time spent on a certain type of behaviour (in percentage) during the resident intruder test per group. Error bars included. Different behaviours first row left to right: upright, rearing, being attacked. Second row: cage exploration, social exploration, freezing. Third row: washing, being explored by the resident, being chased by the resident. Fourth row: keep an eye on the resident.

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In figure 10 the mean amplitude of the temperature per day is shown. Resident intruder tests took place at day 4 and day 5.

After these tests a decrease in the amplitude is shown for all the groups. After day 5, a period of recovery is shown for all groups where the temperature returns to baseline. In general, Wistar rats show a larger amplitude compared to the WTG groups. The increase of amplitude at day=10 is due to the elevated plus maze experiment.

0 5 10 15 20

0.0 0.2 0.4 0.6 0.8

1.0 Subordinate A

WTG B Wistar B Wistar E WTG F Amplitude

Date

Amplitude temperature

Figure 10. Average amplitude of the temperature per day per group. Amplitude is calculated as the differences between the average of the light phase and the average of the dark phase for each day. Resident intruder test takes place at day=4 and day=5. Elevated plus maze takes place at day=10.

Elevated plus maze

Figure 11 shows the percentages of time spent on the open arm during the elevated plus maze.

There are significant differences between both the WTG rat groups compared to the Wistar control group E. One clear finding is the increase of percentage of time spent on the open arm by control WTG rats from group F compared to the Wistar groups.

EPM

A Domi nant Wista

r

A Subordinate Wista r

B WT G

B Wista r

E Wista r

F WT G 0

10 20 30 40 50

A Subordinate Wistar B WTG

B Wistar E Wistar F WTG A Dominant Wistar

*

**

Groups

Percentage Open Arm

Figure 11. Percentage time spent on the open arm during the elevated plus maze (in %). * significant difference (p<0,05) compared to WTG rats of colony B. ** significant difference (p<0,05) compared to the controls WTG of F.

5-HT1A sensitivity

In figure 12 the average temperature per group in intervals of five minutes represents the sensitivity for the selective 5-HT1A receptor ligand F15599. The 5-HT1A receptor ligand is known to decrease body temperature after injection. At t=0 all rats received the injection, resulting in a reduction in body temperature.

The WTG rats in the control group show the steepest decrease in body temperature after injection (more than two degrees). The group of interest, the Wistar rats from the mixed colonies, are close to the other Wistar groups and show an average decrease of two degrees Celsius. In figure 13 the maximum decrease of the body temperature due to the 5-HT1A agonist injection is shown. There are no significant differences between the groups. The Wistar rats of group F do not show abnormal values as in figure 12.

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Figure 12. The body temperature of the rats after the 5HT1A agonist injection. The injection is given at time point 0. Temperature is measured every 5 minutes.

5HT1a agonist injection

Subordinate A B WT

G B Wista

r

E Wista r

F WT G 0

1 2

3 Subordinate A

B WTG B Wistar E Wistar F WTG

Groups

Decrease in temperature

Figure 13. The maximum decrease in temperature after the 5HT1A agonist injection (in Celsius).

Anhedonia

Sucrose preference

In figure 14 the sucrose versus tap water intake in percentages per 24 hours is shown. While starting at different percentages, all the groups reach about the same common baseline.

Probably rats need to habituate a few days to the sucrose water. On day = 4 most of the groups seem to show a consistent similar percentage of sucrose intake. The resident intruder tests took place at day 4 and 5. After these days, a decrease in sucrose consumption is shown for all the groups. The groups with a steep decrease are the subordinate and dominant Wistar groups from setup A and the WTG control rats. The group Wistar rats from the mixed colony, Wistar rats of B, shows less decrease in consumption after defeat. The

control Wistar rats have the steadiest consumption and do not seem to be influenced by the stressors. All groups show recovery reflected in a return to baseline.

Effect RI on sucrose intake

3 4 5 6 7

75 80 85 90 95 100

A dominant A subordinate B WTG B Wistar E Wistar F WTG

day

% sucrose intake

Figure 14. Average consumption of 1% sucrose solution in percentages of total fluid intake.

Measurements were taken every 24 hours per group. Resident intruder tests took place at day= 4 and day= 5.

Pavlovian conditioning test

In figure 15 the activity increase in percentage is shown. Point zero in the figure is the baseline (activity = 100%), measured 30 minutes prior to the stimuli. At the start of point one the stimuli is given.

This point accounts for the time interval between the stimuli and the reward, named the anticipatory phase. Point two, three, four and five are the average activity recovery points per 15 minutes after the reward. In every group an increase in activity is found after the stimuli.

