Beyond marbles buried: A multidimensional analysis of the marble burying test in the C57BL/6J mouse strain

Restricted and repetitive behaviors (RRBs), defined as responses with high frequency and for unusually long duration, are distinctive features of several psychiatric conditions, including autism spectrum disorder and obsessive-compulsive disorder. A common paradigm to assess RRBs in murine models is the marble burying test (MBT), whose main outcome is the total number of marbles buried under bedding substrate. Despite its widespread application for evaluating RRBs, the behavioral significance of marble burying remains highly debated in the literature. This study aimed to: i) systematically record all behavioral responses displayed by mice during the MBT to identify potential correlations with the number of marbles buried; ii) conduct a correlational analysis between MBT and other behavioral tasks designed to target RRBs; and iii) perform a principal component analysis (PCA) on parameters from MBT and elevated plus maze, a paradigm for anxiety-like behavior, in C57BL/6 J mice of both sexes. Results revealed sex differences in behavioral responses exhibited by mice during MBT, with males exhibiting higher levels of marble-oriented behaviors compared to females. The number of marbles buried was positively associated with both marble-directed digging and general displacement digging, as well as with sniffing behavior. PCA indicated that marble burying loaded on two principal components, highlighting the multidimensional nature of this behavioral response: one relative to anxiety-like behaviors, the other to context-dependent explorative/repetitive behaviors. Overall, our findings suggest that the number of marbles buried is a reliable proxy for the duration of digging behavior and a sensitive measure for detecting sex differences in rodent models.