Webcam pro 9000

An aquarium (112 L, 110 × 36.5 × 28 cm, Fig. 1A) with all sides covered with white plastic sheets on the inner sides was used as the experimental area. The aquarium was divided into three compartments (one experimental compartment and two stimulus compartments) by a removable transparent partition. The experimental compartment (48 × 36.5 × 28 cm) was in the middle of the aquarium, and the two stimulus compartments (31 × 36.5 × 28 cm) were at both ends of the aquarium. These two stimulus compartments either housed a predator or were kept empty alternatively during the measurements. To permit visual and chemical communications between prey and predator, we cut two rows of holes with diameters of approximately 0.5 cm each in the bottom half of the removable transparent partitions. The behavior of the test fish (in the experimental compartment) was recorded by a webcam (above the aquarium; Logitech Webcam, Pro 9000) connected to a remote monitor, and the experimental aquarium was illuminated by carefully placed fluorescent tubes.

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 end^{43 (link),44 (link)}. 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.

Behavioral tests on anxiety-like behavior and exploratory locomotion were performed in the dark period between 12:00 a.m. and 4:30 p.m., i.e., during the animals’ active phase. Individual testing order was randomized on each test day and all equipment used for the tests was cleaned with 70% ethanol between subjects.
Tests were performed in a room separate from the subjects’ housing room, where they were transported in an empty Makrolon type II. Behavior was recorded by a camera (Logitech Webcam Pro 9000) and the animals’ movements were automatically analyzed by the video tracking system ANY-maze (version 4.75, Stoelting Co., Wood Dale, IL, USA).

All behavioral paradigms were performed during the animals' active phase when the lights were off in the housing room (between 1 and 6 p.m.), and mice were tested according to a randomized daily order, which was balanced across treatments. Barrier Test, Novel Cage Test, Interaction Test, and Nest Test were executed under red light conditions in the housing room and evaluated by live observations. For the tests on anxiety-like and exploratory behavior (Elevated Plus Maze Test, Dark Light Test, Open Field Test) and on spatial learning (Labyrinth-Maze Test) the animals were transferred to a separate testing room using a darkened transport box. All tests on anxiety-like and exploratory behaviors were recorded by a camera (Logitech Webcam Pro 9000) and automatically analyzed in real time by the video-tracking system ANY-maze [v. 4.75 (Exp. 1)/v. 5.33 (Exp. 2), Stoelting Co.,Wood Dale, USA], while the Labyrinth-Maze Test was manually evaluated by live observation. Between subjects, all test equipment was cleaned with 70% ethanol. Mice were given a pause of at least 48 h between individual tests.

The EPM was conducted to examine exploratory locomotion and anxiety-like behaviour of the animals^{60 (link)}. The apparatus was elevated 50 cm above the ground and was composed of two opposing open (30 cm × 5 cm) and two opposing closed arms (30 cm × 5 cm) which were connected via a central square (5 cm × 5 cm). The closed arms were surrounded by 20 cm high walls. The EPM was illuminated from above (25 lx in the centre square). After spending 1 min in an empty box protected from light, the mouse was placed on the central platform facing a closed arm and was allowed to freely explore the apparatus for 5 min. During that time, the animal was recorded by a webcam (Webcam Pro 9000, Logitech) in the absence of the experimenter. Outcome measures taken were the relative amount of entries into and the relative time spent in the open arms [i.e. open arm entries or time/(open arm + closed arm entries or time)]. In addition, the number of protected head dips (‘mouse lowers its head over the side of an open arm with its ears protruding over the edge, while at least one paw remains in the closed segment or central platform’; cf.^{33 (link)}) was calculated relative to the total amount of head dips shown.

For courtship assay, virgin iso31 male flies were collected, housed in groups of ~10 in standard food for 1–2 days, and transferred to either 5% sucrose food or standard food. Flies were kept in these conditions for 7 days, with food renewed every 3 days. Virgin iso31 females were kept on standard food for 4–5 days in groups of ~10. Courtship assays were performed between ZT1 and ZT4. A male and female were gently aspirated into a plastic mating chamber (15 mm diameter and 3 mm depth) containing 5% sucrose and 2% agar and were kept separated until a divider was removed ~10 min later. Flies were recorded for 2 hr using a USB webcam (Logitech Webcam Pro 9000) and scored blind to experimental condition. Courtship index was determined as the fraction of total time a male was engaged in courtship activity during a 10 min period or until successful copulation after courtship initiation. Courtship activity included orienting, chasing, singing, and attempted copulation. For simultaneous analysis of courtship and sleep during the night, videos recorded under infrared light were manually scored for courtship and sleep during 5 min periods as described above.