Materials and Reagents

1. Animals
   Laboratory-bred experimentally-naïve male mice. Both CD1 and C57BL/6J strains have been successfully tested. Male mice are housed in groups of 4-5 per cage, kept in an environment with controlled temperature (23 ± 2 °C) and humidity under a 12-12 h light-dark cycle with food and water ad libitum. The use of only one gender reduces inter-individual variation. Males are used instead of females to avoid cycling hormone effects associated to oestrus (Jemiolo et al., 1986).
   Note: All mice must be of equal or similar age (i.e. always less than two weeks apart, as the rate of neurogenesis is age-dependent). When this is not possible, cohorts of similar sizes for each experimental condition can be tested separately at different times, but this schedule results in greater variability and will, therefore, require a higher total number of animals. In most of our experiments a minimum of 7 mice were required to reach statistical significance. However, 10-12 mice per group are recommended to minimize the effects of inter-individual variation.
   
   
2. Odorants
   Odorants must have non-emotional value. We use two synthetic odorants: citralva (geranonitrile, 3,7-dimethyl-2,6-octadien-1-nitrile), a lemon-like citrus smell, and geraniol (3,7-dimethyl-2,6-octadien-1-ol), a sweet rose-like scent, at dilutions that range between 1:160 to 1:10. Both odorants have been tested before in olfactory experiments (Luo et al., 2002; Delgado et al., 2014) although others, i.e. banana odor (isoamyl acetate), almond odor (benzaldehyde), or hexanal, have also been used in similar tests (see, for example, Mandairon et al., 2006; Fleming et al., 2008). Odorants can be obtained from different suppliers, such as Ventós S.A (dealer of International Flavors and Fragrances Inc. Barcelona, Spain). Because synthetic fragrances are usually available in an oleic format, we use mineral oil for their dilution. It is important to keep them always in tightly sealed containers to minimize their evaporation.
   
   
3. Other materials
   1.Mineral oil (Sigma-Aldrich, catalog number: M5904 )
   

Equipment

Olfactory experiments are performed in a dedicated lab room with positive pressure (10-15 gauges) and frequent air reposition (no less than 20 times per hour) for fast removal of odors. Indirect dim lighting is recommended for testing. Essence dilutions should be prepared in a different, not connected room.
The materials used (see Figure 1) are:

1. A 22.5 x 22.5 x 29.5 cm open Plexiglas box with solid light green walls and a hole of 1 cm of diameter in one of the sides, located at 8 cm above the box ground and at 11.2 cm from the lateral corners of the box. A transparent methyl methacrylate solid cover is advisable to avoid essence dissipation during the tests. The video recording system is fixed above the exploration box at a distance of 30-40 cm from the transparent cover in order to video-tape at good resolution without interfering with the test.
   Note: The olfactory exploration box can be custom-made following the description. The box is thoroughly wiped clean with 10% alcohol and dried before and after the experiment, as well as between animals. After each test, the used soiled bedding has to be carefully removed with a household vacuum cleaner and replaced by fresh bedding. Use odorless disposable paper towels and nitrile gloves for cleaning and drying.
   
2. Regular cotton sticks bought in any drugstore
   
3. Lab timer
   
4. Micropipette (2-20 or 20-200 µl-range) and appropriate disposable tips
   
5. Adhesive tape
   
6. 1.5 ml microcentrifuge tubes to preserve the essences and a microcentrifuge tube rack to avoid fluid-containing tubes from tipping over
   
7. A sealed container (optional if the garbage bag is sealed) can be used for disposal of any objects that are impregnated with essences.
   

Procedure