From day 4 onwards rats are trained to wait for 15 minutes after the conditioned sound, before the chocolate is given. At day 6 the Wistar rats of the mixed colony have the same response as the control WTG rats of F. The rest of the groups show a lower increase. Unfortunately this is not consistent. There is no constant pattern during the following days. Each group of rats behaves differently every day. Wistar rats of colony B do not show consistent copying behaviour towards the WTG rats they were housed with during the juvenile stage. However, they do not show consistent different behaviour compared to the other Wistar groups as well.

(14)

Day 6

0 1 2 3 4 5

0 200 400 600 800 1000

Activity increase (in percentage) Day 7

0 1 2 3 4 5

0 200 400 600 800 1000

Activity increase (in percentage)

Day 8

0 1 2 3 4 5

0 500 1000 1500

Activity increase (in percentage) Day 9

0 1 2 3 4 5

0 2000 4000 6000

Activity increase (in percentage)

Day 10

0 1 2 3 4 5

0 100 200 300

Subordinate A B WTG B Wistar E Wistar F WTG

Activity increase (in percentage)

Figure 15. The response in activity increase during different phases of the anhedonia test. On the x scale time is represented in numbers. 0 = 30 minutes before the stimuli, represented as the baseline. 1 = the anticipatory phase between stimuli and reward. 2 = the first 15 minutes after reward. 3 = 15 to 30 minutes after reward. 4 = 30 to 45 minutes after reward. 5 = 45 to 60 minutes after reward. Activity was measured every 5 minutes and summoned for a quarter. Activity is calculated as percentage of the baseline for each quarter of an hour.

(15)

Discussion Growth

Visually, in figure 3 three different sets of groups can be subdivided. Groups of rats with similar behavioural patterns have growth curves close to each other. There seems to be a relation between body weight and dominance (see figure 5). Overall, WTG rats weigh less compared to the Wistar rats, suggesting a faster growth curve for Wistar rats. These current results did not match with the results of previous research from Geerdink et al5. These differences in result can be explained by the differences in age. The WTG rats of this previous research were 8 to 10 days older compared to the Wistar rats when placed in the experimental setup. Besides age, housing condition can play a role in body weight. In a colony condition, more interaction and therefore more activity take place, causing an increase in energy expenditure.

Hierarchy

Hierarchy was determined by scoring the number of attacks and excessive grooming during the first 10 minutes of the dark phase.

To discriminate between rats hair dye was used.

The hair products used had an impact on the behaviour of the rats during the first couple of days after the marking. Wistar rats were given respectively red or black dye on either their hips or shoulders. WTG rats were bleached on either their hips or shoulders. These different dyes and body parts make the rats look different compared to situated in their natural environment. All rats were constantly trying to pull of the dye by grooming themselves and each other. This behaviour might have an impact on hierarchical behaviour. Despite of the marking of the rats, WTG rats were hard to distinguish. The Wistar rats in the mixed colony were expected to behave subordinate to the WTG rats. This was not consequently observed. Perhaps the differences in weight were the cause. Wistar rats were significantly heavier compared to the WTG rats.

Open field

The open field test took place at PND 76 before the resident intruder paradigm, forming a baseline to measure anxiety afterwards. In

previous research WTG rats showed more cautious behaviour compared to Wistar rats 17. This finding corresponds with the observations during the handling of the animals in their home cage; Wistar rats are less shy and more curious compared to the WTG rats. Based on this observation WTG rats can be expected to move less. During the open field test this expectation is confirmed: WTG rats are shyer.

Wistar rats could copy the behaviour of WTG rats by growing up together in the housing condition. On the other hand, these Wistar rats could learn from interactions with the WTG rats and adjust their own behaviour compared to Wistar rats growing up with other Wistar rats.

Replication of behaviour or significant deviant behaviour by these Wistar rats was not shown.

Clearly all the Wistar rat groups were less shy and explored more in the open field test.

The latency time is an indication for the time it takes to reach the middle zone for the first time.

WTG rats take significantly more time to reach this zone compared to all the Wistar groups.

One explanation for this phenomenon is less activity of the WTG, which is not the cause.

The shy behaviour of WTG rats might be the cause of the longer latency time. Housing conditions do not influence the behaviour of Wistar rats in the open field test. Although the Wistar rats that grew up with WTG rats seem to be slightly more careful (reflected by a longer latency time compared to the other Wistar rat groups), this deviant behaviour is not very convincing. Test results could be influenced by turbulent environmental factors during the test.

Resident intruder test

Wistar rats from the mixed colonies are expected to learn from the social interactions with the WTG rats and also learn to show improved defensive aggressive behaviour from the WTG rats. This is not reflected in the scored types of behaviour of the resident intruder tests. The percentage time being attacked is dependent on the resident rat.

WTG rats provoke the aggressive behaviour by the resident rat more and for a longer time.

Wistar rats that grow up with WTG rats do not behave differently in the resident intruder test compared to controls, causing similar behaviour of the resident (the difference of attacks is only

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