Materials and Reagents

1. Animals: C57/BL6N male mice (Janvier Labs)
   
2. Mineral oil (Sigma-Aldrich, catalog number: M-5904 )
   
3. Isoamyl acetate (banana odor) (Sigma-Aldrich, catalog number: W205508 )
   
4. Benzaldehyde (almond odor) (Sigma-Aldrich, catalog number: 418099 )
   
5. 20% ethanol
   Note: Mineral oil was used as a solvent, because of its odorless property as reported in the literature (Linster et al., 2009; Qiu et al., 2014; Slotnick and Restrepo, 2005; Tan et al., 2010). Almond and banana odors were used as novel, yet neutral odors(Yang et al., 2013; Yang and Crawley, 2009). However, other odors are also suitable to test in our conditions, such as hexanal.
   

Equipment

1. Standard individual plexiglass cage for mice (see Figure 1A)
   
2. Water bottle cap with sipper tube made in stainless steel (from now called Odor holder, see Figure 1B) (Tecniplast)
   
3. Filter paper (Thermo Fisher Scientific, see Figure 1C and Figure 2)
   
4. Opaque eppendorfs (1 ml volume) (Eppendorf, see Figure 1C)
   
5. Scissors
   
6. 10 µl pipet
   
7. 10 µl pipet tips
   
8. Tissue paper
   
9. Standard table to place the test cage
   
10. CCTV camera for an aerial view of the test
    
11. Standard computer (PC or Mac)
    
    
    Figure 1. Basic material to perform the olfactory habituation test. A) Test cage made in plexiglass (12 cm width x 30 cm length x 13 cm height), with a circular perforation (5 cm diameter) in one of the extremes. B) Odor holder made in stainless steel (5 cm height from the base to the top of the sipper tube; base of 5 cm diameter; the hole of the sipper tube of 3 mm diameter). C) Standard opaque Eppendorf (left) containing the odor solution and the filter paper (3 cm height x 1 cm width) used to administer the odor. This filter paper is introduced in the sipper tube of B).
    

Software

1. Video recording software (GrabBee, grabbee.software.informer.com)
   
2. Software to analyze exploratory behavior (Behav_Scor_v3.0_beta)
   

Procedure