# Effects of different social experiences on emotional state in mice

Sep 17, 2020

### Animals and housing conditions

The present study was conducted with 24 male C57BL/6J mice, purchased from a professional breeder (Charles River Laboratories, Research Models and Services, Germany GmbH, Sulzfeld, Germany) at the age of five weeks. Upon arrival, mice were housed in same-sex groups of 3 individuals per cage (Makrolon cages type III, 38 × 23 × 15 cm3), since in sub-adult male mice, the occurrence of escalated aggression is very unlikely. However, with the males becoming adult, the probability of escalated agonistic encounters increases. Therefore, at the age of nine weeks, mice were transferred to single housing conditions to avoid any escalated aggressive interactions. Please note that the question whether to house male laboratory mice singly or in groups is under ongoing discussion and there is still no “gold standard” regarding its solution. For current discussions about recommendations for male mouse housing see37,38. Cages were equipped with wood chips as bedding material (TierWohl Super, J. Rettenmaier & Söhne GmbH + Co.KG, Rosenberg, Germany), a wooden stick, a semi-transparent red plastic house (11.1 × 11.1 × 5.5 cm3, Tecniplast Deutschland GmbH, Hohenpeißenberg, Germany), and a paper tissue. Housing rooms were maintained at a reversed 12 h dark/light cycle with lights off at 8 a.m., a temperature of approximately 23 °C, and a relative humidity of about 50%. The animals had ad libitum access to water and food (Altromin 1324, Altromin Spezialfutter GmbH & Co. KG, Lage, Germany) until the beginning of the touchscreen training phase. From then on they were mildly food restricted to 90–95% of their ad libitum feeding weights in order to enhance their motivation to work for food rewards. As neither distinct negative effects of such a restricted feeding protocol39, nor an interference with judgement bias assessment17,18 could be detected in previous studies, we considered this method to not affect the emotional state of the mice itself. Weights were monitored on a daily basis using a digital scale (weighing capacity: 150 g, resolution: 0.1 g; CM 150-1 N, Kern, Ballingen, Germany).

In addition to the experimental animals, 16 group-housed adult female C57BL/6J mice and 5 single-housed adult male NMRI mice, purchased from Charles River Laboratories, were used to provide the test animals with social experiences.

### Ethics statement

All procedures complied with the regulations covering animal experimentation within Germany (Animal Welfare Act), the EU (European Communities Council DIRECTIVE 2010/63/EU), and the fundamental principles of the Basel Declaration, and were approved by the local (Gesundheits- und Veterinäramt Münster, Nordrhein-Westfalen) and federal authorities (Landesamt für Natur, Umwelt und Verbraucherschutz Nordrhein-Westfalen “LANUV NRW”, reference number 84-02.04.2015.A441).

### Experimental design

In this study, the effects of different social experiences on important correlates of animal emotions, comprising cognitive (judgement bias), behavioural (anxiety-like and exploratory behaviour) as well as physiological (stress hormone levels) measures, were investigated. The experiment comprised six phases: a handling phase, a training phase, a first cognitive judgement bias (CJB) test phase, an experience phase, a second CJB test phase, and a behavioural test phase (Fig. 1).

During the handling phase starting at PND 69, mice were first habituated to cup handling for 5 days and thereafter underwent daily training sessions to learn the discrimination task required for CJB testing, starting at PND 76. Afterwards, the animals’ initial CJB was assessed (start test phase 1: PND 223 ± 77; for details on CJB training and testing see following section).

During a subsequent experience phase starting at PND 230 ± 77, mice were exposed to one of three different experiences, each comprising three group-specific encounters, classified as either mildly “adverse”, “beneficial”, or “neutral”. Encounters took place under red light between 2:45 p.m. and 4:35 p.m. on 3 different days, always separated by a gap day. The mildly “adverse experience” group (AE group, n = 8) repeatedly encountered a dominant opponent of the aggressive NMRI strain40, with each confrontation lasting maximally 10 minutes30,41. Confrontations were terminated in cases of high aggression. The “beneficial experience” group (BE group, n = 8) was repeatedly presented with freshly collected urine of an unfamiliar C57BL/6J female for 10 minutes31. To provide all subjects with comparable experiences, we controlled for the females’ oestrus state. Since the time of oestrus in mice is relatively short42, urine from non-oestrous females was used in order to keep the total number of involved females low. The “neutral experience” group (NE group, n = 8) served as a control group and was repeatedly placed into a novel cage containing clean bedding material for 10 min.

