Materials and Reagents

1. Mice should be at least 8-week-old
   Notes:
   1. We used 8-week-old C57BL/6J background mice of both sexes, it is important that the animals have the same age.
      
   2. It is possible to use this protocol with other strains of mice. It might be applicable to rats as well, but needs standardization of procedures, such as: size of the cage, depth of the bedding, size of the chow pellet, as well as food deprivation time.
      
   3. In females, estrus can effect olfactory discrimination. Considering this, we initially did statistical analysis in separating genders: wild-type (4 males and 5 females), heterozygote (3 males and 3 females) and conditional knock-out mice (4 males and 5 females). However, we observed no differences between genders, so in our final results, we used both genders in all groups. We recommend evaluating the differences between genders before deciding to use mixed-gender groups or not.
2. Filtered and autoclaved water
   
3. Chow pellets (Food stimulus)
   Notes:
   1. We used a 2 g pellet of the same chow the animals were regularly fed with.
      
   2. We used AIN-93G chow (for use during rapid growth, pregnancy and lactation, from Rhoster, described in Reeves et al. (1993), because it was the chow type regularly fed to our animals.
      
   3. Other types of pleasant foods have been previously used as stimulus, such as: Oreo cookie, Kellog’s Fruit Loops, chow covered in peanut butter and other. We found, however, that Kellog’s Fruit Loops did not stimulate foraging through olfactory cues in the mice. In our experiments, regular food chow pellets showed better results in foraging behavior than the Fruit Loops. This may be due to novelty induced hypophagia, so we recommend the use of food stimulus that the animals are accustomed to in order to avoid novelty induced hypophagia.

Equipment

1. Experimental animal room
   Note: An adequate procedure room is essential for the successful development of the behavior test, since it is sensitive to external distractions. This requirement should be carefully considered during the planning stages. An adequate procedure room consists of an isolated and silent experimentation room, where it is possible to avoid the entrance of people during the test.
   
2. Clean mouse cage of a regular size (30.5 cm length x 16 cm width x 16 cm height or similar) (Figure 1) (We used the M.I.C.E.^® Animal Care Systems cage [Animal Care Systems, catalog number: M/P 79010 ])
   Note: Do not use regular cage lids. The stainless-steel top which holds food and water interferes with observation and should not be included in the setup. If needed, it is best to use acrylic lids.
   
   
   Figure 1. Layout of the buried food-seeking test. The purpose of this experimental test is to measure the animal's ability to use olfactory cues for foraging. The main parameter measured in this test is the latency to find the hidden food. Latency is defined as the time between when the mouse was placed in the cage and when the mouse uncovered the food pellet. For the test, 8-week-old mice were deprived of food for 24 h but received water ad libitum. Next day, a 2 g pellet of regular chow was buried 8 cm beneath the surface of the fresh bedding in one end of a clean test cage. The site of animal placement and the site at which the pellet was buried remained constant.
   
   
3. Fresh cage bedding to create an 8 cm layer in each cage
   Notes:
   1. For each subject, a clean cage washed and dried by the animal care cage washing facility and clean bedding should be used. Do not re-use cages or bedding.
      
   2. Cage lining: we use Premium Hygienic Animal Bedding LIGNOCEL^® FS-14 to bury the pellet.
4. Portable digital scale (Denver Instrument, model: TR-403 , or equivalent scale)
   Note: To weigh the pellet chow, we used a digital scale (Denver Instrument, TR-403, Max = 410 g, d = 0.001g).
   
5. Digital stopwatch
   Note: The stopwatch is used to monitor the test length.
   
6. Digital video camera (optional, we used Sony Cyber-shot 14.1 mega pixels) (Sony, model: DSC-W330 )
   Notes:
   1. The presence of a human observer may influence animal behavior. The use of a video camera may help reduce human interference during the test.
      
   2. All videos were recorded in .avi format and viewed using VLC media player.

Procedure