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2.3.1 Open field test

The Open Field (OF) test is based on evoking a conflict between the animal’s exploratory drive and its evolutionary innate fear of novel and open areas. The OF apparatus consists of a black, open topped PVC cylinder (60 cm in diameter, 40 cm in height) mounted on a grey PVC floor plate (see Figure 4). The circular test area was divided into a more aversive, inner zone (30 cm in diameter) and a more protective, ring-shaped outer zone near the walls of the OF. The apparati were placed into test chambers surrounded on three sides by black PVC walls, the fourth side could be closed by a black curtain to prevent the experimental animals from visual or auditory cues. In all experiments, the test apparati were dimly and evenly illuminated with 15 lux, thereby reducing the averseness of the test situation since we were not interested in the assessment of anxiety-related behavioural parameters, but rather in the exploratory drive and locomotor activity of the mice. During the 10-min test period, the animals’ total distance of travelling, the time within each zone, as well as the ratio between the path length in the inner and outer zone were automatically assessed utilising the ANY-Maze Video Tracking Software (version 4.72, Stoelting Co., Wood Dale, IL, USA). After testing an animal, the OF was thoroughly cleaned with soapy water and 70% ethanol.

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Figure 4: Experimental setup of the Open Field (OF) test.

2.3.2 Forced swim test

The Forced Swim Test (FST) is widely used in pharmacological research using mice and rats to screen pharmaceutical compounds for their antidepressant activity. In the FST, animals are subjected to a psychological (inescapable), as well as physiological (swimming), stressful situation in order to monitor their depression-like behaviour and individual stress-coping style, respectively. This paradigm is especially demanding for mice since they rarely swim in their natural environment. Being placed in a glass beaker (12 cm in diameter, 23.5 cm in height) filled two thirds with 23 °C warm water (see Figure 5) and unable to touch the bottom with their hind paws or tail, the mice actively try to escape from the situation by attempting to climb out of the tank (struggling), followed by periods of active swimming. Eventually, the animals adopt immobile postures, so called floating (defined as complete immobility of the animals extremities excluding small movements to keep balance in the water), with the amount of immobility representing depression-like behaviour or behavioural despair. In our studies, the duration of struggling, swimming and floating behaviour during a 6-min FST period was scored by a trained observer blind to line and treatment using EVENTLOG software (EVENTLOG Event Recorder, version 1.0, EMCO software).

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Figure 5: Experimental setup of the Forced Swim Test (FST).

2.3.3 Y-Maze test

To assess the spatial, hippocampus-dependent learning and memory of the experimental animals, the Y-Maze test was used. The Y-Maze apparatus used in our tests consisted of three similarly shaped arms made of grey PVC, each 11 cm wide and 30 cm long, surrounded by 15 cm high walls and interconnected through a triangular central zone (see Figure 6). To facilitate the mice’ orientation in the maze, the wall of each arm was tagged with a distinctive symbol in the shape of a bar, a triangle or a plus, respectively. During the 10-min acquisition phase, one arm was blocked by a grey PVC inlay, leaving two arms for the mice to explore. To avoid a preference for one arm during the acquisition phase, the animals were first placed into the central zone facing the partition wall. After returning the animals for 1 h to their home cages (inter-trial interval), they were placed back into the test apparatus (retrieval phase), this time with the opportunity to explore all three arms of the maze. The retrieval phase lasted for 5 min. The apparati were placed in test chambers surrounded by black PVC walls and a black curtain to avoid additional distal orientation cues apart from the proximal cues positioned on the Y-Maze walls. After each animal and each trial the Y-Maze was thoroughly cleaned with soapy water and 70 % ethanol to prevent mutual interference of the test animals.

The retrieval phase of each animal was analysed regarding the time spent in each of the three arms using ANY-Maze Video Tracking Software (version 4.72, Stoelting Co., Wood Dale, IL, USA). If the experimental animals were able to discriminate between the

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familiar arms, i.e. the two arms open during the acquisition phase, and the novel arm, i.e. the newly opened arm during the retrieval phase, this was reflected by an extended amount of time spent in this new arm. In order to counterbalance differences in locomotion and therefore in total time spent in the respective arms, the mean percent time spent in the familiar arms was compared to the percent time spent in the novel arm. Additionally, a ‘discrimination ratio’ (% time spent in the novel arm/familiar arms) was calculated reflecting the accuracy of the animals’ memory performance. The higher the ratio, the more accurately the animals were assumed to remember the ‘old’, previously explored arms.

Figure 6: Experimental setup of the Y-maze test.