Materials and Reagents

1. Paper towels and nitrile laboratory gloves
2. Waterproof marking pen
3. Laboratory rats (3 months old; weight between 200 and 240 g)
   However, individuals of a different age may be used in studies on developmental or aging processes. The rats are housed in groups of 4 per cage and kept in an environment with controlled temperature (22 ± 2 °C) and humidity (45-65%). The animals have a 12-12 h light-dark cycle and are provided with food and water ad libitum
4. Virkon^® S (Bayer) (used for cleaning purposes)
   Virkon^® S is a disinfectant in which the main component (49.4%) is a triple salt of potassium: monoperoxy sulphate, potassium hydrogen sulphate and potassium sulphate. It also contains 18% sodium hexanophosphate, 15% sodium dodecylbenzenesulphonate, 10% malic acid and 5% sulphamic acid

Equipment

1. Home cage (Tecniplast^®(Italy), 1500U Eurostandard Type IV)
2. Night vision video camera (BCS-THC3400IR-E, HD-CVI 4MPX IR 30M)
3. Wide angle infrared (IR) illuminator (IR Lab Ltd., model: LIR-CS32-940)
4. Transporter for rats
   A device made of opaque material, used to move the animals from the home cage to the test arena (Figure 1). In our experiments we used a custom made transporter constructed with two overlapping hard PVC tubes. Lifting the middle tube (with a string) reveals the entrance cut out in the outer tube. It is used to standardize the procedure and ensure the minimum stress level caused by human handling during the experiment. The transporter should be equipped with a pull-up door/gate. The gate must be left open during the whole trial in order to allow (but not force) the animal to leave the transporter or stay in it. It should be noted however, that any other similar solutions may be applied
   
   
   Figure 1. Cylindrical transporter used in our experiment
   Note: The exit gate (70 mm x 120 mm) should match the size of the entrance to the test arena.
   
   
5. Chamber for measuring exploratory behavior (Figure 2)
   This test arena is a large box (width = 800 mm, height = 800 mm, depth = 600 mm). The chamber is divided into three zones (A, B and C) which have been separated by two walls running perpendicularly to its longer side. The front and the top of the chamber are made of transparent walls that can be lifted to gain full access to the test arena. The wooden division walls between the zones have triangular entrances (120 mm x 140 mm) at the bottom, which enables the animals to move freely between the chamber parts. Dividers are placed at an angle, so as to prevent blind spots in the video recording from the top and from the front. The entrance to the chamber is placed in the central zone (A). The remaining zones, B and C, are where the objects allowing experimental manipulations are placed. In our recent studies (e.g., Pisula et al., 2019), we used wooden tunnels for this purpose (length = 240 mm, width = 120 mm, height = 80 mm). The entire chamber is covered with a layer of washable varnish. Wooden tunnels are covered with washable paint
6. Tunnels (Figure 2)
   In contrast to the most frequently used two-dimensional experimental settings, these tunnels provide a complex three-dimensional environment. In our experiments we used tunnels (200 mm x 120 mm x 80 mm) made of hardwood covered with washable paint. The tunnels are placed in zones B and C. The configuration of the tunnels is changed in one of the chamber zones before the first test trial and this change introduces novelty to the experimental plan. Tunnel configuration is changed by manually moving them or adding/subtracting tunnels in two zones of the chambers
   
   
   Figure 2. Chamber for measuring free exploration and reaction to low-stress novelty in rats and other small mammals. Frontal view through the transparent front wall.

Software

1. BORIS event logging software (Friard and Gamba, 2016; http://www.boris.unito.it/), for coding behaviors from recorded materials, or any other software enabling behavior coding on the basis of video recordings

Procedure