Following the experience phase, CJB was assessed again to investigate the influence of the respective experience on the animals’ judgement bias (start test phase 2: PND 237 ± 77). In this second test phase, a so-called reminder was presented immediately before each test session. These reminders were introduced to acutely re-evoke the affective state the mice experienced during the encounters of the treatment phase. Reminders took place immediately before each test session of the second CJB test phase. For this purpose, mice were placed into a cage (Makrolon type II cage; 22 × 16 × 14 cm3) filled with bedding for 3 min. For AE mice, another 25 ml of soiled bedding from the home cage of the last NMRI male encountered were added. For BE mice, the same was done with soiled bedding from the home cage of the last female of which urine had been presented.

On the last day of each CJB test phase, faeces samples were obtained to assess corticosterone metabolite (FCM) concentrations. Finally, animals underwent a battery of standard behavioural tests for anxiety-like behaviour and exploratory locomotion (elevated plus maze test (EPM), dark-light test (DL), and open field test (OF); start: PND 245 ± 77). Before each test session, a reminder was presented again.

The allocation of mice to the treatment groups was pseudo-randomised, so that balanced numbers of mice with different learning speeds were present in each group. The testing order of mice was randomised once before the first CJB test and subsequently maintained for the following CJB and behavioural test sessions. As reminders were provided immediately before CJB testing as well as before the subsequent behavioural tests, blinding of the experimenter was not possible.

### The touchscreen-based cognitive judgement bias test

#### Procedure

The same apparatus as described previously was used28,36 (Bussey-Saksida Mouse Touch Screen Chambers, Model 80614, Campden Instruments Ltd., Loughborough, Leics., UK). Mice underwent daily touchscreen sessions at intervals of approximately 24 h on maximally 6 consecutive days. Before each session, each mouse was taken out of its home cage and weighed. In a red semi-transparent box (21 × 21 × 15 cm3) the animal was then transported to a separate room where it was placed into the touchscreen chamber. The session was started and ended after a maximum number of trials had been performed or after a training step-specific duration. All touchscreen sessions were conducted during the dark phase between 8.15 a.m. and 1 p.m.

The paradigm applied here was the same as described previously with minor modifications36. Briefly, mice were trained to distinguish between a positive and a negative condition (Fig. 2). The positive condition was signalled by a bar at the bottom (5 cm below upper edge) of the cue-presentation field, the negative condition by a bar at the top (1 cm below upper edge). Mice had to touch either the left or right touch field in response to the cues. A correct touch in the positive condition led to the delivery of a large reward (12 μl of sweet condensed milk, diluted 1:4 in tap water, in the following “SCM”). An incorrect touch resulted in the delivery of a small reward (4 μl of SCM). In the negative condition, correct touches led to the delivery of a small reward (4 μl of SCM), while incorrect touches resulted in a mild “punishment” (5 s time out and houselight on). Mice had to learn to touch the high-rewarded side in the positive condition and the small-rewarded side in the negative condition. The small-rewarded touch field was the same in both conditions. The association between condition and correct touch side was the same for each individual but counterbalanced between mice. For a detailed description of the training procedure please see the supplementary material. After successful training, animals underwent CJB testing. The two cognitive bias test phases took place on five consecutive days each. During each CJB test session, three types of ambiguous cues, interspersed with the learned reference cues, were presented. These were bars at three intermediate positions: near positive (NP, 4 cm below upper edge), middle (M, 3 cm below upper edge) and near negative (NN, 2 cm below upper edge). Touches in response to these ambiguous cues resulted in a neutral outcome (neither a reward nor a “punishment”). The animals’ judgements made in response to these cues indicated whether they interpreted them according to the positive (“optimistic” response) or negative (“pessimistic” response) reference cue, serving as a measure of CJB.

Each test session comprised 54 trials. Per session, each type of ambiguous cue was presented twice, interspersed with 48 training trials. Per test phase, each mouse was presented with each ambiguous cue ten times and each trained cue 120 times.

