Methodological approach: using a within-subjects design in the marble burying assay.

In 2016, the National Institutes of Health mandated the use of both male and female mice in funded research. The use of both sexes is an important variable to consider; however, it comes with negative consequences such as increased animal expenses. One way to combat these negatives is to explore the option of using a within-subjects design (repeated measures) in behavioral assays that historically use a between-subjects design. Our study aimed to determine if a within-subjects design can be utilized in the marble burying assay. The marble burying assay is used as a tool for screening putative anxiolytic compounds as the assay is thought to measure obsessive-compulsive disorder- or anxiety-like behaviors. First, we compared the effects of sex and digging medium (corn cob or Sani Chip) on the number of marbles buried using CD-1 mice. Second, we determined if mice would continue to bury marbles after repeated exposures to the test arena. Lastly, we tested three positive controls (buspirone, ketamine, and fluoxetine). We found that mice buried significantly more marbles within Sani Chip digging medium, and no sex differences were observed. Next, the number of marbles buried and locomotor activity remained consistent across four test sessions. The positive controls buspirone (3.2-10 mg/kg) ketamine (32 mg/kg), and fluoxetine (10 mg/kg) decreased the number of marbles buried using the within-subjects design. These data suggest that a within-subjects design is optimal for the marble burying assay as it will reduce the number of animals and increase statistical power.