Materials and Reagents

1. Latex gloves (NIPPON Genetics, catalog number: SLPF-M )
   
2. Paper towels (KCWW, Kimberly-Clark, catalog number: 47000 )
   
3. Filter tips
   10 μl (FUKAEKASEI and WATSON, catalog number: 1252-207CS )
   200 μl (FUKAEKASEI and WATSON, catalog number: 1252-703CS )
   1,000 μl (FUKAEKASEI and WATSON, catalog number: 124-1000S )
   
4. Laboratory-bred mice
   Note: Mice are housed in groups 3-5 per cage, kept in a room with controlled temperature (~23 °C) and humidity under 12 h light/dark cycle (lights on at 8:30 AM) with ad libitum access to food and water.
   
5. Eugenol (6.3 M) (Nacalai Tesque, catalog number: 15806-42 )
   
6. 70% ethanol
   

Equipment

1. Olfactometer with two channels (Matsumi Kagaku Keisoku)
   
2. Gas cylinder (normal air)
   
3. Test cage (25 x 37 x 24 cm, polypropylene)
   
4. Clear acrylic board (which can cover the roof of the test cage)
   
5. Video camera (Sony, catalog number: HDR-CX560V )
   Note: The mouse behavior is recorded under the weak-light condition. This model (Sony Nightshot Camcorder) is equipped with infrared-mode.
   
6. Tripod for camera
   

Note: The gas cylinder containing clean air is connected to the olfactometer. The olfactometer regulates the gas flow (0.5 L/min), and switches flow between clean air and air with an odor by passing the air through a bottle containing eugenol. The olfactometer, connected to the test cage (25 x 37 x 24 cm), can supply either the clean air or the odor through the gas port (0.5-mm diameter hole) on the wall (2-cm height). The roof of test cage is covered with clear acrylic board (Figure 1).


Figure 1. Apparatus of the olfactory habituation-dishabituation test

Software

1. Microsoft Excel (Microsoft)

Procedure