#### Behavioural measures

Responses to ambiguous cues served as a measure of the animals’ CJB. Touches according to the positive condition were defined as “optimistic” choices, touches according to the negative condition were defined as “pessimistic” choices. Out of all responses per condition, a “choice score” was calculated as previously28,36 according to the following formula:

$$Choice,Score = frac{{N,choices ( {text{“}}optimistic{text{”}} ) – N,choices ({text{“}}pessimistic{text{”}})}}{ N,choices ({text{“}}optimistic{text{”}} + {text{“}}pessimistic{text{”}})}$$

The choice score could range between − 1 to + 1. Higher scores indicated a higher proportion of “optimistic” choices and consequently a relatively positive CJB compared to lower scores. Please note that choice scores are not an absolute, but a relative measure of CJB and that the term was chosen for the sake of intuitiveness.

### Anxiety-like behaviour and exploratory locomotion

Mice were tested in three tests on anxiety-like behaviour and exploratory locomotion in the following order: the elevated plus-maze test (EPM), the dark-light test (DL) and the open field test (OF). The sequence of tests followed recommendations to schedule tests that are more sensitive to previous experience at the beginning of such a battery, and to conduct potentially more stressful tests towards the end43,44. Tests were carried out at intervals of at least 48 h and were performed in a room different from the housing room between 12:45 p.m. and 3:35 p.m. Test equipment was cleaned with 70% ethanol between subjects. Behaviour was recorded with a webcam (Logitech Webcam Pro 9000) and the animals’ movements during the EPM and OF were automatically analysed by the video tracking system ANY-maze (ANY-maze version 4.99, Stoelting Co., Wood Dale, IL, USA). Videos of the DL were analysed manually by an experienced observer (Sophie Siestrup). For apparatus descriptions and details about testing procedures see supplementary material.

### Faecal corticosterone metabolites

The basal levels of adrenocortical activity of the subjects were monitored non-invasively by measuring faecal corticosterone metabolites45,46,47 (FCMs). Faeces samples of each individual were collected on the last day of the first CJB test week (= before the experience phase) and on the last day of the second CJB test week (= after the experience phase). During the dark phase, a peak of FCMs can be found in the faeces 4–6 h after the exposure to a stressor45. For this reason, faeces samples were collected 5.5–8.5 h after an individual finished CJB testing to ensure that faeces collection could be finished in the dark phase. For sample collection, mice were placed in Makrolon cages type III with a thin layer of bedding material and clean enrichment items as present in the home cage. Water was available ad libitum. After the sampling period of 3 h, mice were transferred to novel clean cages together with the enrichment items. All faeces produced during this time were collected and frozen at − 20 °C. Faecal samples were dried and homogenised and aliquots of 0.05 g were extracted with 1 ml of 80% methanol. Samples were then analysed using a 5α-pregnane-3β, 11β, 21-triol-20-one enzyme immunoassay (for details see45,46). Intra- and inter-assay coefficients of variation were < 10% and < 12%, respectively.

### Data analysis

To check for the assumptions of parametric analysis, residuals of all data were analysed for heteroscedasticity and normal distribution graphically and using the Shapiro-Wilk normality test. If the assumptions were not met, data were transformed whenever possible (DL: latency to enter light compartment, logarithmic transformation). As CJB test data did not meet the assumptions of parametric analysis even after transformation, untransformed data were analysed using non-parametric tests.

Data from behavioural tests were analysed using a linear mixed effect model (LMM) with “experience” as fixed factor and “age” as random factor, followed by Holm-Bonferroni post hoc comparisons. Faecal corticosterone metabolite data were analysed using an LMM with “experience” and “time” as fixed factors, and “age” and “individual” as random factors.

In order to examine whether mice interpreted the conditions of the CJB test differently, data were pooled across animals for each condition and each test phase and analysed using the Friedman test. Post hoc comparisons between conditions were conducted using the Holm-Bonferroni-corrected Wilcoxon signed-rank test. The Wilcoxon signed-rank test was also used for within-group comparisons of choice scores before and after the experience phase. The Kruskal-Wallis test was used for between-group comparisons of choice scores. Subsequent post hoc comparisons were carried out using the Holm-Bonferroni-corrected Wilcoxon rank-sum test (unpaired).

Differences were considered significant at p ≤ 0.05. Whenever LMMs were used, effect sizes were calculated additionally to p-values as partial eta squared (η2p). Statistical analyses were performed using the software R48 (www.r-project.org, open source). Graphs were created using the software SigmaPlot for Windows (Version 12.5, Build 12.5.0.38, Systat Software, Inc. 2011).