Buried pellet test

The buried pellet test was performed for three consecutive days, with 12 h of overnight fasting in between. Each day of testing, the location of the buried food was changed. The testing paradigm was adapted from Lehmkuhl et al.²¹. The buried pellet testing was performed one week prior to the semi-automated olfactory testing described above.

Three days prior to the first day of testing, food was restricted for 12 h during the active phase for mice (dark cycle). When the mice had access to food, they were also given one piece of sweetened cereal (Cap’n Crunch) to avoid neophobic food behavior during testing. The starting weight of the animals was recorded prior to food restriction; if an animal lost 10% of its starting weight it was exempt from food restriction and subsequent testing.

For testing, animals were moved to individual clean cages containing 2 cm of fresh bedding, covered with a filter top lid, and allowed to acclimate for 5 min. After acclimation, the mouse was put in a clean holding cage while the researcher buried a sweetened cereal item 0.5 cm under the bedding in the testing cage. The mouse was then placed back in the test cage and the latency for the mouse to uncover and put its nose against the food was measured. Once the food was uncovered, the mouse was allowed to eat it. The maximum time allowed to find the food was 5 min; if a mouse was unable to find it within that time period, its latency was recorded as 300 s and it was given the food. Tested mice were not allowed back into their housing cage if it contained untested mice. When all animals in a cage had been tested, they were given free access to food. To avoid outside olfactory cues, the experimenter used double pairs of gloves and changed the outer pair in between mice.

To control for variability in motivation for food seeking behavior, the surface pellet test was also carried out. This was performed as described for the buried pellet test, but instead the food item was placed on the surface of the bedding. If a mouse was uninterested in the food item, it was excluded from subsequent testing. All mice in our study were motivated by this sweetened cereal.