(a-f) Conditioned place preference assay (CPP) under ad libitum fed (AL-fed) condition: DIO mice were treated with celastrol (100 μg/kg, i.p., once a day) for three days. On the fourth day, 1 h after the beginning of the light cycle, the mice were administered vehicle or celastrol (200 μg/kg, i.p.) and the CPP assay was performed 6 h later under AL-fed condition (n=8 for both vehicle- and celastrol-treated groups). (a) Total distance that the mice traveled during the test (P=0.941). (b) Average velocity of movement during the CPP assay (P=0.934). (c) Frequency (numbers during assay) that mice traveled to the food-paired side chamber (P=0.688). (d) Total time the mice spent in the food-paired side chamber (P=0.641). (e) Percentage of total assay time the mice spent in the food-paired side chamber (P=0.641). (f) Frequency (numbers during assay) that the mice traveled to the food-containing zone within the food-paired chamber (P=0.557). (g-l) CPP under 20-hour fasted condition: DIO mice were treated with celastrol (100 μg/kg, i.p.) once a day and fasted for 15 h after the third injection. On the fourth day, 1h after the beginning of the light cycle, the mice were administered vehicle or celastrol (200 μg/kg, i.p.) and the CPP assay was performed 5 h after this injection under fasting condition (n=7 for both vehicle- and celastrol-treated groups). (g) Total distance traveled by the mice during the CPP assay (P=0.02). (h) Average velocity of movement (P=0.02). (i) Frequency with which the mice traveled to the food-paired side chamber (P=0.104). (j) Total time the mice spent in the food-paired side chamber during the test (P=0.02). (k) Percent of total assay time the mice spent in the food-paired side chamber (P=0.02). (l) Frequency with which the mice traveled to the food-containing zone within the food-paired side chamber (P=0.01). (m,n) Representative traces showing movements of individual vehicle- and celastrol-treated mice in the CPP assays conducted under (m) AL-fed or (n) 20-hour fasted state. Values indicate average ± s.e.m. P values were determined by two-tailed Student t test. * P < 0.05, n.s., not significant (P>0.05).