A Novel Automated Approach for Improving Standardization of the Marble Burying Test Enables Quantification of Burying Bouts and Activity Characteristics

Abstract

The marble burying test is a commonly used paradigm to describe phenotypes in mouse models of neurodevelopmental and psychiatric disorders. The current methodological approach relies predominantly on reporting the number of buried marbles at the end of the test. By measuring the proxy of the behavior (buried marbles), many important characteristics regarding the temporal aspect of this assay are lost. Here, we introduce a novel, automated method to quantify mouse behavior during the marble burying test with the focus on the burying bouts and movement dynamics. Using open-source software packages, we trained a supervised machine learning algorithm (the “classifier”) to distinguish burying behavior in freely moving mice. In order to confirm the classifier’s accuracy and characterize burying events in high detail, we performed the marble burying test in three mouse models: Ube3a m-/p+ [Angelman syndrome (AS) model], Shank2 −/− (autism model), and Sapap3 −/− [obsessive-compulsive disorder (OCD) model] mice. The classifier scored burying behavior accurately and consistent with the previously reported phenotype of the Ube3a m-/p+ mice, which showed decreased levels of burying compared with controls. Shank2 −/− mice showed a similar pattern of decreased burying behavior, which was not found in Sapap3 −/− mice. Tracking mouse behavior throughout the test revealed hypoactivity in Ube3a m-/p+ and hyperactivity in the Shank2 −/− mice, indicating that mouse activity is unrelated to burying behavior. Reducing activity with midazolam in Shank2 −/− mice did not alter the burying behavior. Together, we demonstrate that our classifier is an accurate method for the analysis of the marble burying test, providing more information than currently used methods.

Biallelic PAX5 mutations cause hypogammaglobulinemia, sensorimotor deficits, and autism spectrum disorder

“…(6) Elevated-plus maze ( Badura et al, 2018 ). (7) Marble burying test ( Wahl et al, 2022 ). (8) LocoMouse ( Machado et al, 2015 ).…”

Morphological and behavioral analysis of Slc35f1-deficient mice revealed no neurodevelopmental phenotype

“…Moreover, since SLC35F1 deficiency can lead to a phenotype that resembles Rett syndrome (Di Fede et al 2021 ), we analyzed the Slc35f1 mice in the marble-burying test. This test is commonly used to describe phenotypes in mouse models of neurodevelopmental and psychiatric disorders (Wahl et al 2022 ). For instance, hemizygous MeCP2-308 mice (a mouse RTT- model) display decreased digging and buried a lower number of marbles compared their wild-type littermates (De Filippis et al 2014 ).…”

Multi-parametric assays capture sex- and environment-dependent modifiers of behavioral phenotypes in autism mouse models

“…The marble burying test was performed as described previously 62 . In short, animals were transferred to a large cage (26,6 cm x 42,5 cm x 18,5 cm; Eurostand 1291H-type III H) filled with ∼4 cm of bedding and 20 evenly spaced out glass marbles and left for 30 minutes, after which the animals were removed.…”

“…In short, animals were transferred to a large cage (26,6 cm x 42,5 cm x 18,5 cm; Eurostand 1291H-type III H) filled with ∼4 cm of bedding and 20 evenly spaced out glass marbles and left for 30 minutes, after which the animals were removed. Burying behavior during the test was quantified using JAABA 62 